# Patent

## Title
RNA interference mediated inhibition of vascular endothelial growth factor and vascular endothelial growth factor receptor gene expression using short interfering nucleic acid (siNA)

## Abstract

This invention relates to compounds, compositions, and methods useful for modulating VEGF and/or VEGFR gene expression using short interfering nucleic acid (siNA) molecules. This invention also relates to compounds, compositions, and methods useful for modulating the expression and activity of other genes involved in pathways of VEGF and/or VEGFR gene expression and/or activity by RNA interference (RNAi) using small nucleic acid molecules. In particular, the instant invention features small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methods used to modulate the expression of VEGF and/or VEGFR genes. The application also relates to methods of treating diseases and conditions associated with VEGF and/or VEGFR gene expression, such as ocular diseases and conditions, including age related macular degeneration (AMD) and diabetic retinopathy, as well as providing dosing regimens and treatment protocols.

## Background
This application is a continuation-in-part of U.S. patent application Ser. No. 10/962,898, filed Oct. 12, 2004, which is a continuation-in-part of U.S. patent application Ser. No. 10/944,611, filed Sep. 16, 2004, which is a continuation-in-part of U.S. patent application Ser. No. 10/844,076, filed May 11, 2004, which is a continuation-in-part of U.S. patent application Ser. No. 10/831,620, filed Apr. 23, 2004, which is a continuation-in-part of U.S. patent application Ser. No. 10/764,957, filed Jan. 26, 2004, which is a continuation-in-part of U.S. Ser. No. 10/670,011, filed Sep. 23, 2003, which is a continuation-in-part of both U.S. Ser. Nos. 10/665,255 and 10/664,767, filed Sep. 16, 2003, which are continuations-in-part of PCT/US03/05022, filed Feb. 20, 2003, which claims the benefit of U.S. Provisional Application No. 60/393,796 filed Jul. 3, 2002 and claims the benefit of U.S. Provisional Application No. 60/399,348 filed Jul. 29, 2002. This application is also a continuation-in-part of International Patent Application No. PCT/US04/16390, filed May 24, 2004, which is a continuation-in-part of U.S. patent application Ser. No. 10/826,966, filed Apr. 16, 2004, which is continuation-in-part of U.S. patent application Ser. No. 10/757,803, filed Jan. 14, 2004, which is a continuation-in-part of U.S. patent application Ser. No. 10/720,448, filed Nov. 24, 2003, which is a continuation-in-part of U.S. patent application Ser. No. 10/693,059, filed Oct. 23, 2003, which is a continuation-in-part of U.S. patent application Ser. No. 10/444,853, filed May 23, 2003, which is a continuation-in-part of International Patent Application No. PCT/US03/05346, filed Feb. 20, 2003, and a continuation-in-part of International Patent Application No. PCT/US03/05028, filed Feb. 20, 2003, both of which claim the benefit of U.S. Provisional Application No. 60/358,580 filed Feb. 20, 2002, U.S. Provisional Application No. 60/363,124 filed Mar. 11, 2002, U.S. Provisional Application No. 60/386,782 filed Jun. 6, 2002, U.S. Provisional Application No. 60/406,784 filed Aug. 29, 2002, U.S. Provisional Application No. 60/408,378 filed Sep. 5, 2002, U.S. Provisional Application No. 60/409,293 filed Sep. 9, 2002, and U.S. Provisional Application No. 60/440,129 filed Jan. 15, 2003. This application is also a continuation-in-part of International Patent Application No. PCT/US04/13456, filed Apr. 30, 2004, which is a continuation-in-part of U.S. patent application Ser. No. 10/780,447, filed Feb. 13, 2004, which is a continuation-in-part of U.S. patent application No. 10/427,160, filed Apr. 30, 2003, which is a continuation-in-part of International Patent Application No. PCT/US02/15876 filed May 17, 2002, which claims the benefit of U.S. Provisional Application No. 60/292,217, filed May 18, 2001, U.S. Provisional Application No. 60/362,016, filed Mar. 6, 2002, U.S. Provisional Application No. 60/306,883, filed Jul. 20, 2001, and U.S. Provisional Application No. 60/311,865, filed Aug. 13, 2001. This application is also a continuation-in-part of U.S. patent application Ser. No. 10/727,780 filed Dec. 3, 2003. This application is also a continuation-in-part of U.S. patent application Ser. No. 10/922,675 filed Aug. 20, 2004, which is a continuation-in-part of U.S. patent application Ser. No. 10/863,973, filed Jul. 7, 2004, which is a continuation-in-part of International Patent Application No. PCT/US03/04566, filed Feb. 14, 2003. This application also claims the benefit of U.S. Provisional Application No. 60/543,480, filed Feb. 10, 2004. The instant application claims the benefit of all the listed applications, which are hereby incorporated by reference herein in their entireties, including the drawings.


FIELD OF THE INVENTION
The present invention relates to compounds, compositions, and methods for the study, diagnosis, and treatment of traits, diseases and conditions that respond to the modulation of vascular endothelial growth factor (VEGF) and/or vascular endothelial growth factor receptor (e.g., VEGFR1, VEGFR2 and/or VEGFR3, collectively VEGFR) gene expression and/or activity. The present invention is also directed to compounds, compositions, and methods relating to traits, diseases and conditions that respond to the modulation of expression and/or activity of genes involved in vascular endothelial growth factor (VEGF) and/or vascular endothelial growth factor receptor (VEGFR) gene expression pathways or other cellular processes that mediate the maintenance or development of such traits, diseases and conditions. Specifically, the invention relates to small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of mediating or that mediate RNA interference (RNAi) against VEGF and/or VEGFR gene expression. Such small nucleic acid molecules are useful, for example, in providing compositions for treatment of traits, diseases and conditions that can respond to modulation of VEGF and/or VEGFR gene expression in a subject or organism, such proliferative diseases and conditions, ocular diseases and conditions, dermatological diseases and conditions, and any other disease, condition, trait or indication that can respond to the level of VEGF and/or VEGFR gene expression in a cell or tissue.
The sequence listing submitted on compact disc, in compliance with 37 C.F.R. § 1.52(e)(5), in incorporated by reference. Two separate compact discs are submitted, each containing the file “02-742-W SeqList.Txt” (1,529,856 bytes in size), each created on CD on Jun. 6, 2006.
BACKGROUND OF THE INVENTION
The following is a discussion of relevant art pertaining to RNAi. The discussion is provided only for understanding of the invention that follows. The summary is not an admission that any of the work described below is prior art to the claimed invention.
RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Fire et al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286, 950-951; Lin et al., 1999, Nature, 402, 128-129; Sharp, 1999, Genes & Dev., 13:139-141; and Strauss, 1999, Science, 286, 886). The corresponding process in plants (Heifetz et al., International PCT Publication No. WO 99/61631) is commonly referred to as post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes and is commonly shared by diverse flora and phyla (Fire et al., 1999, Trends Genet., 15, 358). Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or from the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA or viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response through a mechanism that has yet to be fully characterized. This mechanism appears to be different from other known mechanisms involving double stranded RNA-specific ribonucleases, such as the interferon response that results from dsRNA-mediated activation of protein kinase PKR and 2′,5′-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L (see for example U.S. Pat. Nos. 6,107,094; 5,898,031; Clemens et al., 1997, J. Interferon & Cytokine Res., 17, 503-524; Adah et al., 2001, Curr. Med. Chem., 8, 1189).
The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer (Bass, 2000, Cell, 101, 235; Zamore et al., 2000, Cell, 101, 25-33; Hammond et al., 2000, Nature, 404, 293). Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Bass, 2000, Cell, 101, 235; Berstein et al., 2001, Nature, 409, 363). Short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes (Zamore et al., 2000, Cell, 101, 25-33; Elbashir et al., 2001, Genes Dev., 15, 188). Dicer has also been implicated in the excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001, Science, 293, 834). The RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).
RNAi has been studied in a variety of systems. Fire et al., 1998, Nature, 391, 806, were the first to observe RNAi in C. elegans. Bahramian and Zarbl, 1999, Molecular and Cellular Biology, 19, 274-283 and Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated by dsRNA in mammalian systems. Hammond et al., 2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001, Nature, 411, 494 and Tuschl et al., International PCT Publication No. WO 01/75164, describe RNAi induced by introduction of duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells. Recent work in Drosophila embryonic lysates (Elbashir et al., 2001, EMBO J., 20, 6877 and Tuschl et al., International PCT Publication No. WO 01/75164) has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies have shown that 21-nucleotide siRNA duplexes are most active when containing 3′-terminal dinucleotide overhangs. Furthermore, complete substitution of one or both siRNA strands with 2′-deoxy (2′-H) or 2′-O-methyl nucleotides abolishes RNAi activity, whereas substitution of the 3′-terminal siRNA overhang nucleotides with 2′-deoxy nucleotides (2′-H) was shown to be tolerated. Single mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity. In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5′-end of the siRNA guide sequence rather than the 3′-end of the guide sequence (Elbashir et al., 2001, EMBO J., 20, 6877). Other studies have indicated that a 5′-phosphate on the target-complementary strand of a siRNA duplex is required for siRNA activity and that ATP is utilized to maintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309).
Studies have shown that replacing the 3′-terminal nucleotide overhanging segments of a 21-mer siRNA duplex having two-nucleotide 3′-overhangs with deoxyribonucleotides does not have an adverse effect on RNAi activity. Replacing up to four nucleotides on each end of the siRNA with deoxyribonucleotides has been reported to be well tolerated, whereas complete substitution with deoxyribonucleotides results in no RNAi activity (Elbashir et al., 2001, EMBO J., 20, 6877 and Tuschl et al., International PCT Publication No. WO 01/75164). In addition, Elbashir et al., supra, also report that substitution of siRNA with 2′-O-methyl nucleotides completely abolishes RNAi activity. Li et al., International PCT Publication No. WO 00/44914, and Beach et al., International PCT Publication No. WO 01/68836 preliminarily suggest that siRNA may include modifications to either the phosphate-sugar backbone or the nucleoside to include at least one of a nitrogen or sulfur heteroatom, however, neither application postulates to what extent such modifications would be tolerated in siRNA molecules, nor provides any further guidance or examples of such modified siRNA. Kreutzer et al., Canadian Patent Application No. 2,359,180, also describe certain chemical modifications for use in dsRNA constructs in order to counteract activation of double-stranded RNA-dependent protein kinase PKR, specifically 2′-amino or 2′-O-methyl nucleotides, and nucleotides containing a 2′-O or 4′-C methylene bridge. However, Kreutzer et al. similarly fails to provide examples or guidance as to what extent these modifications would be tolerated in dsRNA molecules.
Parrish et al., 2000, Molecular Cell, 6, 1077-1087, tested certain chemical modifications targeting the unc-22 gene in C. elegans using long (>25 nt) siRNA transcripts. The authors describe the introduction of thiophosphate residues into these siRNA transcripts by incorporating thiophosphate nucleotide analogs with T7 and T3 RNA polymerase and observed that RNAs with two phosphorothioate modified bases also had substantial decreases in effectiveness as RNAi. Further, Parrish et al. reported that phosphorothioate modification of more than two residues greatly destabilized the RNAs in vitro such that interference activities could not be assayed. Id. at 1081. The authors also tested certain modifications at the 2′-position of the nucleotide sugar in the long siRNA transcripts and found that substituting deoxynucleotides for ribonucleotides produced a substantial decrease in interference activity, especially in the case of Uridine to Thymidine and/or Cytidine to deoxy-Cytidine substitutions. Id. In addition, the authors tested certain base modifications, including substituting, in sense and antisense strands of the siRNA, 4-thiouracil, 5-bromouracil, 5-iodouracil, and 3-(aminoallyl)uracil for uracil, and inosine for guanosine. Whereas 4-thiouracil and 5-bromouracil substitution appeared to be tolerated, Parrish reported that inosine produced a substantial decrease in interference activity when incorporated in either strand. Parrish also reported that incorporation of 5-iodouracil and 3-(aminoallyl)uracil in the antisense strand resulted in a substantial decrease in RNAi activity as well.
The use of longer dsRNA has been described. For example, Beach et al., International PCT Publication No. WO 01/68836, describes specific methods for attenuating gene expression using endogenously-derived dsRNA. Tuschl et al., International PCT Publication No. WO 01/75164, describe a Drosophila in vitro RNAi system and the use of specific siRNA molecules for certain functional genomic and certain therapeutic applications; although Tuschl, 2001, Chem. Biochem., 2, 239-245, doubts that RNAi can be used to cure genetic diseases or viral infection due to the danger of activating interferon response. Li et al., International PCT Publication No. WO 00/44914, describe the use of specific long (141 bp-488 bp) enzymatically synthesized or vector expressed dsRNAs for attenuating the expression of certain target genes. Zernicka-Goetz et al., International PCT Publication No. WO 01/36646, describe certain methods for inhibiting the expression of particular genes in mammalian cells using certain long (550 bp-714 bp), enzymatically synthesized or vector expressed dsRNA molecules. Fire et al., International PCT Publication No. WO 99/32619, describe particular methods for introducing certain long dsRNA molecules into cells for use in inhibiting gene expression in nematodes. Plaetinck et al., International PCT Publication No. WO 00/01846, describe certain methods for identifying specific genes responsible for conferring a particular phenotype in a cell using specific long dsRNA molecules. Mello et al., International PCT Publication No. WO 01/29058, describe the identification of specific genes involved in dsRNA-mediated RNAi. Pachuck et al., International PCT Publication No. WO 00/63364, describe certain long (at least 200 nucleotide) dsRNA constructs. Deschamps Depaillette et al., International PCT Publication No. WO 99/07409, describe specific compositions consisting of particular dsRNA molecules combined with certain anti-viral agents. Waterhouse et al., International PCT Publication No. 99/53050 and 1998, PNAS, 95, 13959-13964, describe certain methods for decreasing the phenotypic expression of a nucleic acid in plant cells using certain dsRNAs. Driscoll et al., International PCT Publication No. WO 01/49844, describe specific DNA expression constructs for use in facilitating gene silencing in targeted organisms.
Others have reported on various RNAi and gene-silencing systems. For example, Parrish et al., 2000, Molecular Cell, 6, 1077-1087, describe specific chemically-modified dsRNA constructs targeting the unc-22 gene of C. elegans. Grossniklaus, International PCT Publication No. WO 01/38551, describes certain methods for regulating polycomb gene expression in plants using certain dsRNAs. Churikov et al., International PCT Publication No. WO 01/42443, describe certain methods for modifying genetic characteristics of an organism using certain dsRNAs. Cogoni et al, International PCT Publication No. WO 01/53475, describe certain methods for isolating a Neurospora silencing gene and uses thereof. Reed et al., International PCT Publication No. WO 01/68836, describe certain methods for gene silencing in plants. Honer et al., International PCT Publication No. WO 01/70944, describe certain methods of drug screening using transgenic nematodes as Parkinson's Disease models using certain dsRNAs. Deak et al., International PCT Publication No. WO 01/72774, describe certain Drosophila-derived gene products that may be related to RNAi in Drosophila. Arndt et al., International PCT Publication No. WO 01/92513 describe certain methods for mediating gene suppression by using factors that enhance RNAi. Tuschl et al., International PCT Publication No. WO 02/44321, describe certain synthetic siRNA constructs. Pachuk et al., International PCT Publication No. WO 00/63364, and Satishchandran et al., International PCT Publication No. WO 01/04313, describe certain methods and compositions for inhibiting the function of certain polynucleotide sequences using certain long (over 250 bp), vector expressed dsRNAs. Echeverri et al., International PCT Publication No. WO 02/38805, describe certain C. elegans genes identified via RNAi. Kreutzer et al., International PCT Publications Nos. WO 02/055692, WO 02/055693, and EP 1144623 B1 describes certain methods for inhibiting gene expression using dsRNA. Graham et al., International PCT Publications Nos. WO 99/49029 and WO 01/70949, and AU 4037501 describe certain vector expressed siRNA molecules. Fire et al., U.S. Pat. No. 6,506,559, describe certain methods for inhibiting gene expression in vitro using certain long dsRNA (299 bp-1033 bp) constructs that mediate RNAi. Martinez et al., 2002, Cell, 110, 563-574, describe certain single stranded siRNA constructs, including certain 5′-phosphorylated single stranded siRNAs that mediate RNA interference in Hela cells. Harborth et al., 2003, Antisense & Nucleic Acid Drug Development, 13, 83-105, describe certain chemically and structurally modified siRNA molecules. Chiu and Rana, 2003, RNA, 9, 1034-1048, describe certain chemically and structurally modified siRNA molecules. Woolf et al., International PCT Publication Nos. WO 03/064626 and WO 03/064625 describe certain chemically modified dsRNA constructs. Hornung et al., 2005, Nature Medicine, 11, 263-270, describe the sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7. Judge et al., 2005, Nature Biotechnology, Published online: 20 Mar. 2005, describe the sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Yuki et al., International PCT Publication Nos. WO 05/049821 and WO 04/048566, describe certain methods for designing short interfering RNA sequences and certain short interfering RNA sequences with optimized activity. Saigo et al., US Patent Application Publication No. US20040539332, describe certain methods of designing oligo- or polynucleotide sequences, including short interfering RNA sequences, for achieving RNA interference. Tei et al., International PCT Publication No. WO 03/044188, describe certain methods for inhibiting expression of a target gene, which comprises transfecting a cell, tissue, or individual organism with a double-stranded polynucleotide comprising DNA and RNA having a substantially identical nucleotide sequence with at least a partial nucleotide sequence of the target gene.
SUMMARY OF THE INVENTION
This invention relates to compounds, compositions, and methods useful for modulating the expression of genes, such as those genes associated with angiogenesis and proliferation, using short interfering nucleic acid (siNA) molecules. This invention further relates to compounds, compositions, and methods useful for modulating the expression and activity of vascular endothelial growth factor (VEGF) and/or vascular endothelial growth factor receptor (e.g., VEGFR1, VEGFR2, VEGFR3) genes, or genes involved in VEGF and/or VEGFR pathways of gene expression and/or VEGF activity by RNA interference (RNAi) using small nucleic acid molecules. In particular, the instant invention features small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methods used to modulate the expression of VEGF and/or VEGFR genes and/or other genes involved in VEGF and/or VEGFR mediated angiogenesis in a subject or organism.
A siNA of the invention can be unmodified or chemically-modified. A siNA of the instant invention can be chemically synthesized, expressed from a vector or enzymatically synthesized. The instant invention also features various chemically-modified synthetic short interfering nucleic acid (siNA) molecules capable of modulating target gene expression or activity in cells by RNA interference (RNAi). The use of chemically-modified siNA improves various properties of native siNA molecules through increased resistance to nuclease degradation in vivo and/or through improved cellular uptake. Further, contrary to earlier published studies, siNA having multiple chemical modifications retains its RNAi activity. The siNA molecules of the instant invention provide useful reagents and methods for a variety of therapeutic, cosmetic, veterinary, diagnostic, target validation, genomic discovery, genetic engineering, and pharmacogenomic applications.
In one embodiment, the invention features one or more siNA molecules and methods that independently or in combination modulate the expression of VEGF and/or VEGFR target genes, such as genes that are associated with the maintenance and/or development of inflammatory diseases and conditions, respiratory diseases and conditions, allergic diseases and conditions, autoimmune diseases and conditions, neurologic diseases and conditions, ocular diseases and conditions, and cancer and other proliferative diseases and conditions, such as genes encoding sequences comprising those sequences referred to by GenBank Accession Nos. shown in Table I, referred to herein generally as VEGF and/or VEGFR, and also those sequences referred to by GenBank Accession Nos. shown in U.S. Ser. Nos. 10/923,536 and 10/923,536, both incorporated by reference herein, referred to herein generally as “target” sequences. The description below of the various aspects and embodiments of the invention is provided with reference to the exemplary VEGF (e.g., VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D) and VEGFR (e.g., VEGFR1, VEGFR2, VEGFR3) genes referred to herein as VEGF and VEGFR respectively. However, the various aspects and embodiments are also directed to other genes, such as VEGF and/or VEGFR gene homologs, transcript variants, and polymorphisms (e.g., single nucleotide polymorphism, (SNPs)) associated with certain VEGF and/or VEGFR genes. The various aspects and embodiments are also directed to other genes that are involved in VEGF and/or VEGFR mediated pathways of signal transduction or gene expression that are involved in the progression, development, and/or maintenance of disease (e.g., cancer, inflammatory disease, allergic disease, autoimmune disease, ocular disease, or other angiogenesis/neovascularization related diseases and conditions), such as interleukins, including for example IL-4, IL-4 receptor, IL-13, and IL-13 receptor. These additional genes can be analyzed for target sites using the methods described for VEGF and/or VEGFR genes herein. Thus, the modulation of other genes and the effects of such modulation of the other genes can be performed, determined, and measured as described herein.
In one embodiment, the invention features a double stranded nucleic acid molecule, such as an siNA molecule, where one of the strands comprises nucleotide sequence having complementarity to a predetermined nucleotide sequence in a target nucleic acid molecule, or a portion thereof. In one embodiment, the predetermined nucleotide sequence is a nucleotide target sequence described herein. In another embodiment, the predetermined nucleotide sequence is a target sequence as is known in the art.
In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a target gene or that directs cleavage of a target RNA, wherein said siNA molecule comprises about 15 to about 28 base pairs.
In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a target RNA, wherein said siNA molecule comprises about 15 to about 28 base pairs.
In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a target RNA via RNA interference (RNAi), wherein the double stranded siNA molecule comprises a first and a second strand, each strand of the siNA molecule is about 18 to about 28 nucleotides in length, the first strand of the siNA molecule comprises nucleotide sequence having sufficient complementarity to the target RNA for the siNA molecule to direct cleavage of the target RNA via RNA interference, and the second strand of said siNA molecule comprises nucleotide sequence that is complementary to the first strand.
In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a target RNA via RNA interference (RNAi), wherein the double stranded siNA molecule comprises a first and a second strand, each strand of the siNA molecule is about 18 to about 23 nucleotides in length, the first strand of the siNA molecule comprises nucleotide sequence having sufficient complementarity to the target RNA for the siNA molecule to direct cleavage of the target RNA via RNA interference, and the second strand of said siNA molecule comprises nucleotide sequence that is complementary to the first strand.
In one embodiment, the invention features a chemically synthesized double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a target RNA via RNA interference (RNAi), wherein each strand of the siNA molecule is about 18 to about 28 nucleotides in length; and one strand of the siNA molecule comprises nucleotide sequence having sufficient complementarity to the target RNA for the siNA molecule to direct cleavage of the target RNA via RNA interference.
In one embodiment, the invention features a chemically synthesized double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a target RNA via RNA interference (RNAi), wherein each strand of the siNA molecule is about 18 to about 23 nucleotides in length; and one strand of the siNA molecule comprises nucleotide sequence having sufficient complementarity to the target RNA for the siNA molecule to direct cleavage of the target RNA via RNA interference.
In one embodiment, the invention features a siNA molecule that down-regulates expression of a target gene or that directs cleavage of a target RNA, for example, wherein the target gene or RNA comprises protein encoding sequence. In one embodiment, the invention features a siNA molecule that down-regulates expression of a target gene or that directs cleavage of a target RNA, for examples wherein the target gene or RNA comprises non-coding sequence or regulatory elements involved in target gene expression (e.g., non-coding RNA).
In one embodiment, a siNA of the invention is used to inhibit the expression of VEGF and/or VEGFR genes or a VEGF and/or VEGFR gene family (e.g., one or more VEGF and/or VEGFR isoforms), wherein the genes or gene family sequences share sequence homology. Such homologous sequences can be identified as is known in the art, for example using sequence alignments. siNA molecules can be designed to target such homologous sequences, for example using perfectly complementary sequences or by incorporating non-canonical base pairs, for example mismatches and/or wobble base pairs, that can provide additional target sequences. In instances where mismatches are identified, non-canonical base pairs (for example, mismatches and/or wobble bases) can be used to generate siNA molecules that target more than one gene sequence. In a non-limiting example, non-canonical base pairs such as UU and CC base pairs are used to generate siNA molecules that are capable of targeting sequences for differing polynucleotide targets that share sequence homology. As such, one advantage of using siNAs of the invention is that a single siNA can be designed to include nucleic acid sequence that is complementary to the nucleotide sequence that is conserved between the homologous genes. In this approach, a single siNA can be used to inhibit expression of more than one gene instead of using more than one siNA molecule to target the different genes.
In one embodiment, the invention features a siNA molecule having RNAi activity against target RNA (e.g., coding or non-coding RNA), wherein the siNA molecule comprises a sequence complementary to any RNA sequence, such as those sequences having GenBank Accession Nos. shown in GenBank Accession Nos. shown in Table I and in U.S. Ser. Nos. 10/923,536 and 10/923,536, both incorporated by reference herein. In another embodiment, the invention features a siNA molecule having RNAi activity against target RNA, wherein the siNA molecule comprises a sequence complementary to an RNA having variant encoding sequence, for example other mutant genes known in the art to be associated with the maintenance and/or development of diseases, traits, disorders, and/or conditions described herein or otherwise known in the art. Chemical modifications as shown in Tables III and IV or otherwise described herein can be applied to any siNA construct of the invention. In another embodiment, a siNA molecule of the invention includes a nucleotide sequence that can interact with nucleotide sequence of a target gene and thereby mediate silencing of target gene expression, for example, wherein the siNA mediates regulation of target gene expression by cellular processes that modulate the chromatin structure or methylation patterns of the target gene and prevent transcription of the target gene.
In one embodiment, the invention features a siNA molecule having RNAi activity against VEGF and/or VEGFR RNA, wherein the siNA molecule comprises a sequence complementary to any RNA having VEGF and/or VEGFR encoding sequence, such as those sequences having VEGF and/or VEGFR GenBank Accession Nos. shown in Table I. In another embodiment, the invention features a siNA molecule having RNAi activity against VEGF and/or VEGFR RNA, wherein the siNA molecule comprises a sequence complementary to an RNA having other VEGF and/or VEGFR encoding sequence, for example, mutant VEGF and/or VEGFR genes, splice variants of VEGF and/or VEGFR genes, VEGF and/or VEGFR variants with conservative substitutions, and homologous VEGF and/or VEGFR ligands and receptors. Chemical modifications as shown in Tables III and IV or otherwise described herein can be applied to any siNA construct of the invention.
In one embodiment, siNA molecules of the invention are used to down regulate or inhibit the expression of proteins arising from VEGF and/or VEGFR haplotype polymorphisms that are associated with a trait, disease or condition. Analysis of genes, or protein or RNA levels can be used to identify subjects with such polymorphisms or those subjects who are at risk of developing traits, conditions, or diseases described herein (see for example Silvestri et al., 2003, Int J Cancer., 104, 310-7). These subjects are amenable to treatment, for example, treatment with siNA molecules of the invention and any other composition useful in treating diseases related to VEGF and/or VEGFR gene expression. As such, analysis of VEGF and/or VEGFR protein or RNA levels can be used to determine treatment type and the course of therapy in treating a subject. Monitoring of VEGF and/or VEGFR protein or RNA levels can be used to predict treatment outcome and to determine the efficacy of compounds and compositions that modulate the level and/or activity of certain VEGF and/or VEGFR proteins associated with a trait, condition, or disease.
In one embodiment, siNA molecules of the invention are used to down regulate or inhibit the expression of soluble VEGF receptors (e.g. sVEGFR1 or sVEGFR2). Analysis of soluble VEGF receptor levels can be used to identify subjects with certain cancer types. These cancers can be amenable to treatment, for example, treatment with siNA molecules of the invention and any other chemotherapeutic composition. As such, analysis of soluble VEGF receptor levels can be used to determine treatment type and the course of therapy in treating a subject. Monitoring of soluble VEGF receptor levels can be used to predict treatment outcome and to determine the efficacy of compounds and compositions that modulate the level and/or activity of VEGF receptors (see for example Pavco U.S. Ser. No. 10/438,493, incorporated by reference herein in its entirety including the drawings).
In one embodiment of the invention a siNA molecule comprises an antisense strand comprising a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof encoding a target VEGF and/or VEGFR protein. The siNA further comprises a sense strand, wherein said sense strand comprises a nucleotide sequence of a target VEGF and/or VEGFR gene or a portion thereof.
In another embodiment, a siNA molecule comprises an antisense region comprising a nucleotide sequence that is complementary to a nucleotide sequence encoding a target VEGF and/or VEGFR protein or a portion thereof. The siNA molecule further comprises a sense region, wherein said sense region comprises a nucleotide sequence of a target VEGF and/or VEGFR gene or a portion thereof.
In another embodiment, the invention features a siNA molecule comprising nucleotide sequence, for example, nucleotide sequence in the antisense region of the siNA molecule that is complementary to a nucleotide sequence or portion of sequence of a target VEGF and/or VEGFR gene. In another embodiment, the invention features a siNA molecule comprising a region, for example, the antisense region of the siNA construct, complementary to a sequence comprising a target VEGF and/or VEGFR gene sequence or a portion thereof.
In one embodiment, the sense region or sense strand of a siNA molecule of the invention is complementary to that portion of the antisense region or antisense strand of the siNA molecule that is complementary to a target VEGF and/or VEGFR polynucleotide sequence.
In yet another embodiment, the invention features a siNA molecule comprising a sequence, for example, the antisense sequence of the siNA construct, complementary to a sequence or portion of sequence comprising sequence represented by GenBank Accession Nos. shown in U.S. Ser. Nos. 10/923,536 and 10/923,536, both incorporated by reference herein. Chemical modifications in Table I and otherwise described herein can be applied to any siNA construct of the invention.
In one embodiment of the invention a siNA molecule comprises an antisense strand having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein the antisense strand is complementary to a target VEGF and/or VEGFR RNA sequence or a portion thereof, and wherein said siNA further comprises a sense strand having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, and wherein said sense strand and said antisense strand are distinct nucleotide sequences where at least about 15 nucleotides in each strand are complementary to the other strand.
In another embodiment of the invention a siNA molecule of the invention comprises an antisense region having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein the antisense region is complementary to a target VEGF and/or VEGFR DNA sequence, and wherein said siNA further comprises a sense region having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein said sense region and said antisense region are comprised in a linear molecule where the sense region comprises at least about 15 nucleotides that are complementary to the antisense region.
In one embodiment, a siNA molecule of the invention has RNAi activity that modulates expression of RNA encoded by one or more genes. Because various genes can share some degree of sequence homology with each other, siNA molecules can be designed to target a class of genes or alternately specific genes (e.g., polymorphic variants) by selecting sequences that are either shared amongst different gene target VEGF and/or VEGFRs or alternatively that are unique for a specific gene target VEGF and/or VEGFR. Therefore, in one embodiment, the siNA molecule can be designed to target conserved regions of target VEGF and/or VEGFR RNA sequences having homology among several gene variants so as to target a class of genes with one siNA molecule. Accordingly, in one embodiment, the siNA molecule of the invention modulates the expression of one or both gene alleles in a subject. In another embodiment, the siNA molecule can be designed to target a sequence that is unique to a specific target VEGF and/or VEGFR RNA sequence (e.g., a single allele or single nucleotide polymorphism (SNP)) due to the high degree of specificity that the siNA molecule requires to mediate RNAi activity.
In one embodiment, nucleic acid molecules of the invention that act as mediators of the RNA interference gene silencing response are double-stranded nucleic acid molecules. In another embodiment, the siNA molecules of the invention consist of duplex nucleic acid molecules containing about 15 to about 30 base pairs between oligonucleotides comprising about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In yet another embodiment, siNA molecules of the invention comprise duplex nucleic acid molecules with overhanging ends of about 1 to about 3 (e.g., about 1, 2, or 3) nucleotides, for example, about 21-nucleotide duplexes with about 19 base pairs and 3′-terminal mononucleotide, dinucleotide, or trinucleotide overhangs. In yet another embodiment, siNA molecules of the invention comprise duplex nucleic acid molecules with blunt ends, where both ends are blunt, or alternatively, where one of the ends is blunt.
In one embodiment, a double stranded nucleic acid (e.g., siNA) molecule comprises nucleotide or non-nucleotide overhangs. By “overhang” is meant a terminal portion of the nucleotide sequence that is not base paired between the two strands of a double stranded nucleic acid molecule (see for example FIG. 6). In one embodiment, a double stranded nucleic acid molecule of the invention can comprise nucleotide or non-nucleotide overhangs at the 3′-end of one or both strands of the double stranded nucleic acid molecule. For example, a double stranded nucleic acid molecule of the invention can comprise a nucleotide or non-nucleotide overhang at the 3′-end of the guide strand or antisense strand/region, the 3′-end of the passenger strand or sense strand/region, or both the guide strand or antisense strand/region and the passenger strand or sense strand/region of the double stranded nucleic acid molecule. In another embodiment, the nucleotide overhang portion of a double stranded nucleic acid (siNA) molecule of the invention comprises 2′-O-methyl, 2′-deoxy, 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, universal base, acyclic, or 5-C-methyl nucleotides. In another embodiment, the non-nucleotide overhang portion of a double stranded nucleic acid (siNA) molecule of the invention comprises glyceryl, abasic, or inverted deoxy abasic non-nucleotides.
In one embodiment, the nucleotides comprising the overhang portions of a double stranded nucleic acid (e.g., siNA) molecule of the invention correspond to the nucleotides comprising the target VEGF and/or VEGFR polynucleotide sequence of the siNA molecule. Accordingly, in such embodiments, the nucleotides comprising the overhang portion of a siNA molecule of the invention comprise sequence based on the target VEGF and/or VEGFR polynucleotide sequence in which nucleotides comprising the overhang portion of the guide strand or antisense strand/region of a siNA molecule of the invention can be complementary to nucleotides in the target VEGF and/or VEGFR polynucleotide sequence and nucleotides comprising the overhang portion of the passenger strand or sense strand/region of a siNA molecule of the invention can comprise the nucleotides in the target VEGF and/or VEGFR polynucleotide sequence. Such nucleotide overhangs comprise sequence that would result from Dicer processing of a native dsRNA into siRNA.
In one embodiment, the nucleotides comprising the overhang portion of a double stranded nucleic acid (e.g., siNA) molecule of the invention are complementary to the target VEGF and/or VEGFR polynucleotide sequence and are optionally chemically modified as described herein. As such, in one embodiment, the nucleotides comprising the overhang portion of the guide strand or antisense strand/region of a siNA molecule of the invention can be complementary to nucleotides in the target VEGF and/or VEGFR polynucleotide sequence, i.e. those nucleotide positions in the target VEGF and/or VEGFR polynucleotide sequence that are complementary to the nucleotide positions of the overhang nucleotides in the guide strand or antisense strand/region of a siNA molecule. In another embodiment, the nucleotides comprising the overhang portion of the passenger strand or sense strand/region of a siNA molecule of the invention can comprise the nucleotides in the target VEGF and/or VEGFR polynucleotide sequence, i.e. those nucleotide positions in the target VEGF and/or VEGFR polynucleotide sequence that correspond to same the nucleotide positions of the overhang nucleotides in the passenger strand or sense strand/region of a siNA molecule. In one embodiment, the overhang comprises a two nucleotide (e.g., 3′-GA; 3′-GU; 3′-GG; 3′GC; 3′-CA; 3′-CU; 3′-CG; 3′CC; 3′-UA; 3′-UU; 3′-UG; 3′UC; 3′-AA; 3′-AU; 3′-AG; 3′-AC; 3′-TA; 3′-TU; 3′-TG; 3′-TC; 3′-AT; 3′-UT; 3′-GT; 3′-CT) overhang that is complementary to a portion of the target VEGF and/or VEGFR polynucleotide sequence. In one embodiment, the overhang comprises a two nucleotide (e.g., 3′-GA; 3′-GU; 3′-GG; 3′GC; 3′-CA; 3′-CU; 3′-CG; 3′CC; 3′-UA; 3′-UU; 3′-UG; 3′UC; 3′-AA; 3′-AU; 3′-AG; 3′-AC; 3′-TA; 3′-TU; 3′-TG; 3′-TC; 3′-AT; 3′-UT; 3′-GT; 3′-CT) overhang that is not complementary to a portion of the target VEGF and/or VEGFR polynucleotide sequence. In another embodiment, the overhang nucleotides of a siNA molecule of the invention are 2′-O-methyl nucleotides and/or 2′-deoxy-2′-fluoro nucleotides. In another embodiment, the overhang nucleotides of a siNA molecule of the invention are 2′-O-methyl nucleotides in the event the overhang nucleotides are purine nucleotides and/or 2′-deoxy-2′-fluoro nucleotides in the event the overhang nucleotides are pyrimidines nucleotides. In another embodiment, the purine nucleotide (when present) in an overhang of siNA molecule of the invention is 2′-O-methyl nucleotides. In another embodiment, the pyrimidine nucleotide (when present) in an overhang of siNA molecule of the invention is 2′-deoxy-2′-fluoro nucleotides nucleotide.
In one embodiment, the nucleotides comprising the overhang portion of a double stranded nucleic acid (e.g., siNA) molecule of the invention are not complementary to the target VEGF and/or VEGFR polynucleotide sequence and are optionally chemically modified as described herein. In one embodiment, the overhang comprises a 3′-UU overhang that is not complementary to a portion of the target VEGF and/or VEGFR polynucleotide sequence. In another embodiment, the nucleotides comprising the overhanging portion of a siNA molecule of the invention are 2′-O-methyl nucleotides and/or 2′-deoxy-2′-fluoro nucleotides.
In one embodiment, the double stranded nucleic molecule (e.g. siNA) of the invention comprises a two or three nucleotide overhang, wherein the nucleotides in the overhang are same or different. In one embodiment, the double stranded nucleic molecule (e.g. siNA) of the invention comprises a two or three nucleotide overhang, wherein the nucleotides in the overhang are the same or different and wherein one or more nucleotides in the overhang are chemically modified at the base, sugar and/or phosphate backbone.
In one embodiment, the invention features one or more chemically-modified siNA constructs having specificity for target VEGF and/or VEGFR nucleic acid molecules, such as DNA, or RNA encoding a protein or non-coding RNA associated with the expression of target VEGF and/or VEGFR genes. In one embodiment, the invention features a RNA based siNA molecule (e.g., a siNA comprising 2′-OH nucleotides) having specificity for nucleic acid molecules that includes one or more chemical modifications described herein. Non-limiting examples of such chemical modifications include without limitation phosphorothioate internucleotide linkages, 2′-deoxyribonucleotides, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, 4′-thio ribonucleotides, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides (see for example U.S. Ser. No. 10/981,966 filed Nov. 5, 2004, incorporated by reference herein), “universal base” nucleotides, “acyclic” nucleotides, 5-C-methyl nucleotides, and terminal glyceryl and/or inverted deoxy abasic residue incorporation. These chemical modifications, when used in various siNA constructs, (e.g., RNA based siNA constructs), are shown to preserve RNAi activity in cells while at the same time, dramatically increasing the serum stability of these compounds. Furthermore, contrary to the data published by Parrish et al., supra, applicant demonstrates that multiple (greater than one) phosphorothioate substitutions are well-tolerated and confer substantial increases in serum stability for modified siNA constructs.
In one embodiment, a siNA molecule of the invention comprises modified nucleotides while maintaining the ability to mediate RNAi. The modified nucleotides can be used to improve in vitro or in vivo characteristics such as stability, activity, toxicity, immune response, and/or bioavailability. For example, a siNA molecule of the invention can comprise modified nucleotides as a percentage of the total number of nucleotides present in the siNA molecule. As such, a siNA molecule of the invention can generally comprise about 5% to about 100% modified nucleotides (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides). For example, in one embodiment, between about 5% to about 100% (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides) of the nucleotide positions in a siNA molecule of the invention comprise a nucleic acid sugar modification, such as a 2′-sugar modification, e.g., 2′-O-methyl nucleotides, 2′-deoxy-2′-fluoro nucleotides, 2′-O-methoxyethyl nucleotides, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides, or 2′-deoxy nucleotides. In another embodiment, between about 5% to about 100% (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides) of the nucleotide positions in a siNA molecule of the invention comprise a nucleic acid base modification, such as inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), or propyne modifications. In another embodiment, between about 5% to about 100% (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides) of the nucleotide positions in a siNA molecule of the invention comprise a nucleic acid backbone modification, such as a backbone modification having Formula I herein. In another embodiment, between about 5% to about 100% (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides) of the nucleotide positions in a siNA molecule of the invention comprise a nucleic acid sugar, base, or backbone modification or any combination thereof (e.g., any combination of nucleic acid sugar, base, backbone or non-nucleotide modifications herein). The actual percentage of modified nucleotides present in a given siNA molecule will depend on the total number of nucleotides present in the siNA. If the siNA molecule is single stranded, the percent modification can be based upon the total number of nucleotides present in the single stranded siNA molecules. Likewise, if the siNA molecule is double stranded, the percent modification can be based upon the total number of nucleotides present in the sense strand, antisense strand, or both the sense and antisense strands.
A siNA molecule of the invention can comprise modified nucleotides at various locations within the siNA molecule. In one embodiment, a double stranded siNA molecule of the invention comprises modified nucleotides at internal base paired positions within the siNA duplex. For example, internal positions can comprise positions from about 3 to about 19 nucleotides from the 5′-end of either sense or antisense strand or region of a 21 nucleotide siNA duplex having 19 base pairs and two nucleotide 3′-overhangs. In another embodiment, a double stranded siNA molecule of the invention comprises modified nucleotides at non-base paired or overhang regions of the siNA molecule. By “non-base paired” is meant, the nucleotides are not base paired between the sense strand or sense region and the antisense strand or antisense region or the siNA molecule. The overhang nucleotides can be complementary or base paired to a corresponding target VEGF and/or VEGFR polynucleotide sequence (see for example FIG. 6C). For example, overhang positions can comprise positions from about 20 to about 21 nucleotides from the 5′-end of either sense or antisense strand or region of a 21 nucleotide siNA duplex having 19 base pairs and two nucleotide 3′-overhangs. In another embodiment, a double stranded siNA molecule of the invention comprises modified nucleotides at terminal positions of the siNA molecule. For example, such terminal regions include the 3′-position, 5′-position, for both 3′ and 5′-positions of the sense and/or antisense strand or region of the siNA molecule. In another embodiment, a double stranded siNA molecule of the invention comprises modified nucleotides at base-paired or internal positions, non-base paired or overhang regions, and/or terminal regions, or any combination thereof.
One aspect of the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a target VEGF and/or VEGFR gene or that directs cleavage of a target VEGF and/or VEGFR RNA. In one embodiment, the double stranded siNA molecule comprises one or more chemical modifications and each strand of the double-stranded siNA is about 21 nucleotides long. In one embodiment, the double-stranded siNA molecule does not contain any ribonucleotides. In another embodiment, the double-stranded siNA molecule comprises one or more ribonucleotides. In one embodiment, each strand of the double-stranded siNA molecule independently comprises about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein each strand comprises about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that are complementary to the nucleotides of the other strand. In one embodiment, one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof of the target VEGF and/or VEGFR gene, and the second strand of the double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence of the target VEGF and/or VEGFR gene or a portion thereof.
In another embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a target VEGF and/or VEGFR gene or that directs cleavage of a target VEGF and/or VEGFR RNA, comprising an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of the target VEGF and/or VEGFR gene or a portion thereof, and a sense region, wherein the sense region comprises a nucleotide sequence substantially similar to the nucleotide sequence of the target VEGF and/or VEGFR gene or a portion thereof. In one embodiment, the antisense region and the sense region independently comprise about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein the antisense region comprises about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that are complementary to nucleotides of the sense region.
In another embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a target VEGF and/or VEGFR gene or that directs cleavage of a target VEGF and/or VEGFR RNA, comprising a sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by the target VEGF and/or VEGFR gene or a portion thereof and the sense region comprises a nucleotide sequence that is complementary to the antisense region.
In one embodiment, a siNA molecule of the invention comprises blunt ends, i.e., ends that do not include any overhanging nucleotides. For example, a siNA molecule comprising modifications described herein (e.g., comprising nucleotides having Formulae I-VII or siNA constructs comprising “Stab 00”-“Stab 34” or “Stab 3F”-“Stab 34F” (Table IV) or any combination thereof (see Table IV)) and/or any length described herein can comprise blunt ends or ends with no overhanging nucleotides.
In one embodiment, any siNA molecule of the invention can comprise one or more blunt ends, i.e. where a blunt end does not have any overhanging nucleotides. In one embodiment, the blunt ended siNA molecule has a number of base pairs equal to the number of nucleotides present in each strand of the siNA molecule. In another embodiment, the siNA molecule comprises one blunt end, for example wherein the 5′-end of the antisense strand and the 3′-end of the sense strand do not have any overhanging nucleotides. In another example, the siNA molecule comprises one blunt end, for example wherein the 3′-end of the antisense strand and the 5′-end of the sense strand do not have any overhanging nucleotides. In another example, a siNA molecule comprises two blunt ends, for example wherein the 3′-end of the antisense strand and the 5′-end of the sense strand as well as the 5′-end of the antisense strand and 3′-end of the sense strand do not have any overhanging nucleotides. A blunt ended siNA molecule can comprise, for example, from about 15 to about 30 nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides). Other nucleotides present in a blunt ended siNA molecule can comprise, for example, mismatches, bulges, loops, or wobble base pairs to modulate the activity of the siNA molecule to mediate RNA interference.
By “blunt ends” is meant symmetric termini or termini of a double stranded siNA molecule having no overhanging nucleotides. The two strands of a double stranded siNA molecule align with each other without over-hanging nucleotides at the termini. For example, a blunt ended siNA construct comprises terminal nucleotides that are complementary between the sense and antisense regions of the siNA molecule.
In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a target VEGF and/or VEGFR gene or that directs cleavage of a target VEGF and/or VEGFR RNA, wherein the siNA molecule is assembled from two separate oligonucleotide fragments wherein one fragment comprises the sense region and the second fragment comprises the antisense region of the siNA molecule. The sense region can be connected to the antisense region via a linker molecule, such as a polynucleotide linker or a non-nucleotide linker.
In one embodiment, a double stranded nucleic acid molecule (e.g., siNA) molecule of the invention comprises ribonucleotides at positions that maintain or enhance RNAi activity. In one embodiment, ribonucleotides are present in the sense strand or sense region of the siNA molecule, which can provide for RNAi activity by allowing cleavage of the sense strand or sense region by an enzyme within the RISC (e.g., ribonucleotides present at the position of passenger strand, sense strand or sense region cleavage, such as position 9 of the passenger strand of a 19 base-pair duplex is cleaved in the RISC by AGO2 enzyme, see for example Matranga et al., 2005, Cell, 123:1-114 and Rand et al., 2005, Cell, 123:621-629). In another embodiment, one or more (for example 1, 2, 3, 4 or 5) nucleotides at the 5′-end of the guide strand or guide region (also known as antisense strand or antisense region) of the siNA molecule are ribonucleotides.
In one embodiment, a double stranded nucleic acid molecule (e.g., siNA) molecule of the invention comprises one or more ribonucleotides at positions within the passenger strand or passenger region (also known as the sense strand or sense region) that allows cleavage of the passenger strand or passenger region by an enzyme in the RISC complex, (e.g., ribonucleotides present at the position of passenger strand such as position 9 of the passenger strand of a 19 base-pair duplex is cleaved in the RISC by AGO2 enzyme, see for example Matranga et al., 2005, Cell, 123:1-114 and Rand et al., 2005, Cell, 123:621-629).
In one embodiment, a siNA molecule of the invention contains at least 2, 3, 4, 5, or more chemical modifications that can be the same of different. In another embodiment, a siNA molecule of the invention contains at least 2, 3, 4, 5, or more different chemical modifications.
In one embodiment, a siNA molecule of the invention is a double-stranded short interfering nucleic acid (siNA), wherein the double stranded nucleic acid molecule comprises about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) of the nucleotide positions in each strand of the siNA molecule comprises a chemical modification. In another embodiment, the siNA contains at least 2, 3, 4, 5, or more different chemical modifications.
In one embodiment, the invention features double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a target VEGF and/or VEGFR gene or that directs cleavage of a target VEGF and/or VEGFR RNA, wherein the siNA molecule comprises about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein each strand of the siNA molecule comprises one or more chemical modifications. In one embodiment, each strand of the double stranded siNA molecule comprises at least two (e.g., 2, 3, 4, 5, or more) different chemical modifications, e.g., different nucleotide sugar, base, or backbone modifications. In another embodiment, one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of a target VEGF and/or VEGFR gene or a portion thereof, and the second strand of the double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence or a portion thereof of the target VEGF and/or VEGFR gene. In another embodiment, one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of a target VEGF and/or VEGFR gene or portion thereof, and the second strand of the double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence or portion thereof of the target VEGF and/or VEGFR gene. In another embodiment, each strand of the siNA molecule comprises about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, and each strand comprises at least about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that are complementary to the nucleotides of the other strand. The target VEGF and/or VEGFR gene can comprise, for example, sequences referred to herein or incorporated herein by reference.
In one embodiment, each strand of a double stranded siNA molecule of the invention comprises a different pattern of chemical modifications, such as any “Stab 00”-“Stab 34” or “Stab 3F”-“Stab 34F” (Table IV) modification patterns herein or any combination thereof (see Table IV). Non-limiting examples of sense and antisense strands of such siNA molecules having various modification patterns are shown in Table III.
In one embodiment, a siNA molecule of the invention comprises no ribonucleotides. In another embodiment, a siNA molecule of the invention comprises ribonucleotides.
In one embodiment, a siNA molecule of the invention comprises an antisense region comprising a nucleotide sequence that is complementary to a nucleotide sequence of a target VEGF and/or VEGFR gene or a portion thereof, and the siNA further comprises a sense region comprising a nucleotide sequence substantially similar to the nucleotide sequence of the target VEGF and/or VEGFR gene or a portion thereof. In another embodiment, the antisense region and the sense region each comprise about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides and the antisense region comprises at least about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that are complementary to nucleotides of the sense region. In one embodiment, each strand of the double stranded siNA molecule comprises at least two (e.g., 2, 3, 4, 5, or more) different chemical modifications, e.g., different nucleotide sugar, base, or backbone modifications. The target VEGF and/or VEGFR gene can comprise, for example, sequences referred to herein or incorporated by reference herein. In another embodiment, the siNA is a double stranded nucleic acid molecule, where each of the two strands of the siNA molecule independently comprise about 15 to about 40 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23, 33, 34, 35, 36, 37, 38, 39, or 40) nucleotides, and where one of the strands of the siNA molecule comprises at least about 15 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 or more) nucleotides that are complementary to the nucleic acid sequence of the target VEGF and/or VEGFR gene or a portion thereof.
In one embodiment, a siNA molecule of the invention comprises a sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by a target VEGF and/or VEGFR gene, or a portion thereof, and the sense region comprises a nucleotide sequence that is complementary to the antisense region. In one embodiment, the siNA molecule is assembled from two separate oligonucleotide fragments, wherein one fragment comprises the sense region and the second fragment comprises the antisense region of the siNA molecule. In another embodiment, the sense region is connected to the antisense region via a linker molecule. In another embodiment, the sense region is connected to the antisense region via a linker molecule, such as a nucleotide or non-nucleotide linker. In one embodiment, each strand of the double stranded siNA molecule comprises at least two (e.g., 2, 3, 4, 5, or more) different chemical modifications, e.g., different nucleotide sugar, base, or backbone modifications. The target VEGF and/or VEGFR gene can comprise, for example, sequences referred herein or incorporated by reference herein
In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a target VEGF and/or VEGFR gene or that directs cleavage of a target VEGF and/or VEGFR RNA, comprising a sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by the target VEGF and/or VEGFR gene or a portion thereof and the sense region comprises a nucleotide sequence that is complementary to the antisense region, and wherein the siNA molecule has one or more modified pyrimidine and/or purine nucleotides. In one embodiment, each strand of the double stranded siNA molecule comprises at least two (e.g., 2, 3, 4, 5, or more) different chemical modifications, e.g., different nucleotide sugar, base, or backbone modifications. In one embodiment, the pyrimidine nucleotides in the sense region are 2′-O-methyl pyrimidine nucleotides or 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-deoxy purine nucleotides. In another embodiment, the pyrimidine nucleotides in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-O-methyl purine nucleotides. In another embodiment, the pyrimidine nucleotides in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-deoxy purine nucleotides. In one embodiment, the pyrimidine nucleotides in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the antisense region are 2′-O-methyl or 2′-deoxy purine nucleotides. In another embodiment of any of the above-described siNA molecules, any nucleotides present in a non-complementary region of the sense strand (e.g. overhang region) are 2′-deoxy nucleotides.
In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a target VEGF and/or VEGFR gene or that directs cleavage of a target VEGF and/or VEGFR RNA, wherein the siNA molecule is assembled from two separate oligonucleotide fragments wherein one fragment comprises the sense region and the second fragment comprises the antisense region of the siNA molecule, and wherein the fragment comprising the sense region includes a terminal cap moiety at the 5′-end, the 3′-end, or both of the 5′ and 3′ ends of the fragment. In one embodiment, the terminal cap moiety is an inverted deoxy abasic moiety or glyceryl moiety. In one embodiment, each of the two fragments of the siNA molecule independently comprise about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In another embodiment, each of the two fragments of the siNA molecule independently comprise about 15 to about 40 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23, 33, 34, 35, 36, 37, 38, 39, or 40) nucleotides. In a non-limiting example, each of the two fragments of the siNA molecule comprise about 21 nucleotides.
In one embodiment, the invention features a siNA molecule comprising at least one modified nucleotide, wherein the modified nucleotide is a 2′-deoxy-2′-fluoro nucleotide, 2′-O-trifluoromethyl nucleotide, 2′-O-ethyl-trifluoromethoxy nucleotide, or 2′-O-difluoromethoxy-ethoxy nucleotide or any other modified nucleoside/nucleotide described herein and in U.S. Ser. No. 10/981,966, filed Nov. 5, 2004, incorporated by reference herein. In one embodiment, the invention features a siNA molecule comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) modified nucleotides, wherein the modified nucleotide is selected from the group consisting of 2′-deoxy-2′-fluoro nucleotide, 2′-O-trifluoromethyl nucleotide, 2′-O-ethyl-trifluoromethoxy nucleotide, or 2′-O-difluoromethoxy-ethoxy nucleotide or any other modified nucleoside/nucleotide described herein and in U.S. Ser. No. 10/981,966, filed Nov. 5, 2004, incorporated by reference herein. The modified nucleotide/nucleoside can be the same or different. The siNA can be, for example, about 15 to about 40 nucleotides in length. In one embodiment, all pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy, 4′-thio pyrimidine nucleotides. In one embodiment, the modified nucleotides in the siNA include at least one 2′-deoxy-2′-fluoro cytidine or 2′-deoxy-2′-fluoro uridine nucleotide. In another embodiment, the modified nucleotides in the siNA include at least one 2′-fluoro cytidine and at least one 2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all uridine nucleotides present in the siNA are 2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all cytidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro cytidine nucleotides. In one embodiment, all adenosine nucleotides present in the siNA are 2′-deoxy-2′-fluoro adenosine nucleotides. In one embodiment, all guanosine nucleotides present in the siNA are 2′-deoxy-2′-fluoro guanosine nucleotides. The siNA can further comprise at least one modified internucleotidic linkage, such as phosphorothioate linkage. In one embodiment, the 2′-deoxy-2′-fluoronucleotides are present at specifically selected locations in the siNA that are sensitive to cleavage by ribonucleases, such as locations having pyrimidine nucleotides.
In one embodiment, the invention features a method of increasing the stability of a siNA molecule against cleavage by ribonucleases comprising introducing at least one modified nucleotide into the siNA molecule, wherein the modified nucleotide is a 2′-deoxy-2′-fluoro nucleotide. In one embodiment, all pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides. In one embodiment, the modified nucleotides in the siNA include at least one 2′-deoxy-2′-fluoro cytidine or 2′-deoxy-2′-fluoro uridine nucleotide. In another embodiment, the modified nucleotides in the siNA include at least one 2′-fluoro cytidine and at least one 2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all uridine nucleotides present in the siNA are 2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all cytidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro cytidine nucleotides. In one embodiment, all adenosine nucleotides present in the siNA are 2′-deoxy-2′-fluoro adenosine nucleotides. In one embodiment, all guanosine nucleotides present in the siNA are 2′-deoxy-2′-fluoro guanosine nucleotides. The siNA can further comprise at least one modified internucleotidic linkage, such as a phosphorothioate linkage. In one embodiment, the 2′-deoxy-2′-fluoronucleotides are present at specifically selected locations in the siNA that are sensitive to cleavage by ribonucleases, such as locations having pyrimidine nucleotides.
In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a target VEGF and/or VEGFR gene or that directs cleavage of a target VEGF and/or VEGFR RNA, comprising a sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by the target VEGF and/or VEGFR gene or a portion thereof and the sense region comprises a nucleotide sequence that is complementary to the antisense region, and wherein the purine nucleotides present in the antisense region comprise 2′-deoxy-purine nucleotides. In an alternative embodiment, the purine nucleotides present in the antisense region comprise 2′-O-methyl purine nucleotides. In either of the above embodiments, the antisense region can comprise a phosphorothioate internucleotide linkage at the 3′ end of the antisense region. Alternatively, in either of the above embodiments, the antisense region can comprise a glyceryl modification at the 3′ end of the antisense region. In another embodiment of any of the above-described siNA molecules, any nucleotides present in a non-complementary region of the antisense strand (e.g. overhang region) are 2′-deoxy nucleotides.
In one embodiment, the antisense region of a siNA molecule of the invention comprises sequence complementary to a portion of an endogenous transcript having sequence unique to a particular disease or trait related allele in a subject or organism, such as sequence comprising a single nucleotide polymorphism (SNP) associated with the disease or trait specific allele. As such, the antisense region of a siNA molecule of the invention can comprise sequence complementary to sequences that are unique to a particular allele to provide specificity in mediating selective RNAi against the disease, condition, or trait related allele.
In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a target VEGF and/or VEGFR gene or that directs cleavage of a target VEGF and/or VEGFR RNA, wherein the siNA molecule is assembled from two separate oligonucleotide fragments wherein one fragment comprises the sense region and the second fragment comprises the antisense region of the siNA molecule. In one embodiment, each strand of the double stranded siNA molecule is about 21 nucleotides long where about 19 nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule, wherein at least two 3′ terminal nucleotides of each fragment of the siNA molecule are not base-paired to the nucleotides of the other fragment of the siNA molecule. In another embodiment, the siNA molecule is a double stranded nucleic acid molecule, where each strand is about 19 nucleotide long and where the nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule to form at least about 15 (e.g., 15, 16, 17, 18, or 19) base pairs, wherein one or both ends of the siNA molecule are blunt ends. In one embodiment, each of the two 3′ terminal nucleotides of each fragment of the siNA molecule is a 2′-deoxy-pyrimidine nucleotide, such as a 2′-deoxy-thymidine. In another embodiment, all nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule. In another embodiment, the siNA molecule is a double stranded nucleic acid molecule of about 19 to about 25 base pairs having a sense region and an antisense region, where about 19 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the target VEGF and/or VEGFR gene. In another embodiment, about 21 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the target VEGF and/or VEGFR gene. In any of the above embodiments, the 5′-end of the fragment comprising said antisense region can optionally include a phosphate group.
In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits the expression of a target VEGF and/or VEGFR RNA sequence, wherein the siNA molecule does not contain any ribonucleotides and wherein each strand of the double-stranded siNA molecule is about 15 to about 30 nucleotides. In one embodiment, the siNA molecule is 21 nucleotides in length. Examples of non-ribonucleotide containing siNA constructs are combinations of stabilization chemistries shown in Table I in any combination of Sense/Antisense chemistries, such as Stab 7/8, Stab 7/11, Stab 8/8, Stab 18/8, Stab 18/11, Stab 12/13, Stab 7/13, Stab 18/13, Stab 7/19, Stab 8/19, Stab 18/19, Stab 7/20, Stab 8/20, Stab 18/20, Stab 7/32, Stab 8/32, or Stab 18/32 (e.g., any siNA having Stab 7, 8, 11, 12, 13, 14, 15, 17, 18, 19, 20, or 32 sense or antisense strands or any combination thereof). Herein, numeric Stab chemistries can include both 2′-fluoro and 2′-OCF3 versions of the chemistries shown in Table I. For example, “Stab 7/8” refers to both Stab 7/8 and Stab 7F/8F etc. In one embodiment, the invention features a chemically synthesized double stranded RNA molecule that directs cleavage of a target VEGF and/or VEGFR RNA via RNA interference, wherein each strand of said RNA molecule is about 15 to about 30 nucleotides in length; one strand of the RNA molecule comprises nucleotide sequence having sufficient complementarity to the target VEGF and/or VEGFR RNA for the RNA molecule to direct cleavage of the target VEGF and/or VEGFR RNA via RNA interference; and wherein at least one strand of the RNA molecule optionally comprises one or more chemically modified nucleotides described herein, such as without limitation deoxynucleotides, 2′-O-methyl nucleotides, 2′-deoxy-2′-fluoro nucleotides, 2′-O-methoxyethyl nucleotides, 4′-thio nucleotides, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides, etc.
In one embodiment, a target VEGF and/or VEGFR RNA of the invention comprises sequence encoding a protein.
In one embodiment, target VEGF and/or VEGFR RNA of the invention comprises non-coding RNA sequence (e.g., miRNA, snRNA, siRNA etc.), see for example Mattick, 2005, Science, 309, 1527-1528 and Claverie, 2005, Science, 309, 1529-1530.
In one embodiment, the invention features a medicament comprising a siNA molecule of the invention.
In one embodiment, the invention features an active ingredient comprising a siNA molecule of the invention.
In one embodiment, the invention features the use of a double-stranded short interfering nucleic acid (siNA) molecule to inhibit, down-regulate, or reduce expression of a target VEGF and/or VEGFR gene, wherein the siNA molecule comprises one or more chemical modifications and each strand of the double-stranded siNA is independently about 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more) nucleotides long. In one embodiment, the siNA molecule of the invention is a double stranded nucleic acid molecule comprising one or more chemical modifications, where each of the two fragments of the siNA molecule independently comprise about 15 to about 40 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23, 33, 34, 35, 36, 37, 38, 39, or 40) nucleotides and where one of the strands comprises at least 15 nucleotides that are complementary to nucleotide sequence of target VEGF and/or VEGFR encoding RNA or a portion thereof. In a non-limiting example, each of the two fragments of the siNA molecule comprise about 21 nucleotides. In another embodiment, the siNA molecule is a double stranded nucleic acid molecule comprising one or more chemical modifications, where each strand is about 21 nucleotide long and where about 19 nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule, wherein at least two 3′ terminal nucleotides of each fragment of the siNA molecule are not base-paired to the nucleotides of the other fragment of the siNA molecule. In another embodiment, the siNA molecule is a double stranded nucleic acid molecule comprising one or more chemical modifications, where each strand is about 19 nucleotide long and where the nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule to form at least about 15 (e.g., 15,16, 17, 18, or 19) base pairs, wherein one or both ends of the siNA molecule are blunt ends. In one embodiment, each of the two 3′ terminal nucleotides of each fragment of the siNA molecule is a 2′-deoxy-pyrimidine nucleotide, such as a 2′-deoxy-thymidine. In another embodiment, all nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule. In another embodiment, the siNA molecule is a double stranded nucleic acid molecule of about 19 to about 25 base pairs having a sense region and an antisense region and comprising one or more chemical modifications, where about 19 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the target VEGF and/or VEGFR gene. In another embodiment, about 21 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the target VEGF and/or VEGFR gene. In any of the above embodiments, the 5′-end of the fragment comprising said antisense region can optionally include a phosphate group.
In one embodiment, the invention features the use of a double-stranded short interfering nucleic acid (siNA) molecule that inhibits, down-regulates, or reduces expression of a target VEGF and/or VEGFR gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of target VEGF and/or VEGFR RNA or a portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand. In one embodiment, each strand has at least two (e.g., 2, 3, 4, 5, or more) chemical modifications, which can be the same or different, such as nucleotide, sugar, base, or backbone modifications. In one embodiment, a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification. In one embodiment, a majority of the purine nucleotides present in the double-stranded siNA molecule comprises a sugar modification.
In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits, down-regulates, or reduces expression of a target VEGF and/or VEGFR gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of target VEGF and/or VEGFR RNA or a portion thereof, wherein the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand. In one embodiment, each strand has at least two (e.g., 2, 3, 4, 5, or more) chemical modifications, which can be the same or different, such as nucleotide, sugar, base, or backbone modifications. In one embodiment, a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification. In one embodiment, a majority of the purine nucleotides present in the double-stranded siNA molecule comprises a sugar modification.
In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits, down-regulates, or reduces expression of a target VEGF and/or VEGFR gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of target VEGF and/or VEGFR RNA that encodes a protein or portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification. In one embodiment, each strand of the siNA molecule comprises about 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides, wherein each strand comprises at least about 15 nucleotides that are complementary to the nucleotides of the other strand. In one embodiment, the siNA molecule is assembled from two oligonucleotide fragments, wherein one fragment comprises the nucleotide sequence of the antisense strand of the siNA molecule and a second fragment comprises nucleotide sequence of the sense region of the siNA molecule. In one embodiment, the sense strand is connected to the antisense strand via a linker molecule, such as a polynucleotide linker or a non-nucleotide linker. In a further embodiment, the pyrimidine nucleotides present in the sense strand are 2′-deoxy-2′fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-deoxy purine nucleotides. In another embodiment, the pyrimidine nucleotides present in the sense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-O-methyl purine nucleotides. In still another embodiment, the pyrimidine nucleotides present in the antisense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and any purine nucleotides present in the antisense strand are 2′-deoxy purine nucleotides. In another embodiment, the antisense strand comprises one or more 2′-deoxy-2′-fluoro pyrimidine nucleotides and one or more 2′-O-methyl purine nucleotides. In another embodiment, the pyrimidine nucleotides present in the antisense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and any purine nucleotides present in the antisense strand are 2′-O-methyl purine nucleotides. In a further embodiment the sense strand comprises a 3′-end and a 5′-end, wherein a terminal cap moiety (e.g., an inverted deoxy abasic moiety or inverted deoxy nucleotide moiety such as inverted thymidine) is present at the 5′-end, the 3′-end, or both of the 5′ and 3′ ends of the sense strand. In another embodiment, the antisense strand comprises a phosphorothioate internucleotide linkage at the 3′ end of the antisense strand. In another embodiment, the antisense strand comprises a glyceryl modification at the 3′ end. In another embodiment, the 5′-end of the antisense strand optionally includes a phosphate group.
In any of the above-described embodiments of a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a target VEGF and/or VEGFR gene, wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification, each of the two strands of the siNA molecule can comprise about 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides. In one embodiment, about 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides of each strand of the siNA molecule are base-paired to the complementary nucleotides of the other strand of the siNA molecule. In another embodiment, about 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides of each strand of the siNA molecule are base-paired to the complementary nucleotides of the other strand of the siNA molecule, wherein at least two 3′ terminal nucleotides of each strand of the siNA molecule are not base-paired to the nucleotides of the other strand of the siNA molecule. In another embodiment, each of the two 3′ terminal nucleotides of each fragment of the siNA molecule is a 2′-deoxy-pyrimidine, such as 2′-deoxy-thymidine. In one embodiment, each strand of the siNA molecule is base-paired to the complementary nucleotides of the other strand of the siNA molecule. In one embodiment, about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides of the antisense strand are base-paired to the nucleotide sequence of the target VEGF and/or VEGFR RNA or a portion thereof. In one embodiment, about 18 to about 25 (e.g., about 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides of the antisense strand are base-paired to the nucleotide sequence of the target VEGF and/or VEGFR RNA or a portion thereof.
In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a target VEGF and/or VEGFR gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of target VEGF and/or VEGFR RNA or a portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand. In one embodiment, each strand has at least two (e.g., 2, 3, 4, 5, or more) different chemical modifications, such as nucleotide sugar, base, or backbone modifications. In one embodiment, a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification. In one embodiment, a majority of the purine nucleotides present in the double-stranded siNA molecule comprises a sugar modification. In one embodiment, the 5′-end of the antisense strand optionally includes a phosphate group.
In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a target VEGF and/or VEGFR gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of target VEGF and/or VEGFR RNA or a portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification, and wherein the nucleotide sequence or a portion thereof of the antisense strand is complementary to a nucleotide sequence of the untranslated region or a portion thereof of the target VEGF and/or VEGFR RNA.
In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a target VEGF and/or VEGFR gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of target VEGF and/or VEGFR RNA or a portion thereof, wherein the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand, wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification, and wherein the nucleotide sequence of the antisense strand is complementary to a nucleotide sequence of the target VEGF and/or VEGFR RNA or a portion thereof that is present in the target VEGF and/or VEGFR RNA.
In one embodiment, the invention features a composition comprising a siNA molecule of the invention in a pharmaceutically acceptable carrier or diluent.
In a non-limiting example, the introduction of chemically-modified nucleotides into nucleic acid molecules provides a powerful tool in overcoming potential limitations of in vivo stability and bioavailability inherent to native RNA molecules that are delivered exogenously. For example, the use of chemically-modified nucleic acid molecules can enable a lower dose of a particular nucleic acid molecule for a given therapeutic effect since chemically-modified nucleic acid molecules tend to have a longer half-life in serum. Furthermore, certain chemical modifications can improve the bioavailability of nucleic acid molecules by target VEGF and/or VEGFRing particular cells or tissues and/or improving cellular uptake of the nucleic acid molecule. Therefore, even if the activity of a chemically-modified nucleic acid molecule is reduced as compared to a native nucleic acid molecule, for example, when compared to an all-RNA nucleic acid molecule, the overall activity of the modified nucleic acid molecule can be greater than that of the native molecule due to improved stability and/or delivery of the molecule. Unlike native unmodified siNA, chemically-modified siNA can also minimize the possibility of activating interferon activity or immunostimulation in humans.
In any of the embodiments of siNA molecules described herein, the antisense region of a siNA molecule of the invention can comprise a phosphorothioate internucleotide linkage at the 3′-end of said antisense region. In any of the embodiments of siNA molecules described herein, the antisense region can comprise about one to about five phosphorothioate internucleotide linkages at the 5′-end of said antisense region. In any of the embodiments of siNA molecules described herein, the 3′-terminal nucleotide overhangs of a siNA molecule of the invention can comprise ribonucleotides or deoxyribonucleotides that are chemically-modified at a nucleic acid sugar, base, or backbone. In any of the embodiments of siNA molecules described herein, the 3′-terminal nucleotide overhangs can comprise one or more universal base ribonucleotides. In any of the embodiments of siNA molecules described herein, the 3′-terminal nucleotide overhangs can comprise one or more acyclic nucleotides.
One embodiment of the invention provides an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the invention in a manner that allows expression of the nucleic acid molecule. Another embodiment of the invention provides a mammalian cell comprising such an expression vector. The mammalian cell can be a human cell. The siNA molecule of the expression vector can comprise a sense region and an antisense region. The antisense region can comprise sequence complementary to a RNA or DNA sequence encoding a target VEGF and/or VEGFR and the sense region can comprise sequence complementary to the antisense region. The siNA molecule can comprise two distinct strands having complementary sense and antisense regions. The siNA molecule can comprise a single strand having complementary sense and antisense regions.
In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides comprising a backbone modified internucleotide linkage having Formula I:




wherein each R1 and R2 is independently any nucleotide, non-nucleotide, or polynucleotide which can be naturally-occurring or chemically-modified and which can be included in the structure of the siNA molecule or serve as a point of attachment to the siNA molecule, each X and Y is independently O, S, N, alkyl, or substituted alkyl, each Z and W is independently O, S, N, alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, or acetyl and wherein W, X, Y, and Z are optionally not all O. In another embodiment, a backbone modification of the invention comprises a phosphonoacetate and/or thiophosphonoacetate internucleotide linkage (see for example Sheehan et al., 2003, Nucleic Acids Research, 31, 4109-4118).
The chemically-modified internucleotide linkages having Formula I, for example, wherein any Z, W, X, and/or Y independently comprises a sulphur atom, can be present in one or both oligonucleotide strands of the siNA duplex, for example, in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemically-modified internucleotide linkages having Formula I at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified internucleotide linkages having Formula I at the 5′-end of the sense strand, the antisense strand, or both strands. In another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidine nucleotides with chemically-modified internucleotide linkages having Formula I in the sense strand, the antisense strand, or both strands. In yet another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine nucleotides with chemically-modified internucleotide linkages having Formula I in the sense strand, the antisense strand, or both strands. In another embodiment, a siNA molecule of the invention having internucleotide linkage(s) of Formula I also comprises a chemically-modified nucleotide or non-nucleotide having any of Formulae I-VII.
In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotides having Formula II:




wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or a group having any of Formula I, II, III, IV, V, VI and/or VII, any of which can be included in the structure of the siNA molecule or serve as a point of attachment to the siNA molecule; R9 is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such as adenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other non-naturally occurring base that can be complementary or non-complementary to target RNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone, pyridinone, or any other non-naturally occurring universal base that can be complementary or non-complementary to target RNA. In one embodiment, R3 and/or R7 comprises a conjugate moiety and a linker (e.g., a nucleotide or non-nucleotide linker as described herein or otherwise known in the art). Non-limiting examples of conjugate moieties include ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; steroids, and polyamines, such as PEI, spermine or spermidine.

The chemically-modified nucleotide or non-nucleotide of Formula II can be present in one or both oligonucleotide strands of the siNA duplex, for example in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more chemically-modified nucleotides or non-nucleotides of Formula II at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides or non-nucleotides of Formula II at the 5′-end of the sense strand, the antisense strand, or both strands. In anther non-limiting example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides or non-nucleotides of Formula II at the 3′-end of the sense strand, the antisense strand, or both strands.
In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotides having Formula III:




wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or a group having any of Formula I, II, III, IV, V, VI and/or VII, any of which can be included in the structure of the siNA molecule or serve as a point of attachment to the siNA molecule; R9 is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such as adenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other non-naturally occurring base that can be employed to be complementary or non-complementary to target RNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone, pyridinone, or any other non-naturally occurring universal base that can be complementary or non-complementary to target RNA. In one embodiment, R3 and/or R7 comprises a conjugate moiety and a linker (e.g., a nucleotide or non-nucleotide linker as described herein or otherwise known in the art). Non-limiting examples of conjugate moieties include ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; steroids, and polyamines, such as PEI, spermine or spermidine.

The chemically-modified nucleotide or non-nucleotide of Formula III can be present in one or both oligonucleotide strands of the siNA duplex, for example, in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more chemically-modified nucleotides or non-nucleotides of Formula III at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide(s) or non-nucleotide(s) of Formula III at the 5′-end of the sense strand, the antisense strand, or both strands. In anther non-limiting example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide or non-nucleotide of Formula III at the 3′-end of the sense strand, the antisense strand, or both strands.
In another embodiment, a siNA molecule of the invention comprises a nucleotide having Formula II or III, wherein the nucleotide having Formula II or III is in an inverted configuration. For example, the nucleotide having Formula II or III is connected to the siNA construct in a 3′-3′, 3′-2′, 2′-3′, or 5′-5′ configuration, such as at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.
In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) inside a cell or reconstituted in vitro system, wherein the chemical modification comprises a 5′-terminal phosphate group having Formula IV:




wherein each X and Y is independently O, S, N, alkyl, substituted alkyl, or alkylhalo; wherein each Z and W is independently O, S, N, alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, alkylhalo, or acetyl; and wherein W, X, Y and Z are optionally not all O and Y serves as a point of attachment to the siNA molecule.

In one embodiment, the invention features a siNA molecule having a 5′-terminal phosphate group having Formula IV on the target VEGF and/or VEGFR-complementary strand, for example, a strand complementary to a target VEGF and/or VEGFR RNA, wherein the siNA molecule comprises an all RNA siNA molecule. In another embodiment, the invention features a siNA molecule having a 5′-terminal phosphate group having Formula IV on the target VEGF and/or VEGFR-complementary strand wherein the siNA molecule also comprises about 1 to about 3 (e.g., about 1, 2, or 3) nucleotide 3′-terminal nucleotide overhangs having about 1 to about 4 (e.g., about 1, 2, 3, or 4) deoxyribonucleotides on the 3′-end of one or both strands. In another embodiment, a 5′-terminal phosphate group having Formula IV is present on the target VEGF and/or VEGFR-complementary strand of a siNA molecule of the invention, for example a siNA molecule having chemical modifications having any of Formulae I-VII.
In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more phosphorothioate internucleotide linkages. For example, in a non-limiting example, the invention features a chemically-modified short interfering nucleic acid (siNA) having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in one siNA strand. In yet another embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) individually having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in both siNA strands. The phosphorothioate internucleotide linkages can be present in one or both oligonucleotide strands of the siNA duplex, for example in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more phosphorothioate internucleotide linkages at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) consecutive phosphorothioate internucleotide linkages at the 5′-end of the sense strand, the antisense strand, or both strands. In another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidine phosphorothioate internucleotide linkages in the sense strand, the antisense strand, or both strands. In yet another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine phosphorothioate internucleotide linkages in the sense strand, the antisense strand, or both strands.
Each strand of the double stranded siNA molecule can have one or more chemical modifications such that each strand comprises a different pattern of chemical modifications. Several non-limiting examples of modification schemes that could give rise to different patterns of modifications are provided herein.
In one embodiment, the invention features a siNA molecule, wherein the sense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or 2′-deoxy-2′-fluoro nucleotides, with or without one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5-ends, being present in the same or different strand.
In another embodiment, the invention features a siNA molecule, wherein the sense strand comprises about 1 to about 5, specifically about 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or 2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about 5 or more, for example about 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.
In one embodiment, the invention features a siNA molecule, wherein the antisense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or 2′-deoxy-2′-fluoro nucleotides, with or without one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends, being present in the same or different strand.
In another embodiment, the invention features a siNA molecule, wherein the antisense strand comprises about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or 2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about 5, for example about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.
In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule having about 1 to about 5 or more (specifically about 1, 2, 3, 4, 5 or more) phosphorothioate internucleotide linkages in each strand of the siNA molecule.
In another embodiment, the invention features a siNA molecule comprising 2′-5′ internucleotide linkages. The 2′-5′ internucleotide linkage(s) can be at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of one or both siNA sequence strands. In addition, the 2′-5′ internucleotide linkage(s) can be present at various other positions within one or both siNA sequence strands, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of a pyrimidine nucleotide in one or both strands of the siNA molecule can comprise a 2′-5′ internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of a purine nucleotide in one or both strands of the siNA molecule can comprise a 2′-5′ internucleotide linkage.
In another embodiment, a chemically-modified siNA molecule of the invention comprises a duplex having two strands, one or both of which can be chemically-modified, wherein each strand is independently about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length, wherein the duplex has about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the chemical modification comprises a structure having any of Formulae I-VII. For example, an exemplary chemically-modified siNA molecule of the invention comprises a duplex having two strands, one or both of which can be chemically-modified with a chemical modification having any of Formulae I-VII or any combination thereof, wherein each strand consists of about 21 nucleotides, each having a 2-nucleotide 3′-terminal nucleotide overhang, and wherein the duplex has about 19 base pairs. In another embodiment, a siNA molecule of the invention comprises a single stranded hairpin structure, wherein the siNA is about 36 to about 70 (e.g., about 36, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the siNA can include a chemical modification comprising a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises a linear oligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemically-modified with a chemical modification having any of Formulae I-VII or any combination thereof, wherein the linear oligonucleotide forms a hairpin structure having about 19 to about 21 (e.g., 19, 20, or 21) base pairs and a 2-nucleotide 3′-terminal nucleotide overhang. In another embodiment, a linear hairpin siNA molecule of the invention contains a stem loop motif, wherein the loop portion of the siNA molecule is biodegradable. For example, a linear hairpin siNA molecule of the invention is designed such that degradation of the loop portion of the siNA molecule in vivo can generate a double-stranded siNA molecule with 3′-terminal overhangs, such as 3′-terminal nucleotide overhangs comprising about 2 nucleotides.
In another embodiment, a siNA molecule of the invention comprises a hairpin structure, wherein the siNA is about 25 to about 50 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, and wherein the siNA can include one or more chemical modifications comprising a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises a linear oligonucleotide having about 25 to about 35 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that is chemically-modified with one or more chemical modifications having any of Formulae I-VII or any combination thereof, wherein the linear oligonucleotide forms a hairpin structure having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs and a 5′-terminal phosphate group that can be chemically modified as described herein (for example a 5′-terminal phosphate group having Formula IV). In another embodiment, a linear hairpin siNA molecule of the invention contains a stem loop motif, wherein the loop portion of the siNA molecule is biodegradable. In one embodiment, a linear hairpin siNA molecule of the invention comprises a loop portion comprising a non-nucleotide linker.
In another embodiment, a siNA molecule of the invention comprises an asymmetric hairpin structure, wherein the siNA is about 25 to about 50 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, and wherein the siNA can include one or more chemical modifications comprising a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises a linear oligonucleotide having about 25 to about 35 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that is chemically-modified with one or more chemical modifications having any of Formulae I-VII or any combination thereof, wherein the linear oligonucleotide forms an asymmetric hairpin structure having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs and a 5′-terminal phosphate group that can be chemically modified as described herein (for example a 5′-terminal phosphate group having Formula IV). In one embodiment, an asymmetric hairpin siNA molecule of the invention contains a stem loop motif, wherein the loop portion of the siNA molecule is biodegradable. In another embodiment, an asymmetric hairpin siNA molecule of the invention comprises a loop portion comprising a non-nucleotide linker.
In another embodiment, a siNA molecule of the invention comprises an asymmetric double stranded structure having separate polynucleotide strands comprising sense and antisense regions, wherein the antisense region is about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length, wherein the sense region is about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides in length, wherein the sense region and the antisense region have at least 3 complementary nucleotides, and wherein the siNA can include one or more chemical modifications comprising a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises an asymmetric double stranded structure having separate polynucleotide strands comprising sense and antisense regions, wherein the antisense region is about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) nucleotides in length and wherein the sense region is about 3 to about 15 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) nucleotides in length, wherein the sense region the antisense region have at least 3 complementary nucleotides, and wherein the siNA can include one or more chemical modifications comprising a structure having any of Formulae I-VII or any combination thereof. In another embodiment, the asymmetric double stranded siNA molecule can also have a 5′-terminal phosphate group that can be chemically modified as described herein (for example a 5′-terminal phosphate group having Formula IV).
In another embodiment, a siNA molecule of the invention comprises a circular nucleic acid molecule, wherein the siNA is about 38 to about 70 (e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the siNA can include a chemical modification, which comprises a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises a circular oligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemically-modified with a chemical modification having any of Formulae I-VII or any combination thereof, wherein the circular oligonucleotide forms a dumbbell shaped structure having about 19 base pairs and 2 loops.
In another embodiment, a circular siNA molecule of the invention contains two loop motifs, wherein one or both loop portions of the siNA molecule is biodegradable. For example, a circular siNA molecule of the invention is designed such that degradation of the loop portions of the siNA molecule in vivo can generate a double-stranded siNA molecule with 3′-terminal overhangs, such as 3′-terminal nucleotide overhangs comprising about 2 nucleotides.
In one embodiment, a siNA molecule of the invention comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) abasic moiety, for example a compound having Formula V:




wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or a group having any of Formula I, II, III, IV, V, VI and/or VII, any of which can be included in the structure of the siNA molecule or serve as a point of attachment to the siNA molecule; R9 is O, S, CH2, S═O, CHF, or CF2. In one embodiment, R3 and/or R7 comprises a conjugate moiety and a linker (e.g., a nucleotide or non-nucleotide linker as described herein or otherwise known in the art). Non-limiting examples of conjugate moieties include ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; steroids, and polyamines, such as PEI, spermine or spermidine.

In one embodiment, a siNA molecule of the invention comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) inverted abasic moiety, for example a compound having Formula VI:




wherein each R3, R4, R5, R6, R7, R8, R10, R1, R12, and R13 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or a group having any of Formula I, II, III, IV, V, VI and/or VII, any of which can be included in the structure of the siNA molecule or serve as a point of attachment to the siNA molecule; R9 is O, S, CH2, S═O, CHF, or CF2, and either R2, R3, R8 or R13 serve as points of attachment to the siNA molecule of the invention. In one embodiment, R3 and/or R7 comprises a conjugate moiety and a linker (e.g., a nucleotide or non-nucleotide linker as described herein or otherwise known in the art). Non-limiting examples of conjugate moieties include ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; steroids, and polyamines, such as PEI, spermine or spermidine.

In another embodiment, a siNA molecule of the invention comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) substituted polyalkyl moieties, for example a compound having Formula VII:




wherein each n is independently an integer from 1 to 12, each R1, R2 and R3 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or a group having any of Formula I, II, III, IV, V, VI and/or VII, any of which can be included in the structure of the siNA molecule or serve as a point of attachment to the siNA molecule. In one embodiment, R3 and/or R1 comprises a conjugate moiety and a linker (e.g., a nucleotide or non-nucleotide linker as described herein or otherwise known in the art). Non-limiting examples of conjugate moieties include ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; steroids, and polyamines, such as PEI, spermine or spermidine.

By “ZIP code” sequences is meant, any peptide or protein sequence that is involved in cellular topogenic signaling mediated transport (see for example Ray et al., 2004, Science, 306(1501): 1505)
Each nucleotide within the double stranded siNA molecule can independently have a chemical modification comprising the structure of any of Formulae I-VIII. Thus, in one embodiment, one or more nucleotide positions of a siNA molecule of the invention comprises a chemical modification having structure of any of Formulae I-VII or any other modification herein. In one embodiment, each nucleotide position of a siNA molecule of the invention comprises a chemical modification having structure of any of Formulae I-VII or any other modification herein.
In one embodiment, one or more nucleotide positions of one or both strands of a double stranded siNA molecule of the invention comprises a chemical modification having structure of any of Formulae 1-VII or any other modification herein. In one embodiment, each nucleotide position of one or both strands of a double stranded siNA molecule of the invention comprises a chemical modification having structure of any of Formulae I-VII or any other modification herein.
In another embodiment, the invention features a compound having Formula VII, wherein R1 and R2 are hydroxyl (OH) groups, n=1, and R3 comprises O and is the point of attachment to the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of one or both strands of a double-stranded siNA molecule of the invention or to a single-stranded siNA molecule of the invention. This modification is referred to herein as “glyceryl” (for example modification 6 in FIG. 10).
In another embodiment, a chemically modified nucleoside or non-nucleoside (e.g. a moiety having any of Formula V, VI or VII) of the invention is at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of a siNA molecule of the invention. For example, chemically modified nucleoside or non-nucleoside (e.g., a moiety having Formula V, VI or VII) can be present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the antisense strand, the sense strand, or both antisense and sense strands of the siNA molecule. In one embodiment, the chemically modified nucleoside or non-nucleoside (e.g., a moiety having Formula V, VI or VII) is present at the 5′-end and 3′-end of the sense strand and the 3′-end of the antisense strand of a double stranded siNA molecule of the invention. In one embodiment, the chemically modified nucleoside or non-nucleoside (e.g., a moiety having Formula V, VI or VII) is present at the terminal position of the 5′-end and 3′-end of the sense strand and the 3′-end of the antisense strand of a double stranded siNA molecule of the invention. In one embodiment, the chemically modified nucleoside or non-nucleoside (e.g., a moiety having Formula V, VI or VII) is present at the two terminal positions of the 5′-end and 3′-end of the sense strand and the 3′-end of the antisense strand of a double stranded siNA molecule of the invention. In one embodiment, the chemically modified nucleoside or non-nucleoside (e.g., a moiety having Formula V, VI or VII) is present at the penultimate position of the 5′-end and 3′-end of the sense strand and the 3′-end of the antisense strand of a double stranded siNA molecule of the invention. In addition, a moiety having Formula VII can be present at the 3′-end or the 5′-end of a hairpin siNA molecule as described herein.
In another embodiment, a siNA molecule of the invention comprises an abasic residue having Formula V or VI, wherein the abasic residue having Formula VI or VI is connected to the siNA construct in a 3′-3′, 3′-2′, 2′-3′, or 5′-5′ configuration, such as at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.
In one embodiment, a siNA molecule of the invention comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked nucleic acid (LNA) nucleotides, for example, at the 5′-end, the 3′-end, both of the 5′ and 3′-ends, or any combination thereof, of the siNA molecule.
In one embodiment, a siNA molecule of the invention comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 4′-thio nucleotides, for example, at the 5′-end, the 3′-end, both of the 5′ and 3′-ends, or any combination thereof, of the siNA molecule.
In another embodiment, a siNA molecule of the invention comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclic nucleotides, for example, at the 5′-end, the 3′-end, both of the 5′ and 3′-ends, or any combination thereof, of the siNA molecule.
In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the sense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides).
In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the sense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides), wherein any nucleotides comprising a 3′-terminal nucleotide overhang that are present in said sense region are 2′-deoxy nucleotides.
In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the sense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides).
In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), wherein any (e.g., one or more or all) purine nucleotides present in the sense region are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides), and wherein any nucleotides comprising a 3′-terminal nucleotide overhang that are present in said sense region are 2′-deoxy nucleotides.
In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides).
In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides), and wherein any nucleotides comprising a 3′-terminal nucleotide overhang that are present in said antisense region are 2′-deoxy nucleotides.
In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides).
In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides).
In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) inside a cell or reconstituted in vitro system comprising a sense region, wherein one or more pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and one or more purine nucleotides present in the sense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides), and an antisense region, wherein one or more pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and one or more purine nucleotides present in the antisense region are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides). The sense region and/or the antisense region can have a terminal cap modification, such as any modification described herein or shown in FIG. 10, that is optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense and/or antisense sequence. The sense and/or antisense region can optionally further comprise a 3′-terminal nucleotide overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides. The overhang nucleotides can further comprise one or more (e.g., about 1, 2, 3, 4 or more) phosphorothioate, phosphonoacetate, and/or thiophosphonoacetate internucleotide linkages. Non-limiting examples of these chemically-modified siNAs are shown in FIGS. 4 and 5 and Table I herein. In any of these described embodiments, the purine nucleotides present in the sense region are alternatively 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides) and one or more purine nucleotides present in the antisense region are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides). Also, in any of these embodiments, one or more purine nucleotides present in the sense region are alternatively purine ribonucleotides (e.g., wherein all purine nucleotides are purine ribonucleotides or alternately a plurality of purine nucleotides are purine ribonucleotides) and any purine nucleotides present in the antisense region are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides). Additionally, in any of these embodiments, one or more purine nucleotides present in the sense region and/or present in the antisense region are alternatively selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides and 2′-O-methyl nucleotides (e.g., wherein all purine nucleotides are selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides and 2′-O-methyl nucleotides or alternately a plurality of purine nucleotides are selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides and 2′-O-methyl nucleotides).
In another embodiment, any modified nucleotides present in the siNA molecules of the invention, preferably in the antisense strand of the siNA molecules of the invention, but also optionally in the sense and/or both antisense and sense strands, comprise modified nucleotides having properties or characteristics similar to naturally occurring ribonucleotides. For example, the invention features siNA molecules including modified nucleotides having a Northern conformation (e.g., Northern pseudorotation cycle, see for example Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984) otherwise known as a “ribo-like” or “A-form helix” configuration. As such, chemically modified nucleotides present in the siNA molecules of the invention, preferably in the antisense strand of the siNA molecules of the invention, but also optionally in the sense and/or both antisense and sense strands, are resistant to nuclease degradation while at the same time maintaining the capacity to mediate RNAi. Non-limiting examples of nucleotides having a northern configuration include locked nucleic acid (LNA) nucleotides (e.g., 2′-O, 4′-C-methylene-(D-ribofuranosyl) nucleotides); 2′-methoxyethoxy (MOE) nucleotides; 2′-methyl-thio-ethyl, 2′-deoxy-2′-fluoro nucleotides, 2′-deoxy-2′-chloro nucleotides, 2′-azido nucleotides, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides, 4′-thio nucleotides and 2′-O-methyl nucleotides.
In one embodiment, the sense strand of a double stranded siNA molecule of the invention comprises a terminal cap moiety, (see for example FIG. 10) such as an inverted deoxyabaisc moiety, at the 3′-end, 5′-end, or both 3′ and 5′-ends of the sense strand.
In one embodiment, the invention features a chemically-modified short interfering nucleic acid molecule (siNA) capable of mediating RNA interference (RNAi) inside a cell or reconstituted in vitro system, wherein the chemical modification comprises a conjugate covalently attached to the chemically-modified siNA molecule. Non-limiting examples of conjugates contemplated by the invention include conjugates and ligands described in Vargeese et al., U.S. Ser. No. 10/427,160, filed Apr. 30, 2003, incorporated by reference herein in its entirety, including the drawings. In another embodiment, the conjugate is covalently attached to the chemically-modified siNA molecule via a biodegradable linker. In one embodiment, the conjugate molecule is attached at the 3′-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule. In another embodiment, the conjugate molecule is attached at the 5′-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule. In yet another embodiment, the conjugate molecule is attached both the 3′-end and 5′-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule, or any combination thereof. In one embodiment, a conjugate molecule of the invention comprises a molecule that facilitates delivery of a chemically-modified siNA molecule into a biological system, such as a cell. In another embodiment, the conjugate molecule attached to the chemically-modified siNA molecule is a ligand for a cellular receptor, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; steroids, and polyamines, such as PEI, spermine or spermidine. Examples of specific conjugate molecules contemplated by the instant invention that can be attached to chemically-modified siNA molecules are described in Vargeese et al., U.S. Ser. No. 10/201,394, filed Jul. 22, 2002 incorporated by reference herein. The type of conjugates used and the extent of conjugation of siNA molecules of the invention can be evaluated for improved pharmacokinetic profiles, bioavailability, and/or stability of siNA constructs while at the same time maintaining the ability of the siNA to mediate RNAi activity. As such, one skilled in the art can screen siNA constructs that are modified with various conjugates to determine whether the siNA conjugate complex possesses improved properties while maintaining the ability to mediate RNAi, for example in animal models as are generally known in the art.
In one embodiment, the invention features a short interfering nucleic acid (siNA) molecule of the invention, wherein the siNA further comprises a nucleotide, non-nucleotide, or mixed nucleotide/non-nucleotide linker that joins the sense region of the siNA to the antisense region of the siNA. In one embodiment, a nucleotide, non-nucleotide, or mixed nucleotide/non-nucleotide linker is used, for example, to attach a conjugate moiety to the siNA. In one embodiment, a nucleotide linker of the invention can be a linker of ≧2 nucleotides in length, for example about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In another embodiment, the nucleotide linker can be a nucleic acid aptamer. By “aptamer” or “nucleic acid aptamer” as used herein is meant a nucleic acid molecule that binds specifically to a target VEGF and/or VEGFR molecule wherein the nucleic acid molecule has sequence that comprises a sequence recognized by the target VEGF and/or VEGFR molecule in its natural setting. Alternately, an aptamer can be a nucleic acid molecule that binds to a target VEGF and/or VEGFR molecule where the target VEGF and/or VEGFR molecule does not naturally bind to a nucleic acid. The target VEGF and/or VEGFR molecule can be any molecule of interest. For example, the aptamer can be used to bind to a ligand-binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein. This is a non-limiting example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art. (See, for example, Gold et al., 1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J. Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser, 2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287, 820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.)
In yet another embodiment, a non-nucleotide linker of the invention comprises abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g. polyethylene glycols such as those having between 2 and 100 ethylene glycol units). Specific examples include those described by Seela and Kaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic Acids Res. 1987, 15:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 113:6324; Richardson and Schepartz, J. Am. Chem. Soc. 1991, 113:5109; Ma et al., Nucleic Acids Res. 1993, 21:2585 and Biochemistry 1993, 32:1751; Durand et al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides & Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301; Ono et al., Biochemistry 1991, 30:9914; Arnold et al., International Publication No. WO 89/02439; Usman et al., International Publication No. WO 95/06731; Dudycz et al, International Publication No. WO 95/11910 and Ferentz and Verdine, J. Am. Chem. Soc. 1991, 113:4000, all hereby incorporated by reference herein. A “non-nucleotide” further means any group or compound that can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound can be abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine, for example at the C1 position of the sugar.
In one embodiment, the invention features a short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) inside a cell or reconstituted in vitro system, wherein one or both strands of the siNA molecule that are assembled from two separate oligonucleotides do not comprise any ribonucleotides. For example, a siNA molecule can be assembled from a single oligonculeotide where the sense and antisense regions of the siNA comprise separate oligonucleotides that do not have any ribonucleotides (e.g., nucleotides having a 2′-OH group) present in the oligonucleotides. In another example, a siNA molecule can be assembled from a single oligonculeotide where the sense and antisense regions of the siNA are linked or circularized by a nucleotide or non-nucleotide linker as described herein, wherein the oligonucleotide does not have any ribonucleotides (e.g., nucleotides having a 2′-OH group) present in the oligonucleotide. Applicant has surprisingly found that the presense of ribonucleotides (e.g., nucleotides having a 2′-hydroxyl group) within the siNA molecule is not required or essential to support RNAi activity. As such, in one embodiment, all positions within the siNA can include chemically modified nucleotides and/or non-nucleotides such as nucleotides and or non-nucleotides having Formula I, II, III, IV, V, VI, or VII or any combination thereof to the extent that the ability of the siNA molecule to support RNAi activity in a cell is maintained.
In one embodiment, a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity in a cell or reconstituted in vitro system comprising a single stranded polynucleotide having complementarity to a target VEGF and/or VEGFR nucleic acid sequence. In another embodiment, the single stranded siNA molecule of the invention comprises a 5′-terminal phosphate group. In another embodiment, the single stranded siNA molecule of the invention comprises a 5′-terminal phosphate group and a 3′-terminal phosphate group (e.g., a 2′,3′-cyclic phosphate). In another embodiment, the single stranded siNA molecule of the invention comprises about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In yet another embodiment, the single stranded siNA molecule of the invention comprises one or more chemically modified nucleotides or non-nucleotides described herein. For example, all the positions within the siNA molecule can include chemically-modified nucleotides such as nucleotides having any of Formulae I-VII, or any combination thereof to the extent that the ability of the siNA molecule to support RNAi activity in a cell is maintained.
In one embodiment, a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity in a cell or reconstituted in vitro system comprising a single stranded polynucleotide having complementarity to a target VEGF and/or VEGFR nucleic acid sequence, wherein one or more pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any purine nucleotides present in the antisense region are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides), and a terminal cap modification, such as any modification described herein or shown in FIG. 10, that is optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the antisense sequence. The siNA optionally further comprises about 1 to about 4 or more (e.g., about 1, 2, 3, 4 or more) terminal 2′-deoxynucleotides at the 3′-end of the siNA molecule, wherein the terminal nucleotides can further comprise one or more (e.g., 1, 2, 3, 4 or more) phosphorothioate, phosphonoacetate, and/or thiophosphonoacetate internucleotide linkages, and wherein the siNA optionally further comprises a terminal phosphate group, such as a 5′-terminal phosphate group. In any of these embodiments, any purine nucleotides present in the antisense region are alternatively 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides). Also, in any of these embodiments, any purine nucleotides present in the siNA (i.e., purine nucleotides present in the sense and/or antisense region) can alternatively be locked nucleic acid (LNA) nucleotides (e.g., wherein all purine nucleotides are LNA nucleotides or alternately a plurality of purine nucleotides are LNA nucleotides). Also, in any of these embodiments, any purine nucleotides present in the siNA are alternatively 2′-methoxyethyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-methoxyethyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-methoxyethyl purine nucleotides). In another embodiment, any modified nucleotides present in the single stranded siNA molecules of the invention comprise modified nucleotides having properties or characteristics similar to naturally occurring ribonucleotides. For example, the invention features siNA molecules including modified nucleotides having a Northern conformation (e.g., Northern pseudorotation cycle, see for example Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemically modified nucleotides present in the single stranded siNA molecules of the invention are preferably resistant to nuclease degradation while at the same time maintaining the capacity to mediate RNAi.
In one embodiment, a siNA molecule of the invention comprises chemically modified nucleotides or non-nucleotides (e.g., having any of Formulae I-VII, such as 2′-deoxy, 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy or 2′-O-methyl nucleotides) at alternating positions within one or more strands or regions of the siNA molecule. For example, such chemical modifications can be introduced at every other position of a RNA based siNA molecule, starting at either the first or second nucleotide from the 3′-end or 5′-end of the siNA. In a non-limiting example, a double stranded siNA molecule of the invention in which each strand of the siNA is 21 nucleotides in length is featured wherein positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21 of each strand are chemically modified (e.g., with compounds having any of Formulae I-VII, such as such as 2′-deoxy, 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy or 2′-O-methyl nucleotides). In another non-limiting example, a double stranded siNA molecule of the invention in which each strand of the siNA is 21 nucleotides in length is featured wherein positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 of each strand are chemically modified (e.g., with compounds having any of Formulae I-VII, such as such as 2′-deoxy, 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy or 2′-O-methyl nucleotides). In one embodiment, one strand of the double stranded siNA molecule comprises chemical modifications at positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 and chemical modifications at positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21. Such siNA molecules can further comprise terminal cap moieties and/or backbone modifications as described herein.
In one embodiment, a siNA molecule of the invention comprises the following features: if purine nucleotides are present at the 5′-end (e.g., at any of terminal nucleotide positions 1, 2, 3, 4, 5, or 6 from the 5′-end) of the antisense strand or antisense region (otherwise referred to as the guide sequence or guide strand) of the siNA molecule then such purine nucleosides are ribonucleotides. In another embodiment, the purine ribonucleotides, when present, are base paired to nucleotides of the sense strand or sense region (otherwise referred to as the passenger strand) of the siNA molecule. Such purine ribonucleotides can be present in a siNA stabilization motif that otherwise comprises modified nucleotides.
In one embodiment, a siNA molecule of the invention comprises the following features: if pyrimidine nucleotides are present at the 5′-end (e.g., at any of terminal nucleotide positions 1, 2, 3, 4, 5, or 6 from the 5′-end) of the antisense strand or antisense region (otherwise referred to as the guide sequence or guide strand) of the siNA molecule then such pyrimidine nucleosides are ribonucleotides. In another embodiment, the pyrimidine ribonucleotides, when present, are base paired to nucleotides of the sense strand or sense region (otherwise referred to as the passenger strand) of the siNA molecule. Such pyrimidine ribonucleotides can be present in a siNA stabilization motif that otherwise comprises modified nucleotides.
In one embodiment, a siNA molecule of the invention comprises the following features: if pyrimidine nucleotides are present at the 5′-end (e.g., at any of terminal nucleotide positions 1, 2, 3, 4, 5, or 6 from the 5′-end) of the antisense strand or antisense region (otherwise referred to as the guide sequence or guide strand) of the siNA molecule then such pyrimidine nucleosides are modified nucleotides. In another embodiment, the modified pyrimidine nucleotides, when present, are base paired to nucleotides of the sense strand or sense region (otherwise referred to as the passenger strand) of the siNA molecule. Non-limiting examples of modified pyrimidine nucleotides include those having any of Formulae I-VII, such as such as 2′-deoxy, 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy or 2′-O-methyl nucleotides.
In one embodiment, the invention features a double stranded nucleic acid molecule having structure SI:










     B ————————NX3 ————————(N)X2 B -3′




 



B (N)X1————————NX4 —————[N]X5 -5′



 



               SI






    
    
        wherein each N is independently a nucleotide; each B is a terminal cap moiety that can be present or absent; (N) represents non-base paired or overhanging nucleotides which can be unmodified or chemically modified; [N] represents nucleotide positions wherein any purine nucleotides when present are ribonucleotides; X1 and X2 are independently integers from about 0 to about 4; X3 is an integer from about 9 to about 30; X4 is an integer from about 11 to about 30, provided that the sum of X4 and X5 is between 17-36; X5 is an integer from about 1 to about 6; NX3 is complementary to NX4 and NX5, and
        (a) any pyridmidine nucleotides present in the antisense strand (lower strand) are 2′-deoxy-2′-fluoro nucleotides; any purine nucleotides present in the antisense strand (lower strand) other than the purines nucleotides in the [N] nucleotide positions, are independently 2′-O-methyl nucleotides, 2′-deoxyribonucleotides or a combination of 2′-deoxyribonucleotides and 2′-O-methyl nucleotides;
        (b) any pyrimidine nucleotides present in the sense strand (upper strand) are 2′-deoxy-2′-fluoro nucleotides; any purine nucleotides present in the sense strand (upper strand) are independently 2′-deoxyribonucleotides, 2′-O-methyl nucleotides or a combination of 2′-deoxyribonucleotides and 2′-O-methyl nucleotides; and
        (c) any (N) nucleotides are optionally 2′-O-methyl, 2′-deoxy-2′-fluoro, or deoxyribonucleotides.
    
    


In one embodiment, the invention features a double stranded nucleic acid molecule having structure SII:










     B ————————NX3 ————————(N)X2 B -3′




 



B (N)X1————————NX4 —————[N]X5 -5′



 



               SII






    
    
        wherein each N is independently a nucleotide; each B is a terminal cap moiety that can be present or absent; (N) represents non-base paired or overhanging nucleotides which can be unmodified or chemically modified; [N] represents nucleotide positions wherein any purine nucleotides when present are ribonucleotides; X1 and X2 are independently integers from about 0 to about 4; X3 is an integer from about 9 to about 30; X4 is an integer from about 11 to about 30, provided that the sum of X4 and X5 is between 17-36; X5 is an integer from about 1 to about 6; NX3 is complementary to NX4 and NX5, and
        (a) any pyridmidine nucleotides present in the antisense strand (lower strand) are 2′-deoxy-2′-fluoro nucleotides; any purine nucleotides present in the antisense strand (lower strand) other than the purines nucleotides in the [N] nucleotide positions, are 2′-O-methyl nucleotides;
        (b) any pyrimidine nucleotides present in the sense strand (upper strand) are ribonucleotides; any purine nucleotides present in the sense strand (upper strand) are ribonucleotides; and
        (c) any (N) nucleotides are optionally 2′-O-methyl, 2′-deoxy-2′-fluoro, or deoxyribonucleotides.
    
    


In one embodiment, the invention features a double stranded nucleic acid molecule having structure SIII:










     B ————————NX3 ————————(N)X2 B -3′




 



B (N)X1————————NX4 —————[N]X5 -5′



 



               SIII






    
    
        wherein each N is independently a nucleotide; each B is a terminal cap moiety that can be present or absent; (N) represents non-base paired or overhanging nucleotides which can be unmodified or chemically modified; [N] represents nucleotide positions wherein any purine nucleotides when present are ribonucleotides; X1 and X2 are independently integers from about 0 to about 4; X3 is an integer from about 9 to about 30; X4 is an integer from about 11 to about 30, provided that the sum of X4 and X5 is between 17-36; X5 is an integer from about 1 to about 6; NX3 is complementary to NX4 and NX5, and
        (a) any pyridmidine nucleotides present in the antisense strand (lower strand) are 2′-deoxy-2′-fluoro nucleotides; any purine nucleotides present in the antisense strand (lower strand) other than the purines nucleotides in the [N] nucleotide positions, are 2′-O-methyl nucleotides;
        (b) any pyrimidine nucleotides present in the sense strand (upper strand) are 2′-deoxy-2′-fluoro nucleotides; any purine nucleotides present in the sense strand (upper strand) are ribonucleotides; and
        (c) any (N) nucleotides are optionally 2′-O-methyl, 2′-deoxy-2′-fluoro, or deoxyribonucleotides.
    
    


In one embodiment, the invention features a double stranded nucleic acid molecule having structure SIV:










     B ————————NX3 ————————(N)X2 B -3′




 



B (N)X1————————NX4 —————[N]X5 -5′



 



               SIV






    
    
        wherein each N is independently a nucleotide; each B is a terminal cap moiety that can be present or absent; (N) represents non-base paired or overhanging nucleotides which can be unmodified or chemically modified; [N] represents nucleotide positions wherein any purine nucleotides when present are ribonucleotides; X1 and X2 are independently integers from about 0 to about 4; X3 is an integer from about 9 to about 30; X4 is an integer from about 11 to about 30, provided that the sum of X4 and X5 is between 17-36; X5 is an integer from about 1 to about 6; NX3 is complementary to NX4 and NX5, and
        (a) any pyridmidine nucleotides present in the antisense strand (lower strand) are 2′-deoxy-2′-fluoro nucleotides; any purine nucleotides present in the antisense strand (lower strand) other than the purines nucleotides in the [N] nucleotide positions, are 2′-O-methyl nucleotides;
        (b) any pyrimidine nucleotides present in the sense strand (upper strand) are 2′-deoxy-2′-fluoro nucleotides; any purine nucleotides present in the sense strand (upper strand) are deoxyribonucleotides; and
        (c) any (N) nucleotides are optionally 2′-O-methyl, 2′-deoxy-2′-fluoro, or deoxyribonucleotides.
    
    


In one embodiment, the invention features a double stranded nucleic acid molecule having structure SV:










     B ————————NX3 ————————(N)X2 B -3′




 



B (N)X1————————NX4 —————[N]X5 -5′



 



               SV






    
    
        wherein each N is independently a nucleotide; each B is a terminal cap moiety that can be present or absent; (N) represents non-base paired or overhanging nucleotides which can be unmodified or chemically modified; [N] represents nucleotide positions wherein any purine nucleotides when present are ribonucleotides; X1 and X2 are independently integers from about 0 to about 4; X3 is an integer from about 9 to about 30; X4 is an integer from about 11 to about 30, provided that the sum of X4 and X5 is between 17-36; X5 is an integer from about 1 to about 6; NX3 is complementary to NX4 and NX5, and
        (a) any pyridmidine nucleotides present in the antisense strand (lower strand) are nucleotides having a ribo-like configuration (e.g., Northern or A-form helix configuration); any purine nucleotides present in the antisense strand (lower strand) other than the purines nucleotides in the [N] nucleotide positions, are 2′-O-methyl nucleotides;
        (b) any pyrimidine nucleotides present in the sense strand (upper strand) are nucleotides having a ribo-like configuration (e.g., Northern or A-form helix configuration); any purine nucleotides present in the sense strand (upper strand) are 2′-O-methyl nucleotides; and
        (c) any (N) nucleotides are optionally 2′-O-methyl, 2′-deoxy-2′-fluoro, or deoxyribonucleotides.
    
    


In one embodiment, the invention features a double stranded nucleic acid molecule having structure SVI:










     B ————————NX3 ————————(N)X2 B -3′




 



B (N)X1————————NX4 —————[N]X5 -5′



 



               SVI






    
    
        wherein each N is independently a nucleotide; each B is a terminal cap moiety that can be present or absent; (N) represents non-base paired or overhanging nucleotides which can be unmodified or chemically modified; [N] represents nucleotide positions comprising sequence that renders the 5′-end of the antisense strand (lower strand) less thermally stable than the 5′-end of the sense strand (upper strand); X1 and X2 are independently integers from about 0 to about 4; X3 is an integer from about 9 to about 30; X4 is an integer from about 11 to about 30, provided that the sum of X4 and X5 is between 17-36; X5 is an integer from about 1 to about 6; NX3 is complementary to NX4 and NX5, and
        (a) any pyridmidine nucleotides present in the antisense strand (lower strand) are 2′-deoxy-2′-fluoro nucleotides; any purine nucleotides present in the antisense strand (lower strand) other than the purines nucleotides in the [N] nucleotide positions, are independently 2′-O-methyl nucleotides, 2′-deoxyribonucleotides or a combination of 2′-deoxyribonucleotides and 2′-O-methyl nucleotides;
        (b) any pyrimidine nucleotides present in the sense strand (upper strand) are 2′-deoxy-2′-fluoro nucleotides; any purine nucleotides present in the sense strand (upper strand) are independently 2′-deoxyribonucleotides, 2′-O-methyl nucleotides or a combination of 2′-deoxyribonucleotides and 2′-O-methyl nucleotides; and
        (c) any (N) nucleotides are optionally 2′-O-methyl, 2′-deoxy-2′-fluoro, or deoxyribonucleotides.
    
    


In one embodiment, the invention features a double stranded nucleic acid molecule having structure SVII:










     B ————————NX3 ————————(N)X2 B -3′




 



B (N)X1————————NX4 ————————-5′



 



               SVII






    
    
        wherein each N is independently a nucleotide; each B is a terminal cap moiety that can be present or absent; (N) represents non-base paired or overhanging nucleotides; X1 and X2 are independently integers from about 0 to about 4; X3 is an integer from about 9 to about 30; X4 is an integer from about 11 to about 36; NX3 is complementary to NX4, and any (N) nucleotides are 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides.
    
    


In one embodiment, the invention features a double stranded nucleic acid molecule having structure SVIII:




    
    
        wherein each N is independently a nucleotide; each B is a terminal cap moiety that can be present or absent; (N) represents non-base paired or overhanging nucleotides which can be unmodified or chemically modified; [N] represents nucleotide positions comprising sequence that renders the 5′-end of the antisense strand (lower strand) less thermally stable than the 5′-end of the sense strand (upper strand); [N] represents nucleotide positions that are ribonucleotides; X1 and X2 are independently integers from about 0 to about 4; X3 is an integer from about 9 to about 15; X4 is an integer from about 11 to about 30, provided that the sum of X4 and X5 is between 17-36; X5 is an integer from about 1 to about 6; X6 is an integer from about 1 to about 4; X7 is an integer from about 9 to about 15; NX7, NX6, and NX3 are complementary to NX4 and NX5, and
        (a) any pyridmidine nucleotides present in the antisense strand (lower strand) are 2′-deoxy-2′-fluoro nucleotides; any purine nucleotides present in the antisense strand (lower strand) other than the purines nucleotides in the [N] nucleotide positions, are independently 2′-O-methyl nucleotides, 2′-deoxyribonucleotides or a combination of 2′-deoxyribonucleotides and 2′-O-methyl nucleotides;
        (b) any pyrimidine nucleotides present in the sense strand (upper strand) are 2′-deoxy-2′-fluoro nucleotides other than [N] nucleotides; any purine nucleotides present in the sense strand (upper strand) are independently 2′-deoxyribonucleotides, 2′-O-methyl nucleotides or a combination of 2′-deoxyribonucleotides and 2′-O-methyl nucleotides other than [N] nucleotides; and
        (c) any (N) nucleotides are optionally 2′-O-methyl, 2′-deoxy-2′-fluoro, or deoxyribonucleotides.
    
    


In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, SVI, SVII or SVIII comprises a terminal phosphate group at the 5′-end of the antisense strand or antisense region of the nucleic acid molecule.
In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, SVI, SVII or SVIII comprises X5=1, 2, or 3; each XI and X2=1 or 2; X3=12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and X4=15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.
In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, SVI, SVII or SVIII comprises X5=1; each X1 and X2=2; X3=19, and X4=18.
In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, SVI, SVII or SVIII comprises X5=2; each X1 and X2=2; X3=19, and X4=17
In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, SVI, SVII or SVIII comprises X5=3; each X1 and X2=2; X3=19, and X4=16.
In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, SVI, SVII or SVIII comprises B at the 3′ and 5′ ends of the sense strand or sense region.
In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, SVI, SVII or SVIII comprises B at the 3′-end of the antisense strand or antisense region.
In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SII, SIV, SV, SVI, SVII or SVIII comprises B at the 3′ and 5′ ends of the sense strand or sense region and B at the 3′-end of the antisense strand or antisense region.
In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, SVI, SVII or SVIII further comprises one or more phosphorothioate internucleotide linkages at the first terminal (N) on the 3′end of the sense strand, antisense strand, or both sense strand and antisense strands of the nucleic acid molecule. For example, a double stranded nucleic acid molecule can comprise X1 and/or X2=2 having overhanging nucleotide positions with a phosphorothioate internucleotide linkage, e.g., (NsN) where “s” indicates phosphorothioate.
In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, SVI, SVII or SVIII comprises (N) nucleotides that are 2′-O-methyl nucleotides.
In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, SVI, SVII or SVIII comprises (N) nucleotides that are 2′-O-methyl nucleotides.
In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, SVI, SVII or SVIII comprises (N) nucleotides in the antisense strand (lower strand) that are complementary to nucleotides in a target VEGF and/or VEGFR polynucleotide sequence having complementary to the N and [N] nucleotides of the antisense (lower) strand.
In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, SVI, SVII or SVIII comprises (N) nucleotides in the sense strand (upper strand) that comprise nucleotide sequence corresponding a target VEGF and/or VEGFR polynucleotide sequence having complementary to the antisense (lower) strand such that the contiguous (N) and N nucleotide sequence of the sense strand comprises nucleotide sequence of the target VEGF and/or VEGFR nucleic acid sequence.
In one embodiment, a double stranded nucleic acid molecule having any of structure SVIII comprises B only at the 5′-end of the sense (upper) strand of the double stranded nucleic acid molecule.
In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, SVI, SVII or SVIII further comprises an unpaired terminal nucleotide at the 5′-end of the antisense (lower) strand. The unpaired nucleotide is not complementary to the sense (upper) strand. In one embodiment, the unpaired terminal nucleotide is complementary to a target VEGF and/or VEGFR polynucleotide sequence having complementary to the N and [N] nucleotides of the antisense (lower) strand. In another embodiment, the unpaired terminal nucleotide is not complementary to a target VEGF and/or VEGFR polynucleotide sequence having complementary to the N and [N] nucleotides of the antisense (lower) strand.
In one embodiment, a double stranded nucleic acid molecule having any of structure SVIII comprises X6=1 and X3=10.
In one embodiment, a double stranded nucleic acid molecule having any of structure SVIII comprises X6=2 and X3=9.
In one embodiment, the invention features a method for modulating the expression of a target VEGF and/or VEGFR gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified or unmodified, wherein one of the siNA strands comprises a sequence complementary to RNA of the target VEGF and/or VEGFR gene; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the target VEGF and/or VEGFR gene in the cell.
In one embodiment, the invention features a method for modulating the expression of a target VEGF and/or VEGFR gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified or unmodified, wherein one of the siNA strands comprises a sequence complementary to RNA of the target VEGF and/or VEGFR gene and wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequence of the target VEGF and/or VEGFR RNA; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the target VEGF and/or VEGFR gene in the cell.
In another embodiment, the invention features a method for modulating the expression of more than one target VEGF and/or VEGFR gene within a cell comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified or unmodified, wherein one of the siNA strands comprises a sequence complementary to RNA of the target VEGF and/or VEGFR genes; and (b) introducing the siNA molecules into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the target VEGF and/or VEGFR genes in the cell.
In another embodiment, the invention features a method for modulating the expression of two or more target VEGF and/or VEGFR genes within a cell comprising: (a) synthesizing one or more siNA molecules of the invention, which can be chemically-modified or unmodified, wherein the siNA strands comprise sequences complementary to RNA of the target VEGF and/or VEGFR genes and wherein the sense strand sequences of the siNAs comprise sequences identical or substantially similar to the sequences of the target VEGF and/or VEGFR RNAs; and (b) introducing the siNA molecules into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the target VEGF and/or VEGFR genes in the cell.
In another embodiment, the invention features a method for modulating the expression of more than one target VEGF and/or VEGFR gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified or unmodified, wherein one of the siNA strands comprises a sequence complementary to RNA of the target VEGF and/or VEGFR gene and wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequences of the target VEGF and/or VEGFR RNAs; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the target VEGF and/or VEGFR genes in the cell.
In another embodiment, the invention features a method for modulating the expression of a target VEGF and/or VEGFR gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified or unmodified, wherein one of the siNA strands comprises a sequence complementary to RNA of the target VEGF and/or VEGFR gene, wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequences of the target VEGF and/or VEGFR RNA; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the target VEGF and/or VEGFR gene in the cell.
In one embodiment, siNA molecules of the invention are used as reagents in ex vivo applications. For example, siNA reagents are introduced into tissue or cells that are transplanted into a subject for therapeutic effect. The cells and/or tissue can be derived from an organism or subject that later receives the explant, or can be derived from another organism or subject prior to transplantation. The siNA molecules can be used to modulate the expression of one or more genes in the cells or tissue, such that the cells or tissue obtain a desired phenotype or are able to perform a function when transplanted in vivo. In one embodiment, certain target VEGF and/or VEGFR cells from a patient are extracted. These extracted cells are contacted with siNAs target VEGF and/or VEGFRing a specific nucleotide sequence within the cells under conditions suitable for uptake of the siNAs by these cells (e.g. using delivery reagents such as cationic lipids, liposomes and the like or using techniques such as electroporation to facilitate the delivery of siNAs into cells). The cells are then reintroduced back into the same patient or other patients.
In one embodiment, the invention features a method of modulating the expression of a target VEGF and/or VEGFR gene in a tissue explant comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the target VEGF and/or VEGFR gene; and (b) introducing the siNA molecule into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate (e.g., inhibit) the expression of the target VEGF and/or VEGFR gene in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate (e.g., inhibit) the expression of the target VEGF and/or VEGFR gene in that organism.
In one embodiment, the invention features a method of modulating the expression of a target VEGF and/or VEGFR gene in a tissue explant comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the target VEGF and/or VEGFR gene and wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequence of the target VEGF and/or VEGFR RNA; and (b) introducing the siNA molecule into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate (e.g., inhibit) the expression of the target VEGF and/or VEGFR gene in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate (e.g., inhibit) the expression of the target VEGF and/or VEGFR gene in that organism.
In another embodiment, the invention features a method of modulating the expression of more than one target VEGF and/or VEGFR gene in a tissue explant comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the target VEGF and/or VEGFR genes; and (b) introducing the siNA molecules into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate (e.g., inhibit) the expression of the target VEGF and/or VEGFR genes in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate (e.g., inhibit) the expression of the target VEGF and/or VEGFR genes in that organism.
In one embodiment, the invention features a method of modulating the expression of a target VEGF and/or VEGFR gene in a subject or organism comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the target VEGF and/or VEGFR gene; and (b) introducing the siNA molecule into the subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the target VEGF and/or VEGFR gene in the subject or organism. The level of target VEGF and/or VEGFR protein or RNA can be determined using various methods well-known in the art.
In another embodiment, the invention features a method of modulating the expression of more than one target VEGF and/or VEGFR gene in a subject or organism comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the target VEGF and/or VEGFR genes; and (b) introducing the siNA molecules into the subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the target VEGF and/or VEGFR genes in the subject or organism. The level of target VEGF and/or VEGFR protein or RNA can be determined as is known in the art.
In one embodiment, the invention features a method for modulating the expression of a target VEGF and/or VEGFR gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the target VEGF and/or VEGFR gene; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the target VEGF and/or VEGFR gene in the cell.
In another embodiment, the invention features a method for modulating the expression of more than one target VEGF and/or VEGFR gene within a cell comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the target VEGF and/or VEGFR gene; and (b) contacting the cell in vitro or in vivo with the siNA molecule under conditions suitable to modulate (e.g., inhibit) the expression of the target VEGF and/or VEGFR genes in the cell.
In one embodiment, the invention features a method of modulating the expression of a target VEGF and/or VEGFR gene in a tissue explant (e.g., a cochlea, skin, heart, liver, spleen, cornea, lung, stomach, kidney, vein, artery, hair, appendage, or limb transplant, or any other organ, tissue or cell as can be transplanted from one organism to another or back to the same organism from which the organ, tissue or cell is derived) comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the target VEGF and/or VEGFR gene; and (b) contacting a cell of the tissue explant derived from a particular subject or organism with the siNA molecule under conditions suitable to modulate (e.g., inhibit) the expression of the target VEGF and/or VEGFR gene in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the subject or organism the tissue was derived from or into another subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the target VEGF and/or VEGFR gene in that subject or organism.
In another embodiment, the invention features a method of modulating the expression of more than one target VEGF and/or VEGFR gene in a tissue explant (e.g., a cochlear, skin, heart, liver, spleen, cornea, lung, stomach, kidney, vein, artery, hair, appendage, or limb transplant, or any other organ, tissue or cell as can be transplanted from one organism to another or back to the same organism from which the organ, tissue or cell is derived) comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the target VEGF and/or VEGFR gene; and (b) introducing the siNA molecules into a cell of the tissue explant derived from a particular subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the target VEGF and/or VEGFR genes in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the subject or organism the tissue was derived from or into another subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the target VEGF and/or VEGFR genes in that subject or organism.
In one embodiment, the invention features a method of modulating the expression of a target VEGF and/or VEGFR gene in a subject or organism comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the target VEGF and/or VEGFR gene; and (b) introducing the siNA molecule into the subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the target VEGF and/or VEGFR gene in the subject or organism.
In another embodiment, the invention features a method of modulating the expression of more than one target VEGF and/or VEGFR gene in a subject or organism comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the target VEGF and/or VEGFR gene; and (b) introducing the siNA molecules into the subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the target VEGF and/or VEGFR genes in the subject or organism.
In one embodiment, the invention features a method of modulating the expression of a target VEGF and/or VEGFR gene in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate (e.g., inhibit) the expression of the target VEGF and/or VEGFR gene in the subject or organism.
In one embodiment, the invention features a method for treating or preventing a disease, disorder, trait or condition related to gene expression in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the target gene in the subject or organism. The reduction of gene expression and thus reduction in the level of the respective protein/RNA relieves, to some extent, the symptoms of the disease, disorder, trait or condition.
In one embodiment, the invention features a method for treating or preventing ocular disease in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate (e.g., inhibit) the expression of VEGF and/or VEGFR gene expression in the subject or organism. In one embodiment, the ocular disease is age related macular degeneration (e.g., wet or dry AMD). In one embodiment, the ocular disease is diabetic retinopathy.
In one embodiment, the invention features a method for treating or preventing cancer in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the target VEGF and/or VEGFR gene in the subject or organism whereby the treatment or prevention of cancer can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as cancerous cells and tissues. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells involved in the maintenance or development of cancer in a subject or organism. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tissues or cells in the subject or organism. The siNA molecule can be combined with other therapeutic treatments and modalities as are known in the art for the treatment of or prevention of cancer in a subject or organism.
In one embodiment, the invention features a method for treating or preventing a proliferative disease or condition in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the target VEGF and/or VEGFR gene in the subject or organism whereby the treatment or prevention of the proliferative disease or condition can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as cells and tissues involved in proliferative disease. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells involved in the maintenance or development of the proliferative disease or condition in a subject or organism. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tissues or cells in the subject or organism. The siNA molecule can be combined with other therapeutic treatments and modalities as are known in the art for the treatment of or prevention of proliferative diseases, traits, disorders, or conditions in a subject or organism.
In one embodiment, the invention features a method for treating or preventing an inflammatory disease or condition in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the target VEGF and/or VEGFR gene in the subject or organism whereby the treatment or prevention of the inflammatory disease or condition can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as cells and tissues involved in inflammatory disease. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells involved in the maintenance or development of the inflammatory disease or condition in a subject or organism. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tissues or cells in the subject or organism. The siNA molecule can be combined with other therapeutic treatments and modalities as are known in the art for the treatment of or prevention of inflammatory diseases, traits, disorders, or conditions in a subject or organism.
In one embodiment, the invention features a method for treating or preventing an angiogenesis disease or condition in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the target VEGF and/or VEGFR gene in the subject or organism whereby the treatment or prevention of the angiogenesis disease or condition can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as cells and tissues involved in angiogenesis disease. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells involved in the maintenance or development of the angiogenesis disease or condition in a subject or organism. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tissues or cells in the subject or organism. The siNA molecule can be combined with other therapeutic treatments and modalities as are known in the art for the treatment of or prevention of angiogenesis diseases, traits, disorders, or conditions in a subject or organism.
In one embodiment, the invention features a method for treating or preventing an autoimmune disease, disorder, trait or condition in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the target VEGF and/or VEGFR gene in the subject or organism whereby the treatment or prevention of the autoimmune disease, disorder, trait or condition can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as cells and tissues involved in the autoimmune disease, disorder, trait or condition. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells involved in the maintenance or development of the autoimmune disease, disorder, trait or condition in a subject or organism. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tissues or cells in the subject or organism. The siNA molecule can be combined with other therapeutic treatments and modalities as are known in the art for the treatment of or prevention of autoimmune diseases, traits, disorders, or conditions in a subject or organism.
In one embodiment, the siNA molecule or double stranded nucleic acid molecule of the invention is formulated as a composition described in U.S. Provisional patent application No. 60/678,531 and in related U.S. Provisional patent application No. 60/703,946, filed Jul. 29, 2005, and U.S. Provisional patent application No. 60/737,024, filed Nov. 15, 2005 (Vargeese et al.).
In one embodiment, the invention features a method for treating or preventing an age-related disease, disorder, trait or condition in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the target VEGF and/or VEGFR gene in the subject or organism whereby the treatment or prevention of the age-related disease, disorder, trait or condition can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as cells and tissues involved in the age-related disease, disorder, trait or condition. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells involved in the maintenance or development of the age-related disease, disorder, trait or condition in a subject or organism. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tissues or cells in the subject or organism. The siNA molecule can be combined with other therapeutic treatments and modalities as are known in the art for the treatment of or prevention of age-related diseases, traits, disorders, or conditions in a subject or organism.
In one embodiment, the invention features a method for treating or preventing a neurologic or neurodegenerative disease, disorder, trait or condition in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the target VEGF and/or VEGFR gene in the subject or organism whereby the treatment or prevention of the neurologic or neurodegenerative disease, disorder, trait or condition can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as cells and tissues involved in the neurologic or neurodegenerative disease, disorder, trait or condition. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells involved in the maintenance or development of the neurologic or neurodegenerative disease, disorder, trait or condition in a subject or organism. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tissues or cells in the subject or organism. The siNA molecule can be combined with other therapeutic treatments and modalities as are known in the art for the treatment of or prevention of neurologic or neurodegenerative diseases, traits, disorders, or conditions in a subject or organism.
In one embodiment, the invention features a method for treating or preventing a metabolic disease, disorder, trait or condition in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the target VEGF and/or VEGFR gene in the subject or organism whereby the treatment or prevention of the metabolic disease, disorder, trait or condition can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as cells and tissues involved in the metabolic disease, disorder, trait or condition. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells involved in the maintenance or development of the metabolic disease, disorder, trait or condition in a subject or organism. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tissues or cells in the subject or organism. The siNA molecule can be combined with other therapeutic treatments and modalities as are known in the art for the treatment of or prevention of metabolic diseases, traits, disorders, or conditions in a subject or organism.
In one embodiment, the invention features a method for treating or preventing a cardiovascular disease, disorder, trait or condition in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the target VEGF and/or VEGFR gene in the subject or organism whereby the treatment or prevention of the cardiovascular disease, disorder, trait or condition can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as cells and tissues involved in the cardiovascular disease, disorder, trait or condition. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells involved in the maintenance or development of the cardiovascular disease, disorder, trait or condition in a subject or organism. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tissues or cells in the subject or organism. The siNA molecule can be combined with other therapeutic treatments and modalities as are known in the art for the treatment of or prevention of cardiovascular diseases, traits, disorders, or conditions in a subject or organism.
In one embodiment, the invention features a method for treating or preventing a respiratory disease, disorder, trait or condition in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the target VEGF and/or VEGFR gene in the subject or organism whereby the treatment or prevention of the respiratory disease, disorder, trait or condition can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as cells and tissues involved in the respiratory disease, disorder, trait or condition. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells involved in the maintenance or development of the respiratory disease, disorder, trait or condition in a subject or organism. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tissues or cells in the subject or organism. The siNA molecule can be combined with other therapeutic treatments and modalities as are known in the art for the treatment of or prevention of respiratory diseases, traits, disorders, or conditions in a subject or organism.
In one embodiment, the invention features a method for treating or preventing an ocular disease, disorder, trait or condition in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the target VEGF and/or VEGFR gene in the subject or organism whereby the treatment or prevention of the ocular disease, disorder, trait or condition can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as cells and tissues involved in the ocular disease, disorder, trait or condition. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells involved in the maintenance or development of the ocular disease, disorder, trait or condition in a subject or organism. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tissues or cells in the subject or organism. The siNA molecule can be combined with other therapeutic treatments and modalities as are known in the art for the treatment of or prevention of ocular diseases, traits, disorders, or conditions in a subject or organism (e.g., age related macular degeneration, diabetic retinopathy etc.).
In one embodiment, the invention features a method for treating or preventing a dermatological disease, disorder, trait or condition in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the target VEGF and/or VEGFR gene in the subject or organism whereby the treatment or prevention of the dermatological disease, disorder, trait or condition can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as cells and tissues involved in the dermatological disease, disorder, trait or condition. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells involved in the maintenance or development of the dermatological disease, disorder, trait or condition in a subject or organism. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tissues or cells in the subject or organism. The siNA molecule can be combined with other therapeutic treatments and modalities as are known in the art for the treatment of or prevention of dermatological diseases, traits, disorders, or conditions in a subject or organism (e.g., rosacea, acne, dermatitis etc.).
In one embodiment, the invention features a method for treating or preventing a kidney/renal disease, disorder, trait or condition (e.g., polycystic kidney disease etc.) in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the target VEGF and/or VEGFR gene in the subject or organism whereby the treatment or prevention of the kidney/renal disease, disorder, trait or condition can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as kidney/renal cells and tissues involved in the kidney/renal disease, disorder, trait or condition. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells involved in the maintenance or development of the kidney/renal disease, disorder, trait or condition in a subject or organism. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tissues or cells in the subject or organism. The siNA molecule can be combined with other therapeutic treatments and modalities as are known in the art for the treatment of or prevention of kidney diseases, traits, disorders, or conditions in a subject or organism (e.g., polycystic kidney disease).
In one embodiment, the invention features a method for treating or preventing an auditory disease, disorder, trait or condition (e.g., hearing loss, deafness, etc.) in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the target VEGF and/or VEGFR gene in the subject or organism whereby the treatment or prevention of the auditory disease, disorder, trait or condition can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as cells and tissues of the ear, inner hear, or middle ear involved in the auditory disease, disorder, trait or condition. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells involved in the maintenance or development of the auditory disease, disorder, trait or condition in a subject or organism. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tissues or cells in the subject or organism. The siNA molecule can be combined with other therapeutic treatments and modalities as are known in the art for the treatment of or prevention of auditory diseases, traits, disorders, or conditions in a subject or organism.
In another embodiment, the siNA molecules of the invention are used to target conserved sequences corresponding to a gene family or gene families such as VEGF and/or VEGFR family genes. As such, siNA molecules targeting multiple VEGF and/or VEGFR targets can provide increased therapeutic effect. In addition, siNA can be used to characterize pathways of gene function in a variety of applications. For example, the present invention can be used to inhibit the activity of target gene(s) in a pathway to determine the function of uncharacterized gene(s) in gene function analysis, mRNA function analysis, or translational analysis. The invention can be used to determine potential target gene pathways involved in various diseases and conditions toward pharmaceutical development. The invention can be used to understand pathways of gene expression involved in, for example, the progression and/or maintenance of cancer.
In any of the methods of treatment of the invention, the siNA can be administered to the subject as a course of treatment, for example administration at various time intervals, such as once per day over the course of treatment, once every two days over the course of treatment, once every three days over the course of treatment, once every four days over the course of treatment, once every five days over the course of treatment, once every six days over the course of treatment, once per week over the course of treatment, once every other week over the course of treatment, once per month over the course of treatment, etc. In one embodiment, the course of treatment is once every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. In one embodiment, the course of treatment is from about one to about 52 weeks or longer (e.g., indefinitely). In one embodiment, the course of treatment is from about one to about 48 months or longer (e.g., indefinitely).
In one embodiment, a course of treatment involves an initial course of treatment, such as once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks for a fixed interval (e.g., 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10× or more) followed by a maintenance course of treatment, such as once every 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, or more weeks for an additional fixed interval (e.g., 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10× or more).
In any of the methods of treatment of the invention, the siNA can be administered to the subject systemically as described herein or otherwise known in the art, either alone as a monotherapy or in combination with additional therapies as are known in the art. Systemic administration can include, for example, intravenous, subcutaneous, intramuscular, catheterization, nasopharangeal, transdermal, or gastrointestinal administration as is generally known in the art.
In one embodiment, in any of the methods of treatment or prevention of the invention, the siNA can be administered to the subject locally or to local tissues as described herein or otherwise known in the art, either alone as a monotherapy or in combination with additional therapies as are known in the art. Local administration can include, for example, catheterization, implantation, direct injection, dermal/transdermal application, stenting, ear/eye drops, or portal vein administration to relevant tissues, or any other local administration technique, method or procedure, as is generally known in the art.
The siNA molecules of the invention can be designed to down regulate or inhibit target (e.g., VEGF and/or VEGFR) gene expression through RNAi targeting of a variety of RNA molecules. In one embodiment, the siNA molecules of the invention are used to target various DNA corresponding to a target (e.g., VEGF and/or VEGFR) gene, for example via heterochromatic silencing or transcriptional inhibition. In one embodiment, the siNA molecules of the invention are used to target various RNAs corresponding to a target (e.g., VEGF and/or VEGFR) gene, for example via RNA target cleavage or translational inhibition. Non-limiting examples of such RNAs include messenger RNA (mRNA), non-coding RNA (ncRNA) or regulatory elements (see for example Mattick, 2005, Science, 309, 1527-1528 and Clayerie, 2005, Science, 309, 1529-1530) which includes miRNA and other small RNAs, alternate RNA splice variants of target gene(s), post-transcriptionally modified RNA of target gene(s), pre-mRNA of target gene(s), and/or RNA templates. If alternate splicing produces a family of transcripts that are distinguished by usage of appropriate exons, the instant invention can be used to inhibit gene expression through the appropriate exons to specifically inhibit or to distinguish among the functions of gene family members. For example, a protein that contains an alternatively spliced transmembrane domain can be expressed in both membrane bound and secreted forms. Use of the invention to target the exon containing the transmembrane domain can be used to determine the functional consequences of pharmaceutical targeting of membrane bound as opposed to the secreted form of the protein. Non-limiting examples of applications of the invention relating to targeting these RNA molecules include therapeutic pharmaceutical applications, cosmetic applications, veterinary applications, pharmaceutical discovery applications, molecular diagnostic and gene function applications, and gene mapping, for example using single nucleotide polymorphism mapping with siNA molecules of the invention. Such applications can be implemented using known gene sequences or from partial sequences available from an expressed sequence tag (EST).
In another embodiment, the siNA molecules of the invention are used to target conserved sequences corresponding to a gene family or gene families such as VEGF and/or VEGFR family genes. As such, siNA molecules targeting multiple VEGF and/or VEGFR targets can provide increased therapeutic effect. In addition, siNA can be used to characterize pathways of gene function in a variety of applications. For example, the present invention can be used to inhibit the activity of target gene(s) in a pathway to determine the function of uncharacterized gene(s) in gene function analysis, mRNA function analysis, or translational analysis. The invention can be used to determine potential target gene pathways involved in various diseases and conditions toward pharmaceutical development. The invention can be used to understand pathways of gene expression involved in, for example, the progression and/or maintenance of cancer, angiogenesis, etc.
In one embodiment, siNA molecule(s) and/or methods of the invention are used to down regulate the expression of gene(s) that encode RNA referred to by Genbank Accession, for example, VEGF and/or VEGFR genes encoding RNA sequence(s) referred to herein by Genbank Accession number, for example, Genbank Accession Nos. shown in Table I or Genbank Accession Nos. shown in U.S. Ser. Nos. 10/923,536 and 10/923,536, both incorporated by reference herein.
In one embodiment, the invention features a method comprising: (a) generating a library of siNA constructs having a predetermined complexity; and (b) assaying the siNA constructs of (a) above, under conditions suitable to determine RNAi target sites within the target RNA sequence. In one embodiment, the siNA molecules of (a) have strands of a fixed length, for example, about 23 nucleotides in length. In another embodiment, the siNA molecules of (a) are of differing length, for example having strands of about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length. In one embodiment, the assay can comprise a reconstituted in vitro siNA assay as described herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. In another embodiment, fragments of target RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target RNA sequence. The target RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by cellular expression in in vivo systems.
In one embodiment, the invention features a method comprising: (a) generating a randomized library of siNA constructs having a predetermined complexity, such as of 4N, where N represents the number of base paired nucleotides in each of the siNA construct strands (eg. for a siNA construct having 21 nucleotide sense and antisense strands with 19 base pairs, the complexity would be 419); and (b) assaying the siNA constructs of (a) above, under conditions suitable to determine RNAi target sites within the target RNA sequence. In another embodiment, the siNA molecules of (a) have strands of a fixed length, for example about 23 nucleotides in length. In yet another embodiment, the siNA molecules of (a) are of differing length, for example having strands of about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length. In one embodiment, the assay can comprise a reconstituted in vitro siNA assay as described in Example 6 herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. In another embodiment, fragments of target RNA are analyzed for detectable levels of cleavage, for example, by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target RNA sequence. The target RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by cellular expression in in vivo systems.
In another embodiment, the invention features a method comprising: (a) analyzing the sequence of a RNA target encoded by a target gene; (b) synthesizing one or more sets of siNA molecules having sequence complementary to one or more regions of the RNA of (a); and (c) assaying the siNA molecules of (b) under conditions suitable to determine RNAi targets within the target RNA sequence. In one embodiment, the siNA molecules of (b) have strands of a fixed length, for example about 23 nucleotides in length. In another embodiment, the siNA molecules of (b) are of differing length, for example having strands of about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length. In one embodiment, the assay can comprise a reconstituted in vitro siNA assay as described herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. Fragments of target RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target RNA sequence. The target RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by expression in in vivo systems.
By “target site” is meant a sequence within a target RNA that is “targeted” for cleavage mediated by a siNA construct which contains sequences within its antisense region that are complementary to the target sequence.
By “detectable level of cleavage” is meant cleavage of target RNA (and formation of cleaved product RNAs) to an extent sufficient to discern cleavage products above the background of RNAs produced by random degradation of the target RNA. Production of cleavage products from 1-5% of the target RNA is sufficient to detect above the background for most methods of detection.
In one embodiment, the invention features a composition comprising a siNA molecule of the invention, which can be chemically-modified, in a pharmaceutically acceptable carrier or diluent. In another embodiment, the invention features a pharmaceutical composition comprising siNA molecules of the invention, which can be chemically-modified, targeting one or more genes in a pharmaceutically acceptable carrier or diluent. In another embodiment, the invention features a method for diagnosing a disease, trait, or condition in a subject comprising administering to the subject a composition of the invention under conditions suitable for the diagnosis of the disease, trait, or condition in the subject.
In another embodiment, the invention features a method for validating a VEGF and/or VEGFR target gene VEGF and/or VEGFR target, comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands includes a sequence complementary to RNA of a VEGF and/or VEGFR target gene; (b) introducing the siNA molecule into a cell, tissue, subject, or organism under conditions suitable for modulating expression of the VEGF and/or VEGFR target gene in the cell, tissue, subject, or organism; and (c) determining the function of the gene by assaying for any phenotypic change in the cell, tissue, subject, or organism.
In another embodiment, the invention features a method for validating a VEGF and/or VEGFR target comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands includes a sequence complementary to RNA of a VEGF and/or VEGFR target gene; (b) introducing the siNA molecule into a biological system under conditions suitable for modulating expression of the VEGF and/or VEGFR target gene in the biological system; and (c) determining the function of the gene by assaying for any phenotypic change in the biological system.
By “biological system” is meant, material, in a purified or unpurified form, from biological sources, including but not limited to human or animal, wherein the system comprises the components required for RNAi activity. The term “biological system” includes, for example, a cell, tissue, subject, or organism, or extract thereof. The term biological system also includes reconstituted RNAi systems that can be used in an in vitro setting.
By “phenotypic change” is meant any detectable change to a cell that occurs in response to contact or treatment with a nucleic acid molecule of the invention (e.g., siNA). Such detectable changes include, but are not limited to, changes in shape, size, proliferation, motility, protein expression or RNA expression or other physical or chemical changes as can be assayed by methods known in the art. The detectable change can also include expression of reporter genes/molecules such as Green Florescent Protein (GFP) or various tags that are used to identify an expressed protein or any other cellular component that can be assayed.
In one embodiment, the invention features a kit containing a siNA molecule of the invention, which can be chemically-modified, that can be used to modulate the expression of a VEGF and/or VEGFR target gene in a biological system, including, for example, in a cell, tissue, subject, or organism. In another embodiment, the invention features a kit containing more than one siNA molecule of the invention, which can be chemically-modified, that can be used to modulate the expression of more than one VEGF and/or VEGFR target gene in a biological system, including, for example, in a cell, tissue, subject, or organism.
In one embodiment, the invention features a cell containing one or more siNA molecules of the invention, which can be chemically-modified. In another embodiment, the cell containing a siNA molecule of the invention is a mammalian cell. In yet another embodiment, the cell containing a siNA molecule of the invention is a human cell.
In one embodiment, the synthesis of a siNA molecule of the invention, which can be chemically-modified, comprises: (a) synthesis of two complementary strands of the siNA molecule; (b) annealing the two complementary strands together under conditions suitable to obtain a double-stranded siNA molecule. In another embodiment, synthesis of the two complementary strands of the siNA molecule is by solid phase oligonucleotide synthesis. In yet another embodiment, synthesis of the two complementary strands of the siNA molecule is by solid phase tandem oligonucleotide synthesis.
In one embodiment, the invention features a method for synthesizing a siNA duplex molecule comprising: (a) synthesizing a first oligonucleotide sequence strand of the siNA molecule, wherein the first oligonucleotide sequence strand comprises a cleavable linker molecule that can be used as a scaffold for the synthesis of the second oligonucleotide sequence strand of the siNA; (b) synthesizing the second oligonucleotide sequence strand of siNA on the scaffold of the first oligonucleotide sequence strand, wherein the second oligonucleotide sequence strand further comprises a chemical moiety than can be used to purify the siNA duplex; (c) cleaving the linker molecule of (a) under conditions suitable for the two siNA oligonucleotide strands to hybridize and form a stable duplex; and (d) purifying the siNA duplex utilizing the chemical moiety of the second oligonucleotide sequence strand. In one embodiment, cleavage of the linker molecule in (c) above takes place during deprotection of the oligonucleotide, for example, under hydrolysis conditions using an alkylamine base such as methylamine. In one embodiment, the method of synthesis comprises solid phase synthesis on a solid support such as controlled pore glass (CPG) or polystyrene, wherein the first sequence of (a) is synthesized on a cleavable linker, such as a succinyl linker, using the solid support as a scaffold. The cleavable linker in (a) used as a scaffold for synthesizing the second strand can comprise similar reactivity as the solid support derivatized linker, such that cleavage of the solid support derivatized linker and the cleavable linker of (a) takes place concomitantly. In another embodiment, the chemical moiety of (b) that can be used to isolate the attached oligonucleotide sequence comprises a trityl group, for example a dimethoxytrityl group, which can be employed in a trityl-on synthesis strategy as described herein. In yet another embodiment, the chemical moiety, such as a dimethoxytrityl group, is removed during purification, for example, using acidic conditions.
In a further embodiment, the method for siNA synthesis is a solution phase synthesis or hybrid phase synthesis wherein both strands of the siNA duplex are synthesized in tandem using a cleavable linker attached to the first sequence which acts a scaffold for synthesis of the second sequence. Cleavage of the linker under conditions suitable for hybridization of the separate siNA sequence strands results in formation of the double-stranded siNA molecule.
In another embodiment, the invention features a method for synthesizing a siNA duplex molecule comprising: (a) synthesizing one oligonucleotide sequence strand of the siNA molecule, wherein the sequence comprises a cleavable linker molecule that can be used as a scaffold for the synthesis of another oligonucleotide sequence; (b) synthesizing a second oligonucleotide sequence having complementarity to the first sequence strand on the scaffold of (a), wherein the second sequence comprises the other strand of the double-stranded siNA molecule and wherein the second sequence further comprises a chemical moiety than can be used to isolate the attached oligonucleotide sequence; (c) purifying the product of (b) utilizing the chemical moiety of the second oligonucleotide sequence strand under conditions suitable for isolating the full-length sequence comprising both siNA oligonucleotide strands connected by the cleavable linker and under conditions suitable for the two siNA oligonucleotide strands to hybridize and form a stable duplex. In one embodiment, cleavage of the linker molecule in (c) above takes place during deprotection of the oligonucleotide, for example, under hydrolysis conditions. In another embodiment, cleavage of the linker molecule in (c) above takes place after deprotection of the oligonucleotide. In another embodiment, the method of synthesis comprises solid phase synthesis on a solid support such as controlled pore glass (CPG) or polystyrene, wherein the first sequence of (a) is synthesized on a cleavable linker, such as a succinyl linker, using the solid support as a scaffold. The cleavable linker in (a) used as a scaffold for synthesizing the second strand can comprise similar reactivity or differing reactivity as the solid support derivatized linker, such that cleavage of the solid support derivatized linker and the cleavable linker of (a) takes place either concomitantly or sequentially. In one embodiment, the chemical moiety of (b) that can be used to isolate the attached oligonucleotide sequence comprises a trityl group, for example a dimethoxytrityl group.
In another embodiment, the invention features a method for making a double-stranded siNA molecule in a single synthetic process comprising: (a) synthesizing an oligonucleotide having a first and a second sequence, wherein the first sequence is complementary to the second sequence, and the first oligonucleotide sequence is linked to the second sequence via a cleavable linker, and wherein a terminal 5′-protecting group, for example, a 5′-O-dimethoxytrityl group (5′-O-DMT) remains on the oligonucleotide having the second sequence; (b) deprotecting the oligonucleotide whereby the deprotection results in the cleavage of the linker joining the two oligonucleotide sequences; and (c) purifying the product of (b) under conditions suitable for isolating the double-stranded siNA molecule, for example using a trityl-on synthesis strategy as described herein.
In another embodiment, the method of synthesis of siNA molecules of the invention comprises the teachings of Scaringe et al., U.S. Pat. Nos. 5,889,136; 6,008,400; and 6,111,086, incorporated by reference herein in their entirety.
In one embodiment, the invention features siNA constructs that mediate RNAi against a VEGF and/or VEGFR target polynucleotide (e.g., RNA or DNA VEGF and/or VEGFR target), wherein the siNA construct comprises one or more chemical modifications, for example, one or more chemical modifications having any of Formulae I-VII or any combination thereof that increases the nuclease resistance of the siNA construct.
In another embodiment, the invention features a method for generating siNA molecules with increased nuclease resistance comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased nuclease resistance.
In another embodiment, the invention features a method for generating siNA molecules with improved toxicologic profiles (e.g., having attenuated or no immunstimulatory properties) comprising (a) introducing nucleotides having any of Formula I-VII (e.g., siNA motifs referred to in Table IV) or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved toxicologic profiles.
In another embodiment, the invention features a method for generating siNA formulations with improved toxicologic profiles (e.g., having attenuated or no immunstimulatory properties) comprising (a) generating a siNA formulation comprising a siNA molecule of the invention and a delivery vehicle or delivery particle as described herein or as otherwise known in the art, and (b) assaying the siNA formulation of step (a) under conditions suitable for isolating siNA formulations having improved toxicologic profiles.
In another embodiment, the invention features a method for generating siNA molecules that do not stimulate an interferon response (e.g., no interferon response or attenuated interferon response) in a cell, subject, or organism, comprising (a) introducing nucleotides having any of Formula I-VII (e.g., siNA motifs referred to in Table IV) or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules that do not stimulate an interferon response.
In another embodiment, the invention features a method for generating siNA formulations that do not stimulate an interferon response (e.g., no interferon response or attenuated interferon response) in a cell, subject, or organism, comprising (a) generating a siNA formulation comprising a siNA molecule of the invention and a delivery vehicle or delivery particle as described herein or as otherwise known in the art, and (b) assaying the siNA formulation of step (a) under conditions suitable for isolating siNA formulations that do not stimulate an interferon response. In one embodiment, the interferon comprises interferon alpha.
In another embodiment, the invention features a method for generating siNA molecules that do not stimulate an inflammatory or proinflammatory cytokine response (e.g., no cytokine response or attenuated cytokine response) in a cell, subject, or organism, comprising (a) introducing nucleotides having any of Formula I-VII (e.g., siNA motifs referred to in Table IV) or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules that do not stimulate a cytokine response. In one embodiment, the cytokine comprises an interleukin such as interleukin-6 (IL-6) and/or tumor necrosis alpha (TNF-α).
In another embodiment, the invention features a method for generating siNA formulations that do not stimulate an inflammatory or proinflammatory cytokine response (e.g., no cytokine response or attenuated cytokine response) in a cell, subject, or organism, comprising (a) generating a siNA formulation comprising a siNA molecule of the invention and a delivery vehicle or delivery particle as described herein or as otherwise known in the art, and (b) assaying the siNA formulation of step (a) under conditions suitable for isolating siNA formulations that do not stimulate a cytokine response. In one embodiment, the cytokine comprises an interleukin such as interleukin-6 (IL-6) and/or tumor necrosis alpha (TNF-α).
In another embodiment, the invention features a method for generating siNA molecules that do not stimulate Toll-like Receptor (TLR) response (e.g., no TLR response or attenuated TLR response) in a cell, subject, or organism, comprising (a) introducing nucleotides having any of Formula I-VII (e.g., siNA motifs referred to in Table IV) or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules that do not stimulate a TLR response. In one embodiment, the TLR comprises TLR3, TLR7, TLR8 and/or TLR9.
In another embodiment, the invention features a method for generating siNA formulations that do not stimulate a Toll-like Receptor (TLR) response (e.g., no TLR response or attenuated TLR response) in a cell, subject, or organism, comprising (a) generating a siNA formulation comprising a siNA molecule of the invention and a delivery vehicle or delivery particle as described herein or as otherwise known in the art, and (b) assaying the siNA formulation of step (a) under conditions suitable for isolating siNA formulations that do not stimulate a TLR response. In one embodiment, the TLR comprises TLR3, TLR7, TLR8 and/or TLR9.
In one embodiment, the invention features a chemically synthesized double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a VEGF and/or VEGFR target RNA via RNA interference (RNAi), wherein: (a) each strand of said siNA molecule is about 18 to about 38 nucleotides in length; (b) one strand of said siNA molecule comprises nucleotide sequence having sufficient complementarity to said VEGF and/or VEGFR target RNA for the siNA molecule to direct cleavage of the VEGF and/or VEGFR target RNA via RNA interference; and (c) wherein the nucleotide positions within said siNA molecule are chemically modified to reduce the immunostimulatory properties of the siNA molecule to a level below that of a corresponding unmodified siRNA molecule. Such siNA molecules are said to have an improved toxicologic profile compared to an unmodified or minimally modified siNA.
By “improved toxicologic profile”, is meant that the chemically modified or formulated siNA construct exhibits decreased toxicity in a cell, subject, or organism compared to an unmodified or unformulated siNA, or siNA molecule having fewer modifications or modifications that are less effective in imparting improved toxicology. In a non-limiting example, siNA molecules and formulations with improved toxicologic profiles are associated with reduced immunostimulatory properties, such as a reduced, decreased or attenuated immunostimulatory response in a cell, subject, or organism compared to an unmodified or unformulated siNA, or siNA molecule having fewer modifications or modifications that are less effective in imparting improved toxicology. Such an improved toxicologic profile is characterized by abrogated or reduced immunostimulation, such as reduction or abrogation of induction of interferons (e.g., interferon alpha), inflammatory cytokines (e.g., interleukins such as IL-6, and/or TNF-alpha), and/or toll like receptors (e.g., TLR-3, TLR-7, TLR-8, and/or TLR-9). In one embodiment, a siNA molecule or formulation with an improved toxicological profile comprises no ribonucleotides. In one embodiment, a siNA molecule or formulation with an improved toxicological profile comprises less than 5 ribonucleotides (e.g., 1, 2, 3, or 4 ribonucleotides). In one embodiment, a siNA molecule or formulation with an improved toxicological profile comprises Stab 7, Stab 8, Stab 11, Stab 12, Stab 13, Stab 16, Stab 17, Stab 18, Stab 19, Stab 20, Stab 23, Stab 24, Stab 25, Stab 26, Stab 27, Stab 28, Stab 29, Stab 30, Stab 31, Stab 32, Stab 33, Stab 34 or any combination thereof (see Table IV). Herein, numeric Stab chemistries include both 2′-fluoro and 2′-OCF3 versions of the chemistries shown in Table IV. For example, “Stab 7/8” refers to both Stab 7/8 and Stab 7F/8F etc. In one embodiment, a siNA molecule or formulation with an improved toxicological profile comprises a siNA molecule of the invention and a formulation as described in United States Patent Application Publication No. 20030077829, incorporated by reference herein in its entirety including the drawings.
In one embodiment, the level of immunostimulatory response associated with a given siNA molecule can be measured as is described herein or as is otherwise known in the art, for example by determining the level of PKR/interferon response, proliferation, B-cell activation, and/or cytokine production in assays to quantitate the immunostimulatory response of particular siNA molecules (see, for example, Leifer et al., 2003, J Immunother. 26, 313-9; and U.S. Pat. No. 5,968,909, incorporated in its entirety by reference). In one embodiment, the reduced immunostimulatory response is between about 10% and about 100% compared to an unmodified or minimally modified siRNA molecule, e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% reduced immunostimulatory response. In one embodiment, the immunostimulatory response associated with a siNA molecule can be modulated by the degree of chemical modification. For example, a siNA molecule having between about 10% and about 100%, e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the nucleotide positions in the siNA molecule modified can be selected to have a corresponding degree of immunostimulatory properties as described herein.
In one embodiment, the degree of reduced immunostimulatory response is selected for optimized RNAi activity. For example, retaining a certain degree of immunostimulation can be preferred to treat viral infection, where less than 100% reduction in immunostimulation may be preferred for maximal antiviral activity (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduction in immunostimulation) whereas the inhibition of expression of an endogenous gene VEGF and/or VEGFR target may be preferred with siNA molecules that possess minimal immunostimulatory properties to prevent non-specific toxicity or off VEGF and/or VEGFR target effects (e.g., about 90% to about 100% reduction in immunostimulation).
In one embodiment, the invention features a chemically synthesized double stranded siNA molecule that directs cleavage of a VEGF and/or VEGFR target RNA via RNA interference (RNAi), wherein (a) each strand of said siNA molecule is about 18 to about 38 nucleotides in length; (b) one strand of said siNA molecule comprises nucleotide sequence having sufficient complementarity to said VEGF and/or VEGFR target RNA for the siNA molecule to direct cleavage of the VEGF and/or VEGFR target RNA via RNA interference; and (c) wherein one or more nucleotides of said siNA molecule are chemically modified to reduce the immunostimulatory properties of the siNA molecule to a level below that of a corresponding unmodified siNA molecule. In one embodiment, each strand comprises at least about 18 nucleotides that are complementary to the nucleotides of the other strand.
In another embodiment, the siNA molecule comprising modified nucleotides to reduce the immunostimulatory properties of the siNA molecule comprises an antisense region having nucleotide sequence that is complementary to a nucleotide sequence of a VEGF and/or VEGFR target gene or a portion thereof and further comprises a sense region, wherein said sense region comprises a nucleotide sequence substantially similar to the nucleotide sequence of said VEGF and/or VEGFR target gene or portion thereof. In one embodiment thereof, the antisense region and the sense region comprise about 18 to about 38 nucleotides, wherein said antisense region comprises at least about 18 nucleotides that are complementary to nucleotides of the sense region. In one embodiment thereof, the pyrimidine nucleotides in the sense region are 2′-O-methylpyrimidine nucleotides. In another embodiment thereof, the purine nucleotides in the sense region are 2′-deoxy purine nucleotides. In yet another embodiment thereof, the pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides. In another embodiment thereof, the pyrimidine nucleotides of said antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides. In yet another embodiment thereof, the purine nucleotides of said antisense region are 2′-O-methyl purine nucleotides. In still another embodiment thereof, the purine nucleotides present in said antisense region comprise 2′-deoxypurine nucleotides. In another embodiment, the antisense region comprises a phosphorothioate internucleotide linkage at the 3′ end of said antisense region. In another embodiment, the antisense region comprises a glyceryl modification at a 3′ end of said antisense region.
In other embodiments, the siNA molecule comprising modified nucleotides to reduce the immunostimulatory properties of the siNA molecule can comprise any of the structural features of siNA molecules described herein. In other embodiments, the siNA molecule comprising modified nucleotides to reduce the immunostimulatory properties of the siNA molecule can comprise any of the chemical modifications of siNA molecules described herein.
In one embodiment, the invention features a method for generating a chemically synthesized double stranded siNA molecule having chemically modified nucleotides to reduce the immunostimulatory properties of the siNA molecule, comprising (a) introducing one or more modified nucleotides in the siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating an siNA molecule having reduced immunostimulatory properties compared to a corresponding siNA molecule having unmodified nucleotides. Each strand of the siNA molecule is about 18 to about 38 nucleotides in length. One strand of the siNA molecule comprises nucleotide sequence having sufficient complementarity to the VEGF and/or VEGFR target RNA for the siNA molecule to direct cleavage of the VEGF and/or VEGFR target RNA via RNA interference. In one embodiment, the reduced immunostimulatory properties comprise an abrogated or reduced induction of inflammatory or proinflammatory cytokines, such as interleukin-6 (IL-6) or tumor necrosis alpha (TNF-α), in response to the siNA being introduced in a cell, tissue, or organism. In another embodiment, the reduced immunostimulatory properties comprise an abrogated or reduced induction of Toll Like Receptors (TLRs), such as TLR3, TLR7, TLR8 or TLR9, in response to the siNA being introduced in a cell, tissue, or organism. In another embodiment, the reduced immunostimulatory properties comprise an abrogated or reduced induction of interferons, such as interferon alpha, in response to the siNA being introduced in a cell, tissue, or organism.
In one embodiment, the invention features siNA constructs that mediate RNAi against a VEGF and/or VEGFR target polynucleotide, wherein the siNA construct comprises one or more chemical modifications described herein that modulates the binding affinity between the sense and antisense strands of the siNA construct.
In another embodiment, the invention features a method for generating siNA molecules with increased binding affinity between the sense and antisense strands of the siNA molecule comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased binding affinity between the sense and antisense strands of the siNA molecule.
In one embodiment, the invention features siNA constructs that mediate RNAi against a VEGF and/or VEGFR target polynucleotide, wherein the siNA construct comprises one or more chemical modifications described herein that modulates the binding affinity between the antisense strand of the siNA construct and a complementary VEGF and/or VEGFR target RNA sequence within a cell.
In one embodiment, the invention features siNA constructs that mediate RNAi against a VEGF and/or VEGFR target polynucleotide, wherein the siNA construct comprises one or more chemical modifications described herein that modulates the binding affinity between the antisense strand of the siNA construct and a complementary VEGF and/or VEGFR target DNA sequence within a cell.
In another embodiment, the invention features a method for generating siNA molecules with increased binding affinity between the antisense strand of the siNA molecule and a complementary VEGF and/or VEGFR target RNA sequence comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased binding affinity between the antisense strand of the siNA molecule and a complementary VEGF and/or VEGFR target RNA sequence.
In another embodiment, the invention features a method for generating siNA molecules with increased binding affinity between the antisense strand of the siNA molecule and a complementary VEGF and/or VEGFR target DNA sequence comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased binding affinity between the antisense strand of the siNA molecule and a complementary VEGF and/or VEGFR target DNA sequence.
In one embodiment, the invention features siNA constructs that mediate RNAi against a VEGF and/or VEGFR target polynucleotide, wherein the siNA construct comprises one or more chemical modifications described herein that modulate the polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to the chemically-modified siNA construct.
In another embodiment, the invention features a method for generating siNA molecules capable of mediating increased polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to a chemically-modified siNA molecule comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules capable of mediating increased polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to the chemically-modified siNA molecule.
In one embodiment, the invention features chemically-modified siNA constructs that mediate RNAi against a VEGF and/or VEGFR target polynucleotide in a cell, wherein the chemical modifications do not significantly effect the interaction of siNA with a VEGF and/or VEGFR target RNA molecule, DNA molecule and/or proteins or other factors that are essential for RNAi in a manner that would decrease the efficacy of RNAi mediated by such siNA constructs.
In another embodiment, the invention features a method for generating siNA molecules with improved RNAi specificity against polynucleotide VEGF and/or VEGFR targets comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved RNAi specificity. In one embodiment, improved specificity comprises having reduced off VEGF and/or VEGFR target effects compared to an unmodified siNA molecule. For example, introduction of terminal cap moieties at the 3′-end, 5′-end, or both 3′ and 5′-ends of the sense strand or region of a siNA molecule of the invention can direct the siNA to have improved specificity by preventing the sense strand or sense region from acting as a template for RNAi activity against a corresponding VEGF and/or VEGFR target having complementarity to the sense strand or sense region.
In another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against a VEGF and/or VEGFR target polynucleotide comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved RNAi activity.
In yet another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against a VEGF and/or VEGFR target RNA comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved RNAi activity against the VEGF and/or VEGFR target RNA.
In yet another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against a VEGF and/or VEGFR target DNA comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved RNAi activity against the VEGF and/or VEGFR target DNA.
In one embodiment, the invention features siNA constructs that mediate RNAi against a VEGF and/or VEGFR target polynucleotide, wherein the siNA construct comprises one or more chemical modifications described herein that modulates the cellular uptake of the siNA construct, such as cholesterol conjugation of the siNA.
In another embodiment, the invention features a method for generating siNA molecules against a VEGF and/or VEGFR target polynucleotide with improved cellular uptake comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved cellular uptake.
In one embodiment, the invention features siNA constructs that mediate RNAi against a VEGF and/or VEGFR target polynucleotide, wherein the siNA construct comprises one or more chemical modifications described herein that increases the bioavailability of the siNA construct, for example, by attaching polymeric conjugates such as polyethyleneglycol or equivalent conjugates that improve the pharmacokinetics of the siNA construct, or by attaching conjugates that VEGF and/or VEGFR target specific tissue types or cell types in vivo. Non-limiting examples of such conjugates are described in Vargeese et al., U.S. Ser. No. 10/201,394 incorporated by reference herein.
In one embodiment, the invention features a method for generating siNA molecules of the invention with improved bioavailability comprising (a) introducing a conjugate into the structure of a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability. Such conjugates can include ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; cholesterol derivatives, polyamines, such as spermine or spermidine; and others.
In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a VEGF and/or VEGFR target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein said second sequence is chemically modified in a manner that it can no longer act as a guide sequence for efficiently mediating RNA interference and/or be recognized by cellular proteins that facilitate RNAi. In one embodiment, the first nucleotide sequence of the siNA is chemically modified as described herein. In one embodiment, the first nucleotide sequence of the siNA is not modified (e.g., is all RNA).
In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a VEGF and/or VEGFR target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein the second sequence is designed or modified in a manner that prevents its entry into the RNAi pathway as a guide sequence or as a sequence that is complementary to a VEGF and/or VEGFR target nucleic acid (e.g., RNA) sequence. In one embodiment, the first nucleotide sequence of the siNA is chemically modified as described herein. In one embodiment, the first nucleotide sequence of the siNA is not modified (e.g., is all RNA). Such design or modifications are expected to enhance the activity of siNA and/or improve the specificity of siNA molecules of the invention. These modifications are also expected to minimize any off-VEGF and/or VEGFR target effects and/or associated toxicity.
In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a VEGF and/or VEGFR target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein said second sequence is incapable of acting as a guide sequence for mediating RNA interference. In one embodiment, the first nucleotide sequence of the siNA is chemically modified as described herein. In one embodiment, the first nucleotide sequence of the siNA is not modified (e.g., is all RNA).
In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a VEGF and/or VEGFR target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein said second sequence does not have a terminal 5′-hydroxyl (5′-OH) or 5′-phosphate group.
In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a VEGF and/or VEGFR target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein said second sequence comprises a terminal cap moiety at the 5′-end of said second sequence. In one embodiment, the terminal cap moiety comprises an inverted abasic, inverted deoxy abasic, inverted nucleotide moiety, a group shown in FIG. 10, an alkyl or cycloalkyl group, a heterocycle, or any other group that prevents RNAi activity in which the second sequence serves as a guide sequence or template for RNAi.
In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a VEGF and/or VEGFR target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein said second sequence comprises a terminal cap moiety at the 5′-end and 3′-end of said second sequence. In one embodiment, each terminal cap moiety individually comprises an inverted abasic, inverted deoxy abasic, inverted nucleotide moiety, a group shown in FIG. 10, an alkyl or cycloalkyl group, a heterocycle, or any other group that prevents RNAi activity in which the second sequence serves as a guide sequence or template for RNAi.
In one embodiment, the invention features a method for generating siNA molecules of the invention with improved specificity for down regulating or inhibiting the expression of a VEGF and/or VEGFR target nucleic acid (e.g., a DNA or RNA such as a gene or its corresponding RNA), comprising (a) introducing one or more chemical modifications into the structure of a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved specificity. In another embodiment, the chemical modification used to improve specificity comprises terminal cap modifications at the 5′-end, 3′-end, or both 5′ and 3′-ends of the siNA molecule. The terminal cap modifications can comprise, for example, structures shown in FIG. 10 (e.g. inverted deoxyabasic moieties) or any other chemical modification that renders a portion of the siNA molecule (e.g. the sense strand) incapable of mediating RNA interference against an off VEGF and/or VEGFR target nucleic acid sequence. In a non-limiting example, a siNA molecule is designed such that only the antisense sequence of the siNA molecule can serve as a guide sequence for RISC mediated degradation of a corresponding VEGF and/or VEGFR target RNA sequence. This can be accomplished by rendering the sense sequence of the siNA inactive by introducing chemical modifications to the sense strand that preclude recognition of the sense strand as a guide sequence by RNAi machinery. In one embodiment, such chemical modifications comprise any chemical group at the 5′-end of the sense strand of the siNA, or any other group that serves to render the sense strand inactive as a guide sequence for mediating RNA interference. These modifications, for example, can result in a molecule where the 5′-end of the sense strand no longer has a free 5′-hydroxyl (5′-OH) or a free 5′-phosphate group (e.g., phosphate, diphosphate, triphosphate, cyclic phosphate etc.). Non-limiting examples of such siNA constructs are described herein, such as “Stab 9/10”, “Stab 7/8”, “Stab 7/19”, “Stab 17/22”, “Stab 23/24”, “Stab 24/25”, and “Stab 24/26” (e.g., any siNA having Stab 7, 9, 17, 23, or 24 sense strands) chemistries and variants thereof (see Table IV) wherein the 5′-end and 3′-end of the sense strand of the siNA do not comprise a hydroxyl group or phosphate group. Herein, numeric Stab chemistries include both 2′-fluoro and 2′-OCF3 versions of the chemistries shown in Table IV. For example, “Stab 7/8” refers to both Stab 7/8 and Stab 7F/8F etc.
In one embodiment, the invention features a method for generating siNA molecules of the invention with improved specificity for down regulating or inhibiting the expression of a VEGF and/or VEGFR target nucleic acid (e.g., a DNA or RNA such as a gene or its corresponding RNA), comprising introducing one or more chemical modifications into the structure of a siNA molecule that prevent a strand or portion of the siNA molecule from acting as a template or guide sequence for RNAi activity. In one embodiment, the inactive strand or sense region of the siNA molecule is the sense strand or sense region of the siNA molecule, i.e. the strand or region of the siNA that does not have complementarity to the VEGF and/or VEGFR target nucleic acid sequence. In one embodiment, such chemical modifications comprise any chemical group at the 5′-end of the sense strand or region of the siNA that does not comprise a 5′-hydroxyl (5′-OH) or 5′-phosphate group, or any other group that serves to render the sense strand or sense region inactive as a guide sequence for mediating RNA interference. Non-limiting examples of such siNA constructs are described herein, such as “Stab 9/10”, “Stab 7/8”, “Stab 7/19”, “Stab 17/22”, “Stab. 23/24”, “Stab 24/25”, and “Stab 24/26” (e.g., any siNA having Stab 7, 9, 17, 23, or 24 sense strands) chemistries and variants thereof (see Table IV) wherein the 5′-end and 3′-end of the sense strand of the siNA do not comprise a hydroxyl group or phosphate group. Herein, numeric Stab chemistries include both 2′-fluoro and 2′-OCF3 versions of the chemistries shown in Table IV. For example, “Stab 7/8” refers to both Stab 7/8 and Stab 7F/8F etc.
In one embodiment, the invention features a method for screening siNA molecules that are active in mediating RNA interference against a VEGF and/or VEGFR target nucleic acid sequence comprising (a) generating a plurality of unmodified siNA molecules, (b) screening the siNA molecules of step (a) under conditions suitable for isolating siNA molecules that are active in mediating RNA interference against the VEGF and/or VEGFR target nucleic acid sequence, and (c) introducing chemical modifications (e.g. chemical modifications as described herein or as otherwise known in the art) into the active siNA molecules of (b). In one embodiment, the method further comprises re-screening the chemically modified siNA molecules of step (c) under conditions suitable for isolating chemically modified siNA molecules that are active in mediating RNA interference against the VEGF and/or VEGFR target nucleic acid sequence.
In one embodiment, the invention features a method for screening chemically modified siNA molecules that are active in mediating RNA interference against a VEGF and/or VEGFR target nucleic acid sequence comprising (a) generating a plurality of chemically modified siNA molecules (e.g. siNA molecules as described herein or as otherwise known in the art), and (b) screening the siNA molecules of step (a) under conditions suitable for isolating chemically modified siNA molecules that are active in mediating RNA interference against the VEGF and/or VEGFR target nucleic acid sequence.
The term “ligand” refers to any compound or molecule, such as a drug, peptide, hormone, or neurotransmitter, that is capable of interacting with another compound, such as a receptor, either directly or indirectly. The receptor that interacts with a ligand can be present on the surface of a cell or can alternately be an intercellular receptor. Interaction of the ligand with the receptor can result in a biochemical reaction, or can simply be a physical interaction or association.
In another embodiment, the invention features a method for generating siNA molecules of the invention with improved bioavailability comprising (a) introducing an excipient formulation to a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability. Such excipients include polymers such as cyclodextrins, lipids, cationic lipids, polyamines, phospholipids, nanoparticles, receptors, ligands, and others.
In another embodiment, the invention features a method for generating siNA molecules of the invention with improved bioavailability comprising (a) introducing nucleotides having any of Formulae I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability.
In another embodiment, polyethylene glycol (PEG) can be covalently attached to siNA compounds of the present invention. The attached PEG can be any molecular weight, preferably from about 100 to about 50,000 daltons (Da).
The present invention can be used alone or as a component of a kit having at least one of the reagents necessary to carry out the in vitro or in vivo introduction of RNA to test samples and/or subjects. For example, preferred components of the kit include a siNA molecule of the invention and a vehicle that promotes introduction of the siNA into cells of interest as described herein (e.g., using lipids and other methods of transfection known in the art, see for example Beigelman et al, U.S. Pat. No. 6,395,713). The kit can be used for VEGF and/or VEGFR target validation, such as in determining gene function and/or activity, or in drug optimization, and in drug discovery (see for example Usman et al., U.S. Ser. No. 60/402,996). Such a kit can also include instructions to allow a user of the kit to practice the invention.
The term “short interfering nucleic acid”, “siNA”, “short interfering RNA”, “siRNA”, “short interfering nucleic acid molecule”, “short interfering oligonucleotide molecule”, or “chemically-modified short interfering nucleic acid molecule” as used herein refers to any nucleic acid molecule capable of inhibiting or down regulating gene expression or viral replication by mediating RNA interference “RNAi” or gene silencing in a sequence-specific manner. For example the siNA can be a double-stranded nucleic acid molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e., each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure, for example wherein the double stranded region is about 15 to about 30, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs; the antisense strand comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof (e.g., about 15 to about 25 or more nucleotides of the siNA molecule are complementary to the target nucleic acid or a portion thereof). Alternatively, the siNA is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siNA are linked by means of a nucleic acid based or non-nucleic acid-based linker(s). The siNA can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siNA can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siNA molecule capable of mediating RNAi. The siNA can also comprise a single stranded polynucleotide having nucleotide sequence complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof (for example, where such siNA molecule does not require the presence within the siNA molecule of nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), wherein the single stranded polynucleotide can further comprise a terminal phosphate group, such as a 5′-phosphate (see for example Martinez et al., 2002, Cell., 110, 563-574 and Schwarz et al., 2002, Molecular Cell, 10, 537-568), or 5′,3′-diphosphate. In certain embodiments, the siNA molecule of the invention comprises separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic interactions, and/or stacking interactions. In certain embodiments, the siNA molecules of the invention comprise nucleotide sequence that is complementary to nucleotide sequence of a target gene. In another embodiment, the siNA molecule of the invention interacts with nucleotide sequence of a target gene in a manner that causes inhibition of expression of the target gene. As used herein, siNA molecules need not be limited to those molecules containing only RNA, but further encompasses chemically-modified nucleotides and non-nucleotides. In certain embodiments, the short interfering nucleic acid molecules of the invention lack 2′-hydroxy (2′-OH) containing nucleotides. Applicant describes in certain embodiments short interfering nucleic acids that do not require the presence of nucleotides having a 2′-hydroxy group for mediating RNAi and as such, short interfering nucleic acid molecules of the invention optionally do not include any ribonucleotides (e.g., nucleotides having a 2′-OH group). Such siNA molecules that do not require the presence of ribonucleotides within the siNA molecule to support RNAi can however have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2′-OH groups. Optionally, siNA molecules can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions. The modified short interfering nucleic acid molecules of the invention can also be referred to as short interfering modified oligonucleotides “siMON.” As used herein, the term siNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically-modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. Non limiting examples of siNA molecules of the invention are shown in FIGS. 4-6, and Table II herein. Such siNA molecules are distinct from other nucleic acid technologies known in the art that mediate inhibition of gene expression, such as ribozymes, antisense, triplex forming, aptamer, 2,5-A chimera, or decoy oligonucleotides.
By “RNA interference” or “RNAi” is meant a biological process of inhibiting or down regulating gene expression in a cell as is generally known in the art and which is mediated by short interfering nucleic acid molecules, see for example Zamore and Haley, 2005, Science, 309, 1519-1524; Vaughn and Martienssen, 2005, Science, 309, 1525-1526; Zamore et al., 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al., International PCT Publication No. WO 00/44895; Zernicka-Goetz et al., International PCT Publication No. WO 01/36646; Fire, International PCT Publication No. WO 99/32619; Plaetinck et al., International PCT Publication No. WO 00/01846; Mello and Fire, International PCT Publication No. WO 01/29058; Deschamps-Depaillette, International PCT Publication No. WO 99/07409; and Li et al., International PCT Publication No. WO 00/44914; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237; Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002, RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16, 1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831). In addition, as used herein, the term RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, transcriptional inhibition, or epigenetics. For example, siNA molecules of the invention can be used to epigenetically silence genes at both the post-transcriptional level or the pre-transcriptional level. In a non-limiting example, epigenetic modulation of gene expression by siNA molecules of the invention can result from siNA mediated modification of chromatin structure or methylation patterns to alter gene expression (see, for example, Verdel et al., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237). In another non-limiting example, modulation of gene expression by siNA molecules of the invention can result from siNA mediated cleavage of RNA (either coding or non-coding RNA) via RISC, or alternately, translational inhibition as is known in the art. In another embodiment, modulation of gene expression by siNA molecules of the invention can result from transcriptional inhibition (see for example Janowski et al., 2005, Nature Chemical Biology, 1, 216-222).
In one embodiment, a siNA molecule of the invention is a duplex forming oligonucleotide “DFO”, (see for example FIGS. 14-15 and Vaish et al., U.S. Ser. No. 10/727,780 filed Dec. 3, 2003 and International PCT Application No. US04/16390, filed May 24, 2004).
In one embodiment, a siNA molecule of the invention is a multifunctional siNA, (see for example FIGS. 16-21 and Jadhav et al., U.S. Ser. No. 60/543,480 filed Feb. 10, 2004 and International PCT Application No. US04/16390, filed May 24, 2004). In one embodiment, the multifunctional siNA of the invention can comprise sequence targeting, for example, two or more regions of target RNA (see for example target sequences in Table II). In one embodiment, the multifunctional siNA of the invention can comprise sequence targeting one or more VEGF isoforms (e.g., VEGF-A, VEGF-B, VEGF-C, and/or VEGF-D). In one embodiment, the multifunctional siNA of the invention can comprise sequence targeting one or more VEGF receptors (e.g., VEGFR1, VEGFR2, and/or VEGFR3). In one embodiment, the multifunctional siNA of the invention can comprise sequence targeting one or more VEGF isoforms (e.g., VEGF-A, VEGF-B, VEGF-C, and/or VEGF-D) and one or more VEGF receptors, (e.g., VEGFR1, VEGFR2, and/or VEGFR3). In one embodiment, the multifunctional siNA of the invention can comprise sequence targeting one or more VEGF isoforms (e.g., VEGF-A, VEGF-B, VEGF-C, and/or VEGF-D) and one or more interleukins (e.g., IL-4 or IL-13) or one or more interleukin receptors (e.g., IL-4R or IL-13R). In one embodiment, the multifunctional siNA of the invention can comprise sequence targeting one or more VEGF receptors (e.g., VEGFR1, VEGFR2, and/or VEGFR3) and one or more interleukins (e.g., IL-4 or IL-13) or one or more interleukin receptors (e.g., IL-4R or IL-13R). In one embodiment, the multifunctional siNA of the invention can comprise sequence targeting one or more VEGF isoforms (e.g., VEGF-A, VEGF-B, VEGF-C, and/or VEGF-D), one or more VEGF receptors (e.g., VEGFR1, VEGFR2, and/or VEGFR3) and one or more interleukins (e.g., IL-4 or IL-13) or one or more interleukin receptors (e.g., IL-4R or IL-13R).
By “asymmetric hairpin” as used herein is meant a linear siNA molecule comprising an antisense region, a loop portion that can comprise nucleotides or non-nucleotides, and a sense region that comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complementary nucleotides to base pair with the antisense region and form a duplex with loop. For example, an asymmetric hairpin siNA molecule of the invention can comprise an antisense region having length sufficient to mediate RNAi in a cell or in vitro system (e.g. about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and a loop region comprising about 4 to about 12 (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, or 12) nucleotides, and a sense region having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides that are complementary to the antisense region. The asymmetric hairpin siNA molecule can also comprise a 5′-terminal phosphate group that can be chemically modified. The loop portion of the asymmetric hairpin siNA molecule can comprise nucleotides, non-nucleotides, linker molecules, or conjugate molecules as described herein.
By “asymmetric duplex” as used herein is meant a siNA molecule having two separate strands comprising a sense region and an antisense region, wherein the sense region comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complementary nucleotides to base pair with the antisense region and form a duplex. For example, an asymmetric duplex siNA molecule of the invention can comprise an antisense region having length sufficient to mediate RNAi in a cell or in vitro system (e.g., about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and a sense region having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides that are complementary to the antisense region.
By “modulate” is meant that the expression of the gene, or level of a RNA molecule or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the modulator. For example, the term “modulate” can mean “inhibit,” but the use of the word “modulate” is not limited to this definition.
By “inhibit”, “down-regulate”, or “reduce”, it is meant that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, is reduced below that observed in the absence of the nucleic acid molecules (e.g., siNA) of the invention. In one embodiment, inhibition, down-regulation or reduction with an siNA molecule is below that level observed in the presence of an inactive or attenuated molecule. In another embodiment, inhibition, down-regulation, or reduction with siNA molecules is below that level observed in the presence of, for example, an siNA molecule with scrambled sequence or with mismatches. In another embodiment, inhibition, down-regulation, or reduction of gene expression with a nucleic acid molecule of the instant invention is greater in the presence of the nucleic acid molecule than in its absence. In one embodiment, inhibition, down regulation, or reduction of gene expression is associated with post transcriptional silencing, such as RNAi mediated cleavage of a target nucleic acid molecule (e.g. RNA) or inhibition of translation. In one embodiment, inhibition, down regulation, or reduction of gene expression is associated with pretranscriptional silencing, such as by alterations in DNA methylation patterns and DNA chromatin structure.
By “up-regulate”, or “promote”, it is meant that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, is increased above that observed in the absence of the nucleic acid molecules (e.g., siNA) of the invention. In one embodiment, up-regulation or promotion of gene expression with an siNA molecule is above that level observed in the presence of an inactive or attenuated molecule. In another embodiment, up-regulation or promotion of gene expression with siNA molecules is above that level observed in the presence of, for example, an siNA molecule with scrambled sequence or with mismatches. In another embodiment, up-regulation or promotion of gene expression with a nucleic acid molecule of the instant invention is greater in the presence of the nucleic acid molecule than in its absence. In one embodiment, up-regulation or promotion of gene expression is associated with inhibition of RNA mediated gene silencing, such as RNAi mediated cleavage or silencing of a coding or non-coding RNA target that down regulates, inhibits, or silences the expression of the gene of interest to be up-regulated. The down regulation of gene expression can, for example, be induced by a coding RNA or its encoded protein, such as through negative feedback or antagonistic effects. The down regulation of gene expression can, for example, be induced by a non-coding RNA having regulatory control over a gene of interest, for example by silencing expression of the gene via translational inhibition, chromatin structure, methylation, RISC mediated RNA cleavage, or translational inhibition. As such, inhibition or down regulation of targets that down regulate, suppress, or silence a gene of interest can be used to up-regulate or promote expression of the gene of interest toward therapeutic use.
By “gene”, or “target gene” or “target DNA”, is meant a nucleic acid that encodes an RNA, for example, nucleic acid sequences including, but not limited to, structural genes encoding a polypeptide. A gene or target gene can also encode a functional RNA (fRNA) or non-coding RNA (ncRNA), such as small temporal RNA (stRNA), micro RNA (miRNA), small nuclear RNA (snRNA), short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomal RNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof. Such non-coding RNAs can serve as target nucleic acid molecules for siNA mediated RNA interference in modulating the activity of FRNA or ncRNA involved in functional or regulatory cellular processes. Abberant fRNA or ncRNA activity leading to disease can therefore be modulated by siNA molecules of the invention. siNA molecules targeting fRNA and ncRNA can also be used to manipulate or alter the genotype or phenotype of a subject, organism or cell, by intervening in cellular processes such as genetic imprinting, transcription, translation, or nucleic acid processing (e.g., transamination, methylation etc.). The target gene can be a gene derived from a cell, an endogenous gene, a transgene, or exogenous genes such as genes of a pathogen, for example a virus, which is present in the cell after infection thereof. The cell containing the target gene can be derived from or contained in any organism, for example a plant, animal, protozoan, virus, bacterium, or fungus. Non-limiting examples of plants include monocots, dicots, or gymnosperms. Non-limiting examples of animals include vertebrates or invertebrates. Non-limiting examples of fungi include molds or yeasts. For a review, see for example Snyder and Gerstein, 2003, Science, 300, 258-260.
By “non-canonical base pair” is meant any non-Watson Crick base pair, such as mismatches and/or wobble base pairs, including flipped mismatches, single hydrogen bond mismatches, trans-type mismatches, triple base interactions, and quadruple base interactions. Non-limiting examples of such non-canonical base pairs include, but are not limited to, AC reverse Hoogsteen, AC wobble, AU reverse Hoogsteen, GU wobble, AA N7 amino, CC 2-carbonyl-amino(H1)-N-3-amino(H2), GA sheared, UC 4-carbonyl-amino, UU imino-carbonyl, AC reverse wobble, AU Hoogsteen, AU reverse Watson Crick, CG reverse Watson Crick, GC N3-amino-amino N3, AA N1-amino symmetric, AA N7-amino symmetric, GA N7-N1 amino-carbonyl, GA+carbonyl-amino N7-N1, GG N1-carbonyl symmetric, GG N3-amino symmetric, CC carbonyl-amino symmetric, CC N3-amino symmetric, UU 2-carbonyl-imino symmetric, UU 4-carbonyl-imino symmetric, AA amino-N3, AA N1-amino, AC amino 2-carbonyl, AC N3-amino, AC N7-amino, AU amino-4-carbonyl, AU N1-imino, AU N3-imino, AU N7-imino, CC carbonyl-amino, GA amino-N1, GA amino-N7, GA carbonyl-amino, GA N3-amino, GC amino-N3, GC carbonyl-amino, GC N3-amino, GC N7-amino, GG amino-N7, GG carbonyl-imino, GG N7-amino, GU amino-2-carbonyl, GU carbonyl-imino, GU imino-2-carbonyl, GU N7-imino, psiU imino-2-carbonyl, UC 4-carbonyl-amino, UC imino-carbonyl, UU imino-4-carbonyl, AC C2-H-N3, GA carbonyl-C2-H, UU imino-4-carbonyl 2 carbonyl-C5-H, AC amino(A) N3(C)-carbonyl, GC imino amino-carbonyl, Gpsi imino-2-carbonyl amino-2carbonyl, and GU imino amino-2-carbonyl base pairs.
By “target” as used herein is meant, any target protein, peptide, or polypeptide, such as encoded by Genbank Accession Nos. shown in Table I or in U.S. Ser. Nos. 10/923,536 and 10/923,536, both incorporated by reference herein. The term “target” also refers to nucleic acid sequences or target polynucleotide sequence encoding any target protein, peptide, or polypeptide, such as proteins, peptides, or polypeptides encoded by sequences having Genbank Accession Nos. shown in U.S. Ser. Nos. 10/923,536 and 10/923,536. The target of interest can include target polynucleotide sequences, such as target DNA or target RNA. The term “target” is also meant to include other sequences, such as differing isoforms, mutant target genes, splice variants of target polynucleotides, target polymorphisms, and non-coding (e.g., ncRNA, miRNA, sRNA) or other regulatory polynucleotide sequences as described herein. Therefore, in various embodiments of the invention, a double stranded nucleic acid molecule of the invention (e.g., siNA) having complementarity to a target RNA can be used to inhibit or down regulate miRNA or other ncRNA activity. In one embodiment, inhibition of miRNA or ncRNA activity can be used to down regulate or inhibit gene expression (e.g., gene targets described herein or otherwise known in the art) or viral replication (e.g., viral targets described herein or otherwise known in the art) that is dependent on miRNA or ncRNA activity. In another embodiment, inhibition of miRNA or ncRNA activity by double stranded nucleic acid molecules of the invention (e.g. siNA) having complementarity to the miRNA or ncRNA can be used to up regulate or promote target gene expression (e.g., gene targets described herein or otherwise known in the art) where the expression of such genes is down regulated, suppressed, or silenced by the miRNA or ncRNA. Such up-regulation of gene expression can be used to treat diseases and conditions associated with a loss of function or haploinsufficiency as are generally known in the art. In one embodiment, the target is a VEGF and/or VEGFR target, such as a VEGF and/or VEGFR polynucleotide target.
By “VEGF” as used herein is meant, any vascular endothelial growth factor (e.g., VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D) protein, peptide, or polypeptide having vascular endothelial growth factor activity, such as encoded by VEGF Genbank Accession Nos. shown in Table I. The term VEGF also refers to nucleic acid sequences encoding any vascular endothelial growth factor protein, peptide, or polypeptide having vascular endothelial growth factor activity.
By “VEGF-B” is meant, protein, peptide, or polypeptide receptor or a derivative thereof, such as encoded by Genbank Accession No. NM—003377, having vascular endothelial growth factor type B activity. The term VEGF-B also refers to nucleic acid sequences encoding any VEGF-B protein, peptide, or polypeptide having VEGF-B activity.
By “VEGF-C” is meant, protein, peptide, or polypeptide receptor or a derivative thereof, such as encoded by Genbank Accession No. NM—005429, having vascular endothelial growth factor type C activity. The term VEGF-C also refers to nucleic acid sequences encoding any VEGF-C protein, peptide, or polypeptide having VEGF-C activity.
By “VEGF-D” is meant, protein, peptide, or polypeptide receptor or a derivative thereof, such as encoded by Genbank Accession No. NM—004469, having vascular endothelial growth factor type D activity. The term VEGF-D also refers to nucleic acid sequences encoding any VEGF-D protein, peptide, or polypeptide having VEGF-D activity.
By “VEGFR” as used herein is meant, any vascular endothelial growth factor receptor protein, peptide, or polypeptide (e.g., VEGFR1, VEGFR2, or VEGFR3, including both membrane bound and/or soluble forms thereof) having vascular endothelial growth factor receptor activity, such as encoded by VEGFR Genbank Accession Nos. shown in Table I. The term VEGFR also refers to nucleic acid sequences encoding any vascular endothelial growth factor receptor protein, peptide, or polypeptide having vascular endothelial growth factor receptor activity.
By “VEGFR1” is meant, protein, peptide, or polypeptide receptor or a derivative thereof, such as encoded by Genbank Accession No. NM—002019, having vascular endothelial growth factor receptor type 1 (flt) activity, for example, having the ability to bind a vascular endothelial growth factor. The term VEGF1 also refers to nucleic acid sequences encoding any VEGFR1 protein, peptide, or polypeptide having VEGFR1 activity.
By “VEGFR2” is meant, protein, peptide, or polypeptide receptor or a derivative thereof, such as encoded by Genbank Accession No. NM—002253, having vascular endothelial growth factor receptor type 2 (kdr) activity, for example, having the ability to bind a vascular endothelial growth factor. The term VEGF2 also refers to nucleic acid sequences encoding any VEGFR2 protein, peptide, or polypeptide having VEGFR2 activity.
By “VEGFR3” is meant, protein, peptide, or polypeptide receptor or a derivative thereof, such as encoded by Genbank Accession No. NM—002020 having vascular endothelial growth factor receptor type 3 (kdr) activity, for example, having the ability to bind a vascular endothelial growth factor. The term VEGFR3 also refers to nucleic acid sequences encoding any VEGFR3 protein, peptide, or polypeptide having VEGFR3 activity.
By “interleukin” is meant, any interleukin (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, and IL-27) protein, peptide, or polypeptide having any interleukin activity, such as encoded by interleukin Genbank Accession Nos. described in U.S. Ser. No. 10/922,675, filed Aug. 20, 2004 and incorporated by reference herein in its entirety including the drawings. The term interleukin also refers to nucleic acid sequences encoding any interleukin protein, peptide, or polypeptide having interleukin activity. The term “interleukin” is also meant to include other interleukin encoding sequence, such as other interleukin isoforms, mutant interleukin genes, splice variants of interleukin genes, and interleukin gene polymorphisms.
By “interleukin receptor” is meant, any interleukin receptor (e.g., IL-1R, IL-2R, IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, IL-8R, IL-9R, IL-10R, IL-11R, IL-12R, IL-13R, IL-14R, IL-15R, IL-16R, IL-17R, IL-18R, IL-19R, IL-20R, IL-21R, IL-22R, IL-23R, IL-24R, IL-25R, IL-26R, and IL-27R) protein, peptide, or polypeptide having any interleukin receptor activity, such as encoded by interleukin receptor Genbank Accession Nos. described in U.S. Ser. No. 10/922,675, filed Aug. 20, 2004 and incorporated by reference herein in its entirety including the drawings. The term interleukin receptor also refers to nucleic acid sequences encoding any interleukin receptor protein, peptide, or polypeptide having interleukin receptor activity. The term “interleukin receptor” is also meant to include other interleukin receptor encoding sequence, such as other interleukin receptor isoforms, mutant interleukin receptor genes, splice variants of interleukin receptor genes, and interleukin receptor gene polymorphisms.
By “homologous sequence” is meant, a nucleotide sequence that is shared by one or more polynucleotide sequences, such as genes, gene transcripts and/or non-coding polynucleotides. For example, a homologous sequence can be a nucleotide sequence that is shared by two or more genes encoding related but different proteins, such as different members of a gene family, different protein epitopes, different protein isoforms or completely divergent genes, such as a cytokine and its corresponding receptors. A homologous sequence can be a nucleotide sequence that is shared by two or more non-coding polynucleotides, such as noncoding DNA or RNA, regulatory sequences, introns, and sites of transcriptional control or regulation. Homologous sequences can also include conserved sequence regions shared by more than one polynucleotide sequence. Homology does not need to be perfect homology (e.g., 100%), as partially homologous sequences are also contemplated by the instant invention (e.g., 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% etc.).
By “conserved sequence region” is meant, a nucleotide sequence of one or more regions in a polynucleotide does not vary significantly between generations or from one biological system, subject, or organism to another biological system, subject, or organism. The polynucleotide can include both coding and non-coding DNA and RNA.
By “sense region” is meant a nucleotide sequence of a siNA molecule having complementarity to an antisense region of the siNA molecule. In addition, the sense region of a siNA molecule can comprise a nucleic acid sequence having homology with a target nucleic acid sequence. In one embodiment, the sense region of the siNA molecule is referred to as the sense strand or passenger strand.
By “antisense region” is meant a nucleotide sequence of a siNA molecule having complementarity to a target nucleic acid sequence. In addition, the antisense region of a siNA molecule can optionally comprise a nucleic acid sequence having complementarity to a sense region of the siNA molecule. In one embodiment, the antisense region of the siNA molecule is referred to as the antisense strand or guide strand.
By “target nucleic acid” or “target polynucleotide” is meant any nucleic acid sequence whose expression or activity is to be modulated. The target nucleic acid can be DNA or RNA. In one embodiment, a target nucleic acid of the invention is target RNA or DNA.
By “complementarity” is meant that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types as described herein. In one embodiment, a double stranded nucleic acid molecule of the invention, such as an siNA molecule, wherein each strand is between 15 and 30 nucleotides in length, comprises between about 10% and about 100% (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) complementarity between the two strands of the double stranded nucleic acid molecule. In another embodiment, a double stranded nucleic acid molecule of the invention, such as an siNA molecule, where one strand is the sense strand and the other stand is the antisense strand, wherein each strand is between 15 and 30 nucleotides in length, comprises between at least about 10% and about 100% (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) complementarity between the nucleotide sequence in the antisense strand of the double stranded nucleic acid molecule and the nucleotide sequence of its corresponding target nucleic acid molecule, such as a target RNA or target mRNA or viral RNA. In one embodiment, a double stranded nucleic acid molecule of the invention, such as an siNA molecule, where one strand comprises nucleotide sequence that is referred to as the sense region and the other strand comprises a nucleotide sequence that is referred to as the antisense region, wherein each strand is between 15 and 30 nucleotides in length, comprises between about 10% and about 100% (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) complementarity between the sense region and the antisense region of the double stranded nucleic acid molecule. In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785). A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10 nucleotides in the first oligonucleotide being based paired to a second nucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%, 80%, 90%, and 100% complementary respectively). In one embodiment, a siNA molecule of the invention has perfect complementarity between the sense strand or sense region and the antisense strand or antisense region of the siNA molecule. In one embodiment, a siNA molecule of the invention is perfectly complementary to a corresponding target nucleic acid molecule. “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. In one embodiment, a siNA molecule of the invention comprises about 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides that are complementary to one or more target nucleic acid molecules or a portion thereof. In one embodiment, a siNA molecule of the invention has partial complementarity (i.e., less than 100% complementarity) between the sense strand or sense region and the antisense strand or antisense region of the siNA molecule or between the antisense strand or antisense region of the siNA molecule and a corresponding target nucleic acid molecule. For example, partial complementarity can include various mismatches or non-based paired nucleotides (e.g., 1, 2, 3, 4, 5 or more mismatches or non-based paired nucleotides) within the siNA structure which can result in bulges, loops, or overhangs that result between the between the sense strand or sense region and the antisense strand or antisense region of the siNA molecule or between the antisense strand or antisense region of the siNA molecule and a corresponding target nucleic acid molecule.
In one embodiment, a double stranded nucleic acid molecule of the invention, such as siNA molecule, has perfect complementarity between the sense strand or sense region and the antisense strand or antisense region of the nucleic acid molecule. In one embodiment, double stranded nucleic acid molecule of the invention, such as siNA molecule, is perfectly complementary to a corresponding target nucleic acid molecule.
In one embodiment, double stranded nucleic acid molecule of the invention, such as siNA molecule, has partial complementarity (i.e., less than 100% complementarity) between the sense strand or sense region and the antisense strand or antisense region of the double stranded nucleic acid molecule or between the antisense strand or antisense region of the nucleic acid molecule and a corresponding target nucleic acid molecule. For example, partial complementarity can include various mismatches or non-base paired nucleotides (e.g., 1, 2, 3, 4, 5 or more mismatches or non-based paired nucleotides, such as nucleotide bulges) within the double stranded nucleic acid molecule, structure which can result in bulges, loops, or overhangs that result between the sense strand or sense region and the antisense strand or antisense region of the double stranded nucleic acid molecule or between the antisense strand or antisense region of the double stranded nucleic acid molecule and a corresponding target nucleic acid molecule.
In one embodiment, double stranded nucleic acid molecule of the invention is a microRNA (miRNA). By “mircoRNA” or “miRNA” is meant, a small double stranded RNA that regulates the expression of target messenger RNAs either by mRNA cleavage, translational repression/inhibition or heterochromatic silencing (see for example Ambros, 2004, Nature, 431, 350-355; Bartel, 2004, Cell, 116, 281-297; Cullen, 2004, Virus Research., 102, 3-9; He et al., 2004, Nat. Rev. Genet., 5, 522-531; and Ying et al., 2004, Gene, 342, 25-28). In one embodiment, the microRNA of the invention, has partial complementarity (i.e., less than 100% complementarity) between the sense strand or sense region and the antisense strand or antisense region of the miRNA molecule or between the antisense strand or antisense region of the miRNA and a corresponding target nucleic acid molecule. For example, partial complementarity can include various mismatches or non-base paired nucleotides (e.g., 1, 2, 3, 4, 5 or more mismatches or non-based paired nucleotides, such as nucleotide bulges) within the double stranded nucleic acid molecule, structure which can result in bulges, loops, or overhangs that result between the sense strand or sense region and the antisense strand or antisense region of the miRNA or between the antisense strand or antisense region of the miRNA and a corresponding target nucleic acid molecule.
In one embodiment, siNA molecules of the invention that down regulate or reduce target gene expression are used for preventing or treating diseases, disorders, conditions, or traits in a subject or organism as described herein or otherwise known in the art.
In one embodiment siNA molecules of the invention that down regulate or reduce VEGF and/or VEGFR gene expression are used for treating, preventing or reducing ocular disease, cancer, proliferative disease, inflammatory disease, respiratory disease, neurologic disease, allergic disease, renal disease, or angiogenesis in a subject or organism.
By “proliferative disease” or “cancer” as used herein is meant, any disease, condition, trait, genotype or phenotype characterized by unregulated cell growth or replication as is known in the art; including leukemias, for example, acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia, AIDS related cancers such as Kaposi's sarcoma; breast cancers; bone cancers such as Osteosarcoma, Chondrosarcomas, Ewing's sarcoma, Fibrosarcomas, Giant cell tumors, Adamantinomas, and Chordomas; Brain cancers such as Meningiomas, Glioblastomas, Lower-Grade Astrocytomas, Oligodendrocytomas, Pituitary Tumors, Schwannomas, and Metastatic brain cancers; cancers of the head and neck including various lymphomas such as mantle cell lymphoma, non-Hodgkins lymphoma, adenoma, squamous cell carcinoma, laryngeal carcinoma, gallbladder and bile duct cancers, cancers of the retina such as retinoblastoma, cancers of the esophagus, gastric cancers, multiple myeloma, ovarian cancer, uterine cancer, thyroid cancer, testicular cancer, endometrial cancer, melanoma, colorectal cancer, lung cancer, bladder cancer, prostate cancer, lung cancer (including non-small cell lung carcinoma), pancreatic cancer, sarcomas, Wilms' tumor, cervical cancer, head and neck cancer, skin cancers, nasopharyngeal carcinoma, liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladder adeno carcinoma, parotid adenocarcinoma, endometrial sarcoma, multidrug resistant cancers; and proliferative diseases and conditions, such as neovascularization associated with tumor angiogenesis, macular degeneration (e.g., wet/dry AMD), corneal neovascularization, diabetic retinopathy, neovascular glaucoma, myopic degeneration and other proliferative diseases and conditions such as restenosis and polycystic kidney disease, and any other cancer or proliferative disease, condition, trait, genotype or phenotype that can respond to the modulation of disease related gene expression in a cell or tissue, alone or in combination with other therapies.
By “inflammatory disease” or “inflammatory condition” as used herein is meant any disease, condition, trait, genotype or phenotype characterized by an inflammatory or allergic process as is known in the art, such as inflammation, acute inflammation, chronic inflammation, respiratory disease, atherosclerosis, psoriasis, dermatitis, restenosis, asthma, allergic rhinitis, atopic dermatitis, septic shock, rheumatoid arthritis, inflammatory bowl disease, inflammatory pelvic disease, pain, ocular inflammatory disease, celiac disease, Leigh Syndrome, Glycerol Kinase Deficiency, Familial eosinophilia (FE), autosomal recessive spastic ataxia, laryngeal inflammatory disease; Tuberculosis, Chronic cholecystitis, Bronchiectasis, Silicosis and other pneumoconioses, and any other inflammatory disease, condition, trait, genotype or phenotype that can respond to the modulation of disease related gene expression in a cell or tissue, alone or in combination with other therapies.
By “autoimmune disease” or “autoimmune condition” as used herein is meant, any disease, condition, trait, genotype or phenotype characterized by autoimmunity as is known in the art, such as multiple sclerosis, diabetes mellitus, lupus, celiac disease, Crohn's disease, ulcerative colitis, Guillain-Barre syndrome, scleroderms, Goodpasture's syndrome, Wegener's granulomatosis, autoimmune epilepsy, Rasmussen's encephalitis, Primary biliary sclerosis, Sclerosing cholangitis, Autoimmune hepatitis, Addison's disease, Hashimoto's thyroiditis, Fibromyalgia, Menier's syndrome; transplantation rejection (e.g., prevention of allograft rejection) pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome, Grave's disease, and any other autoimmune disease, condition, trait, genotype or phenotype that can respond to the modulation of disease related gene expression in a cell or tissue, alone or in combination with other therapies.
By “neurologic disease” or “neurological disease” is meant any disease, disorder, or condition affecting the central or peripheral nervous system, including ADHD, AIDS-Neurological Complications, Absence of the Septum Pellucidum, Acquired Epileptiform Aphasia, Acute Disseminated Encephalomyelitis, Adrenoleukodystrophy, Agenesis of the Corpus Callosum, Agnosia, Aicardi Syndrome, Alexander Disease, Alpers' Disease, Alternating Hemiplegia, Alzheimer's Disease, Amyotrophic Lateral Sclerosis, Anencephaly, Aneurysm, Angelman Syndrome, Angiomatosis, Anoxia, Aphasia, Apraxia, Arachnoid Cysts, Arachnoiditis, Arnold-Chiari Malformation, Arteriovenous Malformation, Aspartame, Asperger Syndrome, Ataxia Telangiectasia, Ataxia, Attention Deficit-Hyperactivity Disorder, Autism, Autonomic Dysfunction, Back Pain, Barth Syndrome, Batten Disease, Behcet's Disease, Bell's Palsy, Benign Essential Blepharospasm, Benign Focal Amyotrophy, Benign Intracranial Hypertension, Bernhardt-Roth Syndrome, Binswanger's Disease, Blepharospasm, Bloch-Sulzberger Syndrome, Brachial Plexus Birth Injuries, Brachial Plexus Injuries, Bradbury-Eggleston Syndrome, Brain Aneurysm, Brain Injury, Brain and Spinal Tumors, Brown-Sequard Syndrome, Bulbospinal Muscular Atrophy, Canavan Disease, Carpal Tunnel Syndrome, Causalgia, Cavernomas, Cavernous Angioma, Cavernous Malformation, Central Cervical Cord Syndrome, Central Cord Syndrome, Central Pain Syndrome, Cephalic Disorders, Cerebellar Degeneration, Cerebellar Hypoplasia, Cerebral Aneurysm, Cerebral Arteriosclerosis, Cerebral Atrophy, Cerebral Beriberi, Cerebral Gigantism, Cerebral Hypoxia, Cerebral Palsy, Cerebro-Oculo-Facio-Skeletal Syndrome, Charcot-Marie-Tooth Disorder, Chiari Malformation, Chorea, Choreoacanthocytosis, Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), Chronic Orthostatic Intolerance, Chronic Pain, Cockayne Syndrome Type II, Coffin Lowry Syndrome, Coma, including Persistent Vegetative State, Complex Regional Pain Syndrome, Congenital Facial Diplegia, Congenital Myasthenia, Congenital Myopathy, Congenital Vascular Cavernous Malformations, Corticobasal Degeneration, Cranial Arteritis, Craniosynostosis, Creutzfeldt-Jakob Disease, Cumulative Trauma Disorders, Cushing's Syndrome, Cytomegalic Inclusion Body Disease (CIBD), Cytomegalovirus Infection, Dancing Eyes-Dancing Feet Syndrome, Dandy-Walker Syndrome, Dawson Disease, De Morsier's Syndrome, Dejerine-Klumpke Palsy, Dementia-Multi-Infarct, Dementia-Subcortical, Dementia With Lewy Bodies, Dermatomyositis, Developmental Dyspraxia, Devic's Syndrome, Diabetic Neuropathy, Diffuse Sclerosis, Dravet's Syndrome, Dysautonomia, Dysgraphia, Dyslexia, Dysphagia, Dyspraxia, Dystonias, Early Infantile Epileptic Encephalopathy, Empty Sella Syndrome, Encephalitis Lethargica, Encephalitis and Meningitis, Encephaloceles, Encephalopathy, Encephalotrigeminal Angiomatosis, Epilepsy, Erb's Palsy, Erb-Duchenne and Dejerine-Klumpke Palsies, Fabry's Disease, Fahr's Syndrome, Fainting, Familial Dysautonomia, Familial Hemangioma, Familial Idiopathic Basal Ganglia Calcification, Familial Spastic Paralysis, Febrile Seizures (e.g., GEFS and GEFS plus), Fisher Syndrome, Floppy Infant Syndrome, Friedreich's Ataxia, Gaucher's Disease, Gerstmann's Syndrome, Gerstmann-Straussler-Scheinker Disease, Giant Cell Arteritis, Giant Cell Inclusion Disease, Globoid Cell Leukodystrophy, Glossopharyngeal Neuralgia, Guillain-Barre Syndrome, HTLV-1 Associated Myelopathy, Hallervorden-Spatz Disease, Head Injury, Headache, Hemicrania Continua, Hemifacial Spasm, Hemiplegia Alterans, Hereditary Neuropathies, Hereditary Spastic Paraplegia, Heredopathia Atactica Polyneuritiformis, Herpes Zoster Oticus, Herpes Zoster, Hirayama Syndrome, Holoprosencephaly, Huntington's Disease, Hydranencephaly, Hydrocephalus-Normal Pressure, Hydrocephalus, Hydromyelia, Hypercortisolism, Hypersomnia, Hypertonia, Hypotonia, Hypoxia, Immune-Mediated Encephalomyelitis, Inclusion Body Myositis, Incontinentia Pigmenti, Infantile Hypotonia, Infantile Phytanic Acid Storage Disease, Infantile Refsum Disease, Infantile Spasms, Inflammatory Myopathy, Intestinal Lipodystrophy, Intracranial Cysts, Intracranial Hypertension, Isaac's Syndrome, Joubert Syndrome, Kearns-Sayre Syndrome, Kennedy's Disease, Kinsbourne syndrome, Kleine-Levin syndrome, Klippel Feil Syndrome, Klippel-Trenaunay Syndrome (KTS), Klüver-Bucy Syndrome, Korsakoffs Amnesic Syndrome, Krabbe Disease, Kugelberg-Welander Disease, Kuru, Lambert-Eaton Myasthenic Syndrome, Landau-Kleffner Syndrome, Lateral Femoral Cutaneous Nerve Entrapment, Lateral Medullary Syndrome, Learning Disabilities, Leigh's Disease, Lennox-Gastaut Syndrome, Lesch-Nyhan Syndrome, Leukodystrophy, Levine-Critchley Syndrome, Lewy Body Dementia, Lissencephaly, Locked-In Syndrome, Lou Gehrig's Disease, Lupus-Neurological Sequelae, Lyme Disease-Neurological Complications, Machado-Joseph Disease, Macrencephaly, Megalencephaly, Melkersson-Rosenthal Syndrome, Meningitis, Menkes Disease, Meralgia Paresthetica, Metachromatic Leukodystrophy, Microcephaly, Migraine, Miller Fisher Syndrome, Mini-Strokes, Mitochondrial Myopathies, Mobius Syndrome, Monomelic Amyotrophy, Motor Neuron Diseases, Moyamoya Disease, Mucolipidoses, Mucopolysaccharidoses, Multi-Infarct Dementia, Multifocal Motor Neuropathy, Multiple Sclerosis, Multiple System Atrophy with Orthostatic Hypotension, Multiple System Atrophy, Muscular Dystrophy, Myasthenia-Congenital, Myasthenia Gravis, Myelinoclastic Diffuse Sclerosis, Myoclonic Encephalopathy of Infants, Myoclonus, Myopathy-Congenital, Myopathy-Thyrotoxic, Myopathy, Myotonia Congenita, Myotonia, Narcolepsy, Neuroacanthocytosis, Neurodegeneration with Brain Iron Accumulation, Neurofibromatosis, Neuroleptic Malignant Syndrome, Neurological Complications of AIDS, Neurological Manifestations of Pompe Disease, Neuromyelitis Optica, Neuromyotonia, Neuronal Ceroid Lipofuscinosis, Neuronal Migration Disorders, Neuropathy-Hereditary, Neurosarcoidosis, Neurotoxicity, Nevus Cavernosus, Niemann-Pick Disease, O'Sullivan-McLeod Syndrome, Occipital Neuralgia, Occult Spinal Dysraphism Sequence, Ohtahara Syndrome, Olivopontocerebellar Atrophy, Opsoclonus Myoclonus, Orthostatic Hypotension, Overuse Syndrome, Pain-Chronic, Paraneoplastic Syndromes, Paresthesia, Parkinson's Disease, Parmyotonia Congenita, Paroxysmal Choreoathetosis, Paroxysmal Hemicrania, Parry-Romberg, Pelizaeus-Merzbacher Disease, Pena Shokeir II Syndrome, Perineural Cysts, Periodic Paralyses, Peripheral Neuropathy, Periventricular Leukomalacia, Persistent Vegetative State, Pervasive Developmental Disorders, Phytanic Acid Storage Disease, Pick's Disease, Piriformis Syndrome, Pituitary Tumors, Polymyositis, Pompe Disease, Porencephaly, Post-Polio Syndrome, Postherpetic Neuralgia, Postinfectious Encephalomyelitis, Postural Hypotension, Postural Orthostatic Tachycardia Syndrome, Postural Tachycardia Syndrome, Primary Lateral Sclerosis, Prion Diseases, Progressive Hemifacial Atrophy, Progressive Locomotor Ataxia, Progressive Multifocal Leukoencephalopathy, Progressive Sclerosing Poliodystrophy, Progressive Supranuclear Palsy, Pseudotumor Cerebri, Pyridoxine Dependent and Pyridoxine Responsive Siezure Disorders, Ramsay Hunt Syndrome Type I, Ramsay Hunt Syndrome Type II, Rasmussen's Encephalitis and other autoimmune epilepsies, Reflex Sympathetic Dystrophy Syndrome, Refsum Disease-Infantile, Refsum Disease, Repetitive Motion Disorders, Repetitive Stress Injuries, Restless Legs Syndrome, Retrovirus-Associated Myelopathy, Rett Syndrome, Reye's Syndrome, Riley-Day Syndrome, SUNCT Headache, Sacral Nerve Root Cysts, Saint Vitus Dance, Salivary Gland Disease, Sandhoff Disease, Schilder's Disease, Schizencephaly, Seizure Disorders, Septo-Optic Dysplasia, Severe Myoclonic Epilepsy of Infancy (SMEI), Shaken Baby Syndrome, Shingles, Shy-Drager Syndrome, Sjogren's Syndrome, Sleep Apnea, Sleeping Sickness, Soto's Syndrome, Spasticity, Spina Bifida, Spinal Cord Infarction, Spinal Cord Injury, Spinal Cord Tumors, Spinal Muscular Atrophy, Spinocerebellar Atrophy, Steele-Richardson-Olszewski Syndrome, Stiff-Person Syndrome, Striatonigral Degeneration, Stroke, Sturge-Weber Syndrome, Subacute Sclerosing Panencephalitis, Subcortical Arteriosclerotic Encephalopathy, Swallowing Disorders, Sydenham Chorea, Syncope, Syphilitic Spinal Sclerosis, Syringohydromyelia, Syringomyelia, Systemic Lupus Erythematosus, Tabes Dorsalis, Tardive Dyskinesia, Tarlov Cysts, Tay-Sachs Disease, Temporal Arteritis, Tethered Spinal Cord Syndrome, Thomsen Disease, Thoracic Outlet Syndrome, Thyrotoxic Myopathy, Tic Douloureux, Todd's Paralysis, Tourette Syndrome, Transient Ischemic Attack, Transmissible Spongiform Encephalopathies, Transverse Myelitis, Traumatic Brain Injury, Tremor, Trigeminal Neuralgia, Tropical Spastic Paraparesis, Tuberous Sclerosis, Vascular Erectile Tumor, Vasculitis including Temporal Arteritis, Von Economo's Disease, Von Hippel-Lindau disease (VHL), Von Recklinghausen's Disease, Wallenberg's Syndrome, Werdnig-Hoffnan Disease, Wernicke-Korsakoff Syndrome, West Syndrome, Whipple's Disease, Williams Syndrome, Wilson's Disease, X-Linked Spinal and Bulbar Muscular Atrophy, and Zellweger Syndrome.
By “respiratory disease” is meant, any disease or condition affecting the respiratory tract, such as asthma, chronic obstructive pulmonary disease or “COPD”, allergic rhinitis, sinusitis, pulmonary vasoconstriction, inflammation, allergies, impeded respiration, respiratory distress syndrome, cystic fibrosis, pulmonary hypertension, pulmonary vasoconstriction, emphysema, and any other respiratory disease, condition, trait, genotype or phenotype that can respond to the modulation of disease related gene expression in a cell or tissue, alone or in combination with other therapies.
By “cardiovascular disease” is meant and disease or condition affecting the heart and vasculature, including but not limited to, coronary heart disease (CHD), cerebrovascular disease (CVD), aortic stenosis, peripheral vascular disease, atherosclerosis, arteriosclerosis, myocardial infarction (heart attack), cerebrovascular diseases (stroke), transient ischaemic attacks (TIA), angina (stable and unstable), atrial fibrillation, arrhythmia, vavular disease, congestive heart failure, hypercholoesterolemia, type I hyperlipoproteinemia, type II hyperlipoproteinemia, type III hyperlipoproteinemia, type IV hyperlipoproteinemia, type V hyperlipoproteinemia, secondary hypertrigliceridemia, and familial lecithin cholesterol acyltransferase deficiency.
By “ocular disease” as used herein is meant, any disease, condition, trait, genotype or phenotype of the eye and related structures as is known in the art, such as Cystoid Macular Edema, Asteroid Hyalosis, Pathological Myopia and Posterior Staphyloma, Toxocariasis (Ocular Larva Migrans), Retinal Vein Occlusion, Posterior Vitreous Detachment, Tractional Retinal Tears, Epiretinal Membrane, Diabetic Retinopathy, Lattice Degeneration, Retinal Vein Occlusion, Retinal Artery Occlusion, Macular Degeneration (e.g., age related macular degeneration such as wet AMD or dry AMD), Toxoplasmosis, Choroidal Melanoma, Acquired Retinoschisis, Hollenhorst Plaque, Idiopathic Central Serous Chorioretinopathy, Macular Hole, Presumed Ocular Histoplasmosis Syndrome, Retinal Macroaneursym, Retinitis Pigmentosa, Retinal Detachment, Hypertensive Retinopathy, Retinal Pigment Epithelium (RPE) Detachment, Papillophlebitis, Ocular Ischemic Syndrome, Coats' Disease, Leber's Miliary Aneurysm, Conjunctival Neoplasms, Allergic Conjunctivitis, Vernal Conjunctivitis, Acute Bacterial Conjunctivitis, Allergic Conjunctivitis & Vernal Keratoconjunctivitis, Viral Conjunctivitis, Bacterial Conjunctivitis, Chlamydial & Gonococcal Conjunctivitis, Conjunctival Laceration, Episcleritis, Scleritis, Pingueculitis, Pterygium, Superior Limbic Keratoconjunctivitis (SLK of Theodore), Toxic Conjunctivitis, Conjunctivitis with Pseudomembrane, Giant Papillary Conjunctivitis, Terrien's Marginal Degeneration, Acanthamoeba Keratitis, Fungal Keratitis, Filamentary Keratitis, Bacterial Keratitis, Keratitis Sicca/Dry Eye Syndrome, Bacterial Keratitis, Herpes Simplex Keratitis, Sterile Corneal Infiltrates, Phlyctenulosis, Corneal Abrasion & Recurrent Corneal Erosion, Corneal Foreign Body, Chemical Burs, Epithelial Basement Membrane Dystrophy (EBMD), Thygeson's Superficial Punctate Keratopathy, Corneal Laceration, Salzmann's Nodular Degeneration, Fuchs' Endothelial Dystrophy, Crystalline Lens Subluxation, Ciliary-Block Glaucoma, Primary Open-Angle Glaucoma, Pigment Dispersion Syndrome and Pigmentary Glaucoma, Pseudoexfoliation Syndrom and Pseudoexfoliative Glaucoma, Anterior Uveitis, Primary Open Angle Glaucoma, Uveitic Glaucoma & Glaucomatocyclitic Crisis, Pigment Dispersion Syndrome & Pigmentary Glaucoma, Acute Angle Closure Glaucoma, Anterior Uveitis, Hyphema, Angle Recession Glaucoma, Lens Induced Glaucoma, Pseudoexfoliation Syndrome and Pseudoexfoliative Glaucoma, Axenfeld-Rieger Syndrome, Neovascular Glaucoma, Pars Planitis, Choroidal Rupture, Duane's Retraction Syndrome, Toxic/Nutritional Optic Neuropathy, Aberrant Regeneration of Cranial Nerve III, Intracranial Mass Lesions, Carotid-Cavernous Sinus Fistula, Anterior Ischemic Optic Neuropathy, Optic Disc Edema & Papilledema, Cranial Nerve III Palsy, Cranial Nerve IV Palsy, Cranial Nerve VI Palsy, Cranial Nerve VII (Facial Nerve) Palsy, Horner's Syndrome, Internuclear Ophthalmoplegia, Optic Nerve Head Hypoplasia, Optic Pit, Tonic Pupil, Optic Nerve Head Drusen, Demyelinating Optic Neuropathy (Optic Neuritis, Retrobulbar Optic Neuritis), Amaurosis Fugax and Transient Ischemic Attack, Pseudotumor Cerebri, Pituitary Adenoma, Molluscum Contagiosum, Canaliculitis, Verruca and Papilloma, Pediculosis and Pthiriasis, Blepharitis, Hordeolum, Preseptal Cellulitis, Chalazion, Basal Cell Carcinoma, Herpes Zoster Ophthalmicus, Pediculosis & Phthiriasis, Blow-out Fracture, Chronic Epiphora, Dacryocystitis, Herpes Simplex Blepharitis, Orbital Cellulitis, Senile Entropion, and Squamous Cell Carcinoma.
By “metabolic disease” is meant any disease or condition affecting metabolic pathways as in known in the art. Metabolic disease can result in an abnormal metabolic process, either congenital due to inherited enzyme abnormality (inborn errors of metabolism) or acquired due to disease of an endocrine organ or failure of a metabolically important organ such as the liver. In one embodiment, metabolic disease includes obesity, insulin resistance, and diabetes (e.g., type I and/or type II diabetes).
By “dermatological disease” is meany any disease or condition of the skin, dermis, or any substructure therein such as hair, follicle, etc. Dermatological diseases, disorders, conditions, and traits can include rosacea, psoriasis, ectopic dermatitis, skin cancers such as melanoma and basal cell carcinoma, hair loss, hair removal, alterations in pigmentation, and any other disease, condition, or trait associated with the skin, dermis, or structures therein.
By “auditory disease” is meany any disease or condition of the auditory system, including the ear, such as the inner ear, middle ear, outer ear, auditory nerve, and any substructures therein. Auditory diseases, disorders, conditions, and traits can include hearing loss, deafness, tinnitus, Meniere's Disease, vertigo, balance and motion disorders, and any other disease, condition, or trait associated with the ear, or structures therein.
In one embodiment of the present invention, each sequence of a siNA molecule of the invention is independently about 15 to about 30 nucleotides in length, in specific embodiments about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In another embodiment, the siNA duplexes of the invention independently comprise about 15 to about 30 base pairs (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30). In another embodiment, one or more strands of the siNA molecule of the invention independently comprises about 15 to about 30 nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) that are complementary to a target nucleic acid molecule. In yet another embodiment, siNA molecules of the invention comprising hairpin or circular structures are about 35 to about 55 (e.g., about 35, 40, 45, 50 or 55) nucleotides in length, or about 38 to about 44 (e.g., about 38, 39, 40, 41, 42, 43, or 44) nucleotides in length and comprising about 15 to about 25 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs. Exemplary siNA molecules of the invention are shown in Table II and/or FIGS. 4-5.
As used herein “cell” is used in its usual biological sense, and does not refer to an entire multicellular organism, e.g., specifically does not refer to a human. The cell can be present in an organism, e.g., birds, plants and mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell). The cell can be of somatic or germ line origin, totipotent or pluripotent, dividing or non-dividing. The cell can also be derived from or can comprise a gamete or embryo, a stem cell, or a fully differentiated cell.
The siNA molecules of the invention are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through local delivery to the lung, with or without their incorporation in biopolymers. In particular embodiments, the nucleic acid molecules of the invention comprise sequences shown in Tables II-III and/or FIGS. 4-5. Examples of such nucleic acid molecules consist essentially of sequences defined in these tables and figures. Furthermore, the chemically modified constructs described in Table I can be applied to any siNA sequence of the invention.
In another aspect, the invention provides mammalian cells containing one or more siNA molecules of this invention. The one or more siNA molecules can independently be targeted to the same or different sites.
By “RNA” is meant a molecule comprising at least one ribonucleotide residue. By “ribonucleotide” is meant a nucleotide with a hydroxyl group at the 2′ position of a β-D-ribofuranose moiety. The terms include double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the instant invention can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.
By “subject” is meant an organism, which is a donor or recipient of explanted cells or the cells themselves. “Subject” also refers to an organism to which the nucleic acid molecules of the invention can be administered. A subject can be a mammal or mammalian cells, including a human or human cells.
By “chemical modification” as used herein is meant any modification of chemical structure of the nucleotides that differs from nucleotides of native siRNA or RNA. The term “chemical modification” encompasses the addition, substitution, or modification of native siRNA or RNA nucleosides and nucleotides with modified nucleosides and modified nucleotides as described herein or as is otherwise known in the art. Non-limiting examples of such chemical modifications include without limitation phosphorothioate internucleotide linkages, 2′-deoxyribonucleotides, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, 4′-thio ribonucleotides, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides (see for example U.S. Ser. No. 10/981,966 filed Nov. 5, 2004, incorporated by reference herein), “universal base” nucleotides, “acyclic” nucleotides, 5-C-methyl nucleotides, terminal glyceryl and/or inverted deoxy abasic residue incorporation, or a modification having any of Formulae I-VII herein.
The term “phosphorothioate” as used herein refers to an internucleotide linkage having Formula I, wherein Z and/or W comprise a sulfur atom. Hence, the term phosphorothioate refers to both phosphorothioate and phosphorodithioate internucleotide linkages.
The term “phosphonoacetate” as used herein refers to an internucleotide linkage having Formula I, wherein Z and/or W comprise an acetyl or protected acetyl group.
The term “thiophosphonoacetate” as used herein refers to an internucleotide linkage having Formula I, wherein Z comprises an acetyl or protected acetyl group and W comprises a sulfur atom or alternately W comprises an acetyl or protected acetyl group and Z comprises a sulfur atom.
The term “universal base” as used herein refers to nucleotide base analogs that form base pairs with each of the natural DNA/RNA bases with little discrimination between them. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art (see for example Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).
The term “acyclic nucleotide” as used herein refers to any nucleotide having an acyclic ribose sugar, for example where any of the ribose carbons (C1, C2, C3, C4, or C5), are independently or in combination absent from the nucleotide.
The nucleic acid molecules of the instant invention, individually, or in combination or in conjunction with other drugs, can be used to treat, inhibit, reduce, or prevent ocular disease, cancer, proliferative disease, inflammatory disease, respiratory disease, neurologic disease, allergic disease, renal disease, or angiogenesis in a subject or organism. For example, the siNA molecules can be administered to a subject or can be administered to other appropriate cells evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.
In one embodiment, the siNA molecules of the invention can be administered to a subject or can be administered to other appropriate cells evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.
In a further embodiment, the siNA molecules can be used in combination with other known treatments to treat, inhibit, reduce, or prevent ocular disease, cancer, proliferative disease, inflammatory disease, respiratory disease, neurologic disease, allergic disease, renal disease, or angiogenesis in a subject or organism. For example, the described molecules could be used in combination with one or more known compounds, treatments, or procedures to treat, inhibit, reduce, or prevent ocular disease, cancer, proliferative disease, inflammatory disease, respiratory disease, neurologic disease, allergic disease, renal disease, or angiogenesis in a subject or organism as are known in the art.
In one embodiment, the invention features an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the invention, in a manner which allows expression of the siNA molecule. For example, the vector can contain sequence(s) encoding both strands of a siNA molecule comprising a duplex. The vector can also contain sequence(s) encoding a single nucleic acid molecule that is self-complementary and thus forms a siNA molecule. Non-limiting examples of such expression vectors are described in Paul et al., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500; and Novina et al., 2002, Nature Medicine, advance online publication doi:10.1038/nm725.
In another embodiment, the invention features a mammalian cell, for example, a human cell, including an expression vector of the invention.
In yet another embodiment, the expression vector of the invention comprises a sequence for a siNA molecule having complementarity to a RNA molecule referred to by a Genbank Accession numbers, for example Genbank Accession Nos. shown in U.S. Ser. Nos. 10/923,536 and 10/923,536, both incorporated by reference herein.
In one embodiment, an expression vector of the invention comprises a nucleic acid sequence encoding two or more siNA molecules, which can be the same or different.
In another aspect of the invention, siNA molecules that interact with VEGF and/or VEGFR target RNA molecules and down-regulate gene encoding VEGF and/or VEGFR target RNA molecules (for example VEGF and/or VEGFR target RNA molecules referred to by Genbank Accession numbers herein) are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. The recombinant vectors capable of expressing the siNA molecules can be delivered as described herein, and persist in VEGF and/or VEGFR target cells. Alternatively, viral vectors can be used that provide for transient expression of siNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the siNA molecules bind and down-regulate gene function or expression via RNA interference (RNAi). Delivery of siNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired VEGF and/or VEGFR target cell.
By “vectors” is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a non-limiting example of a scheme for the synthesis of siNA molecules. The complementary siNA sequence strands, strand 1 and strand 2, are synthesized in tandem and are connected by a cleavable linkage, such as a nucleotide succinate or abasic succinate, which can be the same or different from the cleavable linker used for solid phase synthesis on a solid support. The synthesis can be either solid phase or solution phase, in the example shown, the synthesis is a solid phase synthesis. The synthesis is performed such that a protecting group, such as a dimethoxytrityl group, remains intact on the terminal nucleotide of the tandem oligonucleotide. Upon cleavage and deprotection of the oligonucleotide, the two siNA strands spontaneously hybridize to form a siNA duplex, which allows the purification of the duplex by utilizing the properties of the terminal protecting group, for example by applying a trityl on purification method wherein only duplexes/oligonucleotides with the terminal protecting group are isolated.
FIG. 2 shows a MALDI-TOF mass spectrum of a purified siNA duplex synthesized by a method of the invention. The two peaks shown correspond to the predicted mass of the separate siNA sequence strands. This result demonstrates that the siNA duplex generated from tandem synthesis can be purified as a single entity using a simple trityl-on purification methodology.
FIG. 3 shows a non-limiting proposed mechanistic representation of target RNA degradation involved in RNAi. Double-stranded RNA (dsRNA), which is generated by RNA-dependent RNA polymerase (RdRP) from foreign single-stranded RNA, for example viral, transposon, or other exogenous RNA, activates the DICER enzyme that in turn generates siNA duplexes. Alternately, synthetic or expressed siNA can be introduced directly into a cell by appropriate means. An active siNA complex forms which recognizes a target RNA, resulting in degradation of the target RNA by the RISC endonuclease complex or in the synthesis of additional RNA by RNA-dependent RNA polymerase (RdRP), which can activate DICER and result in additional siNA molecules, thereby amplifying the RNAi response.
FIG. 4A-F shows non-limiting examples of chemically-modified siNA constructs of the present invention. In the figure, N stands for any nucleotide (adenosine, guanosine, cytosine, uridine, or optionally thymidine, for example thymidine can be substituted in the overhanging regions designated by parenthesis (N N). Various modifications are shown for the sense and antisense strands of the siNA constructs. The (N N) nucleotide positions can be chemically modified as described herein (e.g., 2′-O-methyl, 2′-deoxy-2′-fluoro etc.) and can be either derived from a corresponding target nucleic acid sequence or not (see for example FIG. 6C). Furthermore, the sequences shown in FIG. 4 can optionally include a ribonucleotide at the 9th position from the 5′-end of the sense strand or the 11th position based on the 5′-end of the guide strand by counting 11 nucleotide positions in from the 5′-terminus of the guide strand (see FIG. 6C).
FIG. 4A: The sense strand comprises 21 nucleotides wherein the two terminal 3′-nucleotides are optionally base paired and wherein all nucleotides present are ribonucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all nucleotides present are ribonucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the antisense strand.
FIG. 4B: The sense strand comprises 21 nucleotides wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the sense and antisense strand.
FIG. 4C: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-O-methyl or 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the antisense strand.
FIG. 4D: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein and wherein and all purine nucleotides that may be present are 2′-deoxy nucleotides. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the antisense strand.
FIG. 4E: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the antisense strand.
FIG. 4F: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein and wherein and all purine nucleotides that may be present are 2′-deoxy nucleotides. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and having one 3′-terminal phosphorothioate internucleotide linkage and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-deoxy nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the antisense strand. The antisense strand of constructs A-F comprise sequence complementary to any target nucleic acid sequence of the invention. Furthermore, when a glyceryl moiety (L) is present at the 3′-end of the antisense strand for any construct shown in FIG. 4 A-F, the modified internucleotide linkage is optional.
FIG. 5A-F shows non-limiting examples of specific chemically-modified siNA sequences of the invention. A-F applies the chemical modifications described in FIG. 4A-F to an exemplary VEGF and/or VEGFR target siNA sequence. Such chemical modifications can be applied to any VEGF and/or VEGFR target or other target polynucleotide sequence.
FIG. 6A-C shows non-limiting examples of different siNA constructs of the invention.
The examples shown in FIG. 6A (constructs 1, 2, and 3) have 19 representative base pairs; however, different embodiments of the invention include any number of base pairs described herein. Bracketed regions represent nucleotide overhangs, for example, comprising about 1, 2, 3, or 4 nucleotides in length, preferably about 2 nucleotides. Constructs 1 and 2 can be used independently for RNAi activity. Construct 2 can comprise a polynucleotide or non-nucleotide linker, which can optionally be designed as a biodegradable linker. In one embodiment, the loop structure shown in construct 2 can comprise a biodegradable linker that results in the formation of construct 1 in vivo and/or in vitro. In another example, construct 3 can be used to generate construct 2 under the same principle wherein a linker is used to generate the active siNA construct 2 in vivo and/or in vitro, which can optionally utilize another biodegradable linker to generate the active siNA construct 1 in vivo and/or in vitro. As such, the stability and/or activity of the siNA constructs can be modulated based on the design of the siNA construct for use in vivo or in vitro and/or in vitro.
The examples shown in FIG. 6B represent different variations of double stranded nucleic acid molecule of the invention, such as microRNA, that can include overhangs, bulges, loops, and stem-loops resulting from partial complementarity. Such motifs having bulges, loops, and stem-loops are generally characteristics of miRNA. The bulges, loops, and stem-loops can result from any degree of partial complementarity, such as mismatches or bulges of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides in one or both strands of the double stranded nucleic acid molecule of the invention.
The example shown in FIG. 6C represents a model double stranded nucleic acid molecule of the invention comprising a 19 base pair duplex of two 21 nucleotide sequences having dinucleotide 3′-overhangs. The top strand (1) represents the sense strand (passenger strand), the middle strand (2) represents the antisense (guide strand), and the lower strand (3) represents a target polynucleotide sequence. The dinucleotide overhangs (NN) can comprise sequence derived from the target polynucleotide. For example, the 3′-(NN) sequence in the guide strand can be complementary to the 5′-[NN] sequence of the target polynucleotide. In addition, the 5′-(NN) sequence of the passenger strand can comprise the same sequence as the 5′-[NN] sequence of the target polynucleotide sequence. In other embodiments, the overhangs (NN) are not derived from the target polynucleotide sequence, for example where the 3′-(NN) sequence in the guide strand are not complementary to the 5′-[NN] sequence of the target polynucleotide and the 5′-(NN) sequence of the passenger strand can comprise different sequence from the 5′-[NN] sequence of the target polynucleotide sequence. In additional embodiments, any (NN) nucleotides are chemically modified, e.g., as 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or other modifications herein. Furthermore, the passenger strand can comprise a ribonucleotide position N of the passenger strand. For the representative 19 base pair 21 mer duplex shown, position N can be 9 nucleotides in from the 3′ end of the passenger strand. However, in duplexes of differing length, the position N is determined based on the 5′-end of the guide strand by counting 11 nucleotide positions in from the 5′-terminus of the guide strand and picking the corresponding base paired nucleotide in the passenger strand. Cleavage by Ago2 takes place between positions 10 and 11 as indicated by the arrow. In additional embodiments, there are two ribonucleotides, NN, at positions 10 and 11 based on the 5′-end of the guide strand by counting 10 and 11 nucleotide positions in from the 5′-terminus of the guide strand and picking the corresponding base paired nucleotides in the passenger strand.
FIG. 7A-C is a diagrammatic representation of a scheme utilized in generating an expression cassette to generate siNA hairpin constructs.
FIG. 7A: A DNA oligomer is synthesized with a 5′-restriction site (R1) sequence followed by a region having sequence identical (sense region of siNA) to a predetermined target sequence, wherein the sense region comprises, for example, about 19, 20, 21, or 22 nucleotides (N) in length, which is followed by a loop sequence of defined sequence (X), comprising, for example, about 3 to about 10 nucleotides.
FIG. 7B: The synthetic construct is then extended by DNA polymerase to generate a hairpin structure having self-complementary sequence that will result in a siNA transcript having specificity for a target sequence and having self-complementary sense and antisense regions.
FIG. 7C: The construct is heated (for example to about 95° C.) to linearize the sequence, thus allowing extension of a complementary second DNA strand using a primer to the 3′-restriction sequence of the first strand. The double-stranded DNA is then inserted into an appropriate vector for expression in cells. The construct can be designed such that a 3′-terminal nucleotide overhang results from the transcription, for example, by engineering restriction sites and/or utilizing a poly-U termination region as described in Paul et al., 2002, Nature Biotechnology, 29, 505-508.
FIG. 8A-C is a diagrammatic representation of a scheme utilized in generating an expression cassette to generate double-stranded siNA constructs.
FIG. 8A: A DNA oligomer is synthesized with a 5′-restriction (R1) site sequence followed by a region having sequence identical (sense region of siNA) to a predetermined target sequence, wherein the sense region comprises, for example, about 19, 20, 21, or 22 nucleotides (N) in length, and which is followed by a 3′-restriction site (R2) which is adjacent to a loop sequence of defined sequence (X).
FIG. 8B: The synthetic construct is then extended by DNA polymerase to generate a hairpin structure having self-complementary sequence.
FIG. 8C: The construct is processed by restriction enzymes specific to R1 and R2 to generate a double-stranded DNA which is then inserted into an appropriate vector for expression in cells. The transcription cassette is designed such that a U6 promoter region flanks each side of the dsDNA which generates the separate sense and antisense strands of the siNA. Poly T termination sequences can be added to the constructs to generate U overhangs in the resulting transcript.
FIG. 9A-E is a diagrammatic representation of a method used to determine target sites for siNA mediated RNAi within a particular target nucleic acid sequence, such as messenger RNA.
FIG. 9A: A pool of siNA oligonucleotides are synthesized wherein the antisense region of the siNA constructs has complementarity to target sites across the target nucleic acid sequence, and wherein the sense region comprises sequence complementary to the antisense region of the siNA.
FIGS. 9B&C: (FIG. 9B) The sequences are pooled and are inserted into vectors such that (FIG. 9C) transfection of a vector into cells results in the expression of the siNA.
FIG. 9D: Cells are sorted based on phenotypic change that is associated with modulation of the target nucleic acid sequence.
FIG. 9E: The siNA is isolated from the sorted cells and is sequenced to identify efficacious target sites within the target nucleic acid sequence.
FIG. 10 shows non-limiting examples of different stabilization chemistries (1-10) that can be used, for example, to stabilize the 3′-end of siNA sequences of the invention, including (1) [3-3′]-inverted deoxyribose; (2) deoxyribonucleotide; (3) [5′-3′]-3′-deoxyribonucleotide; (4) [5′-3′]-ribonucleotide; (5) [5′-3′]-3′-O-methyl ribonucleotide; (6) 3′-glyceryl; (7) [3′-5′]-3′-deoxyribonucleotide; (8) [3′-3′]-deoxyribonucleotide; (9) [5′-2′]deoxyribonucleotide; and (10) [5-3′]-dideoxyribonucleotide. In addition to modified and unmodified backbone chemistries indicated in the figure, these chemistries can be combined with different backbone modifications as described herein, for example, backbone modifications having Formula I. In addition, the 2′-deoxy nucleotide shown 5′ to the terminal modifications shown can be another modified or unmodified nucleotide or non-nucleotide described herein, for example modifications having any of Formulae I-VII or any combination thereof.
FIG. 11 shows a non-limiting example of a strategy used to identify chemically modified siNA constructs of the invention that are nuclease resistance while preserving the ability to mediate RNAi activity. Chemical modifications are introduced into the siNA construct based on educated design parameters (e.g. introducing 2′-mofications, base modifications, backbone modifications, terminal cap modifications etc). The modified construct in tested in an appropriate system (e.g. human serum for nuclease resistance, shown, or an animal model for PK/delivery parameters). In parallel, the siNA construct is tested for RNAi activity, for example in a cell culture system such as a luciferase reporter assay). Lead siNA constructs are then identified which possess a particular characteristic while maintaining RNAi activity, and can be further modified and assayed once again. This same approach can be used to identify siNA-conjugate molecules with improved pharmacokinetic profiles, delivery, and RNAi activity.
FIG. 12 shows non-limiting examples of phosphorylated siNA molecules of the invention, including linear and duplex constructs and asymmetric derivatives thereof.
FIG. 13 shows non-limiting examples of chemically modified terminal phosphate groups of the invention.
FIG. 14A shows a non-limiting example of methodology used to design self complementary DFO constructs utilizing palindrome and/or repeat nucleic acid sequences that are identified in a target nucleic acid sequence. (i) A palindrome or repeat sequence is identified in a nucleic acid target sequence. (ii) A sequence is designed that is complementary to the target nucleic acid sequence and the palindrome sequence. (iii) An inverse repeat sequence of the non-palindrome/repeat portion of the complementary sequence is appended to the 3′-end of the complementary sequence to generate a self complementary DFO molecule comprising sequence complementary to the nucleic acid target. (iv) The DFO molecule can self-assemble to form a double stranded oligonucleotide. FIG. 14B shows a non-limiting representative example of a duplex forming oligonucleotide sequence. FIG. 14C shows a non-limiting example of the self assembly schematic of a representative duplex forming oligonucleotide sequence. FIG. 14D shows a non-limiting example of the self assembly schematic of a representative duplex forming oligonucleotide sequence followed by interaction with a target nucleic acid sequence resulting in modulation of gene expression.
FIG. 15 shows a non-limiting example of the design of self complementary DFO constructs utilizing palindrome and/or repeat nucleic acid sequences that are incorporated into the DFO constructs that have sequence complementary to any target nucleic acid sequence of interest. Incorporation of these palindrome/repeat sequences allow the design of DFO constructs that form duplexes in which each strand is capable of mediating modulation of target gene expression, for example by RNAi. First, the target sequence is identified. A complementary sequence is then generated in which nucleotide or non-nucleotide modifications (shown as X or Y) are introduced into the complementary sequence that generate an artificial palindrome (shown as XYXYXY in the Figure). An inverse repeat of the non-palindrome/repeat complementary sequence is appended to the 3′-end of the complementary sequence to generate a self complementary DFO comprising sequence complementary to the nucleic acid target. The DFO can self-assemble to form a double stranded oligonucleotide.
FIG. 16 shows non-limiting examples of multifunctional siNA molecules of the invention comprising two separate polynucleotide sequences that are each capable of mediating RNAi directed cleavage of differing target nucleic acid sequences. FIG. 16A shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first and second complementary regions are situated at the 3′-ends of each polynucleotide sequence in the multifunctional siNA. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. FIG. 16B shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first and second complementary regions are situated at the 5′-ends of each polynucleotide sequence in the multifunctional siNA. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences.
FIG. 17 shows non-limiting examples of multifunctional siNA molecules of the invention comprising a single polynucleotide sequence comprising distinct regions that are each capable of mediating RNAi directed cleavage of differing target nucleic acid sequences. FIG. 17A shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the second complementary region is situated at the 3′-end of the polynucleotide sequence in the multifunctional siNA. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. FIG. 17B shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first complementary region is situated at the 5′-end of the polynucleotide sequence in the multifunctional siNA. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. In one embodiment, these multifunctional siNA constructs are processed in vivo or in vitro to generate multifunctional siNA constructs as shown in FIG. 16.
FIG. 18 shows non-limiting examples of multifunctional siNA molecules of the invention comprising two separate polynucleotide sequences that are each capable of mediating RNAi directed cleavage of differing target nucleic acid sequences and wherein the multifunctional siNA construct further comprises a self complementary, palindrome, or repeat region, thus enabling shorter bifuctional siNA constructs that can mediate RNA interference against differing target nucleic acid sequences. FIG. 18A shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first and second complementary regions are situated at the 3′-ends of each polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. FIG. 18B shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first and second complementary regions are situated at the 5′-ends of each polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences.
FIG. 19 shows non-limiting examples of multifunctional siNA molecules of the invention comprising a single polynucleotide sequence comprising distinct regions that are each capable of mediating RNAi directed cleavage of differing target nucleic acid sequences and wherein the multifunctional siNA construct further comprises a self complementary, palindrome, or repeat region, thus enabling shorter bifuctional siNA constructs that can mediate RNA interference against differing target nucleic acid sequences. FIG. 19A shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the second complementary region is situated at the 3′-end of the polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. FIG. 19B shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first complementary region is situated at the 5′-end of the polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. In one embodiment, these multifunctional siNA constructs are processed in vivo or in vitro to generate multifunctional siNA constructs as shown in FIG. 18.
FIG. 20 shows a non-limiting example of how multifunctional siNA molecules of the invention can target two separate target nucleic acid molecules, such as separate RNA molecules encoding differing proteins, for example, a cytokine and its corresponding receptor, differing viral strains, a virus and a cellular protein involved in viral infection or replication, or differing proteins involved in a common or divergent biologic pathway that is implicated in the maintenance of progression of disease. Each strand of the multifunctional siNA construct comprises a region having complementarity to separate target nucleic acid molecules. The multifunctional siNA molecule is designed such that each strand of the siNA can be utilized by the RISC complex to initiate RNA interference mediated cleavage of its corresponding target. These design parameters can include destabilization of each end of the siNA construct (see for example Schwarz et al., 2003, Cell, 115, 199-208). Such destabilization can be accomplished for example by using guanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), or destabilizing chemically modified nucleotides at terminal nucleotide positions as is known in the art.
FIG. 21 shows a non-limiting example of how multifunctional siNA molecules of the invention can target two separate target nucleic acid sequences within the same target nucleic acid molecule, such as alternate coding regions of a RNA, coding and non-coding regions of a RNA, or alternate splice variant regions of a RNA. Each strand of the multifunctional siNA construct comprises a region having complementarity to the separate regions of the target nucleic acid molecule. The multifunctional siNA molecule is designed such that each strand of the siNA can be utilized by the RISC complex to initiate RNA interference mediated cleavage of its corresponding target region. These design parameters can include destabilization of each end of the siNA construct (see for example Schwarz et al., 2003, Cell, 115, 199-208). Such destabilization can be accomplished for example by using guanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), or destabilizing chemically modified nucleotides at terminal nucleotide positions as is known in the art.
FIG. 22(A-H) shows non-limiting examples of tethered multifunctional siNA constructs of the invention. In the examples shown, a linker (e.g., nucleotide or non-nucleotide linker) connects two siNA regions (e.g., two sense, two antisense, or alternately a sense and an antisense region together. Separate sense (or sense and antisense) sequences corresponding to a first target sequence and second target sequence are hybridized to their corresponding sense and/or antisense sequences in the multifunctional siNA. In addition, various conjugates, ligands, aptamers, polymers or reporter molecules can be attached to the linker region for selective or improved delivery and/or pharmacokinetic properties.
FIG. 23 shows a non-limiting example of various dendrimer based multifunctional siNA designs.
FIG. 24 shows a non-limiting example of various supramolecular multifunctional siNA designs.
FIG. 25 shows a non-limiting example of a dicer enabled multifunctional siNA design using a 30 nucleotide precursor siNA construct. A 30 base pair duplex is cleaved by Dicer into 22 and 8 base pair products from either end (8 b.p. fragments not shown). For ease of presentation the overhangs generated by dicer are not shown—but can be compensated for. Three targeting sequences are shown. The required sequence identity overlapped is indicated by grey boxes. The N's of the parent 30 b.p. siNA are suggested sites of 2′-OH positions to enable Dicer cleavage if this is tested in stabilized chemistries. Note that processing of a 30 mer duplex by Dicer RNase III does not give a precise 22+8 cleavage, but rather produces a series of closely related products (with 22+8 being the primary site). Therefore, processing by Dicer will yield a series of active siNAs.
FIG. 26 shows a non-limiting example of a dicer enabled multifunctional siNA design using a 40 nucleotide precursor siNA construct. A 40 base pair duplex is cleaved by Dicer into 20 base pair products from either end. For ease of presentation the overhangs generated by dicer are not shown—but can be compensated for. Four targeting sequences are shown. The target sequences having homology are enclosed by boxes. This design format can be extended to larger RNAs. If chemically stabilized siNAs are bound by Dicer, then strategically located ribonucleotide linkages can enable designer cleavage products that permit our more extensive repertoire of multifunctional designs. For example cleavage products not limited to the Dicer standard of approximately 22-nucleotides can allow multifunctional siNA constructs with a target sequence identity overlap ranging from, for example, about 3 to about 15 nucleotides.
FIG. 27 shows a non-limiting example of additional multifunctional siNA construct designs of the invention. In one example, a conjugate, ligand, aptamer, label, or other moiety is attached to a region of the multifunctional siNA to enable improved delivery or pharmacokinetic profiling.
FIG. 28 shows a non-limiting example of additional multifunctional siNA construct designs of the invention. In one example, a conjugate, ligand, aptamer, label, or other moiety is attached to a region of the multifunctional siNA to enable improved delivery or pharmacokinetic profiling.
FIG. 29 shows a non-limiting example of a cholesterol linked phosphoramidite that can be used to synthesize cholesterol conjugated siNA molecules of the invention. An example is shown with the cholesterol moiety linked to the 5′-end of the sense strand of a siNA molecule.
FIG. 30 shows a non-limiting example of demonstrated stabilization of visual acuity (VA) in all patients treated with Sirna-027 in a Phase I clinical study, with a variable mean increase of number of letters read for each dose group, and a duration of effect sustained over 8 weeks post-injection.
FIG. 31 shows a non-limiting example of assessment of visual acuity (VA) at 8 weeks post dosing in patients treated with Sirna-027 in a Phase I clinical study, VA (number of lines read) was stable or improved in 100% of the patients, while a clinically significant improvement (equal to or more than 3 lines) was observed in 23% of patients.
FIG. 32 shows a non-limiting example of changes in foveal thickness for the first four dose groups (100, 200, 400 and 800 μg) in patients treated with Sirna-027 in a Phase I clinical study, in which treatment with Sirna-027 demonstrated a decrease of lesion thickness for the 100, 200 and 800 μg dose groups, with maximum effect by 14 days post-injection.



DETAILED DESCRIPTION OF THE INVENTION
Mechanism of Action of Nucleic Acid Molecules of the Invention
The discussion that follows discusses the proposed mechanism of RNA interference mediated by short interfering RNA as is presently known, and is not meant to be limiting and is not an admission of prior art. Applicant demonstrates herein that chemically-modified short interfering nucleic acids possess similar or improved capacity to mediate RNAi as do siRNA molecules and are expected to possess improved stability and activity in vivo; therefore, this discussion is not meant to be limiting only to siRNA and can be applied to siNA as a whole. By “improved capacity to mediate RNAi” or “improved RNAi activity” is meant to include RNAi activity measured in vitro and/or in vivo where the RNAi activity is a reflection of both the ability of the siNA to mediate RNAi and the stability of the siNAs of the invention. In this invention, the product of these activities can be increased in vitro and/or in vivo compared to an all RNA siRNA or a siNA containing a plurality of ribonucleotides. In some cases, the activity or stability of the siNA molecule can be decreased (i.e., less than ten-fold), but the overall activity of the siNA molecule is enhanced in vitro and/or in vivo.
RNA interference refers to the process of sequence specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Fire et al., 1998, Nature, 391, 806). The corresponding process in plants is commonly referred to as post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes which is commonly shared by diverse flora and phyla (Fire et al., 1999, Trends Genet., 15, 358). Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA or viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response though a mechanism that has yet to be fully characterized. This mechanism appears to be different from the interferon response that results from dsRNA-mediated activation of protein kinase PKR and 2′, 5′-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L.
The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as Dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Berstein et al., 2001, Nature, 409, 363). Short interfering RNAs derived from Dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes. Dicer has also been implicated in the excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001, Science, 293, 834). The RNAi response also features an endonuclease complex containing a siRNA, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence homologous to the siRNA. Cleavage of the target RNA takes place in the middle of the region complementary to the guide sequence of the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188). In addition, RNA interference can also involve small RNA (e.g., micro-RNA or miRNA) mediated gene silencing, presumably though cellular mechanisms that regulate chromatin structure and thereby prevent transcription of target gene sequences (see for example Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237). As such, siNA molecules of the invention can be used to mediate gene silencing via interaction with RNA transcripts or alternately by interaction with particular gene sequences, wherein such interaction results in gene silencing either at the transcriptional level or post-transcriptional level.
RNAi has been studied in a variety of systems. Fire et al., 1998, Nature, 391, 806, were the first to observe RNAi in C. elegans. Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated by dsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001, Nature, 411, 494, describe RNAi induced by introduction of duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells. Recent work in Drosophila embryonic lysates has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies have shown that 21 nucleotide siRNA duplexes are most active when containing two 2-nucleotide 3′-terminal nucleotide overhangs. Furthermore, substitution of one or both siRNA strands with 2′-deoxy or 2′-O-methyl nucleotides abolishes RNAi activity, whereas substitution of 3′-terminal siRNA nucleotides with deoxy nucleotides was shown to be tolerated. Mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity. In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5′-end of the siRNA guide sequence rather than the 3′-end (Elbashir et al., 2001, EMBO J., 20, 6877). Other studies have indicated that a 5′-phosphate on the target-complementary strand of a siRNA duplex is required for siRNA activity and that ATP is utilized to maintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309); however, siRNA molecules lacking a 5′-phosphate are active when introduced exogenously, suggesting that 5′-phosphorylation of siRNA constructs may occur in vivo.
Duplex Forming Oligonucleotides (DFO) of the Invention
In one embodiment, the invention features siNA molecules comprising duplex forming oligonucleotides (DFO) that can self-assemble into double stranded oligonucleotides. The duplex forming oligonucleotides of the invention can be chemically synthesized or expressed from transcription units and/or vectors. The DFO molecules of the instant invention provide useful reagents and methods for a variety of therapeutic, diagnostic, agricultural, veterinary, target validation, genomic discovery, genetic engineering and pharmacogenomic applications.
Applicant demonstrates herein that certain oligonucleotides, referred to herein for convenience but not limitation as duplex forming oligonucleotides or DFO molecules, are potent mediators of sequence specific regulation of gene expression. The oligonucleotides of the invention are distinct from other nucleic acid sequences known in the art (e.g., siRNA, miRNA, stRNA, shRNA, antisense oligonucleotides etc.) in that they represent a class of linear polynucleotide sequences that are designed to self-assemble into double stranded oligonucleotides, where each strand in the double stranded oligonucleotides comprises a nucleotide sequence that is complementary to a target nucleic acid molecule. Nucleic acid molecules of the invention can thus self assemble into functional duplexes in which each strand of the duplex comprises the same polynucleotide sequence and each strand comprises a nucleotide sequence that is complementary to a target nucleic acid molecule.
Generally, double stranded oligonucleotides are formed by the assembly of two distinct oligonucleotide sequences where the oligonucleotide sequence of one strand is complementary to the oligonucleotide sequence of the second strand; such double stranded oligonucleotides are assembled from two separate oligonucleotides, or from a single molecule that folds on itself to form a double stranded structure, often referred to in the field as hairpin stem-loop structure (e.g., shRNA or short hairpin RNA). These double stranded oligonucleotides known in the art all have a common feature in that each strand of the duplex has a distinct nucleotide sequence.
Distinct from the double stranded nucleic acid molecules known in the art, the applicants have developed a novel, potentially cost effective and simplified method of forming a double stranded nucleic acid molecule starting from a single stranded or linear oligonucleotide. The two strands of the double stranded oligonucleotide formed according to the instant invention have the same nucleotide sequence and are not covalently linked to each other. Such double-stranded oligonucleotides molecules can be readily linked post-synthetically by methods and reagents known in the art and are within the scope of the invention. In one embodiment, the single stranded oligonucleotide of the invention (the duplex forming oligonucleotide) that forms a double stranded oligonucleotide comprises a first region and a second region, where the second region includes a nucleotide sequence that is an inverted repeat of the nucleotide sequence in the first region, or a portion thereof, such that the single stranded oligonucleotide self assembles to form a duplex oligonucleotide in which the nucleotide sequence of one strand of the duplex is the same as the nucleotide sequence of the second strand. Non-limiting examples of such duplex forming oligonucleotides are illustrated in FIGS. 14 and 15. These duplex forming oligonucleotides (DFOs) can optionally include certain palindrome or repeat sequences where such palindrome or repeat sequences are present in between the first region and the second region of the DFO.
In one embodiment, the invention features a duplex forming oligonucleotide (DFO) molecule, wherein the DFO comprises a duplex forming self complementary nucleic acid sequence that has nucleotide sequence complementary to a target nucleic acid sequence. The DFO molecule can comprise a single self complementary sequence or a duplex resulting from assembly of such self complementary sequences.
In one embodiment, a duplex forming oligonucleotide (DFO) of the invention comprises a first region and a second region, wherein the second region comprises a nucleotide sequence comprising an inverted repeat of nucleotide sequence of the first region such that the DFO molecule can assemble into a double stranded oligonucleotide. Such double stranded oligonucleotides can act as a short interfering nucleic acid (siNA) to modulate gene expression. Each strand of the double stranded oligonucleotide duplex formed by DFO molecules of the invention can comprise a nucleotide sequence region that is complementary to the same nucleotide sequence in a target nucleic acid molecule (e.g., target RNA).
In one embodiment, the invention features a single stranded DFO that can assemble into a double stranded oligonucleotide. The applicant has surprisingly found that a single stranded oligonucleotide with nucleotide regions of self complementarity can readily assemble into duplex oligonucleotide constructs. Such DFOs can assemble into duplexes that can inhibit gene expression in a sequence specific manner. The DFO molecules of the invention comprise a first region with nucleotide sequence that is complementary to the nucleotide sequence of a second region and where the sequence of the first region is complementary to a target nucleic acid. The DFO can form a double stranded oligonucleotide wherein a portion of each strand of the double stranded oligonucleotide comprises a sequence complementary to a target nucleic acid sequence.
In one embodiment, the invention features a double stranded oligonucleotide, wherein the two strands of the double stranded oligonucleotide are not covalently linked to each other, and wherein each strand of the double stranded oligonucleotide comprises a nucleotide sequence that is complementary to the same nucleotide sequence in a target nucleic acid molecule or a portion thereof (e.g., VEGF and/or VEGFR RNA target). In another embodiment, the two strands of the double stranded oligonucleotide share an identical nucleotide sequence of at least about 15, preferably at least about 16, 17, 18, 19, 20, or 21 nucleotides.
In one embodiment, a DFO molecule of the invention comprises a structure having Formula DFO-I:










5′-p-X Z X′-3′







wherein Z comprises a palindromic or repeat nucleic acid sequence optionally with one or more modified nucleotides (e.g., nucleotide with a modified base, such as 2-amino purine, 2-amino-1,6-dihydro purine or a universal base), for example of length about 2 to about 24 nucleotides in even numbers (e.g., about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22 or 24 nucleotides), X represents a nucleic acid sequence, for example of length of about 1 to about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides), X′ comprises a nucleic acid sequence, for example of length about 1 and about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides) having nucleotide sequence complementarity to sequence X or a portion thereof, p comprises a terminal phosphate group that can be present or absent, and wherein sequence X and Z, either independently or together, comprise nucleotide sequence that is complementary to a target nucleic acid sequence or a portion thereof and is of length sufficient to interact (e.g., base pair) with the target nucleic acid sequence or a portion thereof (e.g., VEGF and/or VEGFR RNA target). For example, X independently can comprise a sequence from about 12 to about 21 or more (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more) nucleotides in length that is complementary to nucleotide sequence in a target RNA or a portion thereof. In another non-limiting example, the length of the nucleotide sequence of X and Z together, when X is present, that is complementary to the target RNA or a portion thereof (e.g., VEGF and/or VEGFR RNA target) is from about 12 to about 21 or more nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more). In yet another non-limiting example, when X is absent, the length of the nucleotide sequence of Z that is complementary to the target RNA or a portion thereof is from about 12 to about 24 or more nucleotides (e.g., about 12, 14, 16, 18, 20, 22, 24, or more). In one embodiment X, Z and X′ are independently oligonucleotides, where X and/or Z comprises a nucleotide sequence of length sufficient to interact (e.g., base pair) with a nucleotide sequence in the target RNA or a portion thereof (e.g., VEGF and/or VEGFR RNA target). In one embodiment, the lengths of oligonucleotides X and X′ are identical. In another embodiment, the lengths of oligonucleotides X and X′ are not identical. In another embodiment, the lengths of oligonucleotides X and Z, or Z and X′, or X, Z and X′ are either identical or different.

When a sequence is described in this specification as being of “sufficient” length to interact (i.e., base pair) with another sequence, it is meant that the length is such that the number of bonds (e.g., hydrogen bonds) formed between the two sequences is enough to enable the two sequence to form a duplex under the conditions of interest. Such conditions can be in vitro (e.g., for diagnostic or assay purposes) or in vivo (e.g., for therapeutic purposes). It is a simple and routine matter to determine such lengths.
In one embodiment, the invention features a double stranded oligonucleotide construct having Formula DFO-I(a):










5′-p-X Z X′-3′




 



   3′-X′ Z X-p-5′






wherein Z comprises a palindromic or repeat nucleic acid sequence or palindromic or repeat-like nucleic acid sequence with one or more modified nucleotides (e.g., nucleotides with a modified base, such as 2-amino purine, 2-amino-1,6-dihydro purine or a universal base), for example of length about 2 to about 24 nucleotides in even numbers (e.g., about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 nucleotides), X represents a nucleic acid sequence, for example of length about 1 to about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides), X′ comprises a nucleic acid sequence, for example of length about 1 to about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides) having nucleotide sequence complementarity to sequence X or a portion thereof, p comprises a terminal phosphate group that can be present or absent, and wherein each X and Z independently comprises a nucleotide sequence that is complementary to a target nucleic acid sequence or a portion thereof (e.g., VEGF and/or VEGFR RNA target) and is of length sufficient to interact with the target nucleic acid sequence of a portion thereof (e.g., VEGF and/or VEGFR RNA target). For example, sequence X independently can comprise a sequence from about 12 to about 21 or more nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more) in length that is complementary to a nucleotide sequence in a target RNA or a portion thereof (e.g., VEGF and/or VEGFR RNA target). In another non-limiting example, the length of the nucleotide sequence of X and Z together (when X is present) that is complementary to the target RNA or a portion thereof is from about 12 to about 21 or more nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more). In yet another non-limiting example, when X is absent, the length of the nucleotide sequence of Z that is complementary to the target RNA or a portion thereof is from about 12 to about 24 or more nucleotides (e.g., about 12, 14, 16, 18, 20, 22, 24 or more). In one embodiment X, Z and X′ are independently oligonucleotides, where X and/or Z comprises a nucleotide sequence of length sufficient to interact (e.g., base pair) with nucleotide sequence in the target RNA or a portion thereof (e.g., VEGF and/or VEGFR RNA target). In one embodiment, the lengths of oligonucleotides X and X′ are identical. In another embodiment, the lengths of oligonucleotides X and X′ are not identical. In another embodiment, the lengths of oligonucleotides X and Z or Z and X′ or X, Z and X′ are either identical or different. In one embodiment, the double stranded oligonucleotide construct of Formula I(a) includes one or more, specifically 1, 2, 3 or 4, mismatches, to the extent such mismatches do not significantly diminish the ability of the double stranded oligonucleotide to inhibit target gene expression.

In one embodiment, a DFO molecule of the invention comprises structure having Formula DFO-II:










5′-p-X X′-3′







wherein each X and X′ are independently oligonucleotides of length about 12 nucleotides to about 21 nucleotides, wherein X comprises, for example, a nucleic acid sequence of length about 12 to about 21 nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides), X′ comprises a nucleic acid sequence, for example of length about 12 to about 21 nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides) having nucleotide sequence complementarity to sequence X or a portion thereof, p comprises a terminal phosphate group that can be present or absent, and wherein X comprises a nucleotide sequence that is complementary to a target nucleic acid sequence (e.g., target RNA) or a portion thereof and is of length sufficient to interact (e.g., base pair) with the target nucleic acid sequence of a portion thereof. In one embodiment, the length of oligonucleotides X and X′ are identical. In another embodiment the length of oligonucleotides X and X′ are not identical. In one embodiment, length of the oligonucleotides X and X′ are sufficient to form a relatively stable double stranded oligonucleotide.

In one embodiment, the invention features a double stranded oligonucleotide construct having Formula DFO-II(a):










5′-p-X X′-3′




 



   3′-X′ X-p-5′






wherein each X and X′ are independently oligonucleotides of length about 12 nucleotides to about 21 nucleotides, wherein X comprises a nucleic acid sequence, for example of length about 12 to about 21 nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides), X′ comprises a nucleic acid sequence, for example of length about 12 to about 21 nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides) having nucleotide sequence complementarity to sequence X or a portion thereof, p comprises a terminal phosphate group that can be present or absent, and wherein X comprises nucleotide sequence that is complementary to a target nucleic acid sequence or a portion thereof (e.g., VEGF and/or VEGFR RNA target) and is of length sufficient to interact (e.g., base pair) with the target nucleic acid sequence (e.g., target RNA) or a portion thereof. In one embodiment, the lengths of oligonucleotides X and X′ are identical. In another embodiment, the lengths of oligonucleotides X and X′ are not identical. In one embodiment, the lengths of the oligonucleotides X and X′ are sufficient to form a relatively stable double stranded oligonucleotide. In one embodiment, the double stranded oligonucleotide construct of Formula II(a) includes one or more, specifically 1, 2, 3 or 4, mismatches, to the extent such mismatches do not significantly diminish the ability of the double stranded oligonucleotide to inhibit target gene expression.

In one embodiment, the invention features a DFO molecule having Formula DFO-I(b):










5′-p-Z-3′







where Z comprises a palindromic or repeat nucleic acid sequence optionally including one or more non-standard or modified nucleotides (e.g., nucleotide with a modified base, such as 2-amino purine or a universal base) that can facilitate base-pairing with other nucleotides. Z can be, for example, of length sufficient to interact (e.g., base pair) with nucleotide sequence of a target nucleic acid (e.g., target RNA) molecule, preferably of length of at least 12 nucleotides, specifically about 12 to about 24 nucleotides (e.g., about 12, 14, 16, 18, 20, 22 or 24 nucleotides). p represents a terminal phosphate group that can be present or absent.

In one embodiment, a DFO molecule having any of Formula DFO-I, DFO-I(a), DFO-I(b), DFO-II(a) or DFO-II can comprise chemical modifications as described herein without limitation, such as, for example, nucleotides having any of Formulae I-VII, stabilization chemistries as described in Table IV, or any other combination of modified nucleotides and non-nucleotides as described in the various embodiments herein.
In one embodiment, the palindrome or repeat sequence or modified nucleotide (e.g., nucleotide with a modified base, such as 2-amino purine or a universal base) in Z of DFO constructs having Formula DFO-I, DFO-I(a) and DFO-I(b), comprises chemically modified nucleotides that are able to interact with a portion of the target nucleic acid sequence (e.g., modified base analogs that can form Watson Crick base pairs or non-Watson Crick base pairs).
In one embodiment, a DFO molecule of the invention, for example a DFO having Formula DFO-I or DFO-II, comprises about 15 to about 40 nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides). In one embodiment, a DFO molecule of the invention comprises one or more chemical modifications. In a non-limiting example, the introduction of chemically modified nucleotides and/or non-nucleotides into nucleic acid molecules of the invention provides a powerful tool in overcoming potential limitations of in vivo stability and bioavailability inherent to unmodified RNA molecules that are delivered exogenously. For example, the use of chemically modified nucleic acid molecules can enable a lower dose of a particular nucleic acid molecule for a given therapeutic effect since chemically modified nucleic acid molecules tend to have a longer half-life in serum or in cells or tissues. Furthermore, certain chemical modifications can improve the bioavailability and/or potency of nucleic acid molecules by not only enhancing half-life but also facilitating the targeting of nucleic acid molecules to particular organs, cells or tissues and/or improving cellular uptake of the nucleic acid molecules. Therefore, even if the activity of a chemically modified nucleic acid molecule is reduced in vitro as compared to a native/unmodified nucleic acid molecule, for example when compared to an unmodified RNA molecule, the overall activity of the modified nucleic acid molecule can be greater than the native or unmodified nucleic acid molecule due to improved stability, potency, duration of effect, bioavailability and/or delivery of the molecule.
Multifunctional or Multi-Targeted siNA Molecules of the Invention
In one embodiment, the invention features siNA molecules comprising multifunctional short interfering nucleic acid (multifunctional siNA) molecules that modulate the expression of one or more genes in a biologic system, such as a cell, tissue, or organism. The multifunctional short interfering nucleic acid (multifunctional siNA) molecules of the invention can target more than one region a VEGF and/or VEGFR target nucleic acid sequence or can target sequences of more than one distinct target nucleic acid molecules (e.g., VEGF, VEGFR, interleukin (e.g., IL-4, IL-13), or interleukin receptor (e.g., IL-4R, IL-13R)RNA targets). The multifunctional siNA molecules of the invention can be chemically synthesized or expressed from transcription units and/or vectors. The multifunctional siNA molecules of the instant invention provide useful reagents and methods for a variety of human applications, therapeutic, diagnostic, agricultural, veterinary, target validation, genomic discovery, genetic engineering and pharmacogenomic applications.
Applicant demonstrates herein that certain oligonucleotides, referred to herein for convenience but not limitation as multifunctional short interfering nucleic acid or multifunctional siNA molecules, are potent mediators of sequence specific regulation of gene expression. The multifunctional siNA molecules of the invention are distinct from other nucleic acid sequences known in the art (e.g., siRNA, miRNA, stRNA, shRNA, antisense oligonucleotides, etc.) in that they represent a class of polynucleotide molecules that are designed such that each strand in the multifunctional siNA construct comprises a nucleotide sequence that is complementary to a distinct nucleic acid sequence in one or more target nucleic acid molecules. A single multifunctional siNA molecule (generally a double-stranded molecule) of the invention can thus target more than one (e.g., 2, 3, 4, 5, or more) differing target nucleic acid target molecules. Nucleic acid molecules of the invention can also target more than one (e.g., 2, 3, 4, 5, or more) region of the same target nucleic acid sequence. As such multifunctional siNA molecules of the invention are useful in down regulating or inhibiting the expression of one or more target nucleic acid molecules. For example, a multifunctional siNA molecule of the invention can target nucleic acid molecules encoding a cytokine and its corresponding receptor(s) (e.g., VEGF and VEGF receptors and interleukins (e.g., IL-4, IL-13) and interleukin receptors (e.g., IL-4R, IL-13R) described herein). By reducing or inhibiting expression of more than one target nucleic acid molecule with one multifunctional siNA construct, multifunctional siNA molecules of the invention represent a class of potent therapeutic agents that can provide simultaneous inhibition of multiple targets within a disease or pathogen related pathway. Such simultaneous inhibition can provide synergistic therapeutic treatment strategies without the need for separate preclinical and clinical development efforts or complex regulatory approval process.
Use of multifunctional siNA molecules that target more then one region of a target nucleic acid molecule (e.g., messenger RNA) is expected to provide potent inhibition of gene expression. For example, a single multifunctional siNA construct of the invention can target both conserved and variable regions of a target nucleic acid molecule (e.g., VEGF and/or VEGFR RNA and/or interleukin and/or interleukin receptor RNA), thereby allowing down regulation or inhibition of different splice variants encoded by a single gene, or allowing for targeting of both coding and non-coding regions of a target nucleic acid molecule.
Generally, double stranded oligonucleotides are formed by the assembly of two distinct oligonucleotides where the oligonucleotide sequence of one strand is complementary to the oligonucleotide sequence of the second strand; such double stranded oligonucleotides are generally assembled from two separate oligonucleotides (e.g., siRNA). Alternately, a duplex can be formed from a single molecule that folds on itself (e.g., shRNA or short hairpin RNA). These double stranded oligonucleotides are known in the art to mediate RNA interference and all have a common feature wherein only one nucleotide sequence region (guide sequence or the antisense sequence) has complementarity to a target nucleic acid sequence (e.g., VEGF and/or VEGFR RNA and/or interleukin and/or interleukin receptor RNA) and the other strand (sense sequence) comprises nucleotide sequence that is homologous to the target nucleic acid sequence. Generally, the antisense sequence is retained in the active RISC complex and guides the RISC to the target nucleotide sequence by means of complementary base-pairing of the antisense sequence with the target sequence for mediating sequence-specific RNA interference. It is known in the art that in some cell culture systems, certain types of unmodified siRNAs can exhibit “off target” effects. It is hypothesized that this off-target effect involves the participation of the sense sequence instead of the antisense sequence of the siRNA in the RISC complex (see for example Schwarz et al., 2003, Cell, 115, 199-208). In this instance the sense sequence is believed to direct the RISC complex to a sequence (off-target sequence) that is distinct from the intended target sequence, resulting in the inhibition of the off-target sequence. In these double stranded nucleic acid molecules, each strand is complementary to a distinct target nucleic acid sequence. However, the off-targets that are affected by these dsRNAs are not entirely predictable and are non-specific.
Distinct from the double stranded nucleic acid molecules known in the art, the applicants have developed a novel, potentially cost effective and simplified method of down regulating or inhibiting the expression of more than one target nucleic acid sequence using a single multifunctional siNA construct. The multifunctional siNA molecules of the invention are designed to be double-stranded or partially double stranded, such that a portion of each strand or region of the multifunctional siNA is complementary to a target nucleic acid sequence of choice. As such, the multifunctional siNA molecules of the invention are not limited to targeting sequences that are complementary to each other, but rather to any two differing target nucleic acid sequences. Multifunctional siNA molecules of the invention are designed such that each strand or region of the multifunctional siNA molecule, that is complementary to a given target nucleic acid sequence, is of suitable length (e.g., from about 16 to about 28 nucleotides in length, preferably from about 18 to about 28 nucleotides in length) for mediating RNA interference against the target nucleic acid sequence. The complementarity between the target nucleic acid sequence and a strand or region of the multifunctional siNA must be sufficient (at least about 8 base pairs) for cleavage of the target nucleic acid sequence by RNA interference. multifunctional siNA of the invention is expected to minimize off-target effects seen with certain siRNA sequences, such as those described in (Schwarz et al., supra).
It has been reported that dsRNAs of length between 29 base pairs and 36 base pairs (Tuschl et al., International PCT Publication No. WO 02/44321) do not mediate RNAi. One reason these dsRNAs are inactive may be the lack of turnover or dissociation of the strand that interacts with the target RNA sequence, such that the RISC complex is not able to efficiently interact with multiple copies of the target RNA resulting in a significant decrease in the potency and efficiency of the RNAi process. Applicant has surprisingly found that the multifunctional siNAs of the invention can overcome this hurdle and are capable of enhancing the efficiency and potency of RNAi process. As such, in certain embodiments of the invention, multifunctional siNAs of length of about 29 to about 36 base pairs can be designed such that, a portion of each strand of the multifunctional siNA molecule comprises a nucleotide sequence region that is complementary to a target nucleic acid of length sufficient to mediate RNAi efficiently (e.g., about 15 to about 23 base pairs) and a nucleotide sequence region that is not complementary to the target nucleic acid. By having both complementary and non-complementary portions in each strand of the multifunctional siNA, the multifunctional siNA can mediate RNA interference against a target nucleic acid sequence without being prohibitive to turnover or dissociation (e.g., where the length of each strand is too long to mediate RNAi against the respective target nucleic acid sequence). Furthermore, design of multifunctional siNA molecules of the invention with internal overlapping regions allows the multifunctional siNA molecules to be of favorable (decreased) size for mediating RNA interference and of size that is well suited for use as a therapeutic agent (e.g., wherein each strand is independently from about 18 to about 28 nucleotides in length). Non-limiting examples are illustrated in the enclosed FIGS. 16-21.
In one embodiment, a multifunctional siNA molecule of the invention comprises a first region and a second region, where the first region of the multifunctional siNA comprises a nucleotide sequence complementary to a nucleic acid sequence of a first target nucleic acid molecule, and the second region of the multifunctional siNA comprises nucleic acid sequence complementary to a nucleic acid sequence of a second target nucleic acid molecule. In one embodiment, a multifunctional siNA molecule of the invention comprises a first region and a second region, where the first region of the multifunctional siNA comprises nucleotide sequence complementary to a nucleic acid sequence of the first region of a target nucleic acid molecule, and the second region of the multifunctional siNA comprises nucleotide sequence complementary to a nucleic acid sequence of a second region of a the target nucleic acid molecule. In another embodiment, the first region and second region of the multifunctional siNA can comprise separate nucleic acid sequences that share some degree of complementarity (e.g., from about 1 to about 10 complementary nucleotides). In certain embodiments, multifunctional siNA constructs comprising separate nucleic acid sequences can be readily linked post-synthetically by methods and reagents known in the art and such linked constructs are within the scope of the invention. Alternately, the first region and second region of the multifunctional siNA can comprise a single nucleic acid sequence having some degree of self complementarity, such as in a hairpin or stem-loop structure. Non-limiting examples of such double stranded and hairpin multifunctional short interfering nucleic acids are illustrated in FIGS. 16 and 17 respectively. These multifunctional short interfering nucleic acids (multifunctional siNAs) can optionally include certain overlapping nucleotide sequence where such overlapping nucleotide sequence is present in between the first region and the second region of the multifunctional siNA (see for example FIGS. 18 and 19).
In one embodiment, the invention features a multifunctional short interfering nucleic acid (multifunctional siNA) molecule, wherein each strand of the multifunctional siNA independently comprises a first region of nucleic acid sequence that is complementary to a distinct target nucleic acid sequence and the second region of nucleotide sequence that is not complementary to the target sequence. The target nucleic acid sequence of each strand is in the same target nucleic acid molecule or different target nucleic acid molecules.
In another embodiment, the multifunctional siNA comprises two strands, where: (a) the first strand comprises a region having sequence complementarity to a target nucleic acid sequence (complementary region 1) and a region having no sequence complementarity to the target nucleotide sequence (non-complementary region 1); (b) the second strand of the multifunction siNA comprises a region having sequence complementarity to a target nucleic acid sequence that is distinct from the target nucleotide sequence complementary to the first strand nucleotide sequence (complementary region 2), and a region having no sequence complementarity to the target nucleotide sequence of complementary region 2 (non-complementary region 2); (c) the complementary region 1 of the first strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the non-complementary region 2 of the second strand and the complementary region 2 of the second strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the non-complementary region 1 of the first strand. The target nucleic acid sequence of complementary region 1 and complementary region 2 is in the same target nucleic acid molecule or different target nucleic acid molecules.
In another embodiment, the multifunctional siNA comprises two strands, where: (a) the first strand comprises a region having sequence complementarity to a target nucleic acid sequence derived from a gene (e.g., VEGF, VEGFR, interleukin, and/or interleukin receptor gene) (complementary region 1) and a region having no sequence complementarity to the target nucleotide sequence of complementary region 1 (non-complementary region 1); (b) the second strand of the multifunction siNA comprises a region having sequence complementarity to a target nucleic acid sequence derived from a gene that is distinct from the gene of complementary region 1 (complementary region 2), and a region having no sequence complementarity to the target nucleotide sequence of complementary region 2 (non-complementary region 2); (c) the complementary region 1 of the first strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the non-complementary region 2 of the second strand and the complementary region 2 of the second strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the non-complementary region 1 of the first strand.
In another embodiment, the multifunctional siNA comprises two strands, where: (a) the first strand comprises a region having sequence complementarity to a target nucleic acid sequence derived from a gene (e.g., VEGF, VEGFR, interleukin, and/or interleukin receptor gene) (complementary region 1) and a region having no sequence complementarity to the target nucleotide sequence of complementary region 1 (non-complementary region 1); (b) the second strand of the multifunction siNA comprises a region having sequence complementarity to a target nucleic acid sequence distinct from the target nucleic acid sequence of complementary region 1 (complementary region 2), provided, however, that the target nucleic acid sequence for complementary region 1 and target nucleic acid sequence for complementary region 2 are both derived from the same gene, and a region having no sequence complementarity to the target nucleotide sequence of complementary region 2 (non-complementary region 2); (c) the complementary region 1 of the first strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the non-complementary region 2 of the second strand and the complementary region 2 of the second strand comprises a nucleotide sequence that is complementary to nucleotide sequence in the non-complementary region 1 of the first strand.
In one embodiment, the invention features a multifunctional short interfering nucleic acid (multifunctional siNA) molecule, wherein the multifunctional siNA comprises two complementary nucleic acid sequences in which the first sequence comprises a first region having nucleotide sequence complementary to nucleotide sequence within a target nucleic acid molecule, and in which the second sequence comprises a first region having nucleotide sequence complementary to a distinct nucleotide sequence within the same target nucleic acid molecule. Preferably, the first region of the first sequence is also complementary to the nucleotide sequence of the second region of the second sequence, and where the first region of the second sequence is complementary to the nucleotide sequence of the second region of the first sequence,
In one embodiment, the invention features a multifunctional short interfering nucleic acid (multifunctional siNA) molecule, wherein the multifunctional siNA comprises two complementary nucleic acid sequences in which the first sequence comprises a first region having a nucleotide sequence complementary to a nucleotide sequence within a first target nucleic acid molecule, and in which the second sequence comprises a first region having a nucleotide sequence complementary to a distinct nucleotide sequence within a second target nucleic acid molecule. Preferably, the first region of the first sequence is also complementary to the nucleotide sequence of the second region of the second sequence, and where the first region of the second sequence is complementary to the nucleotide sequence of the second region of the first sequence,
In one embodiment, the invention features a multifunctional siNA molecule comprising a first region and a second region, where the first region comprises a nucleic acid sequence having about 18 to about 28 nucleotides complementary to a nucleic acid sequence within a first target nucleic acid molecule, and the second region comprises nucleotide sequence having about 18 to about 28 nucleotides complementary to a distinct nucleic acid sequence within a second target nucleic acid molecule.
In one embodiment, the invention features a multifunctional siNA molecule comprising a first region and a second region, where the first region comprises nucleic acid sequence having about 18 to about 28 nucleotides complementary to a nucleic acid sequence within a target nucleic acid molecule, and the second region comprises nucleotide sequence having about 18 to about 28 nucleotides complementary to a distinct nucleic acid sequence within the same target nucleic acid molecule.
In one embodiment, the invention features a double stranded multifunctional short interfering nucleic acid (multifunctional siNA) molecule, wherein one strand of the multifunctional siNA comprises a first region having nucleotide sequence complementary to a first target nucleic acid sequence, and the second strand comprises a first region having a nucleotide sequence complementary to a second target nucleic acid sequence. The first and second target nucleic acid sequences can be present in separate target nucleic acid molecules or can be different regions within the same target nucleic acid molecule. As such, multifunctional siNA molecules of the invention can be used to target the expression of different genes, splice variants of the same gene, both mutant and conserved regions of one or more gene transcripts, or both coding and non-coding sequences of the same or differing genes or gene transcripts.
In one embodiment, a target nucleic acid molecule of the invention encodes a single protein. In another embodiment, a target nucleic acid molecule encodes more than one protein (e.g., 1, 2, 3, 4, 5 or more proteins). As such, a multifunctional siNA construct of the invention can be used to down regulate or inhibit the expression of several proteins. For example, a multifunctional siNA molecule comprising a region in one strand having nucleotide sequence complementarity to a first target nucleic acid sequence derived from a gene encoding one protein (e.g., a cytokine, such as vascular endothelial growth factor or VEGF) and the second strand comprising a region with nucleotide sequence complementarity to a second target nucleic acid sequence present in target nucleic acid molecules derived from genes encoding two proteins (e.g., two differing receptors, such as VEGF receptor 1 and VEGF receptor 2, for a single cytokine, such as VEGF) can be used to down regulate, inhibit, or shut down a particular biologic pathway by targeting, for example, a cytokine and receptors for the cytokine, or a ligand and receptors for the ligand.
In one embodiment the invention takes advantage of conserved nucleotide sequences present in different isoforms of cytokines or ligands and receptors for the cytokines or ligands. By designing multifunctional siNAs in a manner where one strand includes a sequence that is complementary to a target nucleic acid sequence conserved among various isoforms of a cytokine and the other strand includes sequence that is complementary to a target nucleic acid sequence conserved among the receptors for the cytokine, it is possible to selectively and effectively modulate or inhibit a biological pathway or multiple genes in a biological pathway using a single multifunctional siNA.
In another nonlimiting example, a multifunctional siNA molecule comprising a region in one strand having a nucleotide sequence complementarity to a first target nucleic acid sequence present in target nucleic acid molecules encoding two proteins (e.g., two isoforms of a cytokine such as VEGF, including for example any of VEGF-A, VEGF-B, VEGF-C, and/or VEGF-D) and the second strand comprising a region with a nucleotide sequence complementarity to a second target nucleic acid sequence present in target nucleotide molecules encoding two additional proteins (e.g., two differing receptors for the cytokine, such as VEGFR1, VEGFR2, and/or VEGFR3) can be used to down regulate, inhibit, or shut down a particular biologic pathway by targeting different isoforms of a cytokine and receptors for such cytokines.
In one embodiment, a multifunctional short interfering nucleic acid (multifunctional siNA) of the invention comprises a region in each strand, wherein the region in one strand comprises nucleotide sequence complementary to a cytokine and the region in the second strand comprises nucleotide sequence complementary to a corresponding receptor for the cytokine. Non-limiting examples of cytokines include vascular endothelial growth factors (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D) and/or interleukins (e.g., IL-4, IL-13) and non-limiting examples of cytokine receptors include VEGFR1, VEGFR2, and VEGFR3 and/or IL-4 and IL-13R.
In one embodiment, a double stranded multifunctional siNA molecule of the invention comprises a structure having Formula MF-I:










5′-p-X Z X′-3′




 



   3′-Y′ Z Y-p-5′






wherein each 5′-p-XZX′-3′ and 5′-p-YZY′-3′ are independently an oligonucleotide of length of about 20 nucleotides to about 300 nucleotides, preferably of about 20 to about 200 nucleotides, about 20 to about 100 nucleotides, about 20 to about 40 nucleotides, about 20 to about 40 nucleotides, about 24 to about 38 nucleotides, or about 26 to about 38 nucleotides; XZ comprises a nucleic acid sequence that is complementary to a first target nucleic acid sequence; YZ is an oligonucleotide comprising nucleic acid sequence that is complementary to a second target nucleic acid sequence; Z comprises nucleotide sequence of length about 1 to about 24 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides) that is self complimentary; X comprises nucleotide sequence of length about 1 to about 100 nucleotides, preferably about 1 to about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides) that is complementary to nucleotide sequence present in region Y′; Y comprises nucleotide sequence of length about 1 to about 100 nucleotides, preferably about 1- about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides) that is complementary to nucleotide sequence present in region X′; each p comprises a terminal phosphate group that is independently present or absent; each XZ and YZ is independently of length sufficient to stably interact (i.e., base pair) with the first and second target nucleic acid sequence, respectively, or a portion thereof. For example, each sequence X and Y can independently comprise sequence from about 12 to about 21 or more nucleotides in length (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more) that is complementary to a target nucleotide sequence in different target nucleic acid molecules, such as target RNAs or a portion thereof. In another non-limiting example, the length of the nucleotide sequence of X and Z together that is complementary to the first target nucleic acid sequence or a portion thereof is from about 12 to about 21 or more nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more). In another non-limiting example, the length of the nucleotide sequence of Y and Z together, that is complementary to the second target nucleic acid sequence or a portion thereof is from about 12 to about 21 or more nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more). In one embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in the same target nucleic acid molecule (e.g., VEGF and/or VEGFR RNA). In another embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in different target nucleic acid molecules (e.g., VEGF, VEGFR, interleukin, and/or interleukin receptor RNA). In one embodiment, Z comprises a palindrome or a repeat sequence. In one embodiment, the lengths of oligonucleotides X and X′ are identical. In another embodiment, the lengths of oligonucleotides X and X′ are not identical. In one embodiment, the lengths of oligonucleotides Y and Y′ are identical. In another embodiment, the lengths of oligonucleotides Y and Y′ are not identical. In one embodiment, the double stranded oligonucleotide construct of Formula I(a) includes one or more, specifically 1, 2, 3 or 4, mismatches, to the extent such mismatches do not significantly diminish the ability of the double stranded oligonucleotide to inhibit target gene expression.

In one embodiment, a multifunctional siNA molecule of the invention comprises a structure having Formula MF-II:










5′-p-X X′-3′




 



   3′-Y′ Y-p-5′






wherein each 5′-p-XX′-3′ and 5′-p-YY′-3′ are independently an oligonucleotide of length of about 20 nucleotides to about 300 nucleotides, preferably about 20 to about 200 nucleotides, about 20 to about 100 nucleotides, about 20 to about 40 nucleotides, about 20 to about 40 nucleotides, about 24 to about 38 nucleotides, or about 26 to about 38 nucleotides; X comprises a nucleic acid sequence that is complementary to a first target nucleic acid sequence; Y is an oligonucleotide comprising nucleic acid sequence that is complementary to a second target nucleic acid sequence; X comprises a nucleotide sequence of length about 1 to about 100 nucleotides, preferably about 1 to about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides) that is complementary to nucleotide sequence present in region Y′; Y comprises nucleotide sequence of length about 1 to about 100 nucleotides, preferably about 1 to about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20 or 21 nucleotides) that is complementary to nucleotide sequence present in region X′; each p comprises a terminal phosphate group that is independently present or absent; each X and Y independently is of length sufficient to stably interact (i.e., base pair) with the first and second target nucleic acid sequence, respectively, or a portion thereof. For example, each sequence X and Y can independently comprise sequence from about 12 to about 21 or more nucleotides in length (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more) that is complementary to a target nucleotide sequence in different target nucleic acid molecules, such as VEGF, VEGFR, interleukin and/or interleukin receptor target RNAs or a portion thereof. In one embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in the same target nucleic acid molecule (e.g., VEGF and/or VEGFR RNA). In another embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in different target nucleic acid molecules (e.g., VEGF, VEGFR, interleukin, and/or interleukin receptor RNA). In one embodiment, Z comprises a palindrome or a repeat sequence. In one embodiment, the lengths of oligonucleotides X and X′ are identical. In another embodiment, the lengths of oligonucleotides X and X′ are not identical. In one embodiment, the lengths of oligonucleotides Y and Y′ are identical. In another embodiment, the lengths of oligonucleotides Y and Y′ are not identical. In one embodiment, the double stranded oligonucleotide construct of Formula I(a) includes one or more, specifically 1, 2, 3 or 4, mismatches, to the extent such mismatches do not significantly diminish the ability of the double stranded oligonucleotide to inhibit target gene expression.

In one embodiment, a multifunctional siNA molecule of the invention comprises a structure having Formula MF-III:










X    X′




 



Y′-W-Y






wherein each X, X′, Y, and Y′ is independently an oligonucleotide of length of about 15 nucleotides to about 50 nucleotides, preferably about 18 to about 40 nucleotides, or about 19 to about 23 nucleotides; X comprises nucleotide sequence that is complementary to nucleotide sequence present in region Y′; X′ comprises nucleotide sequence that is complementary to nucleotide sequence present in region Y; each X and X′ is independently of length sufficient to stably interact (i.e., base pair) with a first and a second target nucleic acid sequence, respectively, or a portion thereof; W represents a nucleotide or non-nucleotide linker that connects sequences Y′ and Y; and the multifunctional siNA directs cleavage of the first and second target sequence via RNA interference. In one embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in the same target nucleic acid molecule (e.g., VEGF and/or VEGFR RNA). In another embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in different target nucleic acid molecules (e.g., VEGF, VEGFR, interleukin, and/or interleukin receptor RNA). In one embodiment, region W connects the 3′-end of sequence Y′ with the 3′-end of sequence Y. In one embodiment, region W connects the 3′-end of sequence Y′ with the 5′-end of sequence Y. In one embodiment, region W connects the 5′-end of sequence Y′ with the 5′-end of sequence Y. In one embodiment, region W connects the 5′-end of sequence Y′ with the 3′-end of sequence Y. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence X. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence X′. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence Y. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence Y′. In one embodiment, W connects sequences Y and Y′ via a biodegradable linker. In one embodiment, W further comprises a conjugate, label, aptamer, ligand, lipid, or polymer.

In one embodiment, a multifunctional siNA molecule of the invention comprises a structure having Formula MF-IV:










X    X′




 



Y′-W-Y






wherein each X, X′, Y, and Y′ is independently an oligonucleotide of length of about 15 nucleotides to about 50 nucleotides, preferably about 18 to about 40 nucleotides, or about 19 to about 23 nucleotides; X comprises nucleotide sequence that is complementary to nucleotide sequence present in region Y′; X′ comprises nucleotide sequence that is complementary to nucleotide sequence present in region Y; each Y and Y′ is independently of length sufficient to stably interact (i.e., base pair) with a first and a second target nucleic acid sequence, respectively, or a portion thereof; W represents a nucleotide or non-nucleotide linker that connects sequences Y′ and Y; and the multifunctional siNA directs cleavage of the first and second target sequence via RNA interference. In one embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in the same target nucleic acid molecule (e.g., VEGF and/or VEGFR RNA). In another embodiment, the first target nucleic acid sequence and the second target nucleic acid sequence are present in different target nucleic acid molecules (e.g., VEGF, VEGFR, interleukin, and/or interleukin receptor RNA). In one embodiment, region W connects the 3′-end of sequence Y′ with the 3′-end of sequence Y. In one embodiment, region W connects the 3′-end of sequence Y′ with the 5′-end of sequence Y. In one embodiment, region W connects the 5′-end of sequence Y′ with the 5′-end of sequence Y. In one embodiment, region W connects the 5′-end of sequence Y′ with the 3′-end of sequence Y. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence X. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence X′. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence Y. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence Y′. In one embodiment, W connects sequences Y and Y′ via a biodegradable linker. In one embodiment, W further comprises a conjugate, label, aptamer, ligand, lipid, or polymer.

In one embodiment, a multifunctional siNA molecule of the invention comprises a structure having Formula MF-V:










X    X′




 



Y′-W-Y






wherein each X, X′, Y, and Y′ is independently an oligonucleotide of length of about 15 nucleotides to about 50 nucleotides, preferably about 18 to about 40 nucleotides, or about 19 to about 23 nucleotides; X comprises nucleotide sequence that is complementary to nucleotide sequence present in region Y′; X′ comprises nucleotide sequence that is complementary to nucleotide sequence present in region Y; each X, X′, Y, or Y′ is independently of length sufficient to stably interact (i.e., base pair) with a first, second, third, or fourth target nucleic acid sequence, respectively, or a portion thereof; W represents a nucleotide or non-nucleotide linker that connects sequences Y′ and Y; and the multifunctional siNA directs cleavage of the first, second, third, and/or fourth target sequence via RNA interference. In one embodiment, the first, second, third and fourth target nucleic acid sequence are all present in the same target nucleic acid molecule (e.g., VEGF and/or VEGFR RNA). In another embodiment, the first, second, third and fourth target nucleic acid sequence are independently present in different target nucleic acid molecules (e.g., VEGF, VEGFR, interleukin, and/or interleukin receptor RNA). In one embodiment, region W connects the 3′-end of sequence Y′ with the 3′-end of sequence Y. In one embodiment, region W connects the 3′-end of sequence Y′ with the 5′-end of sequence Y. In one embodiment, region W connects the 5′-end of sequence Y′ with the 5′-end of sequence Y. In one embodiment, region W connects the 5′-end of sequence Y′ with the 3′-end of sequence Y. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence X. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence X′. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence Y. In one embodiment, a terminal phosphate group is present at the 5′-end of sequence Y′. In one embodiment, W connects sequences Y and Y′ via a biodegradable linker. In one embodiment, W further comprises a conjugate, label, aptamer, ligand, lipid, or polymer.

In one embodiment, regions X and Y of multifunctional siNA molecule of the invention (e.g., having any of Formula MF-I-MF-V), are complementary to different target nucleic acid sequences that are portions of the same target nucleic acid molecule. In one embodiment, such target nucleic acid sequences are at different locations within the coding region of a RNA transcript. In one embodiment, such target nucleic acid sequences comprise coding and non-coding regions of the same RNA transcript. In one embodiment, such target nucleic acid sequences comprise regions of alternately spliced transcripts or precursors of such alternately spliced transcripts.
In one embodiment, a multifunctional siNA molecule having any of Formula MF-I-MF-V can comprise chemical modifications as described herein without limitation, such as, for example, nucleotides having any of Formulae I-VII described herein, stabilization chemistries as described in Table IV, or any other combination of modified nucleotides and non-nucleotides as described in the various embodiments herein.
In one embodiment, the palindrome or repeat sequence or modified nucleotide (e.g., nucleotide with a modified base, such as 2-amino purine or a universal base) in Z of multifunctional siNA constructs having Formula MF-I or MF-II comprises chemically modified nucleotides that are able to interact with a portion of the target nucleic acid sequence (e.g., modified base analogs that can form Watson Crick base pairs or non-Watson Crick base pairs).
In one embodiment, a multifunctional siNA molecule of the invention, for example each strand of a multifunctional siNA having MF-I-MF-V, independently comprises about 15 to about 40 nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides). In one embodiment, a multifunctional siNA molecule of the invention comprises one or more chemical modifications. In a non-limiting example, the introduction of chemically modified nucleotides and/or non-nucleotides into nucleic acid molecules of the invention provides a powerful tool in overcoming potential limitations of in vivo stability and bioavailability inherent to unmodified RNA molecules that are delivered exogenously. For example, the use of chemically modified nucleic acid molecules can enable a lower dose of a particular nucleic acid molecule for a given therapeutic effect since chemically modified nucleic acid molecules tend to have a longer half-life in serum or in cells or tissues. Furthermore, certain chemical modifications can improve the bioavailability and/or potency of nucleic acid molecules by not only enhancing half-life but also facilitating the targeting of nucleic acid molecules to particular organs, cells or tissues and/or improving cellular uptake of the nucleic acid molecules. Therefore, even if the activity of a chemically modified nucleic acid molecule is reduced in vitro as compared to a native/unmodified nucleic acid molecule, for example when compared to an unmodified RNA molecule, the overall activity of the modified nucleic acid molecule can be greater than the native or unmodified nucleic acid molecule due to improved stability, potency, duration of effect, bioavailability and/or delivery of the molecule.
In another embodiment, the invention features multifunctional siNAs, wherein the multifunctional siNAs are assembled from two separate double-stranded siNAs, with one of the ends of each sense strand is tethered to the end of the sense strand of the other siNA molecule, such that the two antisense siNA strands are annealed to their corresponding sense strand that are tethered to each other at one end. The tethers or linkers can be nucleotide-based linkers or non-nucleotide based linkers as generally known in the art and as described herein.
In one embodiment, the invention features a multifunctional siNA, wherein the multifunctional siNA is assembled from two separate double-stranded siNAs, with the 5′-end of one sense strand of the siNA is tethered to the 5′-end of the sense strand of the other siNA molecule, such that the 5′-ends of the two antisense siNA strands, annealed to their corresponding sense strand that are tethered to each other at one end, point away (in the opposite direction) from each other. The tethers or linkers can be nucleotide-based linkers or non-nucleotide based linkers as generally known in the art and as described herein.
In one embodiment, the invention features a multifunctional siNA, wherein the multifunctional siNA is assembled from two separate double-stranded siNAs, with the 3′-end of one sense strand of the siNA is tethered to the 3′-end of the sense strand of the other siNA molecule, such that the 5′-ends of the two antisense siNA strands, annealed to their corresponding sense strand that are tethered to each other at one end, face each other. The tethers or linkers can be nucleotide-based linkers or non-nucleotide based linkers as generally known in the art and as described herein.
In one embodiment, the invention features a multifunctional siNA, wherein the multifunctional siNA is assembled from two separate double-stranded siNAs, with the 5′-end of one sense strand of the siNA is tethered to the 3′-end of the sense strand of the other siNA molecule, such that the 5′-end of the one of the antisense siNA strands annealed to their corresponding sense strand that are tethered to each other at one end, faces the 3′-end of the other antisense strand. The tethers or linkers can be nucleotide-based linkers or non-nucleotide based linkers as generally known in the art and as described herein.
In one embodiment, the invention features a multifunctional siNA, wherein the multifunctional siNA is assembled from two separate double-stranded siNAs, with the 5′-end of one antisense strand of the siNA is tethered to the 3′-end of the antisense strand of the other siNA molecule, such that the 5′-end of the one of the sense siNA strands annealed to their corresponding antisense sense strand that are tethered to each other at one end, faces the 3′-end of the other sense strand. In one embodiment, the linkage between the 5′-end of the first antisense strand and the 3′-end of the second antisense strand is designed in such a way as to be readily cleavable (e.g., biodegradable linker) such that the 5′end of each antisense strand of the multifunctional siNA has a free 5′-end suitable to mediate RNA interefence-based cleavage of the target RNA. The tethers or linkers can be nucleotide-based linkers or non-nucleotide based linkers as generally known in the art and as described herein.
In one embodiment, the invention features a multifunctional siNA, wherein the multifunctional siNA is assembled from two separate double-stranded siNAs, with the 5′-end of one antisense strand of the siNA is tethered to the 5′-end of the antisense strand of the other siNA molecule, such that the 3′-end of the one of the sense siNA strands annealed to their corresponding antisense sense strand that are tethered to each other at one end, faces the 3′-end of the other sense strand. In one embodiment, the linkage between the 5′-end of the first antisense strand and the 5′-end of the second antisense strand is designed in such a way as to be readily cleavable (e.g., biodegradable linker) such that the 5′end of each antisense strand of the multifunctional siNA has a free 5′-end suitable to mediate RNA interference-based cleavage of the target RNA. The tethers or linkers can be nucleotide-based linkers or non-nucleotide based linkers as generally known in the art and as described herein.
In one embodiment, the invention features a multifunctional siNA, wherein the multifunctional siNA is assembled from two separate double-stranded siNAs, with the 3′-end of one antisense strand of the siNA is tethered to the 3′-end of the antisense strand of the other siNA molecule, such that the 5′-end of the one of the sense siNA strands annealed to their corresponding antisense sense strand that are tethered to each other at one end, faces the 3′-end of the other sense strand. In one embodiment, the linkage between the 5′-end of the first antisense strand and the 5′-end of the second antisense strand is designed in such a way as to be readily cleavable (e.g., biodegradable linker) such that the 5′end of each antisense strand of the multifunctional siNA has a free 5′-end suitable to mediate RNA interference-based cleavage of the target RNA. The tethers or linkers can be nucleotide-based linkers or non-nucleotide based linkers as generally known in the art and as described herein.
In any of the above embodiments, a first target nucleic acid sequence or second target nucleic acid sequence can independently comprise VEGF, VEGFR, interleukin, and/or interleukin receptor RNA or a portion thereof. In one embodiment, the first target nucleic acid sequence is a VEGF (e.g., any of VEGF-A, VEGF-B, VEGF-C, and/or VEGF-D) RNA or a portion thereof and the second target nucleic acid sequence is a VEGFR (e.g., any of VEGFR1, VEGFR2, and/or VEGFR3) RNA of a portion thereof. In one embodiment, the first target nucleic acid sequence is a VEGFR (e.g., any of VEGFR1, VEGFR2, and/or VEGFR3) RNA or a portion thereof and the second target nucleic acid sequence is a VEGF (e.g., any of VEGF-A, VEGF-B, VEGF-C, and/or VEGF-D) RNA or a portion thereof. In one embodiment, the first target nucleic acid sequence is a VEGF (e.g., any of VEGF-A, VEGF-B, VEGF-C, and/or VEGF-D) RNA or a portion thereof and the second target nucleic acid sequence is a VEGF (e.g., any of VEGF-A, VEGF-B, VEGF-C, and/or VEGF-D) RNA or a portion thereof. In one embodiment, the first target nucleic acid sequence is a VEGFR (e.g., any of VEGFR1, VEGFR2, and/or VEGFR3) RNA or a portion thereof and the second target nucleic acid sequence is a VEGFR (e.g., any of VEGFR1, VEGFR2, and/or VEGFR3) RNA or a portion thereof. In one embodiment, the first target nucleic acid sequence is a VEGF (e.g., any of VEGF-A, VEGF-B, VEGF-C, and/or VEGF-D) RNA or a portion thereof and the second target nucleic acid sequence is a interleukin (e.g., any of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, and IL-27) RNA or a portion thereof. In one embodiment, the first target nucleic acid sequence is a VEGFR (e.g., any of VEGFR1, VEGFR2, and/or VEGFR3) RNA or a portion thereof and the second target nucleic acid sequence is a interleukin receptor (e.g., any of IL-1R, IL-2R, IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, IL-8R, IL-9R, IL-10R, IL-11R, IL-12R, IL-13R, IL-14R, IL-15R, IL-16R, IL-17R, IL-18R, IL-19R, IL-20R, IL-21R, IL-22R, IL-23R, IL-24R, IL-25R, IL-26R, and IL-27R) RNA or a portion thereof. In one embodiment, the first target nucleic acid sequence is a VEGF (e.g., any of VEGF-A, VEGF-B, VEGF-C, and/or VEGF-D) and VEGFR (e.g., any of VEGFR1, VEGFR2, and/or VEGFR3) RNA or a portion thereof having sequence homology and the second target nucleic acid sequence is a interleukin receptor (e.g., any of IL-1R, IL-2R, IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, IL-8R, IL-9R, IL-10R, IL-11R, IL-12R, IL-13R, IL-14R, IL-15R, IL-16R, IL-17R, IL-18R, IL-19R, IL-20R, IL-21R, IL-22R, IL-23R, IL-24R, IL-25R, IL-26R, and IL-27R)RNA or a portion thereof.
Synthesis of Nucleic Acid Molecules
Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. In this invention, small nucleic acid motifs (“small” refers to nucleic acid motifs no more than 100 nucleotides in length, preferably no more than 80 nucleotides in length, and most preferably no more than 50 nucleotides in length; e.g., individual siNA oligonucleotide sequences or siNA sequences synthesized in tandem) are preferably used for exogenous delivery. The simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of protein and/or RNA structure. Exemplary molecules of the instant invention are chemically synthesized, and others can similarly be synthesized.
Oligonucleotides (e.g., certain modified oligonucleotides or portions of oligonucleotides lacking ribonucleotides) are synthesized using protocols known in the art, for example as described in Caruthers et al., 1992, Methods in Enzymology 211, 3-19, Thompson et al., International PCT Publication No. WO 99/54459, Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol. Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, and Brennan, U.S. Pat. No. 6,001,311. All of these references are incorporated herein by reference. The synthesis of oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 2.5 min coupling step for 2′-O-methylated nucleotides and a 45 second coupling step for 2′-deoxy nucleotides or 2′-deoxy-2′-fluoro nucleotides. Table V outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 μmol scale can be performed on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycle of 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 22-fold excess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite and a 70-fold excess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol) can be used in each coupling cycle of deoxy residues relative to polymer-bound 5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include the following: detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solution is 16.9 mM I2, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems, Inc.). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile) is used.
Deprotection of the DNA-based oligonucleotides is performed as follows: the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aqueous methylamine (1 mL) at 65° C. for 10 minutes. After cooling to −20° C., the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder.
The method of synthesis used for RNA including certain siNA molecules of the invention follows the procedure as described in Usman et al., 1987, J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684 Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5 min coupling step for alkylsilyl protected nucleotides and a 2.5 min coupling step for 2′-O-methylated nucleotides. Table V outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycle of 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol) of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess of S-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in each coupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied. Biosystems, Inc. synthesizer include the following: detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM I2, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems, Inc.). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide0.05 M in acetonitrile) is used.
Deprotection of the RNA is performed using either a two-pot or one-pot protocol. For the two-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10 min. After cooling to −20° C., the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder. The base deprotected oligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300 μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mL TEA.3HF to provide a 1.4 M HF concentration) and heated to 65° C. After 1.5 h, the oligomer is quenched with 1.5 M NH4HCO3.
Alternatively, for the one-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL) at 65° C. for 15 minutes. The vial is brought to room temperature TEA.3HF (0.1 mL) is added and the vial is heated at 65° C. for 15 minutes. The sample is cooled at −20° C. and then quenched with 1.5 M NH4HCO3.
For purification of the trityl-on oligomers, the quenched NH4HCO3 solution is loaded onto a C-18 containing cartridge that had been prewashed with acetonitrile followed by 50 mM TEAA. After washing the loaded cartridge with water, the RNA is detritylated with 0.5% TFA for 13 minutes. The cartridge is then washed again with water, salt exchanged with 1 M NaCl and washed with water again. The oligonucleotide is then eluted with 30% acetonitrile.
The average stepwise coupling yields are typically >98% (Wincott et al., 1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in the art will recognize that the scale of synthesis can be adapted to be larger or smaller than the example described above including but not limited to 96-well format.
Alternatively, the nucleic acid molecules of the present invention can be synthesized separately and joined together post-synthetically, for example, by ligation (Moore et al., 1992, Science 256, 9923; Draper et al., International PCT publication No. WO 93/23569; Shabarova et al., 1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204), or by hybridization following synthesis and/or deprotection.
The siNA molecules of the invention can also be synthesized via a tandem synthesis methodology as described in Example 1 herein, wherein both siNA strands are synthesized as a single contiguous oligonucleotide fragment or strand separated by a cleavable linker which is subsequently cleaved to provide separate siNA fragments or strands that hybridize and permit purification of the siNA duplex. The linker can be a polynucleotide linker or a non-nucleotide linker. The tandem synthesis of siNA as described herein can be readily adapted to both multiwell/multiplate synthesis platforms such as 96 well or similarly larger multi-well platforms. The tandem synthesis of siNA as described herein can also be readily adapted to large scale synthesis platforms employing batch reactors, synthesis columns and the like.
A siNA molecule can also be assembled from two distinct nucleic acid strands or fragments wherein one fragment includes the sense region and the second fragment includes the antisense region of the RNA molecule.
The nucleic acid molecules of the present invention can be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H (for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163). siNA constructs can be purified by gel electrophoresis using general methods or can be purified by high pressure liquid chromatography (HPLC; see Wincott et al., supra, the totality of which is hereby incorporated herein by reference) and re-suspended in water.
In another aspect of the invention, siNA molecules of the invention are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. The recombinant vectors capable of expressing the siNA molecules can be delivered as described herein, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of siNA molecules.
Optimizing Activity of the Nucleic Acid Molecule of the Invention.
Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) can prevent their degradation by serum ribonucleases, which can increase their potency (see e.g., Eckstein et al., International Publication No. WO 92/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman and Cedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162; Sproat, U.S. Pat. No. 5,334,711; Gold et al., U.S. Pat. No. 6,300,074; and Burgin et al., supra; all of which are incorporated by reference herein). All of the above references describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of the nucleic acid molecules described herein. Modifications that enhance their efficacy in cells, and removal of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements are desired.
There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy. For example, oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35, 14090). Sugar modification of nucleic acid molecules have been extensively described in the art (see Eckstein et al., International Publication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344, 565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. International Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al., International PCT publication No. WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al., International PCT Publication No. WO 98/13526; Thompson et al., U.S. Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al., 1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010; all of the references are hereby incorporated in their totality by reference herein). Such publications describe general methods and strategies to determine the location of incorporation of sugar, base and/or phosphate modifications and the like into nucleic acid molecules without modulating catalysis, and are incorporated by reference herein. In view of such teachings, similar modifications can be used as described herein to modify the siNA nucleic acid molecules of the instant invention so long as the ability of siNA to promote RNAi is cells is not significantly inhibited.
While chemical modification of oligonucleotide internucleotide linkages with phosphorothioate, phosphorodithioate, and/or 5′-methylphosphonate linkages improves stability, excessive modifications can cause some toxicity or decreased activity. Therefore, when designing nucleic acid molecules, the amount of these internucleotide linkages should be minimized. The reduction in the concentration of these linkages should lower toxicity, resulting in increased efficacy and higher specificity of these molecules.
Short interfering nucleic acid (siNA) molecules having chemical modifications that maintain or enhance activity are provided. Such a nucleic acid is also generally more resistant to nucleases than an unmodified nucleic acid. Accordingly, the in vitro and/or in vivo activity should not be significantly lowered. In cases in which modulation is the goal, therapeutic nucleic acid molecules delivered exogenously should optimally be stable within cells until translation of the target RNA has been modulated long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state. Improvements in the chemical synthesis of RNA and DNA (Wincott et al., 1995, Nucleic Acids Res. 23, 2677; Caruthers et al., 1992, Methods in Enzymology 211, 3-19 (incorporated by reference herein)) have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability, as described above.
In one embodiment, nucleic acid molecules of the invention include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clamp nucleotides. A G-clamp nucleotide is a modified cytosine analog wherein the modifications confer the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine within a duplex, see for example Lin and Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. A single G-clamp analog substitution within an oligonucleotide can result in substantially enhanced helical thermal stability and mismatch discrimination when hybridized to complementary oligonucleotides. The inclusion of such nucleotides in nucleic acid molecules of the invention results in both enhanced affinity and specificity to nucleic acid targets, complementary sequences, or template strands. In another embodiment, nucleic acid molecules of the invention include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA “locked nucleic acid” nucleotides such as a 2′,4′-C methylene bicyclo nucleotide (see for example Wengel et al., International PCT Publication No. WO 00/66604 and WO 99/14226).
In another embodiment, the invention features conjugates and/or complexes of siNA molecules of the invention. Such conjugates and/or complexes can be used to facilitate delivery of siNA molecules into a biological system, such as a cell. The conjugates and complexes provided by the instant invention can impart therapeutic activity by transferring therapeutic compounds across cellular membranes, altering the pharmacokinetics, and/or modulating the localization of nucleic acid molecules of the invention. The present invention encompasses the design and synthesis of novel conjugates and complexes for the delivery of molecules, including, but not limited to, small molecules, lipids, cholesterol, phospholipids, nucleosides, nucleotides, nucleic acids, antibodies, toxins, negatively charged polymers and other polymers, for example proteins, peptides, hormones, carbohydrates, polyethylene glycols, or polyamines, across cellular membranes. In general, the transporters described are designed to be used either individually or as part of a multi-component system, with or without degradable linkers. These compounds are expected to improve delivery and/or localization of nucleic acid molecules of the invention into a number of cell types originating from different tissues, in the presence or absence of serum (see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of the molecules described herein can be attached to biologically active molecules via linkers that are biodegradable, such as biodegradable nucleic acid linker molecules.
The term “biodegradable linker” as used herein, refers to a nucleic acid or non-nucleic acid linker molecule that is designed as a biodegradable linker to connect one molecule to another molecule, for example, a biologically active molecule to a siNA molecule of the invention or the sense and antisense strands of a siNA molecule of the invention. The biodegradable linker is designed such that its stability can be modulated for a particular purpose, such as delivery to a particular tissue or cell type. The stability of a nucleic acid-based biodegradable linker molecule can be modulated by using various chemistries, for example combinations of ribonucleotides, deoxyribonucleotides, and chemically-modified nucleotides, such as 2′-O-methyl, 2′-fluoro, 2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified or base modified nucleotides. The biodegradable nucleic acid linker molecule can be a dimer, trimer, tetramer or longer nucleic acid molecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprise a single nucleotide with a phosphorus-based linkage, for example, a phosphoramidate or phosphodiester linkage. The biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base modifications.
The term “biodegradable” as used herein, refers to degradation in a biological system, for example, enzymatic degradation or chemical degradation.
The term “biologically active molecule” as used herein refers to compounds or molecules that are capable of eliciting or modifying a biological response in a system. Non-limiting examples of biologically active siNA molecules either alone or in combination with other molecules contemplated by the instant invention include therapeutically active molecules such as antibodies, cholesterol, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers, decoys and analogs thereof. Biologically active molecules of the invention also include molecules capable of modulating the pharmacokinetics and/or pharmacodynamics of other biologically active molecules, for example, lipids and polymers such as polyamines, polyamides, polyethylene glycol and other polyethers.
The term “phospholipid” as used herein, refers to a hydrophobic molecule comprising at least one phosphorus group. For example, a phospholipid can comprise a phosphorus-containing group and saturated or unsaturated alkyl group, optionally substituted with OH, COOH, oxo, amine, or substituted or unsubstituted aryl groups.
Therapeutic nucleic acid molecules (e.g., siNA molecules) delivered exogenously optimally are stable within cells until reverse transcription of the RNA has been modulated long enough to reduce the levels of the RNA transcript. The nucleic acid molecules are resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of nucleic acid molecules described in the instant invention and in the art have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above.
In yet another embodiment, siNA molecules having chemical modifications that maintain or enhance enzymatic activity of proteins involved in RNAi are provided. Such nucleic acids are also generally more resistant to nucleases than unmodified nucleic acids. Thus, in vitro and/or in vivo the activity should not be significantly lowered.
Use of the nucleic acid-based molecules of the invention will lead to better treatments by affording the possibility of combination therapies (e.g., multiple siNA molecules targeted to different genes; nucleic acid molecules coupled with known small molecule modulators; or intermittent treatment with combinations of molecules, including different motifs and/or other chemical or biological molecules). The treatment of subjects with siNA molecules can also include combinations of different types of nucleic acid molecules, such as enzymatic nucleic acid molecules (ribozymes), allozymes, antisense, 2,5-A oligoadenylate, decoys, and aptamers.
In another aspect a siNA molecule of the invention comprises one or more 5′ and/or a 3′-cap structure, for example, on only the sense siNA strand, the antisense siNA strand, or both siNA strands.
By “cap structure” is meant chemical modifications, which have been incorporated at either terminus of the oligonucleotide (see, for example, Adamic et al., U.S. Pat. No. 5,998,203, incorporated by reference herein). These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and may help in delivery and/or localization within a cell. The cap may be present at the 5′-terminus (5′-cap) or at the 3′-terminal (3′-cap) or may be present on both termini. In non-limiting examples, the 5′-cap includes, but is not limited to, glyceryl, inverted deoxy abasic residue (moiety); 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety; 1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety. Non-limiting examples of cap moieties are shown in FIG. 10.
Non-limiting examples of the 3′-cap include, but are not limited to, glyceryl, inverted deoxy abasic residue (moiety), 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasic moiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediol phosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5′-mercapto moieties (for more details see Beaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by reference herein).
By the term “non-nucleotide” is meant any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine and therefore lacks a base at the 1′-position.
An “alkyl” group refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group can be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO2 or N(CH3)2, amino, or SH. The term also includes alkenyl groups that are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12 carbons. More preferably, it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkenyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO2, halogen, N(CH3)2, amino, or SH. The term “alkyl” also includes alkynyl groups that have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has 1 to 12 carbons. More preferably, it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO2 or N(CH3)2, amino or SH.
Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An “aryl” group refers to an aromatic group that has at least one ring having a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted. The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An “alkylaryl” group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above). Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An “amide” refers to an —C(O)—NH—R, where R is either alkyl, aryl, alkylaryl or hydrogen. An “ester” refers to an —C(O)—OR′, where R is either alkyl, aryl, alkylaryl or hydrogen.
By “nucleotide” as used herein is as recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1′ position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see, for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No. WO 92/07065; Usman et al., International PCT Publication No. WO 93/15187; Uhlman & Peyman, supra, all are hereby incorporated by reference herein). There are several examples of modified nucleic acid bases known in the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some of the non-limiting examples of base modifications that can be introduced into nucleic acid molecules include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra). By “modified bases” in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1′ position or their equivalents.
In one embodiment, the invention features modified siNA molecules, with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions. For a review of oligonucleotide backbone modifications, see Hunziker and Leumann, 1995, Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417, and Mesmaeker et al., 1994, Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39.
By “abasic” is meant sugar moieties lacking a nucleobase or having a hydrogen atom (H) or other non-nucleobase chemical groups in place of a nucleobase at the 1′ position of the sugar moiety, see for example Adamic et al., U.S. Pat. No. 5,998,203. In one embodiment, an abasic moiety of the invention is a ribose, deoxyribose, or dideoxyribose sugar.
By “unmodified nucleoside” is meant one of the bases adenine, cytosine, guanine, thymine, or uracil joined to the 1′ carbon of β-D-ribo-furanose.
By “modified nucleoside” is meant any nucleotide base which contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate. Non-limiting examples of modified nucleotides are shown by Formulae I-VII and/or other modifications described herein.
In connection with 2′-modified nucleotides as described for the present invention, by “amino” is meant 2′-NH2 or 2′-O—NH2, which can be modified or unmodified. Such modified groups are described, for example, in Eckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S. Pat. No. 6,248,878, which are both incorporated by reference in their entireties.
Various modifications to nucleic acid siNA structure can be made to enhance the utility of these molecules. Such modifications will enhance shelf-life, half-life in vitro, stability, and ease of introduction of such oligonucleotides to the target site, e.g., to enhance penetration of cellular membranes, and confer the ability to recognize and bind to targeted cells.
Administration of Nucleic Acid Molecules
A siNA molecule of the invention can be adapted for use to treat, prevent, inhibit, or reduce cancer, ocular, proliferative, respiratory, autoimmune, neurologic, allergic, or angiogenesis/neovascularization related diseases, conditions, or disorders, and/or any other trait, disease or condition that is related to or will respond to the levels of VEGF, VEGFR, interleukin, and/or interleukin receptor in a cell or tissue, alone or in combination with other therapies.
In one embodiment, a siNA composition of the invention can comprise a delivery vehicle, including liposomes, for administration to a subject, carriers and diluents and their salts, and/or can be present in pharmaceutically acceptable formulations. Methods for the delivery of nucleic acid molecules are described in Akhtar et al., 1992, Trends Cell Bio., 2, 139; Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al., 1999, Mol. Membr. Biol., 16, 129-140; Hofland and Huang, 1999, Handb. Exp. Pharmacol., 137, 165-192; and Lee et al., 2000, ACS Symp. Ser., 752, 184-192, all of which are incorporated herein by reference. Beigelman et al., U.S. Pat. No. 6,395,713 and Sullivan et al., PCT WO 94/02595 further describe the general methods for delivery of nucleic acid molecules. These protocols can be utilized for the delivery of virtually any nucleic acid molecule. Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins (see for example Gonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074; Wang et al., International PCT publication Nos. WO 03/47518 and WO 03/46185), poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see for example U.S. Pat. No. 6,447,796 and US Patent Application Publication No. US 2002130430), biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (O'Hare and Normand, International PCT Publication No. WO 00/53722). In another embodiment, the nucleic acid molecules of the invention can also be formulated or complexed with polyethyleneimine and derivatives thereof, such as polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives. In one embodiment, the nucleic acid molecules of the invention are formulated as described in United States Patent Application Publication No. 20030077829, incorporated by reference herein in its entirety.
In one embodiment, a siNA molecule of the invention is formulated as a composition described in U.S. Provisional patent application No. 60/678,531 and in related U.S. Provisional patent application No. 60/703,946, filed Jul. 29, 2005, and U.S. Provisional patent application No. 60/737,024, filed Nov. 15, 2005 (Vargeese et al.), all of which are incorporated by reference herein in their entirety. Such siNA formulations are generally referred to as “lipid nucleic acid particles” (LNP).
In one embodiment, a siNA molecule of the invention is complexed with membrane disruptive agents such as those described in U.S. Patent Application Publication No. 20010007666, incorporated by reference herein in its entirety including the drawings. In another embodiment, the membrane disruptive agent or agents and the siNA molecule are also complexed with a cationic lipid or helper lipid molecule, such as those lipids described in U.S. Pat. No. 6,235,310, incorporated by reference herein in its entirety including the drawings.
In one embodiment, a siNA molecule of the invention is complexed with delivery systems as described in U.S. Patent Application Publication No. 2003077829 and International PCT Publication Nos. WO 00/03683 and WO 02/087541, all incorporated by reference herein in their entirety including the drawings.
In one embodiment, a compound, molecule, or composition for the treatment of ocular conditions (e.g., macular degeneration, diabetic retinopathy etc.) is administered to a subject intraocularly or by intraocular means. In another embodiment, a compound, molecule, or composition for the treatment of ocular conditions (e.g., macular degeneration, diabetic retinopathy etc.) is administered to a subject periocularly or by periocular means (see for example Ahlheim et al., International PCT publication No. WO 03/24420). In one embodiment, a siNA molecule and/or formulation or composition thereof is administered to a subject intraocularly or by intraocular means. In another embodiment, a siNA molecule and/or formulation or composition thereof is administered to a subject periocularly or by periocular means. Periocular administration generally provides a less invasive approach to administering siNA molecules and formulation or composition thereof to a subject (see for example Ahlheim et al., International PCT publication No. WO 03/24420). The use of periocular administration also minimizes the risk of retinal detachment, allows for more frequent dosing or administration, provides a clinically relevant route of administration for macular degeneration and other optic conditions, and also provides the possibility of using reservoirs (e.g., implants, pumps or other devices) for drug delivery. In one embodiment, siNA compounds and compositions of the invention are administered locally, e.g., via intraocular or periocular means, such as injection, iontophoresis (see, for example, WO 03/043689 and WO 03/030989), or implant, about every 1-50 weeks (e.g., about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 weeks), alone or in combination with other compounds and/or therapies herein. In one embodiment, siNA compounds and compositions of the invention are administered systemically (e.g., via intravenous, subcutaneous, intramuscular, infusion, pump, implant etc.) about every 1-50 weeks (e.g., about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 weeks), alone or in combination with other compounds and/or therapies described herein and/or otherwise known in the art.
In one embodiment, the nucleic acid molecules of the invention are administered to skeletal tissues (e.g., bone, cartilage, tendon, ligament) or bone metastatic tumors via atelocollagen complexation or conjugation (see for example Takeshita et al., 2005, PNAS, 102, 12177-12182). Therefore, in one embodiment, the instant invention features one or more dsiNA molecules as a composition complexed with atelocollagen. In another embodiment, the instant invention features one or more siNA molecules conjugated to atelocollagen via a linker as described herein or otherwise known in the art.
In one embodiment, the nucleic acid molecules of the invention are administered via pulmonary delivery, such as by inhalation of an aerosol or spray dried formulation administered by an inhalation device or nebulizer, providing rapid local uptake of the nucleic acid molecules into relevant pulmonary tissues. Solid particulate compositions containing respirable dry particles of micronized nucleic acid compositions can be prepared by grinding dried or lyophilized nucleic acid compositions, and then passing the micronized composition through, for example, a 400 mesh screen to break up or separate out large agglomerates. A solid particulate composition comprising the nucleic acid compositions of the invention can optionally contain a dispersant which serves to facilitate the formation of an aerosol as well as other therapeutic compounds. A suitable dispersant is lactose, which can be blended with the nucleic acid compound in any suitable ratio, such as a 1 to 1 ratio by weight.
Aerosols of liquid particles comprising a nucleic acid composition of the invention can be produced by any suitable means, such as with a nebulizer (see for example U.S. Pat. No. 4,501,729). Nebulizers are commercially available devices which transform solutions or suspensions of an active ingredient into a therapeutic aerosol mist either by means of acceleration of a compressed gas, typically air or oxygen, through a narrow venturi orifice or by means of ultrasonic agitation. Suitable formulations for use in nebulizers comprise the active ingredient in a liquid carrier in an amount of up to 40% w/w preferably less than 20% w/w of the formulation. The carrier is typically water or a dilute aqueous alcoholic solution, preferably made isotonic with body fluids by the addition of, for example, sodium chloride or other suitable salts. Optional additives include preservatives if the formulation is not prepared sterile, for example, methyl hydroxybenzoate, anti-oxidants, flavorings, volatile oils, buffering agents and emulsifiers and other formulation surfactants. The aerosols of solid particles comprising the active composition and surfactant can likewise be produced with any solid particulate aerosol generator. Aerosol generators for administering solid particulate therapeutics to a subject produce particles which are respirable, as explained above, and generate a volume of aerosol containing a predetermined metered dose of a therapeutic composition at a rate suitable for human administration.
In one embodiment, a solid particulate aerosol generator of the invention is an insufflator. Suitable formulations for administration by insufflation include finely comminuted powders which can be delivered by means of an insufflator. In the insufflator, the powder, e.g., a metered dose thereof effective to carry out the treatments described herein, is contained in capsules or cartridges, typically made of gelatin or plastic, which are either pierced or opened in situ and the powder delivered by air drawn through the device upon inhalation or by means of a manually-operated pump. The powder employed in the insufflator consists either solely of the active ingredient or of a powder blend comprising the active ingredient, a suitable powder diluent, such as lactose, and an optional surfactant. The active ingredient typically comprises from 0.1 to 100 w/w of the formulation. A second type of illustrative aerosol generator comprises a metered dose inhaler. Metered dose inhalers are pressurized aerosol dispensers, typically containing a suspension or solution formulation of the active ingredient in a liquefied propellant. During use these devices discharge the formulation through a valve adapted to deliver a metered volume to produce a fine particle spray containing the active ingredient. Suitable propellants include certain chlorofluorocarbon compounds, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof. The formulation can additionally contain one or more co-solvents, for example, ethanol, emulsifiers and other formulation surfactants, such as oleic acid or sorbitan trioleate, anti-oxidants and suitable flavoring agents. Other methods for pulmonary delivery are described in, for example US Patent Application No. 20040037780, and U.S. Pat. Nos. 6,592,904; 6,582,728; 6,565,885, all incorporated by reference herein.
In one embodiment, the invention features the use of methods to deliver the nucleic acid molecules of the instant invention to the central nervous system and/or peripheral nervous system. Experiments have demonstrated the efficient in vivo uptake of nucleic acids by neurons. As an example of local administration of nucleic acids to nerve cells, Sommer et al., 1998, Antisense Nuc. Acid Drug Dev., 8, 75, describe a study in which a 15 mer phosphorothioate antisense nucleic acid molecule to c-fos is administered to rats via microinjection into the brain. Antisense molecules labeled with tetramethylrhodamine-isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC) were taken up by exclusively by neurons thirty minutes post-injection. A diffuse cytoplasmic staining and nuclear staining was observed in these cells. As an example of systemic administration of nucleic acid to nerve cells, Epa et al., 2000, Antisense Nuc. Acid Drug Dev., 10, 469, describe an in vivo mouse study in which beta-cyclodextrin-adamantane-oligonucleotide conjugates were used to target the p75 neurotrophin receptor in neuronally differentiated PC12 cells. Following a two week course of IP administration, pronounced uptake of p75 neurotrophin receptor antisense was observed in dorsal root ganglion (DRG) cells. In addition, a marked and consistent down-regulation of p75 was observed in DRG neurons. Additional approaches to the targeting of nucleic acid to neurons are described in Broaddus et al., 1998, J. Neurosurg., 88(4), 734; Karle et al., 1997, Eur. J. Pharmocol., 340(2/3), 153; Bannai et al., 1998, Brain Research, 784(1,2), 304; Rajakumar et al., 1997, Synapse, 26(3), 199; Wu-pong et al., 1999, BioPharm, 12(1), 32; Bannai et al., 1998, Brain Res. Protoc., 3(1), 83; Simantov et al., 1996, Neuroscience, 74(1), 39. Nucleic acid molecules of the invention are therefore amenable to delivery to and uptake by cells that express repeat expansion allelic variants for modulation of RE gene expression. The delivery of nucleic acid molecules of the invention, targeting RE is provided by a variety of different strategies. Traditional approaches to CNS delivery that can be used include, but are not limited to, intrathecal and intracerebroventricular administration, implantation of catheters and pumps, direct injection or perfusion at the site of injury or lesion, injection into the brain arterial system, or by chemical or osmotic opening of the blood-brain barrier. Other approaches can include the use of various transport and carrier systems, for example though the use of conjugates and biodegradable polymers. Furthermore, gene therapy approaches, for example as described in Kaplitt et al., U.S. Pat. No. 6,180,613 and Davidson, WO 04/013280, can be used to express nucleic acid molecules in the CNS.
The delivery of nucleic acid molecules of the invention to the CNS is provided by a variety of different strategies. Traditional approaches to CNS delivery that can be used include, but are not limited to, intrathecal and intracerebroventricular administration, implantation of catheters and pumps, direct injection or perfusion at the site of injury or lesion, injection into the brain arterial system, or by chemical or osmotic opening of the blood-brain barrier. Other approaches can include the use of various transport and carrier systems, for example though the use of conjugates and biodegradable polymers. Furthermore, gene therapy approaches, for example as described in Kaplitt et al., U.S. Pat. No. 6,180,613 and Davidson, WO 04/013280, can be used to express nucleic acid molecules in the CNS.
In one embodiment, a siNA molecule of the invention is administered iontophoretically, for example to a particular organ or compartment (e.g., the eye, back of the eye, heart, liver, kidney, bladder, prostate, tumor, CNS etc.). Non-limiting examples of iontophoretic delivery are described in, for example, WO 03/043689 and WO 03/030989, which are incorporated by reference in their entireties herein.
In one embodiment, the siNA molecules of the invention and formulations or compositions thereof are administered to the liver as is generally known in the art (see for example Wen et al., 2004, World J Gastroenterol., 10, 244-9; Murao et al., 2002, Pharm Res., 19, 1808-14; Liu et al., 2003, Gene Ther., 10, 180-7; Hong et al., 2003, J Pharm Pharmacol., 54, 51-8; Herrmann et al., 2004, Arch Virol., 149, 1611-7; and Matsuno et al., 2003, Gene Ther., 10, 1559-66).
In one embodiment, the invention features the use of methods to deliver the nucleic acid molecules of the instant invention to hematopoietic cells, including monocytes and lymphocytes. These methods are described in detail by Hartmann et al., 1998, J. Phamacol. Exp. Ther., 285(2), 920-928; Kronenwett et al., 1998, Blood, 91(3), 852-862; Filion and Phillips, 1997, Biochim. Biophys. Acta., 1329(2), 345-356; Ma and Wei, 1996, Leuk Res., 20(11/12), 925-930; and Bongartz et al., 1994, Nucleic Acids Research, 22(22), 4681-8. Such methods, as described above, include the use of free oligonucleitide, cationic lipid formulations, liposome formulations including pH sensitive liposomes and immunoliposomes, and bioconjugates including oligonucleotides conjugated to fusogenic peptides, for the transfection of hematopoietic cells with oligonucleotides.
In one embodiment, the siNA molecules and compositions of the invention are administered to the inner ear by contacting the siNA with inner ear cells, tissues, or structures such as the cochlea, under conditions suitable for the administration. In one embodiment, the administration comprises methods and devices as described in U.S. Pat. Nos. 5,421,818, 5,476,446, 5,474,529, 6,045,528, 6,440,102, 6,685,697, 6,120,484; and 5,572,594; all incorporated by reference herein and the teachings of Silverstein, 1999, Ear Nose Throat J., 78, 595-8, 600; and Jackson and Silverstein, 2002, Otolaryngol Clin North Am., 35, 639-53, and adapted for use the siNA molecules of the invention.
In one embodiment, the siNA molecules of the invention and formulations or compositions thereof are administered directly or topically (e.g., locally) to the dermis or follicles as is generally known in the art (see for example Brand, 2001, Curr. Opin. Mol. Ther., 3, 244-8; Regnier et al., 1998, J. Drug Target, 5, 275-89; Kanikkannan, 2002, BioDrugs, 16, 339-47; Wraight et al., 2001, Pharmacol. Ther., 90, 89-104; and Preat and Dujardin, 2001, STP PharmaSciences, 11, 57-68). In one embodiment, the siNA molecules of the invention and formulations or compositions thereof are administered directly or topically using a hydroalcoholic gel formulation comprising an alcohol (e.g., ethanol or isopropanol), water, and optionally including additional agents such isopropyl myristate and carbomer 980.
In one embodiment, delivery systems of the invention include, for example, aqueous and nonaqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone). In one embodiment, the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer. Examples of liposomes which can be used in this invention include the following: (1) CellFectin, 1:1.5 (M/M) liposome formulation of the cationic lipid N,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmit-y-spermine and dioleoyl phosphatidylethanolamine (DOPE) (GIBCO BRL); (2) Cytofectin GSV, 2:1 (M/M) liposome formulation of a cationic lipid and DOPE (Glen Research); (3) DOTAP (N-[1-(2,3-dioleoyloxy)-N,N,N-tri-methyl-ammoniummethylsulfate) (Boehringer Manheim); and (4) Lipofectamine, 3:1 (M/M) liposome formulation of the polycationic lipid DOSPA and the neutral lipid DOPE (GIBCO BRL).
In one embodiment, delivery systems of the invention include patches, tablets, suppositories, pessaries, gels and creams, and can contain excipients such as solubilizers and enhancers (e.g., propylene glycol, bile salts and amino acids), and other vehicles (e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid).
In one embodiment, a siNA molecule of the invention is administered iontophoretically, for example to the dermis or to other relevant tissues such as the inner ear/cochlea. Non-limiting examples of iontophoretic delivery are described in, for example, WO 03/043689 and WO 03/030989, which are incorporated by reference in their entireties herein.
In one embodiment, siNA molecules of the invention are formulated or complexed with polyethylenimine (e.g., linear or branched PEI) and/or polyethylenimine derivatives, including for example grafted PEIs such as galactose PEI, cholesterol PEI, antibody derivatized PEI, and polyethylene glycol PEI (PEG-PEI) derivatives thereof (see for example Ogris et al., 2001, AAPA PharmSci, 3, 1-11; Furgeson et al., 2003, Bioconjugate Chem., 14, 840-847; Kunath et al., 2002, Phramaceutical Research, 19, 810-817; Choi et al., 2001, Bull. Korean Chem. Soc., 22, 46-52; Bettinger et al., 1999, Bioconjugate Chem., 10, 558-561; Peterson et al., 2002, Bioconjugate Chem., 13, 845-854; Erbacher et al., 1999, Journal of Gene Medicine Preprint, 1, 1-18; Godbey et al., 1999., PNAS USA, 96, 5177-5181; Godbey et al., 1999, Journal of Controlled Release, 60, 149-160; Diebold et al., 1999, Journal of Biological Chemistry, 274, 19087-19094; Thomas and Klibanov, 2002, PNAS USA, 99, 14640-14645; and Sagara, U.S. Pat. No. 6,586,524, incorporated by reference herein.
In one embodiment, a siNA molecule of the invention comprises a bioconjugate, for example a nucleic acid conjugate as described in Vargeese et al., U.S. Ser. No. 10/427,160, filed Apr. 30, 2003; U.S. Pat. Nos. 6,528,631; 6,335,434; 6,235,886; 6,153,737; 5,214,136; 5,138,045, all incorporated by reference herein.
Thus, the invention features a pharmaceutical composition comprising one or more nucleic acid(s) of the invention in an acceptable carrier, such as a stabilizer, buffer, and the like. The polynucleotides of the invention can be administered (e.g., RNA, DNA or protein) and introduced to a subject by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition. When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed. The compositions of the present invention can also be formulated and used as creams, gels, sprays, oils and other suitable compositions for topical, dermal, or transdermal administration as is known in the art.
The present invention also includes pharmaceutically acceptable formulations of the compounds described. These formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.
A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic or local administration, into a cell or subject, including for example a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged nucleic acid is desirable for delivery). For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms that prevent the composition or formulation from exerting its effect.
In one embodiment, siNA molecules of the invention are administered to a subject by systemic administration in a pharmaceutically acceptable composition or formulation. By “systemic administration” is meant in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body. Administration routes that lead to systemic absorption include, without limitation: intravenous, subcutaneous, portal vein, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these administration routes exposes the siNA molecules of the invention to an accessible diseased tissue. The rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other drug carrier comprising the compounds of the instant invention can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES). A liposome formulation that can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach can provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells.
By “pharmaceutically acceptable formulation” or “pharmaceutically acceptable composition” is meant, a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity. Non-limiting examples of agents suitable for formulation with the nucleic acid molecules of the instant invention include: P-glycoprotein inhibitors (such as Pluronic P85); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery (Emerich, D F et al, 1999, Cell Transplant, 8, 47-58); and loaded nanoparticles, such as those made of polybutylcyanoacrylate. Other non-limiting examples of delivery strategies for the nucleic acid molecules of the instant invention include material described in Boado et al., 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge et al., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058.
The invention also features the use of a composition comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes) and nucleic acid molecules of the invention. These formulations offer a method for increasing the accumulation of drugs (e.g., siNA) in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011). Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995, Biochim. Biophys. Acta, 1238, 86-90). The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995, 42, 24864-24870; Choi et al., International PCT Publication No. WO 96/10391; Ansell et al., International PCT Publication No. WO 96/10390; Holland et al., International PCT Publication No. WO 96/10392). Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.
The present invention also includes compositions prepared for storage or administration that include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985), hereby incorporated by reference herein. For example, preservatives, stabilizers, dyes and flavoring agents can be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents can be used.
A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer.
The nucleic acid molecules of the invention and formulations thereof can be administered orally, topically, parenterally, by inhalation or spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and/or vehicles. The term parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like. In addition, there is provided a pharmaceutical formulation comprising a nucleic acid molecule of the invention and a pharmaceutically acceptable carrier. One or more nucleic acid molecules of the invention can be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients. The pharmaceutical compositions containing nucleic acid molecules of the invention can be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.
Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients can be, for example, inert diluents; such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets can be uncoated or they can be coated by known techniques. In some cases such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate can be employed.
Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.
Aqueous suspensions contain the active materials in a mixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents can be added to provide palatable oral preparations. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents or suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, can also be present.
Pharmaceutical compositions of the invention can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions can also contain sweetening and flavoring agents.
Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations can also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
The nucleic acid molecules of the invention can also be administered in the form of suppositories, e.g., for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols.
Nucleic acid molecules of the invention can be administered parenterally in a sterile medium. The drug, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle.
Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per subject per day). The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration. Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient.
It is understood that the specific dose level for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
For administration to non-human animals, the composition can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water.
The nucleic acid molecules of the present invention can also be administered to a subject in combination with other therapeutic compounds to increase the overall therapeutic effect. The use of multiple compounds to treat an indication can increase the beneficial effects while reducing the presence of side effects.
In one embodiment, the invention comprises compositions suitable for administering nucleic acid molecules of the invention to specific cell types. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu, 1987, J. Biol. Chem. 262, 4429-4432) is unique to hepatocytes and binds branched galactose-terminal glycoproteins, such as asialoorosomucoid (ASOR). In another example, the folate receptor is overexpressed in many cancer cells. Binding of such glycoproteins, synthetic glycoconjugates, or folates to the receptor takes place with an affinity that strongly depends on the degree of branching of the oligosaccharide chain, for example, triatennary structures are bound with greater affinity than biatenarry or monoatennary chains (Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al., 1982, J. Biol. Chem., 257, 939-945). Lee and Lee, 1987, Glycoconjugate J., 4, 317-328, obtained this high specificity through the use of N-acetyl-D-galactosamine as the carbohydrate moiety, which has higher affinity for the receptor, compared to galactose. This “clustering effect” has also been described for the binding and uptake of mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom et al., 1981, J. Med. Chem., 24, 1388-1395). The use of galactose, galactosamine, or folate based conjugates to transport exogenous compounds across cell membranes can provide a targeted delivery approach to, for example, the treatment of liver disease, cancers of the liver, or other cancers. The use of bioconjugates can also provide a reduction in the required dose of therapeutic compounds required for treatment. Furthermore, therapeutic bioavailability, pharmacodynamics, and pharmacokinetic parameters can be modulated through the use of nucleic acid bioconjugates of the invention. Non-limiting examples of such bioconjugates are described in Vargeese et al., U.S. Ser. No. 10/201,394, filed Aug. 13, 2001; and Matulic-Adamic et al., U.S. Ser. No. 60/362,016, filed Mar. 6, 2002.
Alternatively, certain siNA molecules of the instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci., USA 83, 399; Scanlon et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Dropulic et al., 1992, J. Virol., 66, 1432-41; Weerasinghe et al., 1991, J. Virol., 65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Sarver et al., 1990 Science, 247, 1222-1225; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Good et al., 1997, Gene Therapy, 4, 45. Those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector. The activity of such nucleic acids can be augmented by their release from the primary transcript by a enzymatic nucleic acid (Draper et al., PCT WO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992, Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al., 1993, Nucleic Acids Res., 21, 3249-55; Chowrira et al., 1994, J. Biol. Chem., 269, 25856.
In another aspect of the invention, RNA molecules of the present invention can be expressed from transcription units (see for example Couture et al., 1996, TIG., 12, 510) inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. In another embodiment, pol III based constructs are used to express nucleic acid molecules of the invention (see for example Thompson, U.S. Pats. Nos. 5,902,880 and 6,146,886). The recombinant vectors capable of expressing the siNA molecules can be delivered as described above, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of nucleic acid molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the siNA molecule interacts with the target mRNA and generates an RNAi response. Delivery of siNA molecule expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell (for a review see Couture et al., 1996, TIG., 12, 510).
In one aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the instant invention. The expression vector can encode one or both strands of a siNA duplex, or a single self-complementary strand that self hybridizes into a siNA duplex. The nucleic acid sequences encoding the siNA molecules of the instant invention can be operably linked in a manner that allows expression of the siNA molecule (see for example Paul et al., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500; and Novina et al., 2002, Nature Medicine, advance online publication doi:10.1038/nm725).
In another aspect, the invention features an expression vector comprising: a) a transcription initiation region (e.g., eukaryotic pol I, II or III initiation region); b) a transcription termination region (e.g., eukaryotic pol I, II or III termination region); and c) a nucleic acid sequence encoding at least one of the siNA molecules of the instant invention, wherein said sequence is operably linked to said initiation region and said termination region in a manner that allows expression and/or delivery of the siNA molecule. The vector can optionally include an open reading frame (ORF) for a protein operably linked on the 5′ side or the 3′-side of the sequence encoding the siNA of the invention; and/or an intron (intervening sequences).
Transcription of the siNA molecule sequences can be driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters are expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type depends on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993, Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol. Cell. Biol., 10, 4529-37). Several investigators have demonstrated that nucleic acid molecules expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 6340-4; L'Huillier et al., 1992, EMBO J., 11, 4411-8; Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 8000-4; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech, 1993, Science, 262, 1566). More specifically, transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful in generating high concentrations of desired RNA molecules such as siNA in cells (Thompson et al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al., 1994, Nucleic Acid Res., 22, 2830; Noonberg et al., U.S. Pat. No. 5,624,803; Good et al., 1997, Gene Ther., 4, 45; Beigelman et al., International PCT Publication No. WO 96/18736. The above siNA transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors) (for a review see Couture and Stinchcomb, 1996, supra).
In another aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the siNA molecules of the invention in a manner that allows expression of that siNA molecule. The expression vector comprises in one embodiment; a) a transcription initiation region; b) a transcription termination region; and c) a nucleic acid sequence encoding at least one strand of the siNA molecule, wherein the sequence is operably linked to the initiation region and the termination region in a manner that allows expression and/or delivery of the siNA molecule.
In another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; and d) a nucleic acid sequence encoding at least one strand of a siNA molecule, wherein the sequence is operably linked to the 3′-end of the open reading frame and wherein the sequence is operably linked to the initiation region, the open reading frame and the termination region in a manner that allows expression and/or delivery of the siNA molecule. In yet another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; and d) a nucleic acid sequence encoding at least one siNA molecule, wherein the sequence is operably linked to the initiation region, the intron and the termination region in a manner which allows expression and/or delivery of the nucleic acid molecule.
In another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; and e) a nucleic acid sequence encoding at least one strand of a siNA molecule, wherein the sequence is operably linked to the 3′-end of the open reading frame and wherein the sequence is operably linked to the initiation region, the intron, the open reading frame and the termination region in a manner which allows expression and/or delivery of the siNA molecule.
VEGF and/or VEGFR Biology and Biochemistry
The following discussion is adapted from R&D Systems, Cytokine Mini Reviews, Vascular Endothelial Growth Factor (VEGF), Copyright ©2002 R&D Systems. Angiogenesis is a process of new blood vessel development from pre-existing vasculature. It plays an essential role in embryonic development, normal growth of tissues, wound healing, the female reproductive cycle (i.e., ovulation, menstruation and placental development), as well as a major role in many diseases. Particular interest has focused on cancer, since tumors cannot grow beyond a few millimeters in size without developing a new blood supply. Angiogenesis is also necessary for the spread and growth of tumor cell metastases.
One of the most important growth and survival factors for endothelium is vascular endothelial growth factor (VEGF). VEGF induces angiogenesis and endothelial cell proliferation and plays an important role in regulating vasculogenesis. VEGF is a heparin-binding glycoprotein that is secreted as a homodimer of 45 kDa. Most types of cells, but usually not endothelial cells themselves, secrete VEGF. Since the initially discovered VEGF, VEGF-A, increases vascular permeability, it was known as vascular permeability factor. In addition, VEGF causes vasodilatation, partly through stimulation of nitric oxide synthase in endothelial cells. VEGF can also stimulate cell migration and inhibit apoptosis.
There are several splice variants of VEGF-A. The major ones include: 121, 165, 189 and 206 amino acids (aa), each one comprising a specific exon addition. VEGF165 is the most predominant protein, but transcripts of VEGF 121 may be more abundant. VEGF206 is rarely expressed and has been detected only in fetal liver. Recently, other splice variants of 145 and 183 aa have also been described. The 165, 189 and 206 aa splice variants have heparin-binding domains, which help anchor them in extracellular matrix and are involved in binding to heparin sulfate and presentation to VEGF receptors. Such presentation is a key factor for VEGF potency (i.e., the heparin-binding forms are more active). Several other members of the VEGF family have been cloned including VEGF-B, -C, and -D. Placenta growth factor (PlGF) is also closely related to VEGF-A. VEGF-A, -B, -C, -D, and PlGF are all distantly related to platelet-derived growth factors-A and -B. Less is known about the function and regulation of VEGF-B, -C, and -D, but they do not seem to be regulated by the major pathways that regulate VEGF-A.
VEGF-A transcription is potentiated in response to hypoxia and by activated oncogenes. The transcription factors, hypoxia inducible factor-1a (hif-1a) and -2a, are degraded by proteosomes in normoxia and stabilized in hypoxia. This pathway is dependent on the Von Hippel-Lindau gene product. Hif-1a and hif-2 a heterodimerize with the aryl hydrocarbon nuclear translocator in the nucleus and bind the VEGF promoter/enhancer. This is a key pathway expressed in most types of cells. Hypoxia inducibility, in particular, characterizes VEGF-A versus other members of the VEGF family and other angiogenic factors. VEGF transcription in normoxia is activated by many oncogenes, including H-ras and several transmembrane tyrosine kinases, such as the epidermal growth factor receptor and erbB2. These pathways together account for a marked upregulation of VEGF-A in tumors compared to normal tissues and are often of prognostic importance.
There are three receptors in the VEGF receptor family. They have the common properties of multiple IgG-like extracellular domains and tyrosine kinase activity. The enzyme domains of VEGF receptor 1 (VEGFR1, also known as Flt-1), VEGFR2 (also known as KDR or Flk-1), and VEGFR3 (also known as Flt-4) are divided by an inserted sequence. Endothelial cells also express additional VEGF receptors, Neuropilin-1 and Neuropilin-2. VEGF-A binds to VEGFR1 and VEGFR2 and to Neuropilin-1 and Neuropilin-2. PIGF and VEGF-B bind VEGFR1 and Neuropilin-1. VEGF-C and -D bind VEGFR3 and VEGFR2.
The VEGF-C/VEGFR3 pathway is important for lymphatic proliferation. VEGFR3 is specifically expressed on lymphatic endothelium. A soluble form of Flt-1 can be detected in peripheral blood and is a high affinity ligand for VEGF. Soluble Flt-1 can be used to antagonize VEGF function. VEGFR1 and VEGFR2 are upregulated in tumor and proliferating endothelium, partly by hypoxia and also in response to VEGF-A itself. VEGFR1 and VEGFR2 can interact with multiple downstream signaling pathways via proteins such as PLC-g, Ras, Shc, Nck, PKC and PI3-kinase. VEGFR1 is of higher affinity than VEGFR2 and mediates motility and vascular permeability. VEGFR2 is necessary for proliferation.
VEGF can be detected in both plasma and serum samples of patients, with much higher levels in serum. Platelets release VEGF upon aggregation and may be a major source of VEGF delivery to tumors. Several studies have shown that association of high serum levels of VEGF with poor prognosis in cancer patients may be correlated with an elevated platelet count. Many tumors release cytokines that can stimulate the production of megakaryocytes in the marrow and elevate the platelet count. This can result in an indirect increase of VEGF delivery to tumors.
VEGF is implicated in several other pathological conditions associated with enhanced angiogenesis. For example, VEGF plays a role in both psoriasis and rheumatoid arthritis. Diabetic retinopathy is associated with high intraocular levels of VEGF. Inhibition of VEGF function may result in infertility by blockade of corpus luteum function. Direct demonstration of the importance of VEGF in tumor growth has been achieved using dominant negative VEGF receptors to block in vivo proliferation, as well as blocking antibodies to VEGF39 or to VEGFR2.
Targeting of VEGFR1 can be useful in disrupting VEGF signaling pathways by mediating inhibition of VEGFR1 and VEGFR2 expression. Kou et al., 2005, Biochemistry, 44: 15064-15073 report that inhibition of VEGFR1 using siRNA significantly inhibited VEGFR2 promoter activity as well as VEGFR1 expression.
The use of small interfering nucleic acid molecules targeting VEGF and corresponding receptors and ligands therefore provides a class of novel therapeutic agents that can be used in the diagnosis of and the treatment of inflammatory diseases and conditions, respiratory diseases and conditions, allergic diseases and conditions, autoimmune diseases and conditions, neurologic diseases and conditions, ocular diseases and conditions, and cancer and other proliferative diseases and conditions, or any other disease or condition that responds to modulation of VEGF and/or VEGFR genes or other genes involved in VEGF and/or VEGFR biologic pathways, such as interleukins and interleukin receptors.
EXAMPLES
The following are non-limiting examples showing the selection, isolation, synthesis and activity of nucleic acids of the instant invention.
Example 1
Tandem Synthesis of siNA Constructs
Exemplary siNA molecules of the invention are synthesized in tandem using a cleavable linker, for example, a succinyl-based linker. Tandem synthesis as described herein is followed by a one-step purification process that provides RNAi molecules in high yield. This approach is highly amenable to siNA synthesis in support of high throughput RNAi screening, and can be readily adapted to multi-column or multi-well synthesis platforms.
After completing a tandem synthesis of a siNA oligo and its complement in which the 5′-terminal dimethoxytrityl (5′-O-DMT) group remains intact (trityl on synthesis), the oligonucleotides are deprotected as described above. Following deprotection, the siNA sequence strands are allowed to spontaneously hybridize. This hybridization yields a duplex in which one strand has retained the 5′-O-DMT group while the complementary strand comprises a terminal 5′-hydroxyl. The newly formed duplex behaves as a single molecule during routine solid-phase extraction purification (Trityl-On purification) even though only one molecule has a dimethoxytrityl group. Because the strands form a stable duplex, this dimethoxytrityl group (or an equivalent group, such as other trityl groups or other hydrophobic moieties) is all that is required to purify the pair of oligos, for example, by using a C18 cartridge.
Standard phosphoramidite synthesis chemistry is used up to the point of introducing a tandem linker, such as an inverted deoxy abasic succinate or glyceryl succinate linker (see FIG. 1) or an equivalent cleavable linker. A non-limiting example of linker coupling conditions that can be used includes a hindered base such as diisopropylethylamine (DIPA) and/or DMAP in the presence of an activator reagent such as Bromotripyrrolidinophosphoniumhexaflurorophosphate (PyBrOP). After the linker is coupled, standard synthesis chemistry is utilized to complete synthesis of the second sequence leaving the terminal the 5′-O-DMT intact. Following synthesis, the resulting oligonucleotide is deprotected according to the procedures described herein and quenched with a suitable buffer, for example with 50 mM NaOAc or 1.5M NH4H2CO3.
Purification of the siNA duplex can be readily accomplished using solid phase extraction, for example, using a Waters C18 SepPak 1 g cartridge conditioned with 1 column volume (CV) of acetonitrile, 2 CV H2O, and 2 CV 50 mM NaOAc. The sample is loaded and then washed with 1 CV H2O or 50 mM NaOAc. Failure sequences are eluted with 1 CV 14% ACN (Aqueous with 50 mM NaOAc and 50 mM NaCl). The column is then washed, for example with 1 CV H2O followed by on-column detritylation, for example by passing 1 CV of 1% aqueous trifluoroacetic acid (TFA) over the column, then adding a second CV of 1% aqueous TFA to the column and allowing to stand for approximately 10 minutes. The remaining TFA solution is removed and the column washed with H2O followed by 1 CV 1M NaCl and additional H2O. The siNA duplex product is then eluted, for example, using 1 CV 20% aqueous CAN.
FIG. 2 provides an example of MALDI-TOF mass spectrometry analysis of a purified siNA construct in which each peak corresponds to the calculated mass of an individual siNA strand of the siNA duplex. The same purified siNA provides three peaks when analyzed by capillary gel electrophoresis (CGE), one peak presumably corresponding to the duplex siNA, and two peaks presumably corresponding to the separate siNA sequence strands. Ion exchange HPLC analysis of the same siNA contract only shows a single peak. Testing of the purified siNA construct using a luciferase reporter assay described below demonstrated the same RNAi activity compared to siNA constructs generated from separately synthesized oligonucleotide sequence strands.
Example 2
Identification of Potential siNA Target Sites in any RNA Sequence
The sequence of an RNA target of interest, such as a viral or human mRNA transcript, is screened for target sites, for example by using a computer folding algorithm. In a non-limiting example, the sequence of a gene or RNA gene transcript derived from a database, such as Genbank, is used to generate siNA targets having complementarity to the target. Such sequences can be obtained from a database, or can be determined experimentally as known in the art. Target sites that are known, for example, those target sites determined to be effective target sites based on studies with other nucleic acid molecules, for example ribozymes or antisense, or those targets known to be associated with a disease, trait, or condition such as those sites containing mutations or deletions, can be used to design siNA molecules targeting those sites. Various parameters can be used to determine which sites are the most suitable target sites within the target RNA sequence. These parameters include but are not limited to secondary or tertiary RNA structure, the nucleotide base composition of the target sequence, the degree of homology between various regions of the target sequence, or the relative position of the target sequence within the RNA transcript. Based on these determinations, any number of target sites within the RNA transcript can be chosen to screen siNA molecules for efficacy, for example by using in vitro RNA cleavage assays, cell culture, or animal models. In a non-limiting example, anywhere from 1 to 1000 target sites are chosen within the transcript based on the size of the siNA construct to be used. High throughput screening assays can be developed for screening siNA molecules using methods known in the art, such as with multi-well or multi-plate assays to determine efficient reduction in target gene expression.
Example 3
Selection of siNA Molecule Target Sites in a RNA
The following non-limiting steps can be used to carry out the selection of siNAs targeting a given gene sequence or transcript.
1. The target sequence is parsed in silico into a list of all fragments or subsequences of a particular length, for example 23 nucleotide fragments, contained within the target sequence. This step is typically carried out using a custom Perl script, but commercial sequence analysis programs such as Oligo, MacVector, or the GCG Wisconsin Package can be employed as well.
2. In some instances the siNAs correspond to more than one target sequence; such would be the case for example in targeting different transcripts of the same gene, targeting different transcripts of more than one gene, or for targeting both the human gene and an animal homolog. In this case, a subsequence list of a particular length is generated for each of the targets, and then the lists are compared to find matching sequences in each list. The subsequences are then ranked according to the number of target sequences that contain the given subsequence; the goal is to find subsequences that are present in most or all of the target sequences. Alternately, the ranking can identify subsequences that are unique to a target sequence, such as a mutant target sequence. Such an approach would enable the use of siNA to target specifically the mutant sequence and not effect the expression of the normal sequence.
3. In some instances the siNA subsequences are absent in one or more sequences while present in the desired target sequence; such would be the case if the siNA targets a gene with a paralogous family member that is to remain untargeted. As in case 2 above, a subsequence list of a particular length is generated for each of the targets, and then the lists are compared to find sequences that are present in the target gene but are absent in the untargeted paralog.
4. The ranked siNA subsequences can be further analyzed and ranked according to GC content. A preference can be given to sites containing 30-70% GC, with a further preference to sites containing 40-60% GC.
5. The ranked siNA subsequences can be further analyzed and ranked according to self-folding and internal hairpins. Weaker internal folds are preferred; strong hairpin structures are to be avoided.
6. The ranked siNA subsequences can be further analyzed and ranked according to whether they have runs of GGG or CCC in the sequence. GGG (or even more Gs) in either strand can make oligonucleotide synthesis problematic and can potentially interfere with RNAi activity, so it is avoided whenever better sequences are available. CCC is searched in the target strand because that will place GGG in the antisense strand.
7. The ranked siNA subsequences can be further analyzed and ranked according to whether they have the dinucleotide UU (uridine dinucleotide) on the 3′-end of the sequence, and/or AA on the 5′-end of the sequence (to yield 3′ U on the antisense sequence). These sequences allow one to design siNA molecules with terminal TT thymidine dinucleotides.
8. Four or five target sites are chosen from the ranked list of subsequences as described above. For example, in subsequences having 23 nucleotides, the right 21 nucleotides of each chosen 23-mer subsequence are then designed and synthesized for the upper (sense) strand of the siNA duplex, while the reverse complement of the left 21 nucleotides of each chosen 23-mer subsequence are then designed and synthesized for the lower (antisense) strand of the siNA duplex (see Table II). If terminal TT residues are desired for the sequence (as described in paragraph 7), then the two 3′ terminal nucleotides of both the sense and antisense strands are replaced by TT prior to synthesizing the oligos.
9. The siNA molecules are screened in an in vitro, cell culture or animal model system to identify the most active siNA molecule or the most preferred target site within the target RNA sequence.
10. Other design considerations can be used when selecting target nucleic acid sequences, see, for example, Reynolds et al., 2004, Nature Biotechnology Advanced Online Publication, 1 Feb. 2004, doi:10.1038/nbt936 and Ui-Tei et al., 2004, Nucleic Acids Research, 32, doi:10.1093/nar/gkh247.
In an alternate approach, a pool of siNA constructs specific to a target sequence is used to screen for target sites in cells expressing target RNA, such as cultured Jurkat, HeLa, A549 or 293T cells. The general strategy used in this approach is shown in FIG. 9. Cells expressing the target RNA are transfected with the pool of siNA constructs and cells that demonstrate a phenotype associated with target inhibition are sorted. The pool of siNA constructs can be expressed from transcription cassettes inserted into appropriate vectors (see for example FIG. 7 and FIG. 8). The siNA from cells demonstrating a positive phenotypic change (e.g., decreased proliferation, decreased target mRNA levels or decreased target protein expression), are sequenced to determine the most suitable target site(s) within the target RNA sequence.
Example 4
siNA Design
siNA target sites were chosen by analyzing sequences of the VEGF and/or VEGFR RNA target and optionally prioritizing the target sites on the basis of folding (structure of any given sequence analyzed to determine siNA accessibility to the target), by using a library of siNA molecules as described in Example 3, or alternately by using an in vitro siNA system as described in Example 6 herein siNA molecules were designed that could bind each target and are optionally individually analyzed by computer folding to assess whether the siNA molecule can interact with the target sequence. Varying the length of the siNA molecules can be chosen to optimize activity. Generally, a sufficient number of complementary nucleotide bases are chosen to bind to, or otherwise interact with, the target RNA, but the degree of complementarity can be modulated to accommodate siNA duplexes or varying length or base composition. By using such methodologies, siNA molecules can be designed to target sites within any known RNA sequence, for example those RNA sequences corresponding to the any gene transcript.
Chemically modified siNA constructs are designed to provide nuclease stability for systemic administration in vivo and/or improved pharmacokinetic, localization, and delivery properties while preserving the ability to mediate RNAi activity. Chemical modifications as described herein are introduced synthetically using synthetic methods described herein and those generally known in the art. The synthetic siNA constructs are then assayed for nuclease stability in serum and/or cellular/tissue extracts (e.g. liver extracts). The synthetic siNA constructs are also tested in parallel for RNAi activity using an appropriate assay, such as a luciferase reporter assay as described herein or another suitable assay that can quantity RNAi activity. Synthetic siNA constructs that possess both nuclease stability and RNAi activity can be further modified and re-evaluated in stability and activity assays. The chemical modifications of the stabilized active siNA constructs can then be applied to any siNA sequence targeting any chosen RNA and used, for example, in target screening assays to pick lead siNA compounds for therapeutic development (see for example FIG. 11).
Example 5
Chemical Synthesis and Purification of siNA
siNA molecules can be designed to interact with various sites in the RNA message, for example, target sequences within the RNA sequences described herein. The sequence of one strand of the siNA molecule(s) is complementary to the target site sequences described above. The siNA molecules can be chemically synthesized using methods described herein. Inactive siNA molecules that are used as control sequences can be synthesized by scrambling the sequence of the siNA molecules such that it is not complementary to the target sequence. Generally, siNA constructs can by synthesized using solid phase oligonucleotide synthesis methods as described herein (see for example Usman et al., U.S. Pat. Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098; 6,362,323; 6,437,117; 6,469,158; Scaringe et al, U.S. Pat. Nos. 6,111,086; 6,008,400; 6,111,086 all incorporated by reference herein in their entirety).
In a non-limiting example, RNA oligonucleotides are synthesized in a stepwise fashion using the phosphoramidite chemistry as is known in the art. Standard phosphoramidite chemistry involves the use of nucleosides comprising any of 5′-O-dimethoxytrityl, 2′-O-tert-butyldimethylsilyl, 3′-O-2-Cyanoethyl N,N-diisopropylphos-phoroamidite groups, and exocyclic amine protecting groups (e.g. N6-benzoyl adenosine, N4 acetyl cytidine, and N2-isobutyryl guanosine). Alternately, 2′-O-Silyl Ethers can be used in conjunction with acid-labile 2′-O-orthoester protecting groups in the synthesis of RNA as described by Scaringe supra. Differing 2′ chemistries can require different protecting groups, for example 2′-deoxy-2′-amino nucleosides can utilize N-phthaloyl protection as described by Usman et al., U.S. Pat. No. 5,631,360, incorporated by reference herein in its entirety).
During solid phase synthesis, each nucleotide is added sequentially (3′- to 5′-direction) to the solid support-bound oligonucleotide. The first nucleoside at the 3′-end of the chain is covalently attached to a solid support (e.g., controlled pore glass or polystyrene) using various linkers. The nucleotide precursor, a ribonucleoside phosphoramidite, and activator are combined resulting in the coupling of the second nucleoside phosphoramidite onto the 5′-end of the first nucleoside. The support is then washed and any unreacted 5′-hydroxyl groups are capped with a capping reagent such as acetic anhydride to yield inactive 5′-acetyl moieties. The trivalent phosphorus linkage is then oxidized to a more stable phosphate linkage. At the end of the nucleotide addition cycle, the 5′-O-protecting group is cleaved under suitable conditions (e.g., acidic conditions for trityl-based groups and Fluoride for silyl-based groups). The cycle is repeated for each subsequent nucleotide.
Modification of synthesis conditions can be used to optimize coupling efficiency, for example by using differing coupling times, differing reagent/phosphoramidite concentrations, differing contact times, differing solid supports and solid support linker chemistries depending on the particular chemical composition of the siNA to be synthesized. Deprotection and purification of the siNA can be performed as is generally described in Usman et al., U.S. Pat. Nos. 5,831,071, 6,353,098, 6,437,117, and Bellon et al., U.S. Pat. Nos. 6,054,576, 6,162,909, 6,303,773, or Scaringe supra, incorporated by reference herein in their entireties. Additionally, deprotection conditions can be modified to provide the best possible yield and purity of siNA constructs. For example, applicant has observed that oligonucleotides comprising 2′-deoxy-2′-fluoro nucleotides can degrade under inappropriate deprotection conditions. Such oligonucleotides are deprotected using aqueous methylamine at about 35° C. for 30 minutes. If the 2′-deoxy-2′-fluoro containing oligonucleotide also comprises ribonucleotides, after deprotection with aqueous methylamine at about 35° C. for 30 minutes, TEA-HF is added and the reaction maintained at about 65° C. for an additional 15 minutes.
Example 6
RNAi in vitro Assay to Assess siNA Activity
An in vitro assay that recapitulates RNAi in a cell-free system is used to evaluate siNA constructs targeting VEGF and/or VEGFR RNA targets. The assay comprises the system described by Tuschl et al., 1999, Genes and Development, 13, 3191-3197 and Zamore et al., 2000, Cell, 101, 25-33 adapted for use with a target RNA. A Drosophila extract derived from syncytial blastoderm is used to reconstitute RNAi activity in vitro. Target RNA is generated via in vitro transcription from an appropriate target expressing plasmid using T7 RNA polymerase or via chemical synthesis as described herein. Sense and antisense siNA strands (for example 20 uM each) are annealed by incubation in buffer (such as 100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for 1 minute at 90° C. followed by 1 hour at 37° C., then diluted in lysis buffer (for example 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate). Annealing can be monitored by gel electrophoresis on an agarose gel in TBE buffer and stained with ethidium bromide. The Drosophila lysate is prepared using zero to two-hour-old embryos from Oregon R flies collected on yeasted molasses agar that are dechorionated and lysed. The lysate is centrifuged and the supernatant isolated. The assay comprises a reaction mixture containing 50% lysate [vol/vol], RNA (10-50 pM final concentration), and 10% [vol/vol] lysis buffer containing siNA (10 nM final concentration). The reaction mixture also contains 10 mM creatine phosphate, 10 ug/ml creatine phosphokinase, 100 um GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP, 5 mM DTT, 0.1 U/uL RNasin (Promega), and 100 uM of each amino acid. The final concentration of potassium acetate is adjusted to 100 mM. The reactions are pre-assembled on ice and preincubated at 25° C. for 10 minutes before adding RNA, then incubated at 25° C. for an additional 60 minutes. Reactions are quenched with 4 volumes of 1.25×Passive Lysis Buffer (Promega). Target RNA cleavage is assayed by RT-PCR analysis or other methods known in the art and are compared to control reactions in which siNA is omitted from the reaction.
Alternately, internally-labeled target RNA for the assay is prepared by in vitro transcription in the presence of [alpha-32P] CTP, passed over a G50 Sephadex column by spin chromatography and used as target RNA without further purification. Optionally, target RNA is 5′-32P-end labeled using T4 polynucleotide kinase enzyme. Assays are performed as described above and target RNA and the specific RNA cleavage products generated by RNAi are visualized on an autoradiograph of a gel. The percentage of cleavage is determined by PHOSPHOR IMAGER® (autoradiography) quantitation of bands representing intact control RNA or RNA from control reactions without siNA and the cleavage products generated by the assay.
In one embodiment, this assay is used to determine target sites in the VEGF and/or VEGFR RNA target for siNA mediated RNAi cleavage, wherein a plurality of siNA constructs are screened for RNAi mediated cleavage of the VEGF and/or VEGFR RNA target, for example, by analyzing the assay reaction by electrophoresis of labeled target RNA, or by northern blotting, as well as by other methodology well known in the art.
Example 7
Nucleic Acid Inhibition of VEGF and/or VEGFR Target RNA in vivo
siNA molecules targeted to the human VEGF and/or VEGFR RNA are designed and synthesized as described above. These nucleic acid molecules can be tested for cleavage activity in vivo, for example, using the following procedure. The target sequences and the nucleotide location within the VEGF and/or VEGFR RNA are given in Table II and III.
Two formats are used to test the efficacy of siNAs targeting VEGF and/or VEGFR. First, the reagents are tested in cell culture using, for example, HUVEC, HMVEC, or A375 cells to determine the extent of RNA and protein inhibition siNA reagents (e.g.; see Tables II and III) are selected against the VEGF and/or VEGFR target as described herein. RNA inhibition is measured after delivery of these reagents by a suitable transfection agent to, for example, HUVEC, HMVEC, or A375 cells. Relative amounts of target RNA are measured versus actin using real-time PCR monitoring of amplification (eg., ABI 7700 TAQMAN®). A comparison is made to a mixture of oligonucleotide sequences made to unrelated targets or to a randomized siNA control with the same overall length and chemistry, but randomly substituted at each position. Primary and secondary lead reagents are chosen for the target and optimization performed. After an optimal transfection agent concentration is chosen, a RNA time-course of inhibition is performed with the lead siNA molecule. In addition, a cell-plating format can be used to determine RNA inhibition.
Delivery of siNA to Cells
Cells (e.g., HUVEC, HMVEC, or A375 cells) are seeded, for example, at 1×105 cells per well of a six-well dish in EGM-2 (BioWhittaker) the day before transfection. siNA (final concentration, for example 20 nM) and cationic lipid (e.g., final concentration 2□g/ml) are complexed in EGM basal media (Biowhittaker) at 37° C. for 30 minutes in polystyrene tubes. Following vortexing, the complexed siNA is added to each well and incubated for the times indicated. For initial optimization experiments, cells are seeded, for example, at 1×103 in 96 well plates and siNA complex added as described. Efficiency of delivery of siNA to cells is determined using a fluorescent siNA complexed with lipid. Cells in 6-well dishes are incubated with siNA for 24 hours, rinsed with PBS and fixed in 2% paraformaldehyde for 15 minutes at room temperature. Uptake of siNA is visualized using a fluorescent microscope.
TAQMAN® (Real-Time PCR Monitoring of Amplification) and Lightcycler Quantification of mRNA
Total RNA is prepared from cells following siNA delivery, for example, using Qiagen RNA purification kits for 6-well or Rneasy extraction kits for 96-well assays. For TAQMAN® analysis (real-time PCR monitoring of amplification), dual-labeled probes are synthesized with the reporter dye, FAM or JOE, covalently linked at the 5′-end and the quencher dye TAMRA conjugated to the 3′-end. One-step RT-PCR amplifications are performed on, for example, an ABI PRISM 7700 Sequence Detector using 50 μl reactions consisting of 10 μl total RNA, 100 nM forward primer, 900 nM reverse primer, 100 nM probe, 1× TaqMan PCR reaction buffer (PE-Applied Biosystems), 5.5 mM MgCl2, 300 μM each dATP, dCTP, dGTP, and dTTP, 10U RNase Inhibitor (Promega), 1.25U AMPLITAQ GOLD® (DNA polymerase) (PE-Applied Biosystems) and 10U M-MLV Reverse Transcriptase (Promega). The thermal cycling conditions can consist of 30 minutes at 48° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95° C. and 1 minute at 60° C. Quantitation of mRNA levels is determined relative to standards generated from serially diluted total cellular RNA (300, 100, 33, 11 ng/reaction) and normalizing to β-actin or GAPDH mRNA in parallel TAQMAN® reactions (real-time PCR monitoring of amplification). For each gene of interest an upper and lower primer and a fluorescently labeled probe are designed. Real time incorporation of SYBR Green I dye into a specific PCR product can be measured in glass capillary tubes using a lightcyler. A standard curve is generated for each primer pair using control cRNA. Values are represented as relative expression to GAPDH in each sample.
Western Blotting
Nuclear extracts can be prepared using a standard micro preparation technique (see for example Andrews and Faller, 1991, Nucleic Acids Research, 19, 2499). Protein extracts from supernatants are prepared, for example using TCA precipitation. An equal volume of 20% TCA is added to the cell supernatant, incubated on ice for 1 hour and pelleted by centrifugation for 5 minutes. Pellets are washed in acetone, dried and resuspended in water. Cellular protein extracts are run on a 10% Bis-Tris NuPage (nuclear extracts) or 4-12% Tris-Glycine (supernatant extracts) polyacrylamide gel and transferred onto nitro-cellulose membranes. Non-specific binding can be blocked by incubation, for example, with 5% non-fat milk for 1 hour followed by primary antibody for 16 hour at 4° C. Following washes, the secondary antibody is applied, for example (1:10,000 dilution) for 1 hour at room temperature and the signal detected with SuperSignal reagent (Pierce).
Example 8
Animal Models Useful to Evaluate the Down-Regulation of VEGF and/or VEGFR Gene Expression
There are several animal models in which the anti-angiogenesis effect of nucleic acids of the present invention, such as siRNA, directed against VEGF, VEGFR1, VEGFR2 and/or VEGFR3 mRNAs can be tested. Typically a corneal model has been used to study angiogenesis in rat and rabbit since recruitment of vessels can easily be followed in this normally avascular tissue (Pandey et al., 1995 Science 268: 567-569). In these models, a small Teflon or Hydron disk pretreated with an angiogenesis factor (e.g. bFGF or VEGF) is inserted into a pocket surgically created in the cornea. Angiogenesis is monitored 3 to 5 days later. siRNA directed against VEGF, VEGFR1, VEGFR2 and/or VEGFR3 mRNAs are delivered in the disk as well, or dropwise to the eye over the time course of the experiment. In another eye model, hypoxia has been shown to cause both increased expression of VEGF and neovascularization in the retina (Pierce et al., 1995 Proc. Natl. Acad. Sci. USA. 92: 905-909; Shweiki et al., 1992 J. Clin. Invest. 91: 2235-2243).
In human glioblastomas, it has been shown that VEGF is at least partially responsible for tumor angiogenesis (Plate et al., 1992 Nature 359, 845). Animal models have been developed in which glioblastoma cells are implanted subcutaneously into nude mice and the progress of tumor growth and angiogenesism is studied (Kim et al., 1993 supra; Millauer et al., 1994 supra).
Another animal model that addresses neovascularization involves Matrigel, an extract of basement membrane that becomes a solid gel when injected subcutaneously (Passaniti et al., 1992 Lab. Invest. 67: 519-528). When the Matrigel is supplemented with angiogenesis factors such as VEGF, vessels grow into the Matrigel over a period of 3 to 5 days and angiogenesis can be assessed. Again, nucleic acids directed against VEGFR mRNAs are delivered in the Matrigel.
Several animal models exist for screening of anti-angiogenic agents. These include corneal vessel formation following corneal injury (Burger et al., 1985 Cornea 4: 35-41; Lepri, et al., 1994 J. Ocular Pharmacol. 10: 273-280; Ormerod et al., 1990 Am. J. Pathol. 137: 1243-1252) or intracorneal growth factor implant (Grant et al., 1993 Diabetologia 36: 282-291; Pandey et al. 1995 supra; Zieche et al., 1992 Lab. Invest. 67: 711-715), vessel growth into Matrigel matrix containing growth factors (Passaniti et al., 1992 supra), female reproductive organ neovascularization following hormonal manipulation (Shweiki et al., 1993 Clin. Invest. 91: 2235-2243), several models involving inhibition of tumor growth in highly vascularized solid tumors (O'Reilly et al., 1994 Cell 79: 315-328; Senger et al., 1993 Cancer and Metas. Rev. 12: 303-324; Takahasi et al., 1994 Cancer Res. 54: 4233-4237; Kim et al., 1993 supra), and transient hypoxia-induced neovascularization in the mouse retina (Pierce et al., 1995 Proc. Natl. Acad. Sci. USA. 92: 905-909). Other model systems to study tumor angiogenesis are reviewed by Folkman, 1985 Adv. Cancer. Res. 43, 175.
Ocular Models of Angiogenesis
The cornea model, described in Pandey et al. supra, is the most common and well characterized model for screening anti-angiogenic agent efficacy. This model involves an avascular tissue into which vessels are recruited by a stimulating agent (growth factor, thermal or alkalai burn, endotoxin). The corneal model utilizes the intrastromal corneal implantation of a Teflon pellet soaked in a VEGF-Hydron solution to recruit blood vessels toward the pellet, which can be quantitated using standard microscopic and image analysis techniques. To evaluate their anti-angiogenic efficacy, nucleic acids are applied topically to the eye or bound within Hydron on the Teflon pellet itself. This avascular cornea as well as the Matrigel (see below) provide for low background assays. While the corneal model has been performed extensively in the rabbit, studies in the rat have also been conducted.
The mouse model (Passaniti et al., supra) is a non-tissue model that utilizes Matrigel, an extract of basement membrane (Kleinman et al., 1986) or Millipore® filter disk, which can be impregnated with growth factors and anti-angiogenic agents in a liquid form prior to injection. Upon subcutaneous administration at body temperature, the Matrigel or Millipore® filter disk forms a solid implant. VEGF embedded in the Matrigel or Millipore® filter disk is used to recruit vessels within the matrix of the Matrigel or Millipore® filter disk which can be processed histologically for endothelial cell specific vWF (factor VIII antigen) immunohistochemistry, Trichrome-Masson stain, or hemoglobin content. Like the cornea, the Matrigel or Millipore® filter disk is avascular; however, it is not tissue. In the Matrigel or Millipore® filter disk model, nucleic acids are administered within the matrix of the Matrigel or Millipore® filter disk to test their anti-angiogenic efficacy. Thus, delivery issues in this model, as with delivery of nucleic acids by Hydron-coated Teflon pellets in the rat cornea model, may be less problematic due to the homogeneous presence of the nucleic acid within the respective matrix.
Additionally, siNA molecules of the invention targeting VEGF and/or VEGFR (e.g. VEGFR1, VEGFR2, and/or VEGFR3) can be assessed for activity transgenic mice to determine whether modulation of VEGF and/or VEGFR can inhibit optic neovasculariation. Animal models of choroidal neovascularization are described in, for example, Mori et al., 2001, Journal of Cellular Physiology, 188, 253; Mori et al., 2001, American Journal of Pathology, 159, 313; Ohno-Matsui et al., 2002, American Journal of Pathology, 160, 711; and Kwak et al., 2000, Investigative Ophthalmology & Visual Science, 41, 3158. VEGF plays a central role in causing retinal neovascularization. Increased expression of VEGFR2 in retinal photoreceptors of transgenic mice stimulates neovascularization within the retina, and a blockade of VEGFR2 signaling has been shown to inhibit retinal choroidal neovascularization (CNV) (Mori et al.,2001, J. Cell. Physiol., 188, 253).
CNV is laser induced in, for example, adult C57BL/6 mice. The mice are also given an intravitreous, periocular or a subretinal injection of VEGF and/or VEGFR (e.g., VEGFR2) siNA in each eye. Intravitreous injections are made using a Harvard pump microinjection apparatus and pulled glass micropipets. Then a micropipette is passed through the sclera just behind the limbus into the vitreous cavity. The subretinal injections are made using a condensing lens system on a dissecting microscope. The pipet tip is then passed through the sclera posterior to the limbus and positioned above the retina. Five days after the injection of the vector the mice are anesthetized with ketamine hydrochloride (100 mg/kg body weight), 1% tropicamide is also used to dilate the pupil, and a diode laser photocoagulation is used to rupture Bruch's membrane at three locations in each eye. A slit lamp delivery system and a hand-held cover slide are used for laser photocoagulation. Burns are made in the 9, 12, and 3 o'clock positions 2-3 disc diameters from the optic nerve (Mori et al., supra).
The mice typically develop subretinal neovasculariation due to the expression of VEGF in photoreceptors beginning at prenatal day 7. At prenatal day 21, the mice are anesthetized and perfused with 1 ml of phosphate-buffered saline containing 50 mg/ml of fluorescein-labeled dextran. Then the eyes are removed and placed for 1 hour in a 10% phosphate-buffered formalin. The retinas are removed and examined by fluorescence microscopy (Mori et al., supra).
Fourteen days after the laser induced rupture of Bruch's membrane, the eyes that received intravitreous and subretinal injection of siNA are evaluated for smaller appearing areas of CNV, while control eyes are evaluated for large areas of CNV. The eyes that receive intravitreous injections or a subretinal injection of siNA are also evaluated for fewer areas of neovasculariation on the outer surface of the retina and potential abortive sprouts from deep retinal capillaries that do not reach the retinal surface compared to eyes that did not receive an injection of siNA.
Tumor Models of Angiogenesis
Use of Murine Models
For a typical systemic study involving 10 mice (20 g each) per dose group, 5 doses (1, 3, 10, 30 and 100 mg/kg daily over 14 days continuous administration), approximately 400 mg of siRNA, formulated in saline is used. A similar study in young adult rats (200 g) requires over 4 g. Parallel pharmacokinetic studies involve the use of similar quantities of siRNA further justifying the use of murine models.
Lewis Lung Carcinoma and B-16 Melanoma Murine Models
Identifying a common animal model for systemic efficacy testing of nucleic acids is an efficient way of screening siNA for systemic efficacy.
The Lewis lung carcinoma and B-16 murine melanoma models are well accepted models of primary and metastatic cancer and are used for initial screening of anti-cancer agents. These murine models are not dependent upon the use of immunodeficient mice, are relatively inexpensive, and minimize housing concerns. Both the Lewis lung and B-16 melanoma models involve subcutaneous implantation of approximately 106 tumor cells from metastatically aggressive tumor cell lines (Lewis lung lines 3LL or D122, LLc-LN7; B-16-BL6 melanoma) in C57BL/6J mice. Alternatively, the Lewis lung model can be produced by the surgical implantation of tumor spheres (approximately 0.8 mm in diameter). Metastasis also can be modeled by injecting the tumor cells directly intravenously. In the Lewis lung model, microscopic metastases can be observed approximately 14 days following implantation with quantifiable macroscopic metastatic tumors developing within 21-25 days. The B-16 melanoma exhibits a similar time course with tumor neovascularization beginning 4 days following implantation. Since both primary and metastatic tumors exist in these models after 21-25 days in the same animal, multiple measurements can be taken as indices of efficacy. Primary tumor volume and growth latency as well as the number of micro- and macroscopic metastatic lung foci or number of animals exhibiting metastases can be quantitated. The percent increase in lifespan can also be measured. Thus, these models provide suitable primary efficacy assays for screening systemically administered siRNA nucleic acids and siRNA nucleic acid formulations.
In the Lewis lung and B-16 melanoma models, systemic pharmacotherapy with a wide variety of agents usually begins 1-7 days following tumor implantation/inoculation with either continuous or multiple administration regimens. Concurrent pharmacokinetic studies can be performed to determine whether sufficient tissue levels of siRNA can be achieved for pharmacodynamic effect to be expected. Furthermore, primary tumors and secondary lung metastases can be removed and subjected to a variety of in vitro studies (i.e. target RNA reduction).
Renal Disease Models
In addition, animal models are useful in screening compounds, eg. siNA molecules, for efficacy in treating renal failure, such as a result of autosomal dominant polycystic kidney disease (ADPKD). The Han:SPRD rat model, mice with a targeted mutation in the Pkd2 gene and congenital polycystic kidney (cpk) mice, closely resemble human ADPKD and provide animal models to evaluate the therapeutic effect of siRNA constructs that have the potential to interfere with one or more of the pathogenic elements of ADPKD mediated renal failure, such as angiogenesis. Angiogenesis may be necessary in the progression of ADPKD for growth of cyst cells as well as increased vascular permeability promoting fluid secretion into cysts. Proliferation of cystic epithelium is also a feature of ADPKD because cyst cells in culture produce soluble vascular endothelial growth factor (VEGF). VEGFR1 has also been detected in epithelial cells of cystic tubules but not in endothelial cells in the vasculature of cystic kidneys or normal kidneys. VEGFR2 expression is increased in endothelial cells of cyst vessels and in endothelial cells during renal ischemia-reperfusion. It is proposed that inhibition of VEGF receptors with anti-VEGFR1 and anti-VEGFR2 siRNA molecules would attenuate cyst formation, renal failure and mortality in ADPKD. Anti-VEGFR2 siRNA molecules would therefore be designed to inhibit angiogenesis involved in cyst formation. As VEGFR1 is present in cystic epithelium and not in vascular endothelium of cysts, it is proposed that anti-VEGFR1 siRNA molecules would attenuate cystic epithelial cell proliferation and apoptosis which would in turn lead to less cyst formation. Further, it is proposed that VEGF produced by cystic epithelial cells is one of the stimuli for angiogenesis as well as epithelial cell proliferation and apoptosis. The use of Han:SPRD rats (see for example Kaspareit-Rittinghausen et al., 1991, Am. J. Pathol. 139, 693-696), mice with a targeted mutation in the Pkd2 gene (Pkd2−/− mice, see for example Wu et al., 2000, Nat. Genet. 24, 75-78) and cpk mice (see for example Woo et al., 1994, Nature, 368, 750-753) all provide animal models to study the efficacy of siRNA molecules of the invention against VEGFR1 and VEGFR2 mediated renal failure.
VEGF, VEGFR1 VGFR2 and/or VEGFR3 protein levels can be measured clinically or experimentally by FACS analysis. VEGF, VEGFR1 VGFR2 and/or VEGFR3 encoded mRNA levels are assessed by Northern analysis, RNase-protection, primer extension analysis and/or quantitative RT-PCR. siRNA nucleic acids that block VEGF, VEGFR1 VGFR2 and/or VEGFR3 protein encoding mRNAs and therefore result in decreased levels of VEGF, VEGFR1 VGFR2 and/or VEGFR3 activity by more than 20% in vitro can be identified.
Respiratory Disease Models
Exaggerated levels of VEGF are present in subjects with asthma, but the role of VEGF in normal and asthmatic lungs has not been well defined. Lee et al., 2004, Nature Medicine, 10, 1095-1103, generated lung-targeted VEGF165 transgenic mice and evaluated the role of VEGF in T-helper type 2 cell (TH2)-mediated inflammation in the lungs of these animals. In these mice, VEGF induced, through IL-13-dependent and independent pathways, an asthma-like phenotype characterized by inflammation, parenchymal and vascular remodeling, edema, mucus metaplasia, myocyte hyperplasia and airway hyper-responsiveness. VEGF was also found to enhance respiratory antigen sensitization and TH2 inflammation and increased the number of activated DC2 dendritic cells in the mice. In antigen-induced inflammation, VEGF was produced predominantly by epithelial cells and preferentially by TH2 as opposed to TH1 cells. In this setting, VEGF demonstrated a critical role in TH2 inflammation, cytokine production and physiologic dysregulation. Thus, VEGF is a mediator of vascular and extravascular remodeling, inflammation, and vascular permeability/edema that enhances antigen sensitization and is crucial in adaptive TH2 inflammation. Disruption of VEGF is therefore expected to be of therapeutic significance in the treatment of asthma and other TH2 disorders. The transgenic mice described by Lee et al., 2004, Nature Medicine, 10, 1095-1103 can be used in preclinical models of asthma and other respiratory diseases that utilize treatment of such mice with siNA molecules of the invention, for example via pulmonary delivery approaches as a known in the art to evaluate the efficacy of siNA molecules in the treatment of respiratory disease. Such studies would be useful in the pre-clinical setting to establish parameters of use in treating human subjects.
Other animal models are useful in evaluating siNA molecules of the invention in the treatment of respiratory disease. For example, Kuperman et al., 2002, Nature Medicine, 8, 885-9, describe an animal model of IL-13 mediated asthma response animal models of allergic asthma in which blockade of IL-13 markedly inhibits allergen-induced asthma. Venkayya et al., 2002, Am J Respir Cell Mol. Biol., 26, 202-8 and Yang et al., 2001, Am J Respir Cell Mol. Biol., 25, 522-30 describe animal models of airway inflammation and airway hyperresponsiveness (AHR) in which IL-4/IL-4R and IL-13 mediate asthma. These models can be used to evaluate the efficacy of siNA molecules of the invention targeting, for example, IL-4, IL-4R, IL-13, and/or IL-13R for use is treating asthma.
Example 9
A Phase I, Open-Label, Dose Escalation Trial of a Single Intravitreal Injection of Sirna-027TM in Patients with Subfoveal Choroidal Neovascularization (CNV) Secondary to Age-Related Macular Degeneration (AMD)
A Phase I study was conducted to demonstrate the safety and tolerability of single ascending doses via intravitreal injection of Sirna-027, (Compound No. 31270/31273, Table III) and as proof of principle of biological activity of Sirna-027 in patients with CNV secondary to AMD. Preclinical findings suggest that blocking the VEGF pathway may result in meaningful responses in animal models of CNV. It is believed that the direct delivery of Sirna-027 into the vitreous humor to block the VEGF pathway may also result in clinical responses.
Sirna-027 was studied as a single agent in patients with CNV. The rationale for the doses in this Phase I trial was supported by the preclinical toxicology program, particularly from data obtained in monkeys. Sirna-027 had no significant adverse effects at doses up to 750 μg/eye. The benign preclinical safety profile, combined with the efficacy suggested by multiple rodent studies, justifies the conduct of a Phase I study in AMD patients.
Study Objectives
The Study Objectives Include:

    
    
        To assess the safety, tolerability, and dose-limiting toxicity (DLT) of a single dose of Sirna-027 when administered by intravitreal injection
        To assess the plasma concentrations of Sirna-027 following a single administration by intravitreal injection
        To determine the range of doses appropriate for use in Phase II trials of Sirna-027
        To assess anatomical changes in exudation, retinal thickening and size of CNV
        To assess changes in visual acuity

Inclusion Criteria

    
    


The following inclusion criteria are required for study entry:

    
    
        1. Patients presenting with subfoveal CNV secondary to wet AMD as confirmed by fundus microscopy and fluorescein angiography
        2. ≧50 years and in general good health. Women must be post-menopausal or otherwise not of childbearing potential.
        3. Best corrected visual acuity in the study eye of equal to or worse than 20/100 but not worse than 20/800 on the ETDRS chart at 4 meters
        4. Uncorrected visual acuity in fellow eye not worse than 20/800
        5. Clear ocular media and adequate pupillary dilatation to permit good quality stereoscopic fundus photography
        6. Subfoveal CNV (classic and/or occult CNV)
        7. CNV lesion thickness ≧250 μm by OCT assessment
        8. Intraocular pressure of 25 mmHg or less
        9. Patients with concomitant eye disease such as controlled glaucoma (25 mmHg or less on treatment) may be enrolled
        10. Patients not eligible for or who refuse standard treatment

Exclusion Criteria

    
    


The following will exclude a patient from entry onto the study:

    
    
        1. Women of childbearing potential
        2. Significant media opacities including cataract, which might interfere with visual acuity, assessment of toxicity, or fundus photography
        3. Presence of other causes of CNV including pathologic myopia (spherical equivalent of −8 diopters or more), the ocular histoplasmosis syndrome, angioid streaks, choroidal rupture, and multifocal choroiditis
        4. Any intraocular surgery or treatment of AMD with Visudyne, or other standard or experimental treatments, within 3 months of study entry
        5. Blood occupying >50% of the lesion
        6. Presence of subfoveal scarring >50% of lesion
        7. CNV lesion ≧12 MPS disc area
        8. Previous or concomitant therapy with another investigational agent to treat AMD (except multivitamins and trace minerals) within the last 90 days
        9. Any of the following underlying systemic diseases, including uncontrolled diabetes mellitus or presence of diabetic retinopathy, cardiac disease including myocardial infarction within 12 months before study entry, coronary disease associated with clinical symptoms, stroke (within 12 months of study entry), active bleeding disorders, any major surgical procedure within 1 month of study entry, active peptic ulcer disease with bleeding within 6 months of study entry, and concomitant systemic therapy with corticosteroids (e.g. oral prednisone) or other antiangiogenic drugs (e.g. thalidomide).
        10. Patients on anti-coagulant therapy. However, if the anti-coagulant can be withheld for 3-7 days prior to injection of Sirna-027, patient may be enrolled. Patient eligibility and length of time that anti-coagulant therapy is withheld must be obtained in consultation with the treating physician.

Treatments

Administration and Criteria for Dose Escalation

    
    


Cohorts of 3 patients were treated at each dose level (see below). Cohort 1 received a single intravitreal injection of Sirna-027 of 100 μg. The dose was increased for patients in subsequent cohorts according to the series of preplanned steps described below. Dose escalation for subsequent patients occurred only after sufficient time had passed to observe any acute toxic effects for patients treated at lower doses. Patients in subsequent cohorts received a single intravitreal injection of Sirna-027 at the next planned higher dose only after review of patient data through Study Day 14 of the cohort.
If no DLT was seen at a dose level then the dose can be escalated for the next cohort, or at the Investigator's option and by agreement with the Sponsor, additional patients may be treated at the current dose
If a DLT was seen in one patient in the 3-patient dose cohort, then three more patients would be treated at the same dose level. If no further cases of DLT are seen in these additional patients, then the dose level would escalated for the next cohort. Otherwise dose escalation was stopped.
If 2 or more patients in the 3-patient dose cohort have a DLT, then dose escalation will stop
If DLTs are seen, at the Investigators' option and by agreement with the Sponsor, additional patients may be treated at current, lower, or intermediate doses










Planned and potential dose levels











Planned or Potential Dose
Cohort
Dose







Planned
1
 100 μg



Planned
2
 200 μg



Planned
3
 400 μg



Planned
4
 800 μg



Potential
5
1200 μg



Potential
6
1800 μg




















Safety margin for clinical doses based on the NOAEL in monkey











Single-Dose NOAEL in




Planned or
Monkey Eye1
Clinical Dose2
Safety












Potential

Monkey Vitreal

Human Vitreal
Margin


Dose
Dose
Concentration
Dose
Concentration
(ratio)















Planned
750 μg
234 μg/mL
 100 μg
 25 μg/mL
9.43


Planned
750 μg
234 μg/mL
 200 μg
 50 μg/mL
4.7


Planned
750 μg
234 μg/mL
 400 μg
100 μg/mL
2.3


Planned
750 μg
234 μg/mL
 800 μg
200 μg/mL
1.2


Potential
750 μg
234 μg/mL
1200 μg
300 μg/mL
0.8


Potential
750 μg
234 μg/mL
1800 μg
450 μg/mL
0.5





1750 μg in 3.2 mL vitreous volume (monkey) = 234 μg/mL


2Human vitreous volume of 4.0 mL was used


3Reported as 9.2 in Error! Reference source not found. and in the IDB as a result of rounding differences when mg vs μg are used






List of Potential DLT(s) Observed at Day 14 Visit

An ophthalmic DLT is defined as any toxicity (AE or SAE) determined by the Investigator to be related to the study drug and of such intensity or severity to preclude dose escalation.
By Clinical Examination:


    
    
        Formation of cataract as judged by Investigator to be secondary to the injection of study drug.
        Severe clinically significant inflammation that obscures visualization of the retinal vasculature and/or threatens the patient's vision.
        Other ocular abnormalities not usually seen in patients with AMD, such as retinal, arterial, or venous occlusion, acute retinal detachment, and diffuse retinal hemorrhage.
        Visual acuity: doubling or worsening of the visual angle (loss of ≧15 letters), unless the loss of vision is due to a vitreous hemorrhage related to the injection procedure.
        Tonometry: increase from baseline of intraocular pressure by ≧25 mmHg on two separate examinations at least 1 day apart or a sustained pressure of 30 mmHg for more than a week despite pharmacologic intervention.

By Fluorescein Angiogram:

        Significant retinal or choroidal vascular abnormalities not seen at baseline, such as:
        Choroidal non-perfusion (affecting one or more quadrants)
        Delay in arteriovenous transit times (>15 seconds)
        Retinal arterial or venous occlusion (any deviation from baseline)
        Diffuse retinal permeability alteration affecting retinal circulation in the absence of intraocular inflammation
        Systemic DLT(s) to include Grade III (severe) or IV (life-threatening) toxicities or any significant severe toxicity deemed related to study drug by the Investigator.

Identity of Investigational Product

    
    


Sirna-027 was manufactured by Sirna Therapeutics. The drug product was supplied in 5 mL glass vials that were stored frozen, −25 to −15° C. (−13 to +5° F.). The drug storage area was secure and have limited access. Labeled drug was shipped to the site by the Sponsor or an approved vendor once the necessary regulatory documents were received from the site.
Stability studies to support drug storage under these conditions were conducted by the Sponsor. The Sponsor will continue to monitor the stability and alert the site if a lot needs to be replaced for stability reasons. A Material Safety Data Sheet was provided for Sirna-027.
Sample label text for the drug product is shown below.













Sirna-027 TM
5 mg Lyophilized Powder


Lot # xxxxxx
Single use vial









Store frozen, −25 to −15° C. (−13 to +5° F.)



CAUTION: New Drug - Limited by Federal law to investigational



use.



Sirna Therapeutics, Inc., Boulder, CO USA










Reconstitution for Dosing

Sirna-027 was supplied as a 5 mg lyophilized powder in a 5 mL vial. A phosphate buffered saline solution supplied by Sirna was added to each vial to reconstitute to the appropriate concentration for each dose level. Injection volume was 100 μL for all doses.










Reconstitution schema













Phosphate Buffered





Saline Solution



Dose Group
Clinical Dose
(to add to vial)







Cohort 1
100 μg
 5.0 mL



Cohort 2
200 μg
 2.5 mL



Cohort 3
400 μg
 1.25 mL



Cohort 4
800 μg
0.625 mL



Cohort 5
1200 μg 
0.417 mL



Cohort 6
1800 μg 
0.277 mL










Method for Assigning Patients to the Treatment Group

Patient numbers were assigned sequentially by site. The first two numbers of the five-digit patient number identify the site and the last three numbers were assigned sequentially to the patients being placed on the study.
Selection of Doses in the Study
The starting dose was chosen to be approximately one-tenth of the NOAEL seen in the single dose monkey toxicity study. Dose escalation was based on a doubling of the planned doses up to 800 μg, and 50% incremental increase if additional patients receive doses above 800 μg. Planned dosing was in 4 cohorts of 3 patients each. Impact of possible DLT on dose selection is given above.
Administration of Sirna-027
Procedures were implemented to minimize the risk of potential adverse events associated with intraocular injection (e.g., retinal detachment and endophthalmitis). Aseptic technique was observed by clinic staff involved in the injection tray assembly, anesthetic preparation and administration, and study drug preparation and administration. In addition to the procedures outlined in the protocol, added safety measures in adherence to specific institutional policies associated with intraocular injections were observed, including a complete ophthalmic surgical prep and drape.
Sirna-027 was administered in the study eye only. Following the slit-lamp examination, intravitreal injection of Sirna-027 was performed by a qualified ophthalmologist in either a surgical suite or an appropriately equipped procedure room. The patient was placed in a supine position on a surgical bed or examination chair. After thorough cleansing of the lid, lashes, and periorbital area with an antiseptic, a lid speculum was inserted. Local anesthesia and topical antimicrobials were administered prior to study drug injection.
A 30-gauge, 1⁄2-inch needle, attached to a low-volume (e.g., tuberculin) syringe containing 100 μL of various concentrations of Sirna-027 was inserted through the pre-anesthetized conjunctiva and sclera, approximately 3.5-4.0 mm posterior to the limbus, avoiding the horizontal meridian and aiming toward the center of the globe. The injection volume was delivered slowly. The needle was then be removed slowly to ensure that all drug solution remained in the eye.
Following the intraocular injection, the patients remained at the study site for at least 4 hours. Indirect ophthalmoscopy was performed to determine status of optic nerve perfusion and presence of any retinal pathology such as hemorrhage or tear. Intraocular pressure was measured before and 30 minutes after the Sirna-027 injection. Any patient who developed significantly raised intraocular pressure (≧28 mmHg) at any time during the study was monitored and managed (i.e. paracentesis) according to the physician's clinical judgment and could undergo additional measurements of intraocular pressure beyond those specified in the protocol. If there were no safety concerns in the 4 hours following the injection, the patient could leave the clinic. If any concern or immediate toxicity is noted, the patient would remain at the clinic and would be treated according to the physician's clinical judgment. Patients returned the day after the injection to be evaluated for change in vision, measurement of intraocular pressure, and assessment of eye pain, unusual redness, or any other new ocular symptoms. At any time during the study, if it was determined necessary by the evaluating physician, the patient was asked to return to the clinic as soon as possible for an unscheduled study visit and was evaluated by the ophthalmologist. Patients were instructed to contact their physician at any time should they have health-related concerns.
In addition, antimicrobial drops for the eye (i.e. ofloxacin) were administered immediately after the intravitreal injection, and the patient was instructed to self-administer antimicrobial drops four times daily for 3 days following the intraocular injection of Sirna-027.
Concomitant Medications
Patients already taking multivitamins and trace minerals for AMD could continue these treatments. Patients could not begin these therapies during the first 84 days of this trial. Patients were allowed to continue topical medications for other existing conditions (i.e. glaucoma drops). Subsequent to the injection of Sirna-027, patients were instructed to use topical antibiotics eye drops for three days.
Treatment Compliance
This was a single injection administered by the Investigators at the study sites.
Study Procedures
The various assessments that were conducted during this study are described in this section in narrative form, listed by study visit, and in tabular form (see below).
A complete ophthalmic evaluation with fundus photography, fluorescein angiography, OCT, and Visual Acuity Test was performed prior to study start and at the Study Day 7, 14, 28, and 84. An eye exam including visual acuity, tonometry, slit lamp biomicroscopy and OCT was performed at Study Day 56. A streamlined physical examination (heart, lungs, abdomen, neurology) was performed prior to study start was repeated at Study Day 84 or in case of premature termination from the study.
Patients were monitored for adverse events (AEs) through Study Day 84. Adverse events beginning within this evaluation period (84 days) of this study were followed to resolution. Patients are monitored for serious adverse events (SAEs) through month 24.
Patient safety follow-up consists of routine eye examinations at 6, 9, 12, 18 and 24 months following injection. At these visits patient will be queried regarding any untoward events related to their eye health.
It was anticipated that patients would not need additional medical treatment for AMD during the 84 days of the study evaluation period. During the safety follow-up period (months 3 to 24), patients are allowed to receive medically-indicated treatment (i.e. PDT, laser photocoagulation) as deemed necessary by the evolution of the disease and by the judgment of the treating physician.
Screening Evaluations
Within Two Weeks of the Day 1 Visit


    
    
        Signed informed consent (informed consent must be obtained prior to performance of any study specific tests or evaluations)
        Complete ophthalmic examination (to include visual acuity assessment, slit lamp biomicroscopy-evaluation of lashes, lids, conjunctivae, cornea, anterior chamber, iris, lens, vitreous, and the performance of tonometry, fundus photography, FA and OCT)
        Assess eligibility—confirmation of eligibility to be determined by review of fundus photography, fluorescein angiography and OCT by the Wilmer Retinal Imaging Research and Reading Center
        Demographics
        Streamlined physical exam (heart, lungs, abdomen, neurology)
        Vital signs
        Medical history
        Clinical labs

Evaluations

Baseline/Day 1

        Medical History Update (prior to injection)
        Vital signs (prior to injection)
        Visual acuity test (prior to injection)
        Tonometry (prior to injection)
        Adverse events (including injection to 4 hours post-injection)
        PK blood draws will be done prior to the intravitreal injection of Sirna-027 and at hours 1 and 4 following the injection
        Administer Sirna-027 intravitreal injection, observe patient for 4 hours after injection. (Patient's study eye will be examined at 30 minutes post injection to ensure adequate ocular perfusion and normal intraocular pressure. If pressure exceeds 28 mmHg, the physician may choose to perform a post-injection paracentesis.)

Day 2

        Vital signs, adverse event and concomitant medication assessment
        Eye examination to include visual acuity, tonometry, slit lamp biomicroscopy, and assessment of eye pain, unusual redness, or other new ocular symptoms
        PK blood draw (24 hours post administration)

Day 7

        Vital signs, adverse event and concomitant medication assessment
        PK blood draw
        Complete ophthalmic examination

Day 14

        Vital signs, adverse event and concomitant medication assessment
        Complete ophthalmic examination

Day 28

        Vital signs, adverse events and concomitant medication assessment
        Complete ophthalmic examination

Day 56

        Vital signs, adverse events and concomitant medication assessment
        Eye examination to include visual acuity, tonometry, slit lamp biomicroscopy and OCT

Day 84/Early Termination Visit (Prior to Day 84)

        Streamlined physical exam (heart, lungs, abdomen, neurology)
        Vital signs, adverse events and concomitant medication assessment
        Clinical labs
        Complete ophthalmic examination

Follow-Up Visit-Month 6

        Routine eye exam (to include visual acuity assessment, slit lamp biomicroscopy—evaluation of lashes, lids, conjunctivae, cornea, anterior chamber, iris, lens, vitreous, applanation tonometry and dilated fundus examination) and fundus photography.

Follow-Up Visit-Month 9

        Routine eye exam

Follow-Up Visit-Month 12

        Routine eye exam and fundus photography

Follow-Up Visit-Month 18

        Routine eye exam and fundus photography

Follow-Up Visit-Month 24

        Routine eye exam and fundus photography
    
    












Schedule of procedures/assessments





























Follow-
Follow-
Follow-
Follow-
Follow-




Baseline/





Day 84/
up
up
up
up
up




Day
Day
Day
Day
Day
Day
Early
Month
Month
Month
Month
Month


PROCEDURES
Screen
1
2
71
142
282
562
Term.2
6
9
12
18
24





Administer

X













Intravitreal Injection


of Sirna-027


Sign Informed
X


Consent


Assess Eligibility
X


Demographics
X


Physical Exam
X






X


Vital Signs
X
X
X
X
X
X
X
X


Concomitant Meds


X
X
X
X
X
X


Adverse Event

X
X
X
X
X
X
X


Medical History
X
X


Clinical Labs
X






X


Visual Acuity Test
X
X
X
X
X
X
X
X
X
X
X
X
X


Tonometry
X
X
X
X
X
X
X
X
X
X
X
X
X


Complete ophthalmic
X


X
X
X

X


examination3


Slit lamp


X



X


biomicroscopy


Routine Eye Exam4








X5
X
X5
X5
X3


OCT






X


PK Blood Draws

X6
X7
X





1±1 day


2±2 days


3Includes visual acuity assessment, slit lamp biomicroscopy (evaluation of lashes, lids, conjunctivae, cornea, anterior chamber, iris, lens, vitreous), and the performance of tonometry, fundus photography, FA and OCT.


4Includes visual acuity assessment, slit lamp biomicroscopy (evaluation of lashes, lids, conjunctivae, cornea, anterior chamber, iris, lens, vitreous), applanation tonometry and dilated fundus examination.


5Plus fundus photography


6Pre-injection, 1 and 4 hours post-injection


724 hours post-injection






Safety Assessments

Adverse Events (AEs)

It is the responsibility of the Investigator to document all AEs that occur after the patient receives their single dose of Sirna-027. An AE includes any noxious, pathological, or unintended change in anatomical, physiological, or metabolic functions as indicated by physical signs, symptoms, and/or laboratory changes occurring in any phase of the clinical study after the single administration of study drug whether associated with the study drug and whether or not considered drug related. This includes an exacerbation of pre-existing medical conditions or events, intercurrent illnesses, nonallergic reactions, drug interactions, or the significant worsening of the disease under investigation (AMD). Anticipated day-to-day fluctuations of pre-existing conditions that do not represent a clinically significant exacerbation or worsening need not be considered AEs.
An AE is considered to be associated with the use of the study drug if the relationship between the AE and the drug is classified by the Investigator as “possibly related” or “probably related.” An unexpected AE is any adverse drug experience that is not consistent with the specificity or severity as stated in the current Investigational Drug Brochure.
Assessment of Severity
Intensity should be assigned using severity grades as outlined in the Case Report Form Booklets (to be provided separately).
Assessment of Causality
The degree of certainty with which an AE is attributed to drug treatment (or alternative causes, e.g., natural history of the underlying diseases, concomitant therapy, etc.) will be determined by how well the event can be understood in terms of one or more of the following:

    
    
        Known pharmacology of the investigational drug
        Reaction of similar nature being previously observed with the investigational drug or the class of drug
        The event having been often reported in literature for a similar drug as drug related (e.g., skin rashes, and blood dyscrasia)
        The event being temporally related to study drug exposure, including termination of the event after study drug withdrawal (dechallenge) or recurrence on rechallenge with study drug
        Every effort should be made by the Investigator to explain each AE and assess its relationship, if any, to study drug treatment. Due to the nature of the intravitreal injection, an AE or SAE may be related to the procedure, but not to the study drug. Judgment of relatedness to procedure or study drug will be at the discretion of the Investigator. Causality should be assessed using the following categories: not related, possibly related, probably related, and related. The definitions of these categories are:

Not Related:

    
    


If there is no exposure to investigational drug, then, the AE is not related; OR, the occurrence of the AE is not reasonably related in time, and may be due to other factors such as an intercurrent illness, concomitant medication, or underlying disease state
Possibly Related:
The administration of the investigational drug and AE are considered reasonably related in time AND the AE could be explained by causes other than exposure to the investigational drug
Probably Related:
Exposure to the investigational drug and AE are reasonably related in time AND the investigational drug is more likely than other causes to be responsible for the AE, OR is the most likely cause of the AE
Related:
A reaction from administration of the investigational drug that follows a known response pattern to the suspected investigational drug and can be confirmed with a positive re-challenge test or supporting laboratory data
Follow up of Adverse Events
Investigators should follow up patients with AEs until one of the following:

    
    
        The event has subsided or disappeared
        The condition has stabilized
        The event is otherwise resolved
        The patient is lost to follow-up
        The study ends
    
    


AEs not resolved at Study Day 84 will be followed to resolution. After Study Day 84, only AEs qualifying as serious (SAEs) will be captured and followed. Beyond Study Day 84, patient safety follow-up will consist of the routine ophthalmic evaluation at Months 6, 9, 12, 18 and 24. If, during the follow-up period, a patient requires medically-indicated ocular treatment, e.g. PDT or laser coagulation, then the patient will be considered to have completed his/her participation in this study and no further follow-up will be done.
Pregnancies
Pregnancies are not considered to be AEs or SAEs; however, pregnancies will be followed through outcome. In pregnancies that progress to term, any congenital abnormalities in the offspring of a patient who received study drug should be reported as an SAE.
Serious Adverse Events
Definition of a Serious Adverse Events:
A serious adverse event is defined as any untoward medical occurrence that at any dose:

    
    
        Results in death
        Is life threatening (i.e., the patient was, in the opinion of the Investigator, at immediate risk of death from the event as it occurred)
        Requires or prolongs inpatient hospitalization
        Results in persistent or significant disability/incapacity, (i.e., the event causes a substantial disruption of a person's ability to conduct normal life functions)
        Is a congenital anomaly/birth defect
        Is an important and significant medical event that, based upon appropriate medical judgment, may jeopardize the patient or may require medical or surgical intervention to prevent one of the other outcomes defining serious

Reporting Serious Adverse Experiences

    
    


The Investigator must report any SAEs that occur from study entry through end of study follow-up (Month 24), whether or not considered related to the study drug, to Sponsor by fax within 24 hours. If, during the follow-up period, a patient requires medically indicated ocular treatment, e.g. PDT or laser coagulation, then the patient will be considered to have completed his/her participation in this study and no further reporting of SAEs is required. Specific reporting information and forms are found in the Clinical Study Binder.
Investigators should not wait to receive additional information to fully document the event before notifying Sirna Therapeutics of an SAE. A full written summary detailing relevant aspects of the SAEs should follow the fax report in question. Where applicable, information from relevant hospital case records and autopsy reports should be obtained. The SAE should be recorded on the AE page of the patient's case report form (CRF).
Any deaths that occur between the date of the administration of Sirna-027 through the Month 24 visit are to be reported as SAEs.
The Investigator is responsible for informing his/her Institutional Review Board/Institutional Ethics Committee (IRB/IEC) as soon as possible, but in no event later than 15 working days after the discovery of death from any cause, or SAEs, or problems of medical significance that may reasonably be regarded as caused by or associated with the study drug.
Study Outcome
Twenty-three patients have been enrolled and dosed in Cohorts 1 through 6 (100 μg-1600 μg). Patient demographics are shown below. Seven of the patients were enrolled at the Cole Eye Institute and the other sixteen were enrolled at the Wilmer Eye Institute.










Sirna 0401 Patient Demographics












Dose Cohort
Patient
Gender
Age







100 μg
03-001
F
78




01-001
F
93




03-002
F
84




01-002
M
83



200 μg
01-003
F
75




01-004
M
82




01-005
M
68



400 μg
01-006
F
89




01-007
M
85




01-008
F
85



800 μg
01-009
M
77




01-010
F
70




03-003
F
87




03-004
M
81




01-011
F
76




01-012
F
82



1200 μg 
01-013
F
87




01-014
F
84




03-005
F
82




03-006
F
80




01-015
F
81




01-016
M
84



1600 μg 
03-008
F
85










Safety

There have been no dose limiting toxicities or safety issues to date with any of the 23 patients. Of the adverse events recorded, all but one (a moderate case of diarrhea not related to the study drug) have been considered mild. They were all reversible and none have been definitively attributed to the drug.
Effect on Visual Acuity (VA)
Preliminary review of data on the effect of Sirna-027 on VA for 22 patients was conducted which demonstrated stabilization of VA in all patients, a variable mean increase of number of letters read for each dose group, and a duration of effect sustained over 8 weeks post-injection (FIG. 30). At the same assessment point of 8 weeks, VA (number of lines read) was stable or improved in 100% of the patients, while a clinically significant improvement (equal to or more than 3 lines) was observed in 23% of patients (FIG. 31).
OCT Results
Preliminary data on central foveal thickness for the first four dose groups (100, 200, 400 and 800 μg) showed a decrease of the lesion thickness for the 100, 200 and 800 μg doses, with maximum effect by 14 days post-injection (FIG. 32).
Example 10
A Phase IIa, Multi-Center, Randomized, Masked, Sham- or Positive Controlled, Dose Finding Trial of Multiple Intravitreal Injections of Sirna-027TM in Patients with Subfoveal Choroidal Neovascularization (CNV Secondary to Age-Related Macular Degeneration (AMD)
This study is a Phase IIa multicenter study consisting of 112-day investigational period following 3 injections of Sirna-027 with a follow-up for a total of 24 months. The primary objectives of the study are:

    
    
        To assess the safety, tolerability, and dose-limiting toxicity (DLT) of multiple doses of Sirna-027 when administered by multiple intravitreal injections.
        To determine dose appropriate for use in confirmatory Phase IIb/III trials of Sirna-027 based on changes in visual acuity, and anatomical changes in exudation, retinal thickening and size of CNV in a sub-population of patients.
        To assess the plasma concentrations of Sirna-027 following administration of multiple intravitreal injections.
    
    


The methodolgy of the study consists of:

    
    
        Masked, sham-controlled, (or positive-control) randomized, dose-finding trial.
        All patients will receive 3 intravitreal injection of Sirna-027 or 3 sham procedures (or positive control) at 4, 6, or 8-week intervals.
        Patients will be enrolled in 4 dose-cohorts of 30 evaluable patients each (120 patients total). Treatment in each dose cohort will consist of either 3 sham-procedures (or 3 injection of positive control), or 3 injections of Sirna-027 (both masked to the patient). Enrollment into these 4 dose-cohorts will be by a centralized randomization. The 3 dose levels of Sirna-027 will be the MTD established in Phase I and two lower doses.
        Patient's blood will be assayed for Sirna-027 at Baseline/Study Day 1 (prior to injection, hours 1 and 4 following injection), Study Day 2 (24 hours following injection), Study Day 7, and Study Day 56 following the 3rd injection.
        Patient's visual acuity will be measured beginning at a distance of 4 meters using the Early Treatment Diabetic Retinopathy Study (ETDRS) chart at every study visit.
        Patient's study eye will be examined (fundus and tonometry) at 30 minutes post injection to ensure adequate ocular perfusion and normal intraocular pressure. If pressure exceeds 28 mmHg, the patient will be re-examined at 60 minutes. If the pressure still exceeds 28 mmHg, the physician may choose to perform a post-injection paracentesis. Patients will be observed for 4 hours following the first injection. Remaining injections 2 and 3 require the 30 and 60 minute post injections examination, but will not require the extended 4 hour observation period.
        Patients will receive an eye exam including visual acuity, tonometry, slit lamp biomicroscopy and a complete ophthalmic evaluation, and fundus photography, at screening/baseline and at Study Days 7, 14, 28, 56, 84, and 112 Patients will receive a routine eye exam at the safety follow-up visits at months 6, 9, 12, 18 and 24.
        Fundus photography, OCT, and fluorescein angiography evaluations will be done by an independent reading center on a sub-population of patients.
    
    


Opthalmic Inclusion Criteria for the Study Include:

    
    
        Subfoveal CNV (classic and/or occult), secondary to AMD as confirmed by fundus microscopy and fluorescein angiography, with a total lesion size (including blood, scar/atrophy and neovascularization) of ≦12 MPS disc areas, of which at least 50% has to be active CNV.
        BCVA in the study eye between 20/80 and 20/400, and better than or equal to 20/800 in the fellow eye.
        Clear ocular media and adequate papiliary dilatation to permit good quality stereoscopic fundus photography.
        Subretinal hemorrhage less than 50% of total lesion size.
        CNV lesion thickness ≧250 μm by OCT assessment.
        Intraocular pressure of 25 mmHg or less
        Patients with concomitant eye disease such as controlled glaucoma (25 mmHg or less on treatment) may be enrolled.
        Patients not eligible for or who refuse standard treatment.
    
    


General Inclusion Criteria for the Study Include:

    
    
        ≧50 years and in general good health. Women must be post-menopausal or otherwise not of childbearing potential.
        Written informed consent.
        The ability to return for all study visits.
    
    


Exclusion Criteria for the Study Include:

    
    
        Women of childbearing potential
        Significant media opacities including cataract, which might interfere with visual acuity, assessment of toxicity, or fundus photography
        Presence of other causes of CNV including pathologic myopia (spherical equivalent of −8 diopters or more), the ocular histoplasmosis syndrome, angioid streaks, choroidal rupture, and multifocal choroiditis
        Any intraocular surgery or treatment of AMD with Visudyne, Macugen, or other standard or experimental treatments, within 3 months of study entry
        Blood occupying >50% of the lesion
        Presence of subfoveal scarring >50% of lesion
        CNV lesion ≧12 MPS disc area
        Previous or concomitant therapy with another investigational agent to treat AMD (except multivitamins and trace minerals) within the last 90 days
        Any of the following underlying systemic diseases:
        
            Uncontrolled diabetes mellitus
            Diabetic retinopathy
            Cardiac disease including myocardial infarction within 12 months before study entry
            Coronary disease associated with clinical symptoms
            Stroke (within 12 months of study entry)
            Active bleeding disorders
            Any major surgical procedure within 1 month of study entry
            Active peptic ulcer disease with bleeding within 6 months of study entry
            Concomitant systemic therapy with corticosteroids (e.g. oral prednisone) or other antiangiogenic drugs (e.g. thalidomide).
        
        
        If patient is on anti-coagulant therapy, the anti-coagulant may be withheld for 3-7 days prior to each injection of Sirna-027. Patient eligibility and length of time that anti-coagulant therapy is withheld must be obtained in consultation with the treating physician.
    
    


Test Product: Sirna-027 will be supplied as a lyophilized powder in a vial. A phosphate buffered solution will be added to each vial to reconstitute to the appropriate concentration for each dose level. Injection volume will be 100 μL
Duration of Treatment: Treatment with Sirna-027 is 3 intravitreal injections at 4, 6, or 8-week intervals. Patients will be followed for 24 months from the date of the first injection.
Criteria for Evaluation: For safety evaluation, the incidence of serious and other adverse events will be determined along with pharmacokinetic analysis to test for the accumulation of drug after 3 injections. For determination of efficacy and dose selection, changes in visual acuity as measured by performance on the ETDRS chart beginning at 4 meters will be determined as well as changes in CNV size, exudation, retinal thickness/elevation at any time following administration of Sirna-027 as measured by fundus photography, fluorescein angiography, and OCT in a sub-population of patients.
Statistical Methods: Serious and other adverse events will be reported as the number of patients experiencing an event (incidence) and by probable relatedness to the drug and/or the injection procedure. These events will be classified according to the MedDRA medical dictionary and presented by dose cohort. Results from the fundus photography, fluorescein angiography and OCT will be analyzed by masked readers in a central reading center. Individual and average changes in CNV size, exudation, and retinal thickness/elevation will be displayed by dose cohort. Changes in visual acuity scores (ETDRS) post administration of multiple injections of Sirna-027 will be presented by dose. Appropriate algorithms will be used to select the dose for study in Phase III. Lastly, any association of changes in visual acuity and changes in exudation, CNV size, and/or retinal thickness will be described.
Example 11
Indications
The present body of knowledge in VEGF and/or VEGFR research indicates the need for methods to assay VEGF and/or VEGFR activity and for compounds that can regulate VEGF and/or VEGFR expression for research, diagnostic, and therapeutic use. As described herein, the nucleic acid molecules of the present invention can be used in assays to diagnose disease state related of VEGF and/or VEGFR levels. In addition, the nucleic acid molecules can be used to treat disease state related to VEGF and/or VEGFR levels.
Particular conditions and disease states that can be associated with VEGF and/or VEGFR expression modulation include, but are not limited to:
1) Tumor angiogenesis: Angiogenesis has been shown to be necessary for tumors to grow into pathological size (Folkman, 1971, PNAS 76, 5217-5221; Wellstein & Czubayko, 1996, Breast Cancer Res and Treatment 38, 109-119). In addition, it allows tumor cells to travel through the circulatory system during metastasis. Increased levels of gene expression of a number of angiogenic factors such as vascular endothelial growth factor (VEGF) have been reported in vascularized and edema-associated brain tumors (Berkman et al., 1993 J. Clini. Invest. 91, 153). A more direct demostration of the role of VEGF in tumor angiogenesis was demonstrated by Jim Kim et al., 1993 Nature 362,841 wherein, monoclonal antibodies against VEGF were successfully used to inhibit the growth of rhabdomyosarcoma, glioblastoma multiforme cells in nude mice. Similarly, expression of a dominant negative mutated form of the flt-1 VEGF receptor inhibits vascularization induced by human glioblastoma cells in nude mice (Millauer et al., 1994, Nature 367, 576). Specific tumor/cancer types that can be targeted using the nucleic acid molecules of the invention include but are not limited to the tumor/cancer types described herein.
2) Ocular diseases: Neovascularization has been shown to cause or exacerbate ocular diseases including, but not limited to, macular degeneration, including age related macular degeneration (AMD), dry AMD, wet AMD, predominantly classic AMD (PD AMD), minimally classic AMD (MC AMD), and occult AMD; neovascular glaucoma, diabetic retinopathy, including diabetic macular edema (DME) and proliferative diabetic retinopathy; myopic degeneration, uveitis, and trachoma (Norrby, 1997, APMIS 105, 417-437). Aiello et al., 1994 New Engl. J. Med. 331, 1480, showed that the ocular fluid of a majority of patients suffering from diabetic retinopathy and other retinal disorders contains a high concentration of VEGF. Miller et al., 1994 Am. J. Pathol. 145, 574, reported elevated levels of VEGF mRNA in patients suffering from retinal ischemia. These observations support a direct role for VEGF in ocular diseases. Other factors, including those that stimulate VEGF synthesis, may also contribute to these indications.
3) Dermatological Disorders: Many indications have been identified which may beangiogenesis dependent, including but not limited to, psoriasis, verruca vulgaris, angiofibroma of tuberous sclerosis, pot-wine stains, Sturge Weber syndrome, Kippel-Trenaunay-Weber syndrome, and Osler-Weber-Rendu syndrome (Norrby, supra). Intradermal injection of the angiogenic factor b-FGF demonstrated angiogenesis in nude mice (Weckbecker et al., 1992, Angiogenesis: Key principles-Science-Technology-Medicine, ed R. Steiner). Detmar et al., 1994 J. Exp. Med. 180, 1141 reported that VEGF and its receptors were over-expressed in psoriatic skin and psoriatic dermal microvessels, suggesting that VEGF plays a significant role in psoriasis.
4) Rheumatoid arthritis: Immunohistochemistry and in situ hybridization studies on tissues from the joints of patients suffering from rheumatoid arthritis show an increased level of VEGF and its receptors (Fava et al., 1994 J. Exp. Med. 180, 341). Additionally, Koch et al., 1994 J. Immunol. 152, 4149, found that VEGF-specific antibodies were able to significantly reduce the mitogenic activity of synovial tissues from patients suffering from rheumatoid arthritis. These observations support a direct role for VEGF in rheumatoid arthritis. Other angiogenic factors including those of the present invention may also be involved in arthritis.
5) Endometriosis: Various studies indicate that VEGF is directly implicated in endometriosis. In one study, VEGF concentrations measured by ELISA in peritoneal fluid were found to be significantly higher in women with endometriosis than in women without endometriosis (24.1±15 ng/ml vs 13.3±7.2 ng/ml in normals). In patients with endometriosis, higher concentrations of VEGF were detected in the proliferative phase of the menstrual cycle (33±13 ng/ml) compared to the secretory phase (10.7±5 ng/ml). The cyclic variation was not noted in fluid from normal patients (McLaren et al., 1996, Human Reprod. 11, 220-223). In another study, women with moderate to severe endometriosis had significantly higher concentrations of peritoneal fluid VEGF than women without endometriosis. There was a positive correlation between the severity of endometriosis and the concentration of VEGF in peritoneal fluid. In human endometrial biopsies, VEGF expression increased relative to the early proliferative phase approximately 1.6-, 2-, and 3.6-fold in midproliferative, late proliferative, and secretory endometrium (Shifren et al., 1996, J. Clin. Endocrinol. Metab. 81, 3112-3118). In a third study, VEGF-positive staining of human ectopic endometrium was shown to be localized to macrophages (double immunofluorescent staining with CD14 marker). Peritoneal fluid macrophages demonstrated VEGF staining in women with and without endometriosis. However, increased activation of macrophages (acid phosphates activity) was demonstrated in fluid from women with endometriosis compared with controls. Peritoneal fluid macrophage conditioned media from patients with endometriosis resulted in significantly increased cell proliferation ([3H] thymidine incorporation) in HUVEC cells compared to controls. The percentage of peritoneal fluid macrophages with VEGFR2 mRNA was higher during the secretory phase, and significantly higher in fluid from women with endometriosis (80±15%) compared with controls (32±20%). Flt-mRNA was detected in peritoneal fluid macrophages from women with and without endometriosis, but there was no difference between the groups or any evidence of cyclic dependence (McLaren et al., 1996, J. Clin. Invest. 98, 482-489). In the early proliferative phase of the menstrual cycle, VEGF has been found to be expressed in secretory columnar epithelium (estrogen-responsive) lining both the oviducts and the uterus in female mice. During the secretory phase, VEGF expression was shown to have shifted to the underlying stroma composing the functional endometrium. In addition to examining the endometium, neovascularization of ovarian follicles and the corpus luteum, as well as angiogenesis in embryonic implantation sites have been analyzed. For these processes, VEGF was expressed in spatial and temporal proximity to forming vasculature (Shweiki et al., 1993, J. Clin. Invest. 91, 2235-2243).
6) Kidney disease: Autosomal dominant polycystic kidney disease (ADPKD) is the most common life threatening hereditary disease in the USA. It affects about 1:400 to 1:1000 people and approximately 50% of people with ADPKD develop renal failure. ADPKD accounts for about 5-10% of end-stage renal failure in the USA, requiring dialysis and renal transplantation. Angiogenesis is implicated in the progression of ADPKD for growth of cyst cells, as well as increased vascular permeability promoting fluid secretion into cysts. Proliferation of cystic epithelium is a feature of ADPKD because cyst cells in culture produce soluble vascular endothelial growth factor (VEGF). VEGFR1 has been detected in epithelial cells of cystic tubules but not in endothelial cells in the vasculature of cystic kidneys or normal kidneys. VEGFR2 expression is increased in endothelial cells of cyst vessels and in endothelial cells during renal ischemia-reperfusion.
7) Respiratory/Inflammatory Disease: Exaggerated levels of VEGF are present in subjects with asthma, but the role of VEGF in normal and asthmatic lungs has not been well defined. Lee et al., 2004, Nature Medicine, 10, 1095-1103, generated lung-targeted VEGF165 transgenic mice and evaluated the role of VEGF in T-helper type 2 cell (TH2)-mediated inflammation in the lungs of these animals. In these mice, VEGF induced, through IL-13-dependent and independent pathways, an asthma-like phenotype characterized by inflammation, parenchymal and vascular remodeling, edema, mucus metaplasia, myocyte hyperplasia and airway hyper-responsiveness. VEGF was also found to enhance respiratory antigen sensitization and TH2 inflammation and increased the number of activated DC2 dendritic cells in the mice. In antigen-induced inflammation, VEGF was produced predominantly by epithelial cells and preferentially by TH2 as opposed to TH1 cells. In this setting, VEGF demonstrated a critical role in TH2 inflammation, cytokine production and physiologic dysregulation. Thus, VEGF is a mediator of vascular and extravascular remodeling, inflammation, and vascular permeability/edema that enhances antigen sensitization and is crucial in adaptive TH2 inflammation. Disruption of VEGF is therefore expected to be of therapeutic significance in the treatment of asthma and other TH2 disorders including allergic rhinitis, COPD, and airway sensitization/inflammation.
The use of radiation treatments and chemotherapeutics, such as Gemcytabine and cyclophosphamide, are non-limiting examples of chemotherapeutic agents that can be combined with or used in conjunction with the nucleic acid molecules (e.g. siNA molecules) of the instant invention. Those skilled in the art will recognize that other anti-cancer compounds and therapies can similarly be readily combined with the nucleic acid molecules of the instant invention (e.g. siNA molecules) and are hence within the scope of the instant invention. Such compounds and therapies are well known in the art (see for example Cancer: Principles and Practice of Oncology, Volumes 1 and 2, eds Devita, V. T., Hellman, S., and Rosenberg, S. A., J. B. Lippincott Company, Philadelphia, USA; incorporated herein by reference) and include, without limitation, folates, antifolates, pyrimidine analogs, fluoropyrimidines, purine analogs, adenosine analogs, topoisomerase I inhibitors, anthrapyrazoles, retinoids, antibiotics, anthacyclins, platinum analogs, alkylating agents, nitrosoureas, plant derived compounds such as vinca alkaloids, epipodophyllotoxins, tyrosine kinase inhibitors, taxols, radiation therapy, surgery, nutritional supplements, gene therapy, radiotherapy, for example 3D-CRT, immunotoxin therapy, for example ricin, and monoclonal antibodies. Specific examples of chemotherapeutic compounds that can be combined with or used in conjuction with the nucleic acid molecules of the invention include, but are not limited to, Paclitaxel; Docetaxel; Methotrexate; Doxorubin; Edatrexate; Vinorelbine; Tomaxifen; Leucovorin; 5-fluoro uridine (5-FU); Ionotecan; Cisplatin; Carboplatin; Amsacrine; Cytarabine; Bleomycin; Mitomycin C; Dactinomycin; Mithramycin; Hexamethylmelamine; Dacarbazine; L-asperginase; Nitrogen mustard; Melphalan, Chlorambucil; Busulfan; Ifosfamide; 4-hydroperoxycyclophosphamide; Thiotepa; Irinotecan (CAMPTOSAR®, CPT-11, Camptothecin-11, Campto) Tamoxifen; Herceptin; IMC C225; ABX-EGF; and combinations thereof. Non-limiting examples of therapies and compounds that can be used in combination with siNA molecules of the invention for ocular based diseases and conditions include submacular surgery, focal laser retinal photocoagulation, limited macular translocation surgery, retina and retinal pigment epithelial transplantation, retinal microchip prosthesis, feeder vessel CNVM laser photocoagulation, interferon alpha treatment, intravitreal steroid therapy, transpupillary thermotherapy, membrane differential filtration therapy, aptamers targeting VEGF (e.g., MacugenTM) and/or VEGF receptors, antibodies targeting VEGF (e.g., LucentisTM) and/or VEGF receptors, VisudyneTM (e.g. use in photodynamic therapy, PDT), anti-imflammatory compounds such as CelebrexTM or anecortave acetate (e.g., RetaaneTM), angiostatic steroids such as glucocorticoids, intravitreal implants such as PosurdexTM, FGF2 modulators, antiangiogenic compounds such as squalamine, and/or VEGF traps and other cytokine traps (see for example Economides et al., 2003, Nature Medicine, 9, 47-52).
The use of anticholinergic agents, anti-inflammatories, bronchodilators, adenosine inhibitors, adenosine A1 receptor inhibitors, non-selective M3 receptor antagonists such as atropine, ipratropium brominde and selective M3 receptor antagonists such as darifenacin and revatropate are all non-limiting examples of agents that can be combined with or used in conjunction with the nucleic acid molecules (e.g. siNA molecules) of the instant invention in treating inflammatory, allergic, or respiratory diseases and conditions.
The above list of compounds are non-limiting examples of compounds and/or methods that can be combined with or used in conjunction with the nucleic acid molecules (e.g. siNA) of the instant invention. Those skilled in the art will recognize that other drug compounds and therapies can similarly be readily combined with the nucleic acid molecules of the instant invention (e.g., siNA molecules) are hence within the scope of the instant invention.
Example 12
Multifunctional siNA Inhibition of Target RNA Expression
Multifunctional siNA Design
Once target sites have been identified for multifunctional siNA constructs, each strand of the siNA is designed with a complementary region of length, for example, of about 18 to about 28 nucleotides, that is complementary to a different target nucleic acid sequence. Each complementary region is designed with an adjacent flanking region of about 4 to about 22 nucleotides that is not complementary to the target sequence, but which comprises complementarity to the complementary region of the other sequence (see for example FIG. 16). Hairpin constructs can likewise be designed (see for example FIG. 17). Identification of complementary, palindrome or repeat sequences that are shared between the different target nucleic acid sequences can be used to shorten the overall length of the multifunctional siNA constructs (see for example FIGS. 18 and 19).
In a non-limiting example, three additional categories of additional multifunctional siNA designs are presented that allow a single siNA molecule to silence multiple targets. The first method utilizes linkers to join siNAs (or multiunctional siNAs) in a direct manner. This can allow the most potent siNAs to be joined without creating a long, continuous stretch of RNA that has potential to trigger an interferon response. The second method is a dendrimeric extension of the overlapping or the linked multifunctional design; or alternatively the organization of siNA in a supramolecular format. The third method uses helix lengths greater than 30 base pairs. Processing of these siNAs by Dicer will reveal new, active 5′ antisense ends. Therefore, the long siNAs can target the sites defined by the original 5′ ends and those defined by the new ends that are created by Dicer processing. When used in combination with traditional multifunctional siNAs (where the sense and antisense strands each define a target) the approach can be used for example to target 4 or more sites.
I. Tethered Bifunctional siNAs
The basic idea is a novel approach to the design of multifunctional siNAs in which two antisense siNA strands are annealed to a single sense strand. The sense strand oligonucleotide contains a linker (e.g., non-nulcoetide linker as described herein) and two segments that anneal to the antisense siNA strands (see FIG. 22). The linkers can also optionally comprise nucleotide-based linkers. Several potential advantages and variations to this approach include, but are not limited to:

    1. The two antisense siNAs are independent. Therefore, the choice of target sites is not constrained by a requirement for sequence conservation between two sites. Any two highly active siNAs can be combined to form a multifunctional siNA.
    2. When used in combination with target sites having homology, siNAs that target a sequence present in two genes (e.g., different isoforms), the design can be used to target more than two sites. A single multifunctional siNA can be for example, used to target RNA of two different target RNAs.
    3. Multifunctional siNAs that use both the sense and antisense strands to target a gene can also be incorporated into a tethered multifuctional design. This leaves open the possibility of targeting 6 or more sites with a single complex.
    4. It can be possible to anneal more than two antisense strand siNAs to a single tethered sense strand.
    5. The design avoids long continuous stretches of dsRNA. Therefore, it is less likely to initiate an interferon response.
    6. The linker (or modifications attached to it, such as conjugates described herein) can improve the pharmacokinetic properties of the complex or improve its incorporation into liposomes. Modifications introduced to the linker should not impact siNA activity to the same extent that they would if directly attached to the siNA (see for example FIGS. 27 and 28).
    7. The sense strand can extend beyond the annealed antisense strands to provide additional sites for the attachment of conjugates.
    8. The polarity of the complex can be switched such that both of the antisense 3′ ends are adjacent to the linker and the 5′ ends are distal to the linker or combination thereof.

Dendrimer and Supramolecular siNAs



In the dendrimer siNA approach, the synthesis of siNA is initiated by first synthesizing the dendrimer template followed by attaching various functional siNAs. Various constructs are depicted in FIG. 23. The number of functional siNAs that can be attached is only limited by the dimensions of the dendrimer used.
Supramolecular Approach to Multifunctional siNA
The supramolecular format simplifies the challenges of dendrimer synthesis. In this format, the siNA strands are synthesized by standard RNA chemistry, followed by annealing of various complementary strands. The individual strand synthesis contains an antisense sense sequence of one siNA at the 5′-end followed by a nucleic acid or synthetic linker, such as hexaethyleneglyol, which in turn is followed by sense strand of another siNA in 5′ to 3′ direction. Thus, the synthesis of siNA strands can be carried out in a standard 3′ to 5′ direction. Representative examples of trifunctional and tetrafunctional siNAs are depicted in FIG. 24. Based on a similar principle, higher functionality siNA constucts can be designed as long as efficient annealing of various strands is achieved.
Dicer Enabled Multifunctional siNA
Using bioinformatic analysis of multiple targets, stretches of identical sequences shared between differeing target sequences can be identified ranging from about two to about fourteen nucleotides in length. These identical regions can be designed into extended siNA helixes (e.g., >30 base pairs) such that the processing by Dicer reveals a secondary functional 5′-antisense site (see for example FIG. 25). For example, when the first 17 nucleotides of a siNA antisense strand (e.g., 21 nucleotide strands in a duplex with 3′-TT overhangs) are complementary to a target RNA, robust silencing was observed at 25 nM. 80% silencing was observed with only 16 nucleotide complementarity in the same format.
Incorporation of this property into the designs of siNAs of about 30 to 40 or more base pairs results in additional multifunctional siNA constructs. The example in FIG. 25 illustrates how a 30 base-pair duplex can target three distinct sequences after processing by Dicer-RNaseIII; these sequences can be on the same mRNA or separate RNAs, such as viral and host factor messages, or multiple points along a given pathway (e.g., inflammatory cascades). Furthermore, a 40 base-pair duplex can combine a bifunctional design in tandem, to provide a single duplex targeting four target sequences. An even more extensive approach can include use of homologous sequences to enable five or six targets silenced for one multifunctional duplex. The example in FIG. 25 demonstrates how this can be achieved. A 30 base pair duplex is cleaved by Dicer into 22 and 8 base pair products from either end (8 b.p. fragments not shown). For ease of presentation the overhangs generated by dicer are not shown—but can be compensated for. Three targeting sequences are shown. The required sequence identity overlapped is indicated by grey boxes. The N's of the parent 30 b.p. siNA are suggested sites of 2′-OH positions to enable Dicer cleavage if this is tested in stabilized chemistries. Note that processing of a 30 mer duplex by Dicer RNase III does not give a precise 22+8 cleavage, but rather produces a series of closely related products (with 22+8 being the primary site). Therefore, processing by Dicer will yield a series of active siNAs. Another non-limiting example is shown in FIG. 26. A 40 base pair duplex is cleaved by Dicer into 20 base pair products from either end. For ease of presentation the overhangs generated by dicer are not shown—but can be compensated for. Four targeting sequences are shown in four colors, blue, light-blue and red and orange. The required sequence identity overlapped is indicated by grey boxes. This design format can be extended to larger RNAs. If chemically stabilized siNAs are bound by Dicer, then strategically located ribonucleotide linkages can enable designer cleavage products that permit our more extensive repertoire of multiifunctional designs. For example cleavage products not limited to the Dicer standard of approximately 22-nucleotides can allow multifunctional siNA constructs with a target sequence identity overlap ranging from, for example, about 3 to about 15 nucleotides.
Example 13
Diagnostic Uses
The siNA molecules of the invention can be used in a variety of diagnostic applications, such as in the identification of molecular targets (e.g., RNA) in a variety of applications, for example, in clinical, industrial, environmental, agricultural and/or research settings. Such diagnostic use of siNA molecules involves utilizing reconstituted RNAi systems, for example, using cellular lysates or partially purified cellular lysates. siNA molecules of this invention can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of endogenous or exogenous, for example viral, RNA in a cell. The close relationship between siNA activity and the structure of the target RNA allows the detection of mutations in any region of the molecule, which alters the base-pairing and three-dimensional structure of the target RNA. By using multiple siNA molecules described in this invention, one can map nucleotide changes, which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with siNA molecules can be used to inhibit gene expression and define the role of specified gene products in the progression of disease or infection. In this manner, other genetic targets can be defined as important mediators of the disease. These experiments will lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple siNA molecules targeted to different genes, siNA molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations siNA molecules and/or other chemical or biological molecules). Other in vitro uses of siNA molecules of this invention are well known in the art, and include detection of the presence of mRNAs associated with a disease, infection, or related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a siNA using standard methodologies, for example, fluorescence resonance emission transfer (FRET).
In a specific example, siNA molecules that cleave only wild-type or mutant forms of the target RNA are used for the assay. The first siNA molecules (i.e., those that cleave only wild-type forms of target RNA) are used to identify wild-type RNA present in the sample and the second siNA molecules (i.e., those that cleave only mutant forms of target RNA) are used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA are cleaved by both siNA molecules to demonstrate the relative siNA efficiencies in the reactions and the absence of cleavage of the “non-targeted” RNA species. The cleavage products from the synthetic substrates also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population. Thus, each analysis requires two siNA molecules, two substrates and one unknown sample, which is combined into six reactions. The presence of cleavage products is determined using an RNase protection assay so that fill-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells. The expression of mRNA whose protein product is implicated in the development of the phenotype (i.e., disease related or infection related) is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels is adequate and decreases the cost of the initial diagnosis. Higher mutant form to wild-type ratios are correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.
All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.
One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims.
It will be readily apparent to one skilled in the art that varying substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, such additional embodiments are within the scope of the present invention and the following claims. The present invention teaches one skilled in the art to test various combinations and/or substitutions of chemical modifications described herein toward generating nucleic acid constructs with improved activity for mediating RNAi activity. Such improved activity can comprise improved stability, improved bioavailability, and/or improved activation of cellular responses mediating RNAi. Therefore, the specific embodiments described herein are not limiting and one skilled in the art can readily appreciate that specific combinations of the modifications described herein can be tested without undue experimentation toward identifying siNA molecules with improved RNAi activity.
The invention illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of”, and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims.
In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.







TABLE I





VEGF and/or VEGFR Accession Numbers

















NM_005429



Homo sapiens vascular endothelial growth factor C



(VEGFC), mRNA



gi|19924300|ref|NM_005429.2|[19924300]



NM_003376



Homo sapiens vascular endothelial growth factor



(VEGF), mRNA



gi|19923239|ref|NM_003376.2|[19923239]



AF095785



Homo sapiens vascular endothelial growth factor (VEGF)



gene, promoter region and partial cds



gi|4154290|gb|AF095785.1|[4154290]



NM_003377



Homo sapiens vascular endothelial growth factor B



(VEGFB), mRNA



gi|20070172|ref|NM_003377.2|[20070172]



AF486837



Homo sapiens vascular endothelial growth factor



isoform VEGF165 (VEGF) mRNA, complete cds



gi|19909064|gb|AF486837.1|[19909064]



AF468110



Homo sapiens vascular endothelial growth factor B



isoform (VEGFB) gene, complete cds, alternatively spliced



gi|18766397|gb|AF468110.1|[18766397]



AF437895



Homo sapiens vascular endothelial growth factor (VEGF)



gene, partial cds



gi|16660685|gb|AF437895.1|AF437895[16660685]



AY047581



Homo sapiens vascular endothelial growth factor (VEGF)



mRNA, complete cds



gi|15422108|gb|AY047581.1|[15422108]



AF063657



Homo sapiens vascular endothelial growth factor



receptor (FLT1) mRNA, complete cds



gi|3132830|gb|AF063657.1|AF063657[3132830]



AF092127



Homo sapiens vascular endothelial growth factor (VEGF)



gene, partial sequence



gi|4139168|gb|AF092127.1|AF092127[4139168]



AF092126



Homo sapiens vascular endothelial growth factor (VEGF)



gene, 5′ UTR



gi|4139167|gb|AF092126.1|AF092126[4139167]



AF092125



Homo sapiens vascular endothelial growth factor (VEGF)



gene, partial cds



gi|4139165|gb|AF092125.1|AF092125[4139165]



E15157



Human VEGF mRNA



gi|5709840|dbj|E15157.1||pat|JP|1998052285|2[5709840]



E15156



Human VEGF mRNA



gi|5709839|dbj|E15156.1||pat|JP|1998052285|1[5709839]



E14233



Human mRNA for vascular endothelial growth factor



(VEGF), complete cds



gi|57089161|dbj|E14233.1||pat|JP|1997286795|1[5708916]



AF024710



Homo sapiens vascular endothelial growth factor (VEGF)



mRNA, 3′UTR



gi|2565322|gb|AF024710.1|AF024710[2565322]



AJ010438



Homo sapiens mRNA for vascular endothelial growth



factor, splicing variant



VEGF183



gi|3647280|emb|AJ010438.1|HSA010438[3647280]



AF098331



Homo sapiens vascular endothelial growth factor (VEGF)



gene, promoter, partial sequence



gi|4235431|gb|AF098331.1|AF098331[4235431]



AF022375



Homo sapiens vascular endothelial growth factor mRNA,



complete cds



gi|3719220|gb|AF022375.1|AF022375[3719220]



AH006909



vascular endothelial growth factor {alternative



splicing} [human, Genomic, 414 nt 5 segments]



gi|1680143|gb|AH006909.1||bbm|191843[1680143]



U01134



Human soluble vascular endothelial cell growth factor



receptor (sflt) mRNA, complete cds



gi|451321|gb|U01134.1|U01134[451321]



E14000



Human mRNA for FLT



gi|3252767|dbj|E14000.1||pat|JP|1997255700|1[3252767]



E13332



cDNA encoding vascular endodermal cell growth factor



VEGF



gi|3252137|dbj|E13332.1||pat|JP|1997173075|1[3252137]



E13256



Human mRNA for FLT, complete cds



gi|3252061|dbj|E13256.1||pat|JP|1997154588|1[3252061]



AF063658



Homo sapiens vascular endothelial growth factor



receptor 2 (KDR) mRNA, complete cds



gi|3132832|gb|AF063658.1|AF063658[3132832]



AJ000185



Homo Sapiens mRNA for vascular endothelial growth



factor-D



gi|2879833|emb|AJ000185.1|HSAJ185[2879833]



D89630



Homo sapiens mRNA for VEGF-D, complete cds



gi|2780339|dbj|D89630.1|[2780339]



AF035121



Homo sapiens KDR/flk-1 protein mRNA, complete cds



gi|2655411|gb|AF035121.1|AF035121[2655411]



AF020393



Homo sapiens vascular endothelial growth factor C



gene, partial cds and 5′ upstream region



gi|2582366|gb|AF020393.1|AF020393[2582366]



Y08736



H. sapiens vegf gene, 3′UTR



gi|1619596|emb|Y08736.1|HSVEGF3UT[1619596]



X62568



H. sapiens vegf gene for vascular endothelial growth



factor



gi|37658|emb|X62568.1|HSVEGF[37658]



X94216



H. sapiens mRNA for VEGF-C protein



gi|1177488|emb|X94216.1|HSVEGFC[1177488]



NM_002020



Homo sapiens fms-related tyrosine kinase 4 (FLT4),



mRNA



gi|4503752|ref|NM_002020.1|[4503752]



NM_002253



Homo sapiens kinase insert domain receptor (a type III



receptor tyrosine kinase) (KDR), mRNA



gi|11321596|ref|NM_002253.1|[11321596]

















TABLE II





VEGF and/or VEGFR siNA AND TARGET SEQUENCES


























Seq


Seq


Seq



Pos
Target Sequence
ID
UPos
Upper seq
ID
LPos
Lower seq
ID










VEGFR1/FLT1 NM_002019.1
















1
GCGGACACUCCUCUCGGCU
1
1
GCGGACACUCCUCUCGGCU
1
19
AGCCGAGAGGAGUGUCCGC
428



19
UCCUCCCCGGCAGCGGCGG
2
19
UCCUCCCCGGCAGCGGCGG
2
37
CCGCCGCUGCCGGGGAGGA
429


37
GCGGCUCGGAGCGGGCUCC
3
37
GCGGCUCGGAGCGGGCUCC
3
55
GGAGCCCGCUCCGAGCCGC
430


55
CGGGGCUCGGGUGCAGCGG
4
55
CGGGGCUCGGGUGCAGCGG
4
73
CCGCUGCACCCGAGCCCCG
431


73
GCCAGCGGGCCUGGCGGCG
5
73
GCCAGCGGGCCUGGCGGCG
5
91
CGCCGCCAGGCCCGCUGGC
432


91
GAGGAUUACCCGGGGAAGU
6
91
GAGGAUUACCCGGGGAAGU
6
109
ACUUCCCCGGGUAAUCCUC
433


109
UGGUUGUCUCCUGGCUGGA
7
109
UGGUUGUCUCCUGGCUGGA
7
127
UCCAGCCAGGAGACAACCA
434


127
AGCCGCGAGACGGGCGCUC
8
127
AGCCGCGAGACGGGCGCUC
8
145
GAGCGCCCGUCUCGCGGCU
435


145
CAGGGCGCGGGGCCGGCGG
9
145
CAGGGCGCGGGGCCGGCGG
9
163
CCGCCGGCCCCGCGCCCUG
436


163
GCGGCGAACGAGAGGACGG
10
163
GCGGCGAACGAGAGGACGG
10
181
CCGUCCUCUCGUUCGCCGC
437


181
GACUCUGGCGGCCGGGUCG
11
181
GACUCUGGCGGCCGGGUCG
11
199
CGACCCGGCCGCCAGAGUC
438


199
GUUGGCCGGGGGAGCGCGG
12
199
GUUGGCCGGGGGAGCGCGG
12
217
CCGCGCUCCCCCGGCCAAC
439


217
GGCACCGGGCGAGCAGGCC
13
217
GGCACCGGGCGAGCAGGCC
13
235
GGCCUGCUCGCCCGGUGCC
440


235
CGCGUCGCGCUCACCAUGG
14
235
CGCGUCGCGCUCACCAUGG
14
253
CCAUGGUGAGCGCGACGCG
441


253
GUCAGCUACUGGGACACCG
15
253
GUCAGCUACUGGGACACCG
15
271
CGGUGUCCCAGUAGCUGAC
442


271
GGGGUCCUGCUGUGCGCGC
16
271
GGGGUCCUGCUGUGCGCGC
16
289
GCGCGCACAGCAGGACCCC
443


289
CUGCUCAGCUGUCUGCUUC
17
289
CUGCUCAGCUGUCUGCUUC
17
307
GAAGCAGACAGCUGAGCAG
444


307
CUCACAGGAUCUAGUUCAG
18
307
CUCACAGGAUCUAGUUCAG
18
325
CUGAACUAGAUCCUGUGAG
445


325
GGUUCAAAAUUAAAAGAUC
19
325
GGUUCAAAAUUAAAAGAUC
19
343
GAUCUUUUAAUUUUGAACC
446


343
CCUGAACUGAGUUUAAAAG
20
343
CCUGAACUGAGUUUAAAAG
20
361
CUUUUAAACUCAGUUCAGG
447


361
GGCACCCAGCACAUCAUGC
21
361
GGCACCCAGCACAUCAUGC
21
379
GCAUGAUGUGCUGGGUGCC
448


379
CAAGCAGGCCAGACACUGC
22
379
CAAGCAGGCCAGACACUGC
22
397
GCAGUGUCUGGCCUGCUUG
449


397
CAUCUCCAAUGCAGGGGGG
23
397
CAUCUCCAAUGCAGGGGGG
23
415
CCCCCCUGCAUUGGAGAUG
450


415
GAAGCAGCCCAUAAAUGGU
24
415
GAAGCAGCCCAUAAAUGGU
24
433
ACCAUUUAUGGGCUGCUUC
451


433
UCUUUGCCUGAAAUGGUGA
25
433
UCUUUGCCUGAAAUGGUGA
25
451
UCACCAUUUCAGGCAAAGA
452


451
AGUAAGGAAAGCGAAAGGC
26
451
AGUAAGGAAAGCGAAAGGC
26
469
GCCUUUCGCUUUCCUUACU
453


469
CUGAGCAUAACUAAAUCUG
27
469
CUGAGCAUAACUAAAUCUG
27
487
CAGAUUUAGUUAUGCUCAG
454


487
GCCUGUGGAAGAAAUGGCA
28
487
GCCUGUGGAAGAAAUGGCA
28
505
UGCCAUUUCUUCCACAGGC
455


505
AAACAAUUCUGCAGUACUU
29
505
AAACAAUUCUGCAGUACUU
29
523
AAGUACUGCAGAAUUGUUU
456


523
UUAACCUUGAACACAGCUC
30
523
UUAACCUUGAACACAGCUC
30
541
GAGCUGUGUUCAAGGUUAA
457


541
CAAGCAAACCACACUGGCU
31
541
CAAGCAAACCACACUGGCU
31
559
AGCCAGUGUGGUUUGCUUG
458


559
UUCUACAGCUGCAAAUAUC
32
559
UUCUACAGCUGCAAAUAUC
32
577
GAUAUUUGCAGCUGUAGAA
459


577
CUAGCUGUACCUACUUCAA
33
577
CUAGCUGUACCUACUUCAA
33
595
UUGAAGUAGGUACAGCUAG
460


595
AAGAAGAAGGAAACAGAAU
34
595
AAGAAGAAGGAAACAGAAU
34
613
AUUCUGUUUCCUUCUUCUU
461


613
UCUGCAAUCUAUAUAUUUA
35
613
UCUGCAAUCUAUAUAUUUA
35
631
UAAAUAUAUAGAUUGCAGA
462


631
AUUAGUGAUACAGGUAGAC
36
631
AUUAGUGAUACAGGUAGAC
36
649
GUCUACCUGUAUCACUAAU
463


649
CCUUUCGUAGAGAUGUACA
37
649
CCUUUCGUAGAGAUGUACA
37
667
UGUACAUCUCUACGAAAGG
464


667
AGUGAAAUCCCCGAAAUUA
38
667
AGUGAAAUCCCCGAAAUUA
38
685
UAAUUUCGGGGAUUUCACU
465


685
AUACACAUGACUGAAGGAA
39
685
AUACACAUGACUGAAGGAA
39
703
UUCCUUCAGUCAUGUGUAU
466


703
AGGGAGCUCGUCAUUCCCU
40
703
AGGGAGCUCGUCAUUCCCU
40
721
AGGGAAUGACGAGCUCCCU
467


721
UGCCGGGUUACGUCACCUA
41
721
UGCCGGGUUACGUCACCUA
41
739
UAGGUGACGUAACCCGGCA
468


739
AACAUCACUGUUACUUUAA
42
739
AACAUCACUGUUACUUUAA
42
757
UUAAAGUAACAGUGAUGUU
469


757
AAAAAGUUUCCACUUGACA
43
757
AAAAAGUUUCCACUUGACA
43
775
UGUCAAGUGGAAACUUUUU
470


775
ACUUUGAUCCCUGAUGGAA
44
775
ACUUUGAUCCCUGAUGGAA
44
793
UUCCAUCAGGGAUCAAAGU
471


793
AAACGCAUAAUCUGGGACA
45
793
AAACGCAUAAUCUGGGACA
45
811
UGUCCCAGAUUAUGCGUUU
472


811
AGUAGAAAGGGCUUCAUCA
46
811
AGUAGAAAGGGCUUCAUCA
46
829
UGAUGAAGCCCUUUCUACU
473


829
AUAUCAAAUGCAACGUACA
47
829
AUAUCAAAUGCAACGUACA
47
847
UGUACGUUGCAUUUGAUAU
474


847
AAAGAAAUAGGGCUUCUGA
48
847
AAAGAAAUAGGGCUUCUGA
48
865
UCAGAAGCCCUAUUUCUUU
475


865
ACCUGUGAAGCAACAGUCA
49
865
ACCUGUGAAGCAACAGUCA
49
883
UGACUGUUGCUUCACAGGU
476


883
AAUGGGCAUUUGUAUAAGA
50
883
AAUGGGCAUUUGUAUAAGA
50
901
UCUUAUACAAAUGCCCAUU
477


901
ACAAACUAUCUCACACAUC
51
901
ACAAACUAUCUCACACAUC
51
919
GAUGUGUGAGAUAGUUUGU
478


919
CGACAAACCAAUACAAUCA
52
919
CGACAAACCAAUACAAUCA
52
937
UGAUUGUAUUGGUUUGUCG
479


937
AUAGAUGUCCAAAUAAGCA
53
937
AUAGAUGUCCAAAUAAGCA
53
955
UGCUUAUUUGGACAUCUAU
480


955
ACACCACGCCCAGUCAAAU
54
955
ACACCACGCCCAGUCAAAU
54
973
AUUUGACUGGGCGUGGUGU
481


973
UUACUUAGAGGCCAUACUC
55
973
UUACUUAGAGGCCAUACUC
55
991
GAGUAUGGCCUCUAAGUAA
482


991
CUUGUCCUCAAUUGUACUG
56
991
CUUGUCCUCAAUUGUACUG
56
1009
CAGUACAAUUGAGGACAAG
483


1009
GCUACCACUCCCUUGAACA
57
1009
GCUACCACUCCCUUGAACA
57
1027
UGUUCAAGGGAGUGGUAGC
484


1027
ACGAGAGUUCAAAUGACCU
58
1027
ACGAGAGUUCAAAUGACCU
58
1045
AGGUCAUUUGAACUCUCGU
485


1045
UGGAGUUACCCUGAUGAAA
59
1045
UGGAGUUACCCUGAUGAAA
59
1063
UUUCAUCAGGGUAACUCCA
486


1063
AAAAAUAAGAGAGCUUCCG
60
1063
AAAAAUAAGAGAGCUUCCG
60
1081
CGGAAGCUCUCUUAUUUUU
487


1081
GUAAGGCGACGAAUUGACC
61
1081
GUAAGGCGACGAAUUGACC
61
1099
GGUCAAUUCGUCGCCUUAC
488


1099
CAAAGCAAUUCCCAUGCCA
62
1099
CAAAGCAAUUCCCAUGCCA
62
1117
UGGCAUGGGAAUUGCUUUG
489


1117
AACAUAUUCUACAGUGUUC
63
1117
AACAUAUUCUACAGUGUUC
63
1135
GAACACUGUAGAAUAUGUU
490


1135
CUUACUAUUGACAAAAUGC
64
1135
CUUACUAUUGACAAAAUGC
64
1153
GCAUUUUGUCAAUAGUAAG
491


1153
CAGAACAAAGACAAAGGAC
65
1153
CAGAACAAAGACAAAGGAC
65
1171
GUCCUUUGUCUUUGUUCUG
492


1171
CUUUAUACUUGUCGUGUAA
66
1171
CUUUAUACUUGUCGUGUAA
66
1189
UUACACGACAAGUAUAAAG
493


1189
AGGAGUGGACCAUCAUUCA
67
1189
AGGAGUGGACCAUCAUUCA
67
1207
UGAAUGAUGGUCCACUCCU
494


1207
AAAUCUGUUAACACCUCAG
68
1207
AAAUCUGUUAACACCUCAG
68
1225
CUGAGGUGUUAACAGAUUU
495


1225
GUGCAUAUAUAUGAUAAAG
69
1225
GUGCAUAUAUAUGAUAAAG
69
1243
CUUUAUCAUAUAUAUGCAC
496


1243
GCAUUCAUCACUGUGAAAC
70
1243
GCAUUCAUCACUGUGAAAC
70
1261
GUUUCACAGUGAUGAAUGC
497


1261
CAUCGAAAACAGCAGGUGC
71
1261
CAUCGAAAACAGCAGGUGC
71
1279
GCACCUGCUGUUUUCGAUG
498


1279
CUUGAAACCGUAGCUGGCA
72
1279
CUUGAAACCGUAGCUGGCA
72
1297
UGCCAGCUACGGUUUCAAG
499


1297
AAGCGGUCUUACCGGCUCU
73
1297
AAGCGGUCUUACCGGCUCU
73
1315
AGAGCCGGUAAGACCGCUU
500


1315
UCUAUGAAAGUGAAGGCAU
74
1315
UCUAUGAAAGUGAAGGCAU
74
1333
AUGCCUUCACUUUCAUAGA
501


1333
UUUCCCUCGCCGGAAGUUG
75
1333
UUUCCCUCGCCGGAAGUUG
75
1351
CAACUUCCGGCGAGGGAAA
502


1351
GUAUGGUUAAAAGAUGGGU
76
1351
GUAUGGUUAAAAGAUGGGU
76
1369
ACCCAUCUUUUAACCAUAC
503


1369
UUACCUGCGACUGAGAAAU
77
1369
UUACCUGCGACUGAGAAAU
77
1387
AUUUCUCAGUCGCAGGUAA
504


1387
UCUGCUCGCUAUUUGACUC
78
1387
UCUGCUCGCUAUUUGACUC
78
1405
GAGUCAAAUAGCGAGCAGA
505


1405
CGUGGCUACUCGUUAAUUA
79
1405
CGUGGCUACUCGUUAAUUA
79
1423
UAAUUAACGAGUAGCCACG
506


1423
AUCAAGGACGUAACUGAAG
80
1423
AUCAAGGACGUAACUGAAG
80
1441
CUUCAGUUACGUCCUUGAU
507


1441
GAGGAUGCAGGGAAUUAUA
81
1441
GAGGAUGCAGGGAAUUAUA
81
1459
UAUAAUUCCCUGCAUCCUC
508


1459
ACAAUCUUGCUGAGCAUAA
82
1459
ACAAUCUUGCUGAGCAUAA
82
1477
UUAUGCUCAGCAAGAUUGU
509


1477
AAACAGUCAAAUGUGUUUA
83
1477
AAACAGUCAAAUGUGUUUA
83
1495
UAAACACAUUUGACUGUUU
510


1495
AAAAACCUCACUGCCACUC
84
1495
AAAAACCUCACUGCCACUC
84
1513
GAGUGGCAGUGAGGUUUUU
511


1513
CUAAUUGUCAAUGUGAAAC
85
1513
CUAAUUGUCAAUGUGAAAC
85
1531
GUUUCACAUUGACAAUUAG
512


1531
CCCCAGAUUUACGAAAAGG
86
1531
CCCCAGAUUUACGAAAAGG
86
1549
CCUUUUCGUAAAUCUGGGG
513


1549
GCCGUGUCAUCGUUUCCAG
87
1549
GCCGUGUCAUCGUUUCCAG
87
1567
CUGGAAACGAUGACACGGC
514


1567
GACCCGGCUCUCUACCCAC
88
1567
GACCCGGCUCUCUACCCAC
88
1585
GUGGGUAGAGAGCCGGGUC
515


1585
CUGGGCAGCAGACAAAUCC
89
1585
CUGGGCAGCAGACAAAUCC
89
1603
GGAUUUGUCUGCUGCCCAG
516


1603
CUGACUUGUACCGCAUAUG
90
1603
CUGACUUGUACCGCAUAUG
90
1621
CAUAUGCGGUACAAGUCAG
517


1621
GGUAUCCCUCAACCUACAA
91
1621
GGUAUCCCUCAACCUACAA
91
1639
UUGUAGGUUGAGGGAUACC
518


1639
AUCAAGUGGUUCUGGCACC
92
1639
AUCAAGUGGUUCUGGCACC
92
1657
GGUGCCAGAACCACUUGAU
519


1657
CCCUGUAACCAUAAUCAUU
93
1657
CCCUGUAACCAUAAUCAUU
93
1675
AAUGAUUAUGGUUACAGGG
520


1675
UCCGAAGCAAGGUGUGACU
94
1675
UCCGAAGCAAGGUGUGACU
94
1693
AGUCACACCUUGCUUCGGA
521


1693
UUUUGUUCCAAUAAUGAAG
95
1693
UUUUGUUCCAAUAAUGAAG
95
1711
CUUCAUUAUUGGAACAAAA
522


1711
GAGUCCUUUAUCCUGGAUG
96
1711
GAGUCCUUUAUCCUGGAUG
96
1729
CAUCCAGGAUAAAGGACUC
523


1729
GCUGACAGCAACAUGGGAA
97
1729
GCUGACAGCAACAUGGGAA
97
1747
UUCCCAUGUUGCUGUCAGC
524


1747
AACAGAAUUGAGAGCAUCA
98
1747
AACAGAAUUGAGAGCAUCA
98
1765
UGAUGCUCUCAAUUCUGUU
525


1765
ACUCAGCGCAUGGCAAUAA
99
1765
ACUCAGCGCAUGGCAAUAA
99
1783
UUAUUGCCAUGCGCUGAGU
526


1783
AUAGAAGGAAAGAAUAAGA
100
1783
AUAGAAGGAAAGAAUAAGA
100
1801
UCUUAUUCUUUCCUUCUAU
527


1801
AUGGCUAGCACCUUGGUUG
101
1801
AUGGCUAGCACCUUGGUUG
101
1819
CAACCAAGGUGCUAGCCAU
528


1819
GUGGCUGACUCUAGAAUUU
102
1819
GUGGCUGACUCUAGAAUUU
102
1837
AAAUUCUAGAGUCAGCCAC
529


1837
UCUGGAAUCUACAUUUGCA
103
1837
UCUGGAAUCUACAUUUGCA
103
1855
UGCAAAUGUAGAUUCCAGA
530


1855
AUAGCUUCCAAUAAAGUUG
104
1855
AUAGCUUCCAAUAAAGUUG
104
1873
CAACUUUAUUGGAAGCUAU
531


1873
GGGACUGUGGGAAGAAACA
105
1873
GGGACUGUGGGAAGAAACA
105
1891
UGUUUCUUCCCACAGUCCC
532


1891
AUAAGCUUUUAUAUCACAG
106
1891
AUAAGCUUUUAUAUCACAG
106
1909
CUGUGAUAUAAAAGCUUAU
533


1909
GAUGUGCCAAAUGGGUUUC
107
1909
GAUGUGCCAAAUGGGUUUC
107
1927
GAAACCCAUUUGGCACAUC
534


1927
CAUGUUAACUUGGAAAAAA
108
1927
CAUGUUAACUUGGAAAAAA
108
1945
UUUUUUCCAAGUUAACAUG
535


1945
AUGCCGACGGAAGGAGAGG
109
1945
AUGCCGACGGAAGGAGAGG
109
1963
CCUCUCCUUCCGUCGGCAU
536


1963
GACCUGAAACUGUCUUGCA
110
1963
GACCUGAAACUGUCUUGCA
110
1981
UGCAAGACAGUUUCAGGUC
537


1981
ACAGUUAACAAGUUCUUAU
111
1981
ACAGUUAACAAGUUCUUAU
111
1999
AUAAGAACUUGUUAACUGU
538


1999
UACAGAGACGUUACUUGGA
112
1999
UACAGAGACGUUACUUGGA
112
2017
UCCAAGUAACGUCUCUGUA
539


2017
AUUUUACUGCGGACAGUUA
113
2017
AUUUUACUGCGGACAGUUA
113
2035
UAACUGUCCGCAGUAAAAU
540


2035
AAUAACAGAACAAUGCACU
114
2035
AAUAACAGAACAAUGCACU
114
2053
AGUGCAUUGUUCUGUUAUU
541


2053
UACAGUAUUAGCAAGCAAA
115
2053
UACAGUAUUAGCAAGCAAA
115
2071
UUUGCUUGCUAAUACUGUA
542


2071
AAAAUGGCCAUCACUAAGG
116
2071
AAAAUGGCCAUCACUAAGG
116
2089
CCUUAGUGAUGGCCAUUUU
543


2089
GAGCACUCCAUCACUCUUA
117
2089
GAGCACUCCAUCACUCUUA
117
2107
UAAGAGUGAUGGAGUGCUC
544


2107
AAUCUUACCAUCAUGAAUG
118
2107
AAUCUUACCAUCAUGAAUG
118
2125
CAUUCAUGAUGGUAAGAUU
545


2125
GUUUCCCUGCAAGAUUCAG
119
2125
GUUUCCCUGCAAGAUUCAG
119
2143
CUGAAUCUUGCAGGGAAAC
546


2143
GGCACCUAUGCCUGCAGAG
120
2143
GGCACCUAUGCCUGCAGAG
120
2161
CUCUGCAGGCAUAGGUGCC
547


2161
GCCAGGAAUGUAUACACAG
121
2161
GCCAGGAAUGUAUACACAG
121
2179
CUGUGUAUACAUUCCUGGC
548


2179
GGGGAAGAAAUCCUCCAGA
122
2179
GGGGAAGAAAUCCUCCAGA
122
2197
UCUGGAGGAUUUCUUCCCC
549


2197
AAGAAAGAAAUUACAAUCA
123
2197
AAGAAAGAAAUUACAAUCA
123
2215
UGAUUGUAAUUUCUUUCUU
550


2215
AGAGAUCAGGAAGCACCAU
124
2215
AGAGAUCAGGAAGCACCAU
124
2233
AUGGUGCUUCCUGAUCUCU
551


2233
UACCUCCUGCGAAACCUCA
125
2233
UACCUCCUGCGAAACCUCA
125
2251
UGAGGUUUCGCAGGAGGUA
552


2251
AGUGAUCACACAGUGGCCA
126
2251
AGUGAUCACACAGUGGCCA
126
2269
UGGCCACUGUGUGAUCACU
553


2269
AUCAGCAGUUCCACCACUU
127
2269
AUCAGCAGUUCCACCACUU
127
2287
AAGUGGUGGAACUGCUGAU
554


2287
UUAGACUGUCAUGCUAAUG
128
2287
UUAGACUGUCAUGCUAAUG
128
2305
CAUUAGCAUGACAGUCUAA
555


2305
GGUGUCCCCGAGCCUCAGA
129
2305
GGUGUCCCCGAGCCUCAGA
129
2323
UCUGAGGCUCGGGGACACC
556


2323
AUCACUUGGUUUAAAAACA
130
2323
AUCACUUGGUUUAAAAACA
130
2341
UGUUUUUAAACCAAGUGAU
557


2341
AACCACAAAAUACAACAAG
131
2341
AACCACAAAAUACAACAAG
131
2359
CUUGUUGUAUUUUGUGGUU
558


2359
GAGCCUGGAAUUAUUUUAG
132
2359
GAGCCUGGAAUUAUUUUAG
132
2377
CUAAAAUAAUUCCAGGCUC
559


2377
GGACCAGGAAGCAGCACGC
133
2377
GGACCAGGAAGCAGCACGC
133
2395
GCGUGCUGCUUCCUGGUCC
560


2395
CUGUUUAUUGAAAGAGUCA
134
2395
CUGUUUAUUGAAAGAGUCA
134
2413
UGACUCUUUCAAUAAACAG
561


2413
ACAGAAGAGGAUGAAGGUG
135
2413
ACAGAAGAGGAUGAAGGUG
135
2431
CACCUUCAUCCUCUUCUGU
562


2431
GUCUAUCACUGCAAAGCCA
136
2431
GUCUAUCACUGCAAAGCCA
136
2449
UGGCUUUGCAGUGAUAGAC
563


2449
ACCAACCAGAAGGGCUCUG
137
2449
ACCAACCAGAAGGGCUCUG
137
2467
CAGAGCCCUUCUGGUUGGU
564


2467
GUGGAAAGUUCAGCAUACC
138
2467
GUGGAAAGUUCAGCAUACC
138
2485
GGUAUGCUGAACUUUCCAC
565


2485
CUCACUGUUCAAGGAACCU
139
2485
CUCACUGUUCAAGGAACCU
139
2503
AGGUUCCUUGAACAGUGAG
566


2503
UCGGACAAGUCUAAUCUGG
140
2503
UCGGACAAGUCUAAUCUGG
140
2521
CCAGAUUAGACUUGUCCGA
567


2521
GAGCUGAUCACUCUAACAU
141
2521
GAGCUGAUCACUCUAACAU
141
2539
AUGUUAGAGUGAUCAGCUC
568


2539
UGCACCUGUGUGGCUGCGA
142
2539
UGCACCUGUGUGGCUGCGA
142
2557
UCGCAGCCACACAGGUGCA
569


2557
ACUCUCUUCUGGCUCCUAU
143
2557
ACUCUCUUCUGGCUCCUAU
143
2575
AUAGGAGCCAGAAGAGAGU
570


2575
UUAACCCUCCUUAUCCGAA
144
2575
UUAACCCUCCUUAUCCGAA
144
2593
UUCGGAUAAGGAGGGUUAA
571


2593
AAAAUGAAAAGGUCUUCUU
145
2593
AAAAUGAAAAGGUCUUCUU
145
2611
AAGAAGACCUUUUCAUUUU
572


2611
UCUGAAAUAAAGACUGACU
146
2611
UCUGAAAUAAAGACUGACU
146
2629
AGUCAGUCUUUAUUUCAGA
573


2629
UACCUAUCAAUUAUAAUGG
147
2629
UACCUAUCAAUUAUAAUGG
147
2647
CCAUUAUAAUUGAUAGGUA
574


2647
GACCCAGAUGAAGUUCCUU
148
2647
GACCCAGAUGAAGUUCCUU
148
2665
AAGGAACUUCAUCUGGGUC
575


2665
UUGGAUGAGCAGUGUGAGC
149
2665
UUGGAUGAGCAGUGUGAGC
149
2683
GCUCACACUGCUCAUCCAA
576


2683
CGGCUCCCUUAUGAUGCCA
150
2683
CGGCUCCCUUAUGAUGCCA
150
2701
UGGCAUCAUAAGGGAGCCG
577


2701
AGCAAGUGGGAGUUUGCCC
151
2701
AGCAAGUGGGAGUUUGCCC
151
2719
GGGCAAACUCCCACUUGCU
578


2719
CGGGAGAGACUUAAACUGG
152
2719
CGGGAGAGACUUAAACUGG
152
2737
CCAGUUUAAGUCUCUCCCG
579


2737
GGCAAAUCACUUGGAAGAG
153
2737
GGCAAAUCACUUGGAAGAG
153
2755
CUCUUCCAAGUGAUUUGCC
580


2755
GGGGCUUUUGGAAAAGUGG
154
2755
GGGGCUUUUGGAAAAGUGG
154
2773
CCACUUUUCCAAAAGCCCC
581


2773
GUUCAAGCAUCAGCAUUUG
155
2773
GUUCAAGCAUCAGCAUUUG
155
2791
CAAAUGCUGAUGCUUGAAC
582


2791
GGCAUUAAGAAAUCACCUA
156
2791
GGCAUUAAGAAAUCACCUA
156
2809
UAGGUGAUUUCUUAAUGCC
583


2809
ACGUGCCGGACUGUGGCUG
157
2809
ACGUGCCGGACUGUGGCUG
157
2827
CAGCCACAGUCCGGCACGU
584


2827
GUGAAAAUGCUGAAAGAGG
158
2827
GUGAAAAUGCUGAAAGAGG
158
2845
CCUCUUUCAGCAUUUUCAC
585


2845
GGGGCCACGGCCAGCGAGU
159
2845
GGGGCCACGGCCAGCGAGU
159
2863
ACUCGCUGGCCGUGGCCCC
586


2863
UACAAAGCUCUGAUGACUG
160
2863
UACAAAGCUCUGAUGACUG
160
2881
CAGUCAUCAGAGCUUUGUA
587


2881
GAGCUAAAAAUCUUGACCC
161
2881
GAGCUAAAAAUCUUGACCC
161
2899
GGGUCAAGAUUUUUAGCUC
568


2899
CACAUUGGCCACCAUCUGA
162
2899
CACAUUGGCCACCAUCUGA
162
2917
UCAGAUGGUGGCCAAUGUG
589


2917
AACGUGGUUAACCUGCUGG
163
2917
AACGUGGUUAACCUGCUGG
163
2935
CCAGCAGGUUAACCACGUU
590


2935
GGAGCCUGCAGCAAGCAAG
164
2935
GGAGCCUGCACCAAGCAAG
164
2953
CUUGCUUGGUGCAGGCUCC
591


2953
GGAGGGCCUCUGAUGGUGA
165
2953
GGAGGGCCUCUGAUGGUGA
165
2971
UCACCAUCAGAGGCCCUCC
592


2971
AUUGUUGAAUACUGCAAAU
166
2971
AUUGUUGAAUACUGCAAAU
166
2989
AUUUGCAGUAUUCAACAAU
593


2989
UAUGGAAAUCUCUCCAACU
167
2989
UAUGGAAAUCUCUCCAACU
167
3007
AGUUGGAGAGAUUUCCAUA
594


3007
UACCUCAAGAGCAAACGUG
168
3007
UACCUCAAGAGCAAACGUG
168
3025
CACGUUUGCUCUUGAGGUA
595


3025
GACUUAUUUUUUCUCAACA
169
3025
GACUUAUUUUUUCUCAACA
169
3043
UGUUGAGAAAAAAUAAGUC
596


3043
AAGGAUGCAGCACUACACA
170
3043
AAGGAUGCAGCACUACACA
170
3061
UGUGUAGUGCUGCAUCCUU
597


3061
AUGGAGCCUAAGAAAGAAA
171
3061
AUGGAGCCUAAGAAAGAAA
171
3079
UUUCUUUCUUAGGCUCCAU
598


3079
AAAAUGGAGCCAGGCCUGG
172
3079
AAAAUGGAGCCAGGCCUGG
172
3097
CCAGGCCUGGCUCCAUUUU
599


3097
GAACAAGGCAAGAAACCAA
173
3097
GAACAAGGCAAGAAACCAA
173
3115
UUGGUUUCUUGCCUUGUUC
600


3115
AGACUAGAUAGCGUCACCA
174
3115
AGACUAGAUAGCGUCACCA
174
3133
UGGUGACGCUAUCUAGUCU
601


3133
AGCAGCGAAAGCUUUGCGA
175
3133
AGCAGCGAAAGCUUUGCGA
175
3151
UCGCAAAGCUUUCGCUGCU
602


3151
AGCUCCGGCUUUCAGGAAG
176
3151
AGCUCCGGCUUUCAGGAAG
176
3169
CUUCCUGAAAGCCGGAGCU
603


3169
GAUAAAAGUCUGAGUGAUG
177
3169
GAUAAAAGUCUGAGUGAUG
177
3187
CAUCACUCAGACUUUUAUC
604


3187
GUUGAGGAAGAGGAGGAUU
178
3187
GUUGAGGAAGAGGAGGAUU
178
3205
AAUCCUCCUCUUCCUCAAC
605


3205
UCUGACGGUUUCUACAAGG
179
3205
UCUGACGGUUUCUACAAGG
179
3223
CCUUGUAGAAACCGUCAGA
606


3223
GAGCCCAUCACUAUGGAAG
180
3223
GAGCCCAUCACUAUGGAAG
180
3241
CUUCCAUAGUGAUGGGCUC
607


3241
GAUCUGAUUUCUUACAGUU
181
3241
GAUCUGAUUUCUUACAGUU
181
3259
AACUGUAAGAAAUCAGAUC
608


3259
UUUCAAGUGGCCAGAGGCA
182
3259
UUUCAAGUGGCCAGAGGCA
182
3277
UGCCUCUGGCCACUUGAAA
609


3277
AUGGAGUUCCUGUCUUCCA
183
3277
AUGGAGUUCCUGUCUUCCA
183
3295
UGGAAGACAGGAACUCCAU
610


3295
AGAAAGUGCAUUCAUCGGG
184
3295
AGAAAGUGCAUUCAUCGGG
184
3313
CCCGAUGAAUGCACUUUCU
611


3313
GACCUGGCAGCGAGAAACA
185
3313
GACCUGGCAGCGAGAAACA
185
3331
UGUUUCUCGCUGCCAGGUC
612


3331
AUUCUUUUAUCUGAGAACA
186
3331
AUUCUUUUAUCUGAGAACA
186
3349
UGUUCUCAGAUAAAAGAAU
613


3349
AACGUGGUGAAGAUUUGUG
187
3349
AACGUGGUGAAGAUUUGUG
187
3367
CACAAAUCUUCACCACGUU
614


3367
GAUUUUGGCCUUGCCCGGG
188
3367
GAUUUUGGCCUUGCCCGGG
188
3385
CCCGGGCAAGGCCAAAAUC
615


3385
GAUAUUUAUAAGAACCCCG
189
3385
GAUAUUUAUAAGAACCCCG
189
3403
CGGGGUUCUUAUAAAUAUC
616


3403
GAUUAUGUGAGAAAAGGAG
190
3403
GAUUAUGUGAGAAAAGGAG
190
3421
CUCCUUUUCUCACAUAAUC
617


3421
GAUACUCGACUUCCUCUGA
191
3421
GAUACUCGACUUCCUCUGA
191
3439
UCAGAGGAAGUCGAGUAUC
618


3439
AAAUGGAUGGCUCCCGAAU
192
3439
AAAUGGAUGGCUCCCGAAU
192
3457
AUUCGGGAGCCAUCCAUUU
619


3457
UCUAUCUUUGACAAAAUCU
193
3457
UCUAUCUUUGACAAAAUCU
193
3475
AGAUUUUGUCAAAGAUAGA
620


3475
UACAGCACCAAGAGCGACG
194
3475
UACAGCACCAAGAGCGACG
194
3493
CGUCGCUCUUGGUGCUGUA
621


3493
GUGUGGUCUUACGGAGUAU
195
3493
GUGUGGUCUUACGGAGUAU
195
3511
AUACUCCGUAAGACCACAC
622


3511
UUGCUGUGGGAAAUCUUCU
196
3511
UUGCUGUGGGAAAUCUUCU
196
3529
AGAAGAUUUCCCACAGCAA
623


3529
UCCUUAGGUGGGUCUCCAU
197
3529
UCCUUAGGUGGGUCUCCAU
197
3547
AUGGAGACCCACCUAAGGA
624


3547
UACCCAGGAGUACAAAUGG
198
3547
UACCCAGGAGUACAAAUGG
198
3565
CCAUUUGUACUCCUGGGUA
625


3565
GAUGAGGACUUUUGCAGUC
199
3565
GAUGAGGACUUUUGCAGUC
199
3583
GACUGCAAAAGUCCUCAUC
626


3583
CGCCUGAGGGAAGGCAUGA
200
3583
CGCCUGAGGGAAGGCAUGA
200
3601
UCAUGCCUUCCCUCAGGCG
627


3601
AGGAUGAGAGCUCCUGAGU
201
3601
AGGAUGAGAGCUCCUGAGU
201
3619
ACUCAGGAGCUCUCAUCCU
628


3619
UACUCUACUCCUGAAAUCU
202
3619
UACUCUACUCCUGAAAUCU
202
3637
AGAUUUCAGGAGUAGAGUA
629


3637
UAUCAGAUCAUGCUGGACU
203
3637
UAUCAGAUCAUGCUGGACU
203
3655
AGUCCAGCAUGAUCUGAUA
630


3655
UGCUGGCACAGAGACCCAA
204
3655
UGCUGGCACAGAGACCCAA
204
3673
UUGGGUCUCUGUGCCAGCA
631


3673
AAAGAAAGGCCAAGAUUUG
205
3673
AAAGAAAGGCCAAGAUUUG
205
3691
CAAAUCUUGGCCUUUCUUU
632


3691
GCAGAACUUGUGGAAAAAC
206
3691
GCAGAACUUGUGGAAAAAC
206
3709
GUUUUUCCACAAGUUCUGC
633


3709
CUAGGUGAUUUGCUUCAAG
207
3709
CUAGGUGAUUUGCUUCAAG
207
3727
CUUGAAGCAAAUCACCUAG
634


3727
GCAAAUGUACAACAGGAUG
208
3727
GCAAAUGUACAACAGGAUG
208
3745
CAUCCUGUUGUACAUUUGC
635


3745
GGUAAAGACUACAUCCCAA
209
3745
GGUAAAGACUACAUCCCAA
209
3763
UUGGGAUGUAGUCUUUACC
636


3763
AUCAAUGCCAUACUGACAG
210
3763
AUCAAUGCCAUACUGACAG
210
3781
CUGUCAGUAUGGCAUUGAU
637


3781
GGAAAUAGUGGGUUUACAU
211
3781
GGAAAUAGUGGGUUUACAU
211
3799
AUGUAAACCCACUAUUUCC
638


3799
UACUCAACUCCUGCCUUCU
212
3799
UACUCAACUCCUGCCUUCU
212
3817
AGAAGGCAGGAGUUGAGUA
639


3817
UCUGAGGACUUCUUCAAGG
213
3817
UCUGAGGACUUCUUCAAGG
213
3835
CCUUGAAGAAGUCCUCAGA
640


3835
GAAAGUAUUUCAGCUCCGA
214
3835
GAAAGUAUUUCAGCUCCGA
214
3853
UCGGAGCUGAAAUACUUUC
641


3853
AAGUUUAAUUCAGGAAGCU
215
3853
AAGUUUAAUUCAGGAAGCU
215
3871
AGCUUCCUGAAUUAAACUU
642


3871
UCUGAUGAUGUCAGAUAUG
216
3871
UCUGAUGAUGUCAGAUAUG
216
3889
CAUAUCUGACAUCAUCAGA
643


3889
GUAAAUGCUUUCAAGUUCA
217
3889
GUAAAUGCUUUCAAGUUCA
217
3907
UGAACUUGAAAGCAUUUAC
644


3907
AUGAGCCUGGAAAGAAUCA
218
3907
AUGAGCCUGGAAAGAAUCA
218
3925
UGAUUCUUUCCAGGCUCAU
645


3925
AAAACCUUUGAAGAACUUU
219
3925
AAAACCUUUGAAGAACUUU
219
3943
AAAGUUCUUCAAAGGUUUU
646


3943
UUACCGAAUGCCACCUCCA
220
3943
UUACCGAAUGCCACCUCCA
220
3961
UGGAGGUGGCAUUCGGUAA
647


3961
AUGUUUGAUGACUACCAGG
221
3961
AUGUUUGAUGACUACCAGG
221
3979
CCUGGUAGUCAUCAAACAU
648


3979
GGCGACAGCAGCACUCUGU
222
3979
GGCGACAGCAGCACUCUGU
222
3997
ACAGAGUGCUGCUGUCGCC
649


3997
UUGGCCUCUCCCAUGCUGA
223
3997
UUGGCCUCUCCCAUGCUGA
223
4015
UCAGCAUGGGAGAGGCCAA
650


4015
AAGCGCUUCACCUGGACUG
224
4015
AAGCGCUUCACCUGGACUG
224
4033
CAGUCCAGGUGAAGCGCUU
651


4033
GACAGCAAACCCAAGGCCU
225
4033
GACAGCAAACCCAAGGCCU
225
4051
AGGCCUUGGGUUUGCUGUC
652


4051
UCGCUCAAGAUUGACUUGA
226
4051
UCGCUCAAGAUUGACUUGA
226
4069
UCAAGUCAAUCUUGAGCGA
653


4069
AGAGUAACCAGUAAAAGUA
227
4069
AGAGUAACCAGUAAAAGUA
227
4087
UACUUUUACUGGUUACUCU
654


4087
AAGGAGUCGGGGCUGUCUG
228
4087
AAGGAGUCGGGGCUGUCUG
228
4105
CAGACAGCCCCGACUCCUU
655


4105
GAUGUCAGCAGGCCCAGUU
229
4105
GAUGUCAGCAGGCCCAGUU
229
4123
AACUGGGCCUGCUGACAUC
656


4123
UUCUGCCAUUCCAGCUGUG
230
4123
UUCUGCCAUUCCAGCUGUG
230
4141
CACAGCUGGAAUGGCAGAA
657


4141
GGGCACGUCAGCGAAGGCA
231
4141
GGGCACGUCAGCGAAGGCA
231
4159
UGCCUUCGCUGACGUGCCC
658


4159
AAGCGCAGGUUCACCUACG
232
4159
AAGCGCAGGUUCACCUACG
232
4177
CGUAGGUGAACCUGCGCUU
659


4177
GACCACGCUGAGCUGGAAA
233
4177
GACCACGCUGAGCUGGAAA
233
4195
UUUCCAGCUCAGCGUGGUC
660


4195
AGGAAAAUCGCGUGCUGCU
234
4195
AGGAAAAUCGCGUGCUGCU
234
4213
AGCAGCACGCGAUUUUCCU
661


4213
UCCCCGCCCCCAGACUACA
235
4213
UCCCCGCCCCCAGACUACA
235
4231
UGUAGUCUGGGGGCGGGGA
662


4231
AACUCGGUGGUCCUGUACU
236
4231
AACUCGGUGGUCCUGUACU
236
4249
AGUACAGGACCACCGAGUU
663


4249
UCCACCCCACCCAUCUAGA
237
4249
UCCACCCCACCCAUCUAGA
237
4267
UCUAGAUGGGUGGGGUGGA
664


4267
AGUUUGACACGAAGCCUUA
238
4267
AGUUUGACACGAAGCCUUA
238
4285
UAAGGCUUCGUGUCAAACU
665


4285
AUUUCUAGAAGCACAUGUG
239
4285
AUUUCUAGAAGCACAUGUG
239
4303
CACAUGUGCUUCUAGAAAU
666


4303
GUAUUUAUACCCCCAGGAA
240
4303
GUAUUUAUACCCCCAGGAA
240
4321
UUCCUGGGGGUAUAAAUAC
667


4321
AACUAGCUUUUGCCAGUAU
241
4321
AACUAGCUUUUGCCAGUAU
241
4339
AUACUGGCAAAAGCUAGUU
668


4339
UUAUGCAUAUAUAAGUUUA
242
4339
UUAUGCAUAUAUAAGUUUA
242
4357
UAAACUUAUAUAUGCAUAA
669


4357
ACACCUUUAUCUUUCCAUG
243
4357
ACACCUUUAUCUUUCCAUG
243
4375
CAUGGAAAGAUAAAGGUGU
670


4375
GGGAGCCAGCUGCUUUUUG
244
4375
GGGAGCCAGCUGCUUUUUG
244
4393
CAAAAAGCAGCUGGCUCCC
671


4393
GUGAUUUUUUUAAUAGUGC
245
4393
GUGAUUUUUUUAAUAGUGC
245
4411
GCACUAUUAAAAAAAUCAC
672


4411
CUUUUUUUUUUUGACUAAC
246
4411
CUUUUUUUUUUUGACUAAC
246
4429
GUUAGUCAAAAAAAAAAAG
673


4429
CAAGAAUGUAACUCCAGAU
247
4429
CAAGAAUGUAACUCCAGAU
247
4447
AUCUGGAGUUACAUUCUUG
674


4447
UAGAGAAAUAGUGACAAGU
248
4447
UAGAGAAAUAGUGACAAGU
248
4465
ACUUGUCACUAUUUCUCUA
675


4465
UGAAGAACACUACUGCUAA
249
4465
UGAAGAACACUACUGCUAA
249
4483
UUAGCAGUAGUGUUCUUCA
676


4483
AAUCCUCAUGUUACUCAGU
250
4483
AAUCCUCAUGUUACUCAGU
250
4501
ACUGAGUAACAUGAGGAUU
677


4501
UGUUAGAGAAAUCCUUCCU
251
4501
UGUUAGAGAAAUCCUUCCU
251
4519
AGGAAGGAUUUCUCUAACA
678


4519
UAAACCCAAUGACUUCCCU
252
4519
UAAACCCAAUGACUUCCCU
252
4537
AGGGAAGUCAUUGGGUUUA
679


4537
UGCUCCAACCCCCGCCACC
253
4537
UGCUCCAACCCCCGCCACC
253
4555
GGUGGCGGGGGUUGGAGCA
680


4555
CUCAGGGCACGCAGGACCA
254
4555
CUCAGGGCACGCAGGACCA
254
4573
UGGUCCUGCGUGCCCUGAG
681


4573
AGUUUGAUUGAGGAGCUGC
255
4573
AGUUUGAUUGAGGAGCUGC
255
4591
GCAGCUCCUCAAUCAAACU
682


4591
CACUGAUCACCCAAUGCAU
256
4591
CACUGAUCACCCAAUGCAU
256
4609
AUGCAUUGGGUGAUCAGUG
683


4609
UCACGUACCCCACUGGGCC
257
4609
UCACGUACCCCACUGGGCC
257
4627
GGCCCAGUGGGGUACGUGA
684


4627
CAGCCCUGCAGCCCAAAAC
258
4627
CAGCCCUGCAGCCCAAAAC
258
4645
GUUUUGGGCUGCAGGGCUG
685


4645
CCCAGGGCAACAAGCCCGU
259
4645
CCCAGGGCAACAAGCCCGU
259
4663
ACGGGCUUGUUGCCCUGGG
686


4663
UUAGCCCCAGGGGAUCACU
260
4663
UUAGCCCCAGGGGAUCACU
260
4681
AGUGAUCCCCUGGGGCUAA
687


4681
UGGCUGGCCUGAGCAACAU
261
4681
UGGCUGGCCUGAGCAACAU
261
4699
AUGUUGCUCAGGCCAGCCA
688


4699
UCUCGGGAGUCCUCUAGCA
262
4699
UCUCGGGAGUCCUCUAGCA
262
4717
UGCUAGAGGACUCCCGAGA
689


4717
AGGCCUAAGACAUGUGAGG
263
4717
AGGCGUAAGACAUGUGAGG
263
4735
CCUCACAUGUCUUAGGCCU
690


4735
GAGGAAAAGGAAAAAAAGC
264
4735
GAGGAAAAGGAAAAAAAGC
264
4753
GCUUUUUUUCCUUUUCCUC
691


4753
CAAAAAGCAAGGGAGAAAA
265
4753
CAAAAAGCAAGGGAGAAAA
265
4771
UUUUCUCCCUUGCUUUUUG
692


4771
AGAGAAACCGGGAGAAGGC
266
4771
AGAGAAACCGGGAGAAGGC
266
4789
GCCUUCUCCCGGUUUCUCU
693


4789
CAUGAGAAAGAAUUUGAGA
267
4789
CAUGAGAAAGAAUUUGAGA
267
4807
UCUCAAAUUCUUUCUCAUG
694


4807
ACGCACCAUGUGGGCACGG
268
4807
ACGCACCAUGUGGGCACGG
268
4825
CCGUGCCCACAUGGUGCGU
695


4825
GAGGGGGACGGGGCUCAGC
269
4825
GAGGGGGACGGGGCUCAGC
269
4843
GCUGAGCCCCGUCCCCCUC
696


4843
CAAUGCCAUUUCAGUGGCU
270
4843
CAAUGCCAUUUCAGUGGCU
270
4861
AGCCACUGAAAUGGCAUUG
697


4861
UUCCCAGCUCUGACCCUUC
271
4861
UUCCCAGCUCUGACCCUUC
271
4879
GAAGGGUCAGAGCUGGGAA
698


4879
CUACAUUUGAGGGCCCAGC
272
4879
CUACAUUUGAGGGCCCAGC
272
4897
GCUGGGCCCUCAAAUGUAG
699


4897
CCAGGAGCAGAUGGACAGC
273
4897
CCAGGAGCAGAUGGACAGC
273
4915
GCUGUCCAUCUGCUCCUGG
700


4915
CGAUGAGGGGACAUUUUCU
274
4915
CGAUGAGGGGACAUUUUCU
274
4933
AGAAAAUGUCCCCUCAUCG
701


4933
UGGAUUCUGGGAGGCAAGA
275
4933
UGGAUUCUGGGAGGCAAGA
275
4951
UCUUGCCUCCCAGAAUCCA
702


4951
AAAAGGACAAAUAUCUUUU
276
4951
AAAAGGACAAAUAUCUUUU
276
4969
AAAAGAUAUUUGUCCUUUU
703


4969
UUUGGAACUAAAGCAAAUU
277
4969
UUUGGAACUAAAGCAAAUU
277
4987
AAUUUGCUUUAGUUCCAAA
704


4987
UUUAGACCUUUACCUAUGG
278
4987
UUUAGACCUUUACCUAUGG
278
5005
CCAUAGGUAAAGGUCUAAA
705


5005
GAAGUGGUUCUAUGUCCAU
279
5005
GAAGUGGUUCUAUGUCCAU
279
5023
AUGGACAUAGAACCACUUC
706


5023
UUCUCAUUCGUGGCAUGUU
280
5023
UUCUCAUUCGUGGCAUGUU
280
5041
AACAUGCCACGAAUGAGAA
707


5041
UUUGAUUUGUAGCACUGAG
281
5041
UUUGAUUUGUAGCACUGAG
281
5059
CUCAGUGCUACAAAUCAAA
708


5059
GGGUGGCACUCAACUCUGA
282
5059
GGGUGGCACUCAACUCUGA
282
5077
UCAGAGUUGAGUGCCACCC
709


5077
AGCCCAUACUUUUGGCUCC
283
5077
AGCCCAUACUUUUGGCUCC
283
5095
GGAGCCAAAAGUAUGGGCU
710


5095
CUCUAGUAAGAUGCACUGA
284
5095
CUCUAGUAAGAUGCACUGA
284
5113
UCAGUGCAUCUUACUAGAG
711


5113
AAAACUUAGCCAGAGUUAG
285
5113
AAAACUUAGCCAGAGUUAG
285
5131
CUAACUCUGGCUAAGUUUU
712


5131
GGUUGUCUCCAGGCCAUGA
286
5131
GGUUGUCUCCAGGCCAUGA
286
5149
UCAUGGCCUGGAGACAACC
713


5149
AUGGCCUUACACUGAAAAU
287
5149
AUGGCCUUACACUGAAAAU
287
5167
AUUUUCAGUGUAAGGCCAU
714


5167
UGUCACAUUCUAUUUUGGG
288
5167
UGUCACAUUCUAUUUUGGG
288
5185
CCCAAAAUAGAAUGUGACA
715


5185
GUAUUAAUAUAUAGUCCAG
289
5185
GUAUUAAUAUAUAGUCCAG
289
5203
CUGGACUAUAUAUUAAUAC
716


5203
GACACUUAACUCAAUUUCU
290
5203
GACACUUAACUCAAUUUCU
290
5221
AGAAAUUGAGUUAAGUGUC
717


5221
UUGGUAUUAUUCUGUUUUG
291
5221
UUGGUAUUAUUCUGUUUUG
291
5239
CAAAACAGAAUAAUACCAA
718


5239
GCACAGUUAGUUGUGAAAG
292
5239
GCACAGUUAGUUGUGAAAG
292
5257
CUUUCACAACUAACUGUGC
719


5257
GAAAGCUGAGAAGAAUGAA
293
5257
GAAAGCUGAGAAGAAUGAA
293
5275
UUCAUUCUUCUCAGCUUUC
720


5275
AAAUGCAGUCCUGAGGAGA
294
5275
AAAUGCAGUCCUGAGGAGA
294
5293
UCUCCUCAGGACUGCAUUU
721


5293
AGUUUUCUCCAUAUCAAAA
295
5293
AGUUUUCUCCAUAUCAAAA
295
5311
UUUUGAUAUGGAGAAAACU
722


5311
ACGAGGGCUGAUGGAGGAA
296
5311
ACGAGGGCUGAUGGAGGAA
296
5329
UUCCUCCAUCAGCCCUCGU
723


5329
AAAAGGUCAAUAAGGUCAA
297
5329
AAAAGGUCAAUAAGGUCAA
297
5347
UUGACCUUAUUGACCUUUU
724


5347
AGGGAAGACCCCGUCUCUA
298
5347
AGGGAAGACCCCGUCUCUA
298
5365
UAGAGACGGGGUCUUCCCU
725


5365
AUACCAACCAAACCAAUUC
299
5365
AUACCAACCAAACCAAUUC
299
5383
GAAUUGGUUUGGUUGGUAU
726


5383
CACCAACACAGUUGGGACC
300
5383
CACCAACACAGUUGGGACC
300
5401
GGUCCCAACUGUGUUGGUG
727


5401
CCAAAACACAGGAAGUCAG
301
5401
CCAAAACACAGGAAGUCAG
301
5419
CUGACUUCCUGUGUUUUGG
728


5419
GUCACGUUUCCUUUUCAUU
302
5419
GUCACGUUUCCUUUUCAUU
302
5437
AAUGAAAAGGAAACGUGAC
729


5437
UUAAUGGGGAUUCCACUAU
303
5437
UUAAUGGGGAUUCCACUAU
303
5455
AUAGUGGAAUCCCCAUUAA
730


5455
UCUCACACUAAUCUGAAAG
304
5455
UCUCACACUAAUCUGAAAG
304
5473
CUUUCAGAUUAGUGUGAGA
731


5473
GGAUGUGGAAGAGCAUUAG
305
5473
GGAUGUGGAAGAGCAUUAG
305
5491
CUAAUGCUCUUCCACAUCC
732


5491
GCUGGCGCAUAUUAAGCAC
306
5491
GCUGGCGCAUAUUAAGCAC
306
5509
GUGCUUAAUAUGCGCCAGC
733


5509
CUUUAAGCUCCUUGAGUAA
307
5509
CUUUAAGCUCCUUGAGUAA
307
5527
UUACUCAAGGAGCUUAAAG
734


5527
AAAAGGUGGUAUGUAAUUU
308
5527
AAAAGGUGGUAUGUAAUUU
308
5545
AAAUUACAUACCACCUUUU
735


5545
UAUGCAAGGUAUUUCUCCA
309
5545
UAUGCAAGGUAUUUCUCCA
309
5563
UGGAGAAAUACCUUGCAUA
736


5563
AGUUGGGACUCAGGAUAUU
310
5563
AGUUGGGACUCAGGAUAUU
310
5581
AAUAUCCUGAGUCCCAACU
737


5581
UAGUUAAUGAGCCAUCACU
311
5581
UAGUUAAUGAGCCAUCACU
311
5599
AGUGAUGGCUCAUUAACUA
735


5599
UAGAAGAAAAGCCCAUUUU
312
5599
UAGAAGAAAAGCCCAUUUU
312
5617
AAAAUGGGCUUUUCUUCUA
739


5617
UCAACUGCUUUGAAACUUG
313
5617
UCAACUGCUUUGAAACUUG
313
5635
CAAGUUUCAAAGCAGUUGA
740


5635
GCCUGGGGUCUGAGCAUGA
314
5635
GCCUGGGGUCUGAGCAUGA
314
5653
UCAUGCUCAGACCCCAGGC
741


5653
AUGGGAAUAGGGAGACAGG
315
5653
AUGGGAAUAGGGAGACAGG
315
5671
CCUGUCUCCCUAUUCCCAU
742


5671
GGUAGGAAAGGGCGCCUAC
316
5671
GGUAGGAAAGGGCGCCUAC
316
5689
GUAGGCGCCCUUUCCUACC
743


5689
CUCUUCAGGGUCUAAAGAU
317
5689
CUCUUCAGGGUCUAAAGAU
317
5707
AUCUUUAGACCCUGAAGAG
744


5707
UCAAGUGGGCCUUGGAUCG
318
5707
UCAAGUGGGCCUUGGAUCG
318
5725
CGAUCCAAGGCCCACUUGA
745


5725
GCUAAGCUGGCUCUGUUUG
319
5725
GCUAAGCUGGCUCUGUUUG
319
5743
CAAACAGAGCCAGCUUAGC
746


5743
GAUGCUAUUUAUGCAAGUU
320
5743
GAUGCUAUUUAUGCAAGUU
320
5761
AACUUGCAUAAAUAGCAUC
747


5761
UAGGGUCUAUGUAUUUAGG
321
5761
UAGGGUCUAUGUAUUUAGG
321
5779
CCUAAAUACAUAGACCCUA
748


5779
GAUGCGCCUACUCUUCAGG
322
5779
GAUGCGCCUACUCUUCAGG
322
5797
CCUGAAGAGUAGGCGCAUC
749


5797
GGUCUAAAGAUCAAGUGGG
323
5797
GGUCUAAAGAUCAAGUGGG
323
5815
CCCACUUGAUCUUUAGACC
750


5815
GCCUUGGAUCGCUAAGCUG
324
5815
GCCUUGGAUCGCUAAGCUG
324
5833
CAGCUUAGCGAUCCAAGGC
751


5833
GGCUCUGUUUGAUGCUAUU
325
5833
GGCUCUGUUUGAUGCUAUU
325
5851
AAUAGCAUCAAACAGAGCC
752


5851
UUAUGCAAGUUAGGGUCUA
326
5851
UUAUGCAAGUUAGGGUCUA
326
5869
UAGACCCUAACUUGCAUAA
753


5869
AUGUAUUUAGGAUGUCUGC
327
5869
AUGUAUUUAGGAUGUCUGC
327
5887
GCAGACAUCCUAAAUACAU
754


5887
CACCUUCUGCAGCCAGUCA
328
5887
CACCUUCUGCAGCCAGUCA
328
5905
UGACUGGCUGCAGAAGGUG
755


5905
AGAAGCUGGAGAGGCAACA
329
5905
AGAAGCUGGAGAGGCAACA
329
5923
UGUUGCCUCUCCAGCUUCU
756


5923
AGUGGAUUGCUGCUUCUUG
330
5923
AGUGGAUUGCUGCUUCUUG
330
5941
CAAGAAGCAGCAAUCCACU
757


5941
GGGGAGAAGAGUAUGCUUC
331
5941
GGGGAGAAGAGUAUGCUUC
331
5959
GAAGCAUACUCUUCUCCCC
758


5959
CCUUUUAUCCAUGUAAUUU
332
5959
CCUUUUAUCCAUGUAAUUU
332
5977
AAAUUACAUGGAUAAAAGG
759


5977
UAACUGUAGAACCUGAGCU
333
5977
UAACUGUAGAACCUGAGCU
333
5995
AGCUCAGGUUCUACAGUUA
760


5995
UCUAAGUAACCGAAGAAUG
334
5995
UCUAAGUAACCGAAGAAUG
334
6013
CAUUCUUCGGUUACUUAGA
761


6013
GUAUGCCUCUGUUCUUAUG
335
6013
GUAUGCCUCUGUUCUUAUG
335
6031
CAUAAGAACAGAGGCAUAC
762


6031
GUGCCACAUCCUUGUUUAA
336
6031
GUGCCACAUCCUUGUUUAA
336
6049
UUAAACAAGGAUGUGGCAC
763


6049
AAGGCUCUCUGUAUGAAGA
337
6049
AAGGCUCUCUGUAUGAAGA
337
6067
UCUUCAUACAGAGAGCCUU
764


6067
AGAUGGGACCGUCAUCAGC
338
6067
AGAUGGGACCGUCAUCAGC
338
6085
GCUGAUGACGGUCCCAUCU
765


6085
CACAUUCCCUAGUGAGCCU
339
6085
CACAUUCCCUAGUGAGCCU
339
6103
AGGCUCACUAGGGAAUGUG
766


6103
UACUGGCUCCUGGCAGCGG
340
6103
UACUGGCUCCUGGCAGCGG
340
6121
CCGCUGCCAGGAGCCAGUA
767


6121
GCUUUUGUGGAAGACUCAC
341
6121
GCUUUUGUGGAAGACUCAC
341
6139
GUGAGUCUUCCACAAAAGC
768


6139
CUAGCCAGAAGAGAGGAGU
342
6139
CUAGCCAGAAGAGAGGAGU
342
6157
ACUCCUCUCUUCUGGCUAG
769


6157
UGGGACAGUCCUCUCCACC
343
6157
UGGGACAGUCCUCUCCACC
343
6175
GGUGGAGAGGACUGUCCCA
770


6175
CAAGAUCUAAAUCCAAACA
344
6175
CAAGAUCUAAAUCCAAACA
344
6193
UGUUUGGAUUUAGAUCUUG
771


6193
AAAAGCAGGCUAGAGCCAG
345
6193
AAAAGCAGGCUAGAGCCAG
345
6211
CUGGCUCUAGCCUGCUUUU
772


6211
GAAGAGAGGACAAAUCUUU
346
6211
GAAGAGAGGACAAAUCUUU
346
6229
AAAGAUUUGUCCUCUCUUC
773


6229
UGUUGUUCCUCUUCUUUAC
347
6229
UGUUGUUCCUCUUCUUUAC
347
6247
GUAAAGAAGAGGAACAACA
774


6247
CACAUACGCAAACCACCUG
348
6247
CACAUACGCAAACCACCUG
348
6265
CAGGUGGUUUGCGUAUGUG
775


6265
GUGACAGCUGGCAAUUUUA
349
6265
GUGACAGCUGGCAAUUUUA
349
6283
UAAAAUUGCCAGCUGUCAC
776


6283
AUAAAUCAGGUAACUGGAA
350
6283
AUAAAUCAGGUAACUGGAA
350
6301
UUCCAGUUACCUGAUUUAU
777


6301
AGGAGGUUAAACUCAGAAA
351
6301
AGGAGGUUAAACUCAGAAA
351
6319
UUUCUGAGUUUAACCUCCU
778


6319
AAAAGAAGACCUCAGUCAA
352
6319
AAAAGAAGACCUCAGUCAA
352
6337
UUGACUGAGGUCUUCUUUU
779


6337
AUUCUCUACUUUUUUUUUU
353
6337
AUUCUCUACUUUUUUUUUU
353
6355
AAAAAAAAAAGUAGAGAAU
780


6355
UUUUUUUCCAAAUCAGAUA
354
6355
UUUUUUUCCAAAUCAGAUA
354
6373
UAUCUGAUUUGGAAAAAAA
781


6373
AAUAGCCCAGCAAAUAGUG
355
6373
AAUAGCCCAGCAAAUAGUG
355
6391
CACUAUUUGCUGGGCUAUU
782


6391
GAUAACAAAUAAAACCUUA
356
6391
GAUAACAAAUAAAACCUUA
356
6409
UAAGGUUUUAUUUGUUAUC
783


6409
AGCUGUUCAUGUCUUGAUU
357
6409
AGCUGUUCAUGUCUUGAUU
357
6427
AAUCAAGACAUGAACAGCU
784


6427
UUCAAUAAUUAAUUCUUAA
358
6427
UUCAAUAAUUAAUUCUUAA
358
6445
UUAAGAAUUAAUUAUUGAA
785


6445
AUCAUUAAGAGACCAUAAU
359
6445
AUCAUUAAGAGACCAUAAU
359
6463
AUUAUGGUCUCUUAAUGAU
786


6463
UAAAUACUCCUUUUCAAGA
360
6463
UAAAUACUCCUUUUCAAGA
360
6481
UCUUGAAAAGGAGUAUUUA
787


6481
AGAAAAGCAAAACCAUUAG
361
6481
AGAAAAGCAAAACCAUUAG
361
6499
CUAAUGGUUUUGCUUUUCU
788


6499
GAAUUGUUACUCAGCUCCU
362
6499
GAAUUGUUACUCAGCUCCU
362
6517
AGGAGCUGAGUAACAAUUC
789


6517
UUCAAACUCAGGUUUGUAG
363
6517
UUCAAACUCAGGUUUGUAG
363
6535
CUACAAACCUGAGUUUGAA
790


6535
GCAUACAUGAGUCCAUCCA
364
6535
GCAUACAUGAGUCCAUCCA
364
6553
UGGAUGGACUCAUGUAUGC
791


6553
AUCAGUCAAAGAAUGGUUC
365
6553
AUCAGUCAAAGAAUGGUUC
365
6571
GAACCAUUCUUUGACUGAU
792


6571
CCAUCUGGAGUCUUAAUGU
366
6571
CCAUCUGGAGUCUUAAUGU
366
6589
ACAUUAAGACUCCAGAUGG
793


6589
UAGAAAGAAAAAUGGAGAC
367
6589
UAGAAAGAAAAAUGGAGAC
367
6607
GUCUCCAUUUUUCUUUCUA
794


6607
CUUGUAAUAAUGAGCUAGU
368
6607
CUUGUAAUAAUGAGCUAGU
368
6625
ACUAGCUCAUUAUUACAAG
795


6625
UUACAAAGUGCUUGUUCAU
369
6625
UUACAAAGUGCUUGUUCAU
369
6643
AUGAACAAGCACUUUGUAA
796


6643
UUAAAAUAGCACUGAAAAU
370
6643
UUAAAAUAGCACUGAAAAU
370
6661
AUUUUCAGUGCUAUUUUAA
797


6661
UUGAAACAUGAAUUAACUG
371
6661
UUGAAACAUGAAUUAACUG
371
6679
CAGUUAAUUCAUGUUUCAA
798


6679
GAUAAUAUUCCAAUCAUUU
372
6679
GAUAAUAUUCCAAUCAUUU
372
6697
AAAUGAUUGGAAUAUUAUC
799


6697
UGCCAUUUAUGACAAAAAU
373
6697
UGCCAUUUAUGACAAAAAU
373
6715
AUUUUUGUCAUAAAUGGCA
800


6715
UGGUUGGCACUAACAAAGA
374
6715
UGGUUGGCACUAACAAAGA
374
6733
UCUUUGUUAGUGCCAACCA
801


6733
AACGAGCACUUCCUUUCAG
375
6733
AACGAGCACUUCCUUUCAG
375
6751
CUGAAAGGAAGUGCUCGUU
802


6751
GAGUUUCUGAGAUAAUGUA
376
6751
GAGUUUCUGAGAUAAUGUA
376
6769
UACAUUAUCUCAGAAACUC
803


6769
ACGUGGAACAGUCUGGGUG
377
6769
ACGUGGAACAGUCUGGGUG
377
6787
CACCCAGACUGUUCCACGU
804


6787
GGAAUGGGGCUGAAACCAU
378
6787
GGAAUGGGGCUGAAACCAU
378
6805
AUGGUUUCAGCCCCAUUCC
805


6805
UGUGCAAGUCUGUGUCUUG
379
6805
UGUGCAAGUCUGUGUCUUG
379
6823
CAAGACACAGACUUGCACA
806


6823
GUCAGUCCAAGAAGUGACA
380
6823
GUCAGUCCAAGAAGUGACA
380
6841
UGUCACUUCUUGGACUGAC
807


6841
ACCGAGAUGUUAAUUUUAG
381
6841
ACCGAGAUGUUAAUUUUAG
381
6859
CUAAAAUUAACAUCUCGGU
808


6859
GGGACCCGUGCCUUGUUUC
382
6859
GGGACCCGUGCCUUGUUUC
382
6877
GAAACAAGGCACGGGUCCC
809


6877
CCUAGCCCACAAGAAUGCA
383
6877
CCUAGCCCACAAGAAUGCA
383
6895
UGCAUUCUUGUGGGCUAGG
810


6895
AAACAUCAAACAGAUACUC
384
6895
AAACAUCAAACAGAUACUC
384
6913
GAGUAUCUGUUUGAUGUUU
811


6913
CGCUAGCCUCAUUUAAAUU
385
6913
CGCUAGCCUCAUUUAAAUU
385
6931
AAUUUAAAUGAGGCUAGCG
812


6931
UGAUUAAAGGAGGAGUGCA
386
6931
UGAUUAAAGGAGGAGUGCA
386
6949
UGCACUCCUCCUUUAAUCA
813


6949
AUCUUUGGCCGACAGUGGU
387
6949
AUCUUUGGCCGACAGUGGU
387
6967
ACCACUGUCGGCCAAAGAU
814


6967
UGUAACUGUGUGUGUGUGU
388
6967
UGUAACUGUGUGUGUGUGU
388
6985
ACACACACACACAGUUACA
815


6985
UGUGUGUGUGUGUGUGUGU
389
6985
UGUGUGUGUGUGUGUGUGU
389
7003
ACACACACACACACACACA
816


7003
UGUGUGUGUGUGGGUGUGG
390
7003
UGUGUGUGUGUGGGUGUGG
390
7021
CCACACCCACACACACACA
817


7021
GGUGUAUGUGUGUUUUGUG
391
7021
GGUGUAUGUGUGUUUUGUG
391
7039
CACAAAACACACAUACACC
818


7039
GCAUAACUAUUUAAGGAAA
392
7039
GCAUAACUAUUUAAGGAAA
392
7057
UUUCCUUAAAUAGUUAUGC
819


7057
ACUGGAAUUUUAAAGUUAC
393
7057
ACUGGAAUUUUAAAGUUAC
393
7075
GUAACUUUAAAAUUCCAGU
820


7075
CUUUUAUACAAACCAAGAA
394
7075
CUUUUAUACAAACCAAGAA
394
7093
UUCUUGGUUUGUAUAAAAG
821


7093
AUAUAUGCUACAGAUAUAA
395
7093
AUAUAUGCUACAGAUAUAA
395
7111
UUAUAUCUGUAGCAUAUAU
822


7111
AGACAGACAUGGUUUGGUC
396
7111
AGACAGACAUGGUUUGGUC
396
7129
GACCAAACCAUGUCUGUCU
823


7129
CCUAUAUUUCUAGUCAUGA
397
7129
CCUAUAUUUCUAGUCAUGA
397
7147
UCAUGACUAGAAAUAUAGG
824


7147
AUGAAUGUAUUUUGUAUAC
398
7147
AUGAAUGUAUUUUGUAUAC
398
7165
GUAUACAAAAUACAUUCAU
825


7165
CCAUCUUCAUAUAAUAUAC
399
7165
CCAUCUUCAUAUAAUAUAC
399
7183
GUAUAUUAUAUGAAGAUGG
826


7183
CUUAAAAAUAUUUCUUAAU
400
7183
CUUAAAAAUAUUUCUUAAU
400
7201
AUUAAGAAAUAUUUUUAAG
827


7201
UUGGGAUUUGUAAUCGUAC
401
7201
UUGGGAUUUGUAAUCGUAC
401
7219
GUACGAUUACAAAUCCCAA
828


7219
CCAACUUAAUUGAUAAACU
402
7219
CCAACUUAAUUGAUAAACU
402
7237
AGUUUAUCAAUUAAGUUGG
829


7237
UUGGCAACUGCUUUUAUGU
403
7237
UUGGCAACUGCUUUUAUGU
403
7255
ACAUAAAAGCAGUUGCCAA
830


7255
UUCUGUCUCCUUCCAUAAA
404
7255
UUCUGUCUCCUUCCAUAAA
404
7273
UUUAUGGAAGGAGACAGAA
831


7273
AUUUUUCAAAAUACUAAUU
405
7273
AUUUUUCAAAAUACUAAUU
405
7291
AAUUAGUAUUUUGAAAAAU
832


7291
UCAACAAAGAAAAAGCUCU
406
7291
UCAACAAAGAAAAAGCUCU
406
7309
AGAGCUUUUUCUUUGUUGA
833


7309
UUUUUUUUCCUAAAAUAAA
407
7309
UUUUUUUUCCUAAAAUAAA
407
7327
UUUAUUUUAGGAAAAAAAA
834


7327
ACUCAAAUUUAUCCUUGUU
408
7327
ACUCAAAUUUAUCCUUGUU
408
7345
AACAAGGAUAAAUUUGAGU
835


7345
UUAGAGCAGAGAAAAAUUA
409
7345
UUAGAGCAGAGAAAAAUUA
409
7363
UAAUUUUUCUCUGCUCUAA
836


7363
AAGAAAAACUUUGAAAUGG
410
7363
AAGAAAAACUUUGAAAUGG
410
7381
CCAUUUCAAAGUUUUUCUU
837


7381
GUCUCAAAAAAUUGCUAAA
411
7381
GUCUCAAAAAAUUGCUAAA
411
7399
UUUAGCAAUUUUUUGAGAC
838


7399
AUAUUUUCAAUGGAAAACU
412
7399
AUAUUUUCAAUGGAAAACU
412
7417
AGUUUUCCAUUGAAAAUAU
839


7417
UAAAUGUUAGUUUAGCUGA
413
7417
UAAAUGUUAGUUUAGCUGA
413
7435
UCAGCUAAACUAACAUUUA
840


7435
AUUGUAUGGGGUUUUCGAA
414
7435
AUUGUAUGGGGUUUUCGAA
414
7453
UUCGAAAACCCCAUACAAU
841


7453
ACCUUUCACUUUUUGUUUG
415
7453
ACCUUUCACUUUUUGUUUG
415
7471
CAAACAAAAAGUGAAAGGU
842


7471
GUUUUACCUAUUUCACAAC
416
7471
GUUUUACCUAUUUCACAAC
416
7489
GUUGUGAAAUAGGUAAAAC
843


7489
CUGUGUAAAUUGCCAAUAA
417
7489
CUGUGUAAAUUGCCAAUAA
417
7507
UUAUUGGCAAUUUACACAG
844


7507
AUUCCUGUCCAUGAAAAUG
418
7507
AUUCCUGUCCAUGAAAAUG
418
7525
CAUUUUCAUGGACAGGAAU
845


7525
GCAAAUUAUCCAGUGUAGA
419
7525
GCAAAUUAUCCAGUGUAGA
419
7543
UCUACACUGGAUAAUUUGC
846


7543
AUAUAUUUGACCAUCACCC
420
7543
AUAUAUUUGACCAUCACCC
420
7561
GGGUGAUGGUCAAAUAUAU
847


7561
CUAUGGAUAUUGGCUAGUU
421
7561
CUAUGGAUAUUGGCUAGUU
421
7579
AACUAGCCAAUAUCCAUAG
848


7579
UUUGCCUUUAUUAAGCAAA
422
7579
UUUGCCUUUAUUAAGCAAA
422
7597
UUUGCUUAAUAAAGGCAAA
849


7597
AUUCAUUUCAGCCUGAAUG
423
7597
AUUCAUUUCAGCCUGAAUG
423
7615
CAUUCAGGCUGAAAUGAAU
850


7615
GUCUGCCUAUAUAUUCUCU
424
7615
GUCUGCCUAUAUAUUCUCU
424
7633
AGAGAAUAUAUAGGCAGAC
851


7633
UGCUCUUUGUAUUCUCCUU
425
7633
UGCUCUUUGUAUUCUCCUU
425
7651
AAGGAGAAUACAAAGAGCA
852


7651
UUGAACCCGUUAAAACAUC
426
7651
UUGAACCCGUUAAAACAUC
426
7669
GAUGUUUUAACGGGUUCAA
853


7662
AAAACAUCCUGUGGCACUC
427
7662
AAAACAUCCUGUGGCACUC
427
7680
GAGUGCCACAGGAUGUUUU
854


 







VEGFR2/KDR NM_002253.1
















1
ACUGAGUCCCGGGACCCCG
855
1
ACUGAGUCCCGGGACCCCG
855
19
CGGGGUCCCGGGACUCAGU
1179



19
GGGAGAGCGGUCAGUGUGU
856
19
GGGAGAGCGGUCAGUGUGU
856
37
ACACACUGACCGCUCUCCC
1180


37
UGGUCGCUGCGUUUCCUCU
857
37
UGGUCGCUGCGUUUCCUCU
857
55
AGAGGAAACGCAGCGACCA
1181


55
UGCCUGCGCCGGGCAUCAC
858
55
UGCCUGCGCCGGGCAUCAC
858
73
GUGAUGCCCGGCGCAGGCA
1182


73
CUUGCGCGCCGCAGAAAGU
859
73
CUUGCGCGCCGCAGAAAGU
859
91
ACUUUCUGCGGCGCGCAAG
1183


91
UCCGUCUGGCAGCCUGGAU
860
91
UCCGUCUGGCAGCCUGGAU
860
109
AUCCAGGCUGCCAGACGGA
1184


109
UAUCCUCUCCUACCGGCAC
861
109
UAUCCUCUCCUACCGGCAC
861
127
GUGCCGGUAGGAGAGGAUA
1185


127
CCCGCAGACGCCCCUGCAG
862
127
CCCGCAGACGCCCCUGCAG
862
145
CUGCAGGGGCGUCUGCGGG
1186


145
GCCGCCGGUCGGCGCCCGG
863
145
GCCGCCGGUCGGCGCCCGG
863
163
CCGGGCGCCGACCGGCGGC
1187


163
GGCUCCCUAGCCCUGUGCG
864
163
GGCUCCCUAGCCCUGUGCG
864
181
CGCACAGGGCUAGGGAGCC
1188


181
GCUCAACUGUCCUGCGCUG
865
181
GCUCAACUGUCCUGCGCUG
865
199
CAGCGCAGGACAGUUGAGC
1189


199
GCGGGGUGCCGCGAGUUCC
866
199
GCGGGGUGCCGCGAGUUCC
866
217
GGAACUCGCGGCACCCCGC
1190


217
CACCUCCGCGCCUCCUUCU
867
217
CACCUCCGCGCCUCCUUCU
867
235
AGAAGGAGGCGCGGAGGUG
1191


235
UCUAGACAGGCGCUGGGAG
868
235
UCUAGACAGGCGCUGGGAG
868
253
CUCCCAGCGCCUGUCUAGA
1192


253
GAAAGAACCGGCUCCCGAG
869
253
GAAAGAACCGGCUCCCGAG
869
271
CUCGGGAGCCGGUUCUUUC
1193


271
GUUCUGGGCAUUUCGCCCG
870
271
GUUCUGGGCAUUUCGCCCG
870
289
CGGGCGAAAUGCCCAGAAC
1194


289
GGCUCGAGGUGCAGGAUGC
871
289
GGCUCGAGGUGCAGGAUGC
871
307
GCAUCCUGCACCUCGAGCC
1195


307
CAGAGCAAGGUGCUGCUGG
872
307
CAGAGCAAGGUGCUGCUGG
872
325
CCAGCAGCACCUUGCUCUG
1196


325
GCCGUCGCCCUGUGGCUCU
873
325
GCCGUCGCCCUGUGGCUCU
873
343
AGAGCCACAGGGCGACGGC
1197


343
UGCGUGGAGACCCGGGCCG
874
343
UGCGUGGAGACCCGGGCCG
874
361
CGGCCCGGGUCUCCACGCA
1198


361
GCCUCUGUGGGUUUGCCUA
875
361
GCCUCUGUGGGUUUGCCUA
875
379
UAGGCAAACCCACAGAGGC
1199


379
AGUGUUUCUCUUGAUCUGC
876
379
AGUGUUUCUCUUGAUCUGC
876
397
GCAGAUCAAGAGAAACACU
1200


397
CCCAGGCUCAGCAUACAAA
877
397
CCCAGGCUCAGCAUACAAA
877
415
UUUGUAUGCUGAGCCUGGG
1201


415
AAAGACAUACUUACAAUUA
878
415
AAAGACAUACUUACAAUUA
878
433
UAAUUGUAAGUAUGUCUUU
1202


433
AAGGCUAAUACAACUCUUC
879
433
AAGGCUAAUACAACUCUUC
879
451
GAAGAGUUGUAUUAGCCUU
1203


451
CAAAUUACUUGCAGGGGAC
880
451
CAAAUUACUUGCAGGGGAC
880
469
GUCCCCUGCAAGUAAUUUG
1204


469
CAGAGGGACUUGGACUGGC
881
469
CAGAGGGACUUGGACUGGC
881
487
GCCAGUCCAAGUCCCUCUG
1205


487
CUUUGGCCCAAUAAUCAGA
882
487
CUUUGGCCCAAUAAUCAGA
882
505
UCUGAUUAUUGGGCCAAAG
1206


505
AGUGGCAGUGAGCAAAGGG
883
505
AGUGGCAGUGAGCAAAGGG
883
523
CCCUUUGCUCACUGCCACU
1207


523
GUGGAGGUGACUGAGUGGA
884
523
GUGGAGGUGACUGAGUGGA
884
541
UGCACUCAGUCACCUCCAC
1208


541
AGCGAUGGCCUCUUCUGUA
885
541
AGCGAUGGCCUCUUCUGUA
885
559
UACAGAAGAGGCCAUCGCU
1209


559
AAGACACUCACAAUUCCAA
886
559
AAGACACUCACAAUUCCAA
886
577
UUGGAAUUGUGAGUGUCUU
1210


577
AAAGUGAUCGGAAAUGACA
887
577
AAAGUGAUCGGAAAUGACA
887
595
UGUCAUUUCCGAUCACUUU
1211


595
ACUGGAGCCUACAAGUGCU
888
595
ACUGGAGCCUACAAGUGCU
888
613
AGCACUUGUAGGCUCCAGU
1212


613
UUCUACCGGGAAACUGACU
889
613
UUCUACCGGGAAACUGACU
889
631
AGUCAGUUUCCCGGUAGAA
1213


631
UUGGCCUCGGUCAUUUAUG
890
631
UUGGCCUCGGUCAUUUAUG
890
649
CAUAAAUGACCGAGGCCAA
1214


649
GUCUAUGUUCAAGAUUACA
891
649
GUCUAUGUUCAAGAUUACA
891
667
UGUAAUCUUGAACAUAGAC
1215


667
AGAUCUCCAUUUAUUGCUU
892
667
AGAUCUCCAUUUAUUGCUU
892
685
AAGCAAUAAAUGGAGAUCU
1216


685
UCUGUUAGUGACCAACAUG
893
685
UCUGUUAGUGACCAACAUG
893
703
CAUGUUGGUCACUAACAGA
1217


703
GGAGUCGUGUACAUUACUG
894
703
GGAGUCGUGUACAUUACUG
894
721
CAGUAAUGUACACGACUCC
1218


721
GAGAACAAAAACAAAACUG
895
721
GAGAACAAAAACAAAACUG
895
739
CAGUUUUGUUUUUGUUCUC
1219


739
GUGGUGAUUCCAUGUCUCG
896
739
GUGGUGAUUCCAUGUCUCG
896
757
CGAGACAUGGAAUCACCAC
1220


757
GGGUCCAUUUCAAAUCUCA
897
757
GGGUCCAUUUCAAAUCUCA
897
775
UGAGAUUUGAAAUGGACCC
1221


775
AACGUGUCACUUUGUGCAA
898
775
AACGUGUCACUUUGUGCAA
898
793
UUGCACAAAGUGACACGUU
1222


793
AGAUACCCAGAAAAGAGAU
899
793
AGAUACCCAGAAAAGAGAU
899
811
AUCUCUUUUCUGGGUAUCU
1223


811
UUUGUUCCUGAUGGUAACA
900
811
UUUGUUCCUGAUGGUAACA
900
829
UGUUACCAUCAGGAACAAA
1224


829
AGAAUUUCCUGGGACAGCA
901
829
AGAAUUUCCUGGGACAGCA
901
847
UGCUGUCCCAGGAAAUUCU
1225


847
AAGAAGGGCUUUACUAUUC
902
847
AAGAAGGGCUUUACUAUUC
902
865
GAAUAGUAAAGCCCUUCUU
1226


865
CCCAGCUACAUGAUCAGCU
903
865
CCCAGCUACAUGAUCAGCU
903
883
AGCUGAUCAUGUAGCUGGG
1227


883
UAUGCUGGCAUGGUCUUCU
904
883
UAUGCUGGCAUGGUCUUCU
904
901
AGAAGACCAUGCCAGCAUA
1228


901
UGUGAAGCAAAAAUUAAUG
905
901
UGUGAAGCAAAAAUUAAUG
905
919
CAUUAAUUUUUGCUUCACA
1229


919
GAUGAAAGUUACCAGUCUA
906
919
GAUGAAAGUUACCAGUCUA
906
937
UAGACUGGUAACUUUCAUC
1230


937
AUUAUGUACAUAGUUGUCG
907
937
AUUAUGUACAUAGUUGUCG
907
955
CGACAACUAUGUACAUAAU
1231


955
GUUGUAGGGUAUAGGAUUU
908
955
GUUGUAGGGUAUAGGAUUU
908
973
AAAUCCUAUACCCUACAAC
1232


973
UAUGAUGUGGUUCUGAGUC
909
973
UAUGAUGUGGUUCUGAGUC
909
991
GACUCAGAACCACAUCAUA
1233


991
CCGUCUCAUGGAAUUGAAC
910
991
CCGUCUCAUGGAAUUGAAC
910
1009
GUUCAAUUCCAUGAGACGG
1234


1009
CUAUCUGUUGGAGAAAAGC
911
1009
CUAUCUGUUGGAGAAAAGC
911
1027
GCUUUUCUCCAACAGAUAG
1235


1027
CUUGUCUUAAAUUGUACAG
912
1027
CUUGUCUUAAAUUGUACAG
912
1045
CUGUACAAUUUAAGACAAG
1236


1045
GCAAGAACUGAACUAAAUG
913
1045
GCAAGAACUGAACUAAAUG
913
1063
CAUUUAGUUCAGUUCUUGC
1237


1063
GUGGGGAUUGACUUCAACU
914
1063
GUGGGGAUUGACUUCAACU
914
1081
AGUUGAAGUCAAUCCCCAC
1238


1081
UGGGAAUACCCUUCUUCGA
915
1081
UGGGAAUACCCUUCUUCGA
915
1099
UCGAAGAAGGGUAUUCCCA
1239


1099
AAGCAUCAGCAUAAGAAAC
916
1099
AAGCAUCAGCAUAAGAAAC
916
1117
GUUUCUUAUGCUGAUGCUU
1240


1117
CUUGUAAACCGAGACCUAA
917
1117
CUUGUAAACCGAGACCUAA
917
1135
UUAGGUCUCGGUUUACAAG
1241


1135
AAAACCCAGUCUGGGAGUG
918
1135
AAAACCCAGUCUGGGAGUG
918
1153
CACUCCCAGACUGGGUUUU
1242


1153
GAGAUGAAGAAAUUUUUGA
919
1153
GAGAUGAAGAAAUUUUUGA
919
1171
UCAAAAAUUUCUUCAUCUC
1243


1171
AGCACCUUAACUAUAGAUG
920
1171
AGCACCUUAACUAUAGAUG
920
1189
CAUCUAUAGUUAAGGUGCU
1244


1189
GGUGUAACCCGGAGUGACC
921
1189
GGUGUAACCCGGAGUGACC
921
1207
GGUCACUCCGGGUUACACC
1245


1207
CAAGGAUUGUACACCUGUG
922
1207
CAAGGAUUGUACACCUGUG
922
1225
CACAGGUGUACAAUCCUUG
1246


1225
GCAGCAUCCAGUGGGCUGA
923
1225
GCAGCAUCCAGUGGGCUGA
923
1243
UCAGCCCACUGGAUGCUGC
1247


1243
AUGACCAAGAAGAACAGCA
924
1243
AUGACCAAGAAGAACAGCA
924
1261
UGCUGUUCUUCUUGGUCAU
1248


1261
ACAUUUGUCAGGGUCCAUG
925
1261
ACAUUUGUCAGGGUCCAUG
925
1279
CAUGGACCCUGACAAAUGU
1249


1279
GAAAAACCUUUUGUUGCUU
926
1279
GAAAAACCUUUUGUUGCUU
926
1297
AAGCAACAAAAGGUUUUUC
1250


1297
UUUGGAAGUGGCAUGGAAU
927
1297
UUUGGAAGUGGCAUGGAAU
927
1315
AUUCCAUGCCACUUCCAAA
1251


1315
UCUCUGGUGGAAGCCACGG
928
1315
UCUCUGGUGGAAGCCACGG
928
1333
CCGUGGCUUCCACCAGAGA
1252


1333
GUGGGGGAGCGUGUCAGAA
929
1333
GUGGGGGAGCGUGUCAGAA
929
1351
UUCUGACACGCUCCCCCAC
1253


1351
AUCCCUGCGAAGUACCUUG
930
1351
AUCCCUGCGAAGUACCUUG
930
1369
CAAGGUACUUCGCAGGGAU
1254


1369
GGUUACCCACCCCCAGAAA
931
1369
GGUUACCCACCCCCAGAAA
931
1387
UUUCUGGGGGUGGGUAACC
1255


1387
AUAAAAUGGUAUAAAAAUG
932
1387
AUAAAAUGGUAUAAAAAUG
932
1405
CAUUUUUAUACCAUUUUAU
1256


1405
GGAAUACCCCUUGAGUCCA
933
1405
GGAAUACCCCUUGAGUCCA
933
1423
UGGACUCAAGGGGUAUUCC
1257


1423
AAUCACACAAUUAAAGCGG
934
1423
AAUCACACAAUUAAAGCGG
934
1441
CCGCUUUAAUUGUGUGAUU
1258


1441
GGGCAUGUACUGACGAUUA
935
1441
GGGCAUGUACUGACGAUUA
935
1459
UAAUCGUCAGUACAUGCCC
1259


1459
AUGGAAGUGAGUGAAAGAG
936
1459
AUGGAAGUGAGUGAAAGAG
936
1477
CUCUUUCACUCACUUCCAU
1260


1477
GACACAGGAAAUUACACUG
937
1477
GACACAGGAAAUUACACUG
937
1495
CAGUGUAAUUUCCUGUGUC
1261


1495
GUCAUCCUUACCAAUCCCA
938
1495
GUCAUCCUUACCAAUCCCA
938
1513
UGGGAUUGGUAAGGAUGAC
1262


1513
AUUUCAAAGGAGAAGCAGA
939
1513
AUUUCAAAGGAGAAGCAGA
939
1531
UCUGCUUCUCCUUUGAAAU
1263


1531
AGCCAUGUGGUCUCUCUGG
940
1531
AGCCAUGUGGUCUCUCUGG
940
1549
CCAGAGAGACCACAUGGCU
1264


1549
GUUGUGUAUGUCCCACCCC
941
1549
GUUGUGUAUGUCCCACCCC
941
1567
GGGGUGGGACAUACACAAC
1265


1567
CAGAUUGGUGAGAAAUCUC
942
1567
CAGAUUGGUGAGAAAUCUC
942
1585
GAGAUUUCUCACCAAUCUG
1266


1585
CUAAUCUCUCCUGUGGAUU
943
1585
CUAAUCUCUCCUGUGGAUU
943
1603
AAUCCACAGGAGAGAUUAG
1267


1603
UCCUACCAGUACGGCACCA
944
1603
UCCUACCAGUACGGCACCA
944
1621
UGGUGCCGUACUGGUAGGA
1268


1621
ACUCAAACGCUGACAUGUA
945
1621
ACUCAAACGCUGACAUGUA
945
1639
UACAUGUCAGCGUUUGAGU
1269


1639
ACGGUCUAUGCCAUUCCUC
946
1639
ACGGUCUAUGCCAUUCCUC
946
1657
GAGGAAUGGCAUAGACCGU
1270


1657
CCCCCGCAUCACAUCCACU
947
1657
CCCCCGCAUCACAUCCACU
947
1675
AGUGGAUGUGAUGCGGGGG
1271


1675
UGGUAUUGGCAGUUGGAGG
948
1675
UGGUAUUGGCAGUUGGAGG
948
1693
CCUCCAACUGCCAAUACCA
1272


1693
GAAGAGUGCGCCAACGAGC
949
1693
GAAGAGUGCGCCAACGAGC
949
1711
GCUCGUUGGCGCACUCUUC
1273


1711
CCCAGCCAAGCUGUCUCAG
950
1711
CCCAGCCAAGCUGUCUCAG
950
1729
CUGAGACAGCUUGGCUGGG
1274


1729
GUGACAAACCCAUACCCUU
951
1729
GUGACAAACCCAUACCCUU
951
1747
AAGGGUAUGGGUUUGUCAC
1275


1747
UGUGAAGAAUGGAGAAGUG
952
1747
UGUGAAGAAUGGAGAAGUG
952
1765
CACUUCUCCAUUCUUCACA
1276


1765
GUGGAGGACUUCCAGGGAG
953
1765
GUGGAGGACUUCCAGGGAG
953
1783
CUCCCUGGAAGUCCUCCAC
1277


1783
GGAAAUAAAAUUGAAGUUA
954
1783
GGAAAUAAAAUUGAAGUUA
954
1801
UAACUUCAAUUUUAUUUCC
1278


1801
AAUAAAAAUCAAUUUGCUC
955
1801
AAUAAAAAUCAAUUUGCUC
955
1819
GAGCAAAUUGAUUUUUAUU
1279


1819
CUAAUUGAAGGAAAAAACA
956
1819
CUAAUUGAAGGAAAAAACA
956
1837
UGUUUUUUCCUUCAAUUAG
1280


1837
AAAACUGUAAGUACCCUUG
957
1837
AAAACUGUAAGUACCCUUG
957
1855
CAAGGGUACUUACAGUUUU
1281


1855
GUUAUCCAAGCGGCAAAUG
958
1855
GUUAUCCAAGCGGCAAAUG
958
1873
CAUUUGCCGCUUGGAUAAC
1282


1873
GUGUCAGCUUUGUACAAAU
959
1873
GUGUCAGCUUUGUACAAAU
959
1891
AUUUGUACAAAGCUGACAC
1283


1891
UGUGAAGCGGUCAACAAAG
960
1891
UGUGAAGCGGUCAACAAAG
960
1909
CUUUGUUGACCGCUUCACA
1284


1909
GUCGGGAGAGGAGAGAGGG
961
1909
GUCGGGAGAGGAGAGAGGG
961
1927
CCCUCUCUCCUCUCCCGAC
1285


1927
GUGAUCUCCUUCCACGUGA
962
1927
GUGAUCUCCUUCCACGUGA
962
1945
UCACGUGGAAGGAGAUCAC
1286


1945
ACCAGGGGUCCUGAAAUUA
963
1945
ACCAGGGGUCCUGAAAUUA
963
1963
UAAUUUCAGGACCCCUGGU
1287


1963
ACUUUGCAACCUGACAUGC
964
1963
ACUUUGCAACCUGACAUGC
964
1981
GCAUGUCAGGUUGCAAAGU
1288


1981
CAGCCCACUGAGGAGGAGA
965
1981
CAGCCCACUGAGCAGGAGA
965
1999
UCUCCUGCUCAGUGGGCUG
1289


1999
AGCGUGUCUUUGUGGUGCA
966
1999
AGCGUGUCUUUGUGGUGCA
966
2017
UGCACCACAAAGACACGCU
1290


2017
ACUGCAGACAGAUCUACGU
967
2017
ACUGCAGACAGAUCUACGU
967
2035
ACGUAGAUCUGUCUGCAGU
1291


2035
UUUGAGAACCUCACAUGGU
968
2035
UUUGAGAACCUCACAUGGU
968
2053
ACCAUGUGAGGUUCUCAAA
1292


2053
UACAAGCUUGGCCCACAGC
969
2053
UACAAGCUUGGCCCACAGC
969
2071
GCUGUGGGCCAAGCUUGUA
1293


2071
CCUCUGCCAAUCCAUGUGG
970
2071
CCUCUGCCAAUCCAUGUGG
970
2089
CCACAUGGAUUGGCAGAGG
1294


2089
GGAGAGUUGCCCACACCUG
971
2089
GGAGAGUUGCCCACACCUG
971
2107
CAGGUGUGGGCAACUCUCC
1295


2107
GUUUGCAAGAACUUGGAUA
972
2107
GUUUGCAAGAACUUGGAUA
972
2125
UAUCCAAGUUCUUGCAAAC
1296


2125
ACUCUUUGGAAAUUGAAUG
973
2125
ACUCUUUGGAAAUUGAAUG
973
2143
CAUUCAAUUUCCAAAGAGU
1297


2143
GCCACCAUGUUCUCUAAUA
974
2143
GCCACCAUGUUCUCUAAUA
974
2161
UAUUAGAGAACAUGGUGGC
1298


2161
AGCACAAAUGACAUUUUGA
975
2161
AGCACAAAUGACAUUUUGA
975
2179
UCAAAAUGUCAUUUGUGCU
1299


2179
AUCAUGGAGCUUAAGAAUG
976
2179
AUCAUGGAGCUUAAGAAUG
976
2197
CAUUCUUAAGCUCCAUGAU
1300


2197
GCAUCCUUGCAGGACCAAG
977
2197
GCAUCCUUGCAGGACCAAG
977
2215
CUUGGUCCUGCAAGGAUGC
1301


2215
GGAGACUAUGUCUGCCUUG
978
2215
GGAGACUAUGUCUGCCUUG
978
2233
CAAGGCAGACAUAGUCUCC
1302


2233
GCUCAAGACAGGAAGACCA
979
2233
GCUCAAGACAGGAAGACCA
979
2251
UGGUCUUCCUGUCUUGAGC
1303


2251
AAGAAAAGACAUUGCGUGG
980
2251
AAGAAAAGACAUUGCGUGG
980
2269
CCACGCAAUGUCUUUUCUU
1304


2269
GUCAGGCAGCUCACAGUCC
981
2269
GUCAGGCAGCUCACAGUCC
981
2287
GGACUGUGAGCUGCCUGAC
1305


2287
CUAGAGCGUGUGGCACCCA
982
2287
CUAGAGCGUGUGGCACCCA
982
2305
UGGGUGCCACACGCUCUAG
1306


2305
ACGAUCACAGGAAACCUGG
983
2305
ACGAUCACAGGAAACCUGG
983
2323
CCAGGUUUCCUGUGAUCGU
1307


2323
GAGAAUCAGACGACAAGUA
984
2323
GAGAAUCAGACGACAAGUA
984
2341
UACUUGUCGUCUGAUUCUC
1308


2341
AUUGGGGAAAGCAUCGAAG
985
2341
AUUGGGGAAAGCAUCGAAG
985
2359
CUUCGAUGCUUUCCCCAAU
1309


2359
GUCUCAUGCACGGCAUCUG
986
2359
GUCUCAUGCACGGCAUCUG
986
2377
CAGAUGCCGUGCAUGAGAC
1310


2377
GGGAAUCCCCCUCCACAGA
987
2377
GGGAAUCCCCCUCCACAGA
987
2395
UCUGUGGAGGGGGAUUCCC
1311


2395
AUCAUGUGGUUUAAAGAUA
988
2395
AUCAUGUGGUUUAAAGAUA
988
2413
UAUCUUUAAACCACAUGAU
1312


2413
AAUGAGACCCUUGUAGAAG
989
2413
AAUGAGACCCUUGUAGAAG
989
2431
CUUCUACAAGGGUCUCAUU
1313


2431
GACUCAGGCAUUGUAUUGA
990
2431
GACUCAGGCAUUGUAUUGA
990
2449
UCAAUACAAUGCCUGAGUC
1314


2449
AAGGAUGGGAACCGGAACC
991
2449
AAGGAUGGGAACCGGAACC
991
2467
GGUUCCGGUUCCCAUCCUU
1315


2467
CUCACUAUCCGCAGAGUGA
992
2467
CUCACUAUCCGCAGAGUGA
992
2485
UCACUCUGCGGAUAGUGAG
1316


2485
AGGAAGGAGGACGAAGGCC
993
2485
AGGAAGGAGGACGAAGGCC
993
2503
GGCCUUCGUCCUCCUUCCU
1317


2503
CUCUACACCUGCCAGGCAU
994
2503
CUCUACACCUGCCAGGCAU
994
2521
AUGCCUGGCAGGUGUAGAG
1318


2521
UGCAGUGUUCUUGGCUGUG
995
2521
UGCAGUGUUCUUGGCUGUG
995
2539
CACAGCCAAGAACACUGCA
1319


2539
GCAAAAGUGGAGGCAUUUU
996
2539
GCAAAAGUGGAGGCAUUUU
996
2557
AAAAUGCCUCCACUUUUGC
1320


2557
UUCAUAAUAGAAGGUGCCC
997
2557
UUCAUAAUAGAAGGUGCCC
997
2575
GGGCACCUUCUAUUAUGAA
1321


2575
CAGGAAAAGACGAACUUGG
998
2575
CAGGAAAAGACGAACUUGG
998
2593
CCAAGUUCGUCUUUUCCUG
1322


2593
GAAAUCAUUAUUCUAGUAG
999
2593
GAAAUCAUUAUUCUAGUAG
999
2611
CUACUAGAAUAAUGAUUUC
1323


2611
GGCACGGCGGUGAUUGCCA
1000
2611
GGCACGGCGGUGAUUGCCA
1000
2629
UGGCAAUCACCGCCGUGCC
1324


2629
AUGUUCUUCUGGCUACUUC
1001
2629
AUGUUCUUCUGGCUACUUC
1001
2647
GAAGUAGCCAGAAGAACAU
1325


2647
CUUGUCAUCAUCCUACGGA
1002
2647
CUUGUCAUCAUCCUACGGA
1002
2665
UCCGUAGGAUGAUGACAAG
1326


2665
ACCGUUAAGCGGGCCAAUG
1003
2665
ACCGUUAAGCGGGCCAAUG
1003
2683
CAUUGGCCCGCUUAACGGU
1327


2683
GGAGGGGAACUGAAGACAG
1004
2683
GGAGGGGAACUGAAGACAG
1004
2701
CUGUCUUCAGUUCCCCUCC
1328


2701
GGCUACUUGUCCAUCGUCA
1005
2701
GGCUACUUGUCCAUCGUCA
1005
2719
UGACGAUGGACAAGUAGCC
1329


2719
AUGGAUCCAGAUGAACUCC
1006
2719
AUGGAUCCAGAUGAACUCC
1006
2737
GGAGUUCAUCUGGAUCCAU
1330


2737
CCAUUGGAUGAACAUUGUG
1007
2737
CCAUUGGAUGAACAUUGUG
1007
2755
CACAAUGUUCAUCCAAUGG
1331


2755
GAACGACUGCCUUAUGAUG
1008
2755
GAACGACUGCCUUAUGAUG
1008
2773
CAUCAUAAGGCAGUCGUUC
1332


2773
GCCAGCAAAUGGGAAUUCC
1009
2773
GCCAGCAAAUGGGAAUUCC
1009
2791
GGAAUUCCCAUUUGCUGGC
1333


2791
CCCAGAGACCGGCUGAAGC
1010
2791
CCCAGAGACCGGCUGAAGC
1010
2809
GCUUCAGCCGGUCUCUGGG
1334


2809
CUAGGUAAGCCUCUUGGCC
1011
2809
CUAGGUAAGCCUCUUGGCC
1011
2827
GGCCAAGAGGCUUACCUAG
1335


2827
CGUGGUGCCUUUGGCCAAG
1012
2827
CGUGGUGCCUUUGGCCAAG
1012
2845
CUUGGCCAAAGGCACCACG
1336


2845
GUGAUUGAAGCAGAUGCCU
1013
2845
GUGAUUGAAGCAGAUGCCU
1013
2863
AGGCAUCUGCUUCAAUCAC
1337


2863
UUUGGAAUUGACAAGACAG
1014
2863
UUUGGAAUUGACAAGACAG
1014
2881
CUGUCUUGUCAAUUCCAAA
1338


2881
GCAACUUGCAGGACAGUAG
1015
2881
GCAACUUGCAGGACAGUAG
1015
2899
CUACUGUCCUGCAAGUUGC
1339


2899
GCAGUCAAAAUGUUGAAAG
1016
2899
GCAGUCAAAAUGUUGAAAG
1016
2917
CUUUCAACAUUUUGACUGC
1340


2917
GAAGGAGCAACACACAGUG
1017
2917
GAAGGAGCAACACACAGUG
1017
2935
CACUGUGUGUUGCUCCUUC
1341


2935
GAGCAUCGAGCUCUCAUGU
1018
2935
GAGCAUCGAGCUCUCAUGU
1018
2953
ACAUGAGAGCUCGAUGCUC
1342


2953
UCUGAACUCAAGAUCCUCA
1019
2953
UCUGAACUCAAGAUCCUCA
1019
2971
UGAGGAUCUUGAGUUCAGA
1343


2971
AUUCAUAUUGGUCACCAUC
1020
2971
AUUCAUAUUGGUCACCAUC
1020
2989
GAUGGUGACCAAUAUGAAU
1344


2989
CUCAAUGUGGUCAACCUUC
1021
2989
CUCAAUGUGGUCAACCUUC
1021
3007
GAAGGUUGACCACAUUGAG
1345


3007
CUAGGUGCCUGUACCAAGC
1022
3007
CUAGGUGCCUGUACCAAGC
1022
3025
GCUUGGUACAGGCACCUAG
1346


3025
CCAGGAGGGCCACUCAUGG
1023
3025
CCAGGAGGGCCACUCAUGG
1023
3043
CCAUGAGUGGCCCUCCUGG
1347


3043
GUGAUUGUGGAAUUCUGCA
1024
3043
GUGAUUGUGGAAUUCUGCA
1024
3061
UGCAGAAUUCCACAAUCAC
1348


3061
AAAUUUGGAAACCUGUCCA
1025
3061
AAAUUUGGAAACCUGUCCA
1025
3079
UGGACAGGUUUCCAAAUUU
1349


3079
ACUUACCUGAGGAGCAAGA
1026
3079
ACUUACCUGAGGAGCAAGA
1026
3097
UCUUGCUCCUCAGGUAAGU
1350


3097
AGAAAUGAAUUUGUCCCCU
1027
3097
AGAAAUGAAUUUGUCCCCU
1027
3115
AGGGGACAAAUUCAUUUCU
1351


3115
UACAAGACCAAAGGGGCAC
1028
3115
UACAAGACCAAAGGGGCAC
1028
3133
GUGCCCCUUUGGUCUUGUA
1352


3133
CGAUUCCGUCAAGGGAAAG
1029
3133
CGAUUCCGUCAAGGGAAAG
1029
3151
CUUUCCCUUGACGGAAUCG
1353


3151
GACUACGUUGGAGCAAUCC
1030
3151
GACUACGUUGGAGCAAUCC
1030
3169
GGAUUGCUCCAACGUAGUC
1354


3169
CCUGUGGAUCUGAAACGGC
1031
3169
CCUGUGGAUCUGAAACGGC
1031
3187
GCCGUUUCAGAUCCACAGG
1355


3187
CGCUUGGACAGCAUCACCA
1032
3187
CGCUUGGACAGCAUCACCA
1032
3205
UGGUGAUGCUGUCCAAGCG
1356


3205
AGUAGCCAGAGCUCAGCCA
1033
3205
AGUAGCCAGAGCUCAGCCA
1033
3223
UGGCUGAGCUCUGGCUACU
1357


3223
AGCUCUGGAUUUGUGGAGG
1034
3223
AGCUCUGGAUUUGUGGAGG
1034
3241
CCUCCACAAAUCCAGAGCU
1358


3241
GAGAAGUCCCUCAGUGAUG
1035
3241
GAGAAGUCCCUCAGUGAUG
1035
3259
CAUCACUGAGGGACUUCUC
1359


3259
GUAGAAGAAGAGGAAGCUC
1036
3259
GUAGAAGAAGAGGAAGCUC
1036
3277
GAGCUUCCUCUUCUUCUAC
1360


3277
CCUGAAGAUCUGUAUAAGG
1037
3277
CCUGAAGAUCUGUAUAAGG
1037
3295
CCUUAUACAGAUCUUCAGG
1361


3295
GACUUCCUGACCUUGGAGC
1038
3295
GACUUCCUGACCUUGGAGC
1038
3313
GCUCCAAGGUCAGGAAGUC
1362


3313
CAUCUCAUCUGUUACAGCU
1039
3313
CAUCUCAUCUGUUACAGCU
1039
3331
AGCUGUAACAGAUGAGAUG
1363


3331
UUCCAAGUGGCUAAGGGCA
1040
3331
UUCCAAGUGGCUAAGGGCA
1040
3349
UGCCCUUAGCCACUUGGAA
1364


3349
AUGGAGUUCUUGGCAUCGC
1041
3349
AUGGAGUUCUUGGCAUCGC
1041
3367
GCGAUGCCAAGAACUCCAU
1365


3367
CGAAAGUGUAUCCACAGGG
1042
3367
CGAAAGUGUAUCCACAGGG
1042
3385
CCCUGUGGAUACACUUUCG
1366


3385
GACCUGGCGGCACGAAAUA
1043
3385
GACCUGGCGGCACGAAAUA
1043
3403
UAUUUCGUGCCGCCAGGUC
1367


3403
AUCCUCUUAUCGGAGAAGA
1044
3403
AUCCUCUUAUCGGAGAAGA
1044
3421
UCUUCUCCGAUAAGAGGAU
1368


3421
AACGUGGUUAAAAUCUGUG
1045
3421
AACGUGGUUAAAAUCUGUG
1045
3439
CACAGAUUUUAACCACGUU
1369


3439
GACUUUGGCUUGGCCCGGG
1046
3439
GACUUUGGCUUGGCCCGGG
1046
3457
CCCGGGCCAAGCCAAAGUC
1370


3457
GAUAUUUAUAAAGAUCCAG
1047
3457
GAUAUUUAUAAAGAUCCAG
1047
3475
CUGGAUCUUUAUAAAUAUC
1371


3475
GAUUAUGUCAGAAAAGGAG
1048
3475
GAUUAUGUCAGAAAAGGAG
1048
3493
CUCCUUUUCUGACAUAAUC
1372


3493
GAUGCUCGCCUCCCUUUGA
1049
3493
GAUGCUCGCCUCCCUUUGA
1049
3511
UCAAAGGGAGGCGAGCAUC
1373


3511
AAAUGGAUGGCCCCAGAAA
1050
3511
AAAUGGAUGGCCCCAGAAA
1050
3529
UUUCUGGGGCCAUCCAUUU
1374


3529
ACAAUUUUUGACAGAGUGU
1051
3529
ACAAUUUUUGACAGAGUGU
1051
3547
ACACUCUGUCAAAAAUUGU
1375


3547
UACACAAUCCAGAGUGACG
1052
3547
UACACAAUCCAGAGUGACG
1052
3565
CGUCACUCUGGAUUGUGUA
1376


3565
GUCUGGUCUUUUGGUGUUU
1053
3565
GUCUGGUCUUUUGGUGUUU
1053
3583
AAACACCAAAAGACCAGAC
1377


3583
UUGCUGUGGGAAAUAUUUU
1054
3583
UUGCUGUGGGAAAUAUUUU
1054
3601
AAAAUAUUUCCCACAGCAA
1378


3601
UCCUUAGGUGCUUCUCCAU
1055
3601
UCCUUAGGUGCUUCUCCAU
1055
3619
AUGGAGAAGCACCUAAGGA
1379


3619
UAUCCUGGGGUAAAGAUUG
1056
3619
UAUCCUGGGGUAAAGAUUG
1056
3637
CAAUCUUUACCCCAGGAUA
1380


3637
GAUGAAGAAUUUUGUAGGC
1057
3637
GAUGAAGAAUUUUGUAGGC
1057
3655
GCCUACAAAAUUCUUCAUC
1381


3655
CGAUUGAAAGAAGGAACUA
1058
3655
CGAUUGAAAGAAGGAACUA
1058
3673
UAGUUCCUUCUUUCAAUCG
1382


3673
AGAAUGAGGGCCCCUGAUU
1059
3673
AGAAUGAGGGCCCCUGAUU
1059
3691
AAUCAGGGGCCCUCAUUCU
1383


3691
UAUACUACACCAGAAAUGU
1060
3691
UAUACUACACCAGAAAUGU
1060
3709
ACAUUUCUGGUGUAGUAUA
1384


3709
UACCAGACCAUGCUGGACU
1061
3709
UACCAGACCAUGCUGGACU
1061
3727
AGUCCAGCAUGGUCUGGUA
1385


3727
UGCUGGCACGGGGAGCCCA
1062
3727
UGCUGGCACGGGGAGCCCA
1062
3745
UGGGCUCCCCGUGCCAGCA
1386


3745
AGUCAGAGACCCACGUUUU
1063
3745
AGUCAGAGACCCACGUUUU
1063
3763
AAAACGUGGGUCUCUGACU
1387


3763
UCAGAGUUGGUGGAACAUU
1064
3763
UCAGAGUUGGUGGAACAUU
1064
3781
AAUGUUCCACCAACUCUGA
1388


3781
UUGGGAAAUCUCUUGCAAG
1065
3781
UUGGGAAAUCUCUUGCAAG
1065
3799
CUUGCAAGAGAUUUCCCAA
1389


3799
GCUAAUGCUCAGCAGGAUG
1066
3799
GCUAAUGCUCAGCAGGAUG
1066
3817
CAUCCUGCUGAGCAUUAGC
1390


3817
GGCAAAGACUACAUUGUUC
1067
3817
GGCAAAGACUACAUUGUUC
1067
3835
GAACAAUGUAGUCUUUGCC
1391


3835
CUUCCGAUAUCAGAGACUU
1068
3835
CUUCCGAUAUCAGAGACUU
1068
3853
AAGUCUCUGAUAUCGGAAG
1392


3853
UUGAGCAUGGAAGAGGAUU
1069
3853
UUGAGCAUGGAAGAGGAUU
1069
3871
AAUCCUCUUCCAUGCUCAA
1393


3871
UCUGGACUCUCUCUGCCUA
1070
3871
UCUGGACUCUCUCUGCCUA
1070
3889
UAGGCAGAGAGAGUCCAGA
1394


3889
ACCUCACCUGUUUCCUGUA
1071
3889
ACCUCACCUGUUUCCUGUA
1071
3907
UACAGGAAACAGGUGAGGU
1395


3907
AUGGAGGAGGAGGAAGUAU
1072
3907
AUGGAGGAGGAGGAAGUAU
1072
3925
AUACUUCCUCCUCCUCCAU
1396


3925
UGUGACCCCAAAUUCCAUU
1073
3925
UGUGACCCCAAAUUCCAUU
1073
3943
AAUGGAAUUUGGGGUCACA
1397


3943
UAUGACAACACAGCAGGAA
1074
3943
UAUGACAACACAGCAGGAA
1074
3961
UUCCUGCUGUGUUGUCAUA
1398


3961
AUCAGUCAGUAUCUGCAGA
1075
3961
AUCAGUCAGUAUCUGCAGA
1075
3979
UCUGCAGAUACUGACUGAU
1399


3979
AACAGUAAGCGAAAGAGCC
1076
3979
AACAGUAAGCGAAAGAGCC
1076
3997
GGCUCUUUCGCUUACUGUU
1400


3997
CGGCCUGUGAGUGUAAAAA
1077
3997
CGGCCUGUGAGUGUAAAAA
1077
4015
UUUUUACACUCACAGGCCG
1401


4015
ACAUUUGAAGAUAUCCCGU
1078
4015
ACAUUUGAAGAUAUCCCGU
1078
4033
ACGGGAUAUCUUCAAAUGU
1402


4033
UUAGAAGAACCAGAAGUAA
1079
4033
UUAGAAGAACCAGAAGUAA
1079
4051
UUACUUCUGGUUCUUCUAA
1403


4051
AAAGUAAUCCCAGAUGACA
1080
4051
AAAGUAAUCCCAGAUGACA
1080
4069
UGUCAUCUGGGAUUACUUU
1404


4069
AACCAGACGGACAGUGGUA
1081
4069
AACCAGACGGACAGUGGUA
1081
4087
UACCACUGUCCGUCUGGUU
1405


4087
AUGGUUCUUGCCUCAGAAG
1082
4087
AUGGUUCUUGCCUCAGAAG
1082
4105
CUUCUGAGGCAAGAACCAU
1406


4105
GAGCUGAAAACUUUGGAAG
1083
4105
GAGCUGAAAACUUUGGAAG
1083
4123
CUUCCAAAGUUUUCAGCUC
1407


4123
GACAGAACCAAAUUAUCUC
1084
4123
GACAGAACCAAAUUAUCUC
1084
4141
GAGAUAAUUUGGUUCUGUC
1408


4141
CCAUCUUUUGGUGGAAUGG
1085
4141
CCAUCUUUUGGUGGAAUGG
1085
4159
CCAUUCCACCAAAAGAUGG
1409


4159
GUGCCCAGCAAAAGCAGGG
1086
4159
GUGCCCAGCAAAAGCAGGG
1086
4177
CCCUGCUUUUGCUGGGCAC
1410


4177
GAGUCUGUGGCAUCUGAAG
1087
4177
GAGUCUGUGGCAUCUGAAG
1087
4195
CUUCAGAUGCCACAGACUC
1411


4195
GGCUCAAACCAGACAAGCG
1088
4195
GGCUCAAACCAGACAAGCG
1088
4213
CGCUUGUCUGGUUUGAGCC
1412


4213
GGCUACCAGUCCGGAUAUC
1089
4213
GGCUACCAGUCCGGAUAUC
1089
4231
GAUAUCCGGACUGGUAGCC
1413


4231
CACUCCGAUGACACAGACA
1090
4231
CACUCCGAUGACACAGACA
1090
4249
UGUCUGUGUCAUCGGAGUG
1414


4249
ACCACCGUGUACUCCAGUG
1091
4249
ACCACCGUGUACUCCAGUG
1091
4267
CACUGGAGUACACGGUGGU
1415


4267
GAGGAAGCAGAACUUUUAA
1092
4267
GAGGAAGCAGAACUUUUAA
1092
4285
UUAAAAGUUCUGCUUCCUC
1416


4285
AAGCUGAUAGAGAUUGGAG
1093
4285
AAGCUGAUAGAGAUUGGAG
1093
4303
CUCCAAUCUCUAUCAGCUU
1417


4303
GUGCAAACCGGUAGCACAG
1094
4303
GUGCAAACCGGUAGCACAG
1094
4321
CUGUGCUACCGGUUUGCAC
1418


4321
GCCCAGAUUCUCCAGCCUG
1095
4321
GCCCAGAUUCUCCAGCCUG
1095
4339
CAGGCUGGAGAAUCUGGGC
1419


4339
GACUCGGGGACCACACUGA
1096
4339
GACUCGGGGACCACACUGA
1096
4357
UCAGUGUGGUCCCCGAGUC
1420


4357
AGCUCUCCUCCUGUUUAAA
1097
4357
AGCUCUCCUCCUGUUUAAA
1097
4375
UUUAAACAGGAGGAGAGCU
1421


4375
AAGGAAGCAUCCACACCCC
1098
4375
AAGGAAGCAUCCACACCCC
1098
4393
GGGGUGUGGAUGCUUCCUU
1422


4393
CAACUCCCGGACAUCACAU
1099
4393
CAACUCCCGGACAUCACAU
1099
4411
AUGUGAUGUCCGGGAGUUG
1423


4411
UGAGAGGUCUGCUCAGAUU
1100
4411
UGAGAGGUCUGCUCAGAUU
1100
4429
AAUCUGAGCAGACCUCUCA
1424


4429
UUUGAAGUGUUGUUCUUUC
1101
4429
UUUGAAGUGUUGUUCUUUC
1101
4447
GAAAGAACAACACUUCAAA
1425


4447
CCACCAGCAGGAAGUAGCC
1102
4447
CCACCAGCAGGAAGUAGCC
1102
4465
GGCUACUUCCUGCUGGUGG
1426


4465
CGCAUUUGAUUUUCAUUUC
1103
4465
CGCAUUUGAUUUUCAUUUC
1103
4483
GAAAUGAAAAUCAAAUGCG
1427


4483
CGACAACAGAAAAAGGACC
1104
4483
CGACAACAGAAAAAGGACC
1104
4501
GGUCCUUUUUCUGUUGUCG
1428


4501
CUCGGACUGCAGGGAGCCA
1105
4501
CUCGGACUGCAGGGAGCCA
1105
4519
UGGCUCCCUGCAGUCCGAG
1429


4519
AGUCUUCUAGGCAUAUCCU
1106
4519
AGUCUUCUAGGCAUAUCCU
1106
4537
AGGAUAUGCCUAGAAGACU
1430


4537
UGGAAGAGGCUUGUGACCC
1107
4537
UGGAAGAGGCUUGUGACCC
1107
4555
GGGUCACAAGCCUCUUCCA
1431


4555
CAAGAAUGUGUCUGUGUCU
1108
4555
CAAGAAUGUGUCUGUGUCU
1108
4573
AGACACAGACACAUUCUUG
1432


4573
UUCUCCCAGUGUUGACCUG
1109
4573
UUCUCCCAGUGUUGACCUG
1109
4591
CAGGUCAACACUGGGAGAA
1433


4591
GAUCCUCUUUUUUCAUUCA
1110
4591
GAUCCUCUUUUUUCAUUCA
1110
4609
UGAAUGAAAAAAGAGGAUC
1434


4609
AUUUAAAAAGCAUUAUCAU
1111
4609
AUUUAAAAAGCAUUAUCAU
1111
4627
AUGAUAAUGCUUUUUAAAU
1435


4627
UGCCCCUGCUGCGGGUCUC
1112
4627
UGCCCCUGCUGCGGGUCUC
1112
4645
GAGACCCGCAGCAGGGGCA
1436


4645
CACCAUGGGUUUAGAACAA
1113
4645
CACCAUGGGUUUAGAACAA
1113
4663
UUGUUCUAAACCCAUGGUG
1437


4663
AAGAGCUUCAAGCAAUGGC
1114
4663
AAGAGCUUCAAGCAAUGGC
1114
4681
GCCAUUGCUUGAAGCUCUU
1438


4681
CCCCAUCCUCAAAGAAGUA
1115
4681
CCCCAUCCUCAAAGAAGUA
1115
4699
UACUUCUUUGAGGAUGGGG
1439


4699
AGCAGUACCUGGGGAGCUG
1116
4699
AGCAGUACCUGGGGAGCUG
1116
4717
CAGCUCCCCAGGUACUGCU
1440


4717
GACACUUCUGUAAAACUAG
1117
4717
GACACUUCUGUAAAACUAG
1117
4735
CUAGUUUUACAGAAGUGUC
1441


4735
GAAGAUAAACCAGGCAACG
1118
4735
GAAGAUAAACCAGGCAACG
1118
4753
CGUUGCCUGGUUUAUCUUC
1442


4753
GUAAGUGUUCGAGGUGUUG
1119
4753
GUAAGUGUUCGAGGUGUUG
1119
4771
CAACACCUCGAACACUUAC
1443


4771
GAAGAUGGGAAGGAUUUGC
1120
4771
GAAGAUGGGAAGGAUUUGC
1120
4789
GCAAAUCCUUCCCAUCUUC
1444


4789
CAGGGCUGAGUCUAUCCAA
1121
4789
CAGGGCUGAGUCUAUCCAA
1121
4807
UUGGAUAGACUCAGCCCUG
1445


4807
AGAGGCUUUGUUUAGGACG
1122
4807
AGAGGCUUUGUUUAGGACG
1122
4825
CGUCCUAAACAAAGCCUCU
1446


4825
GUGGGUCCCAAGCCAAGCC
1123
4825
GUGGGUCCCAAGCCAAGCC
1123
4843
GGCUUGGCUUGGGACCCAC
1447


4843
CUUAAGUGUGGAAUUCGGA
1124
4843
CUUAAGUGUGGAAUUCGGA
1124
4861
UCCGAAUUCCACACUUAAG
1448


4861
AUUGAUAGAAAGGAAGACU
1125
4861
AUUGAUAGAAAGGAAGACU
1125
4879
AGUCUUCCUUUCUAUCAAU
1449


4879
UAACGUUACCUUGCUUUGG
1126
4879
UAACGUUACCUUGCUUUGG
1126
4897
CCAAAGCAAGGUAACGUUA
1450


4897
GAGAGUACUGGAGCCUGCA
1127
4897
GAGAGUACUGGAGCCUGCA
1127
4915
UGCAGGCUCCAGUACUCUC
1451


4915
AAAUGCAUUGUGUUUGCUC
1128
4915
AAAUGCAUUGUGUUUGCUC
1128
4933
GAGCAAACACAAUGCAUUU
1452


4933
CUGGUGGAGGUGGGCAUGG
1129
4933
CUGGUGGAGGUGGGCAUGG
1129
4951
CCAUGCCCACCUCCACCAG
1453


4951
GGGUCUGUUCUGAAAUGUA
1130
4951
GGGUCUGUUCUGAAAUGUA
1130
4969
UACAUUUCAGAACAGACCC
1454


4969
AAAGGGUUCAGACGGGGUU
1131
4969
AAAGGGUUCAGACGGGGUU
1131
4987
AACCCCGUCUGAACCCUUU
1455


4987
UUCUGGUUUUAGAAGGUUG
1132
4987
UUCUGGUUUUAGAAGGUUG
1132
5005
CAACCUUCUAAAACCAGAA
1456


5005
GCGUGUUCUUCGAGUUGGG
1133
5005
GCGUGUUCUUCGAGUUGGG
1133
5023
CCCAACUCGAAGAACACGC
1457


5023
GCUAAAGUAGAGUUCGUUG
1134
5023
GCUAAAGUAGAGUUCGUUG
1134
5041
CAACGAACUCUACUUUAGC
1458


5041
GUGCUGUUUCUGACUCCUA
1135
5041
GUGCUGUUUCUGACUCCUA
1135
5059
UAGGAGUCAGAAACAGCAC
1459


5059
AAUGAGAGUUCCUUCCAGA
1136
5059
AAUGAGAGUUCCUUCCAGA
1136
5077
UCUGGAAGGAACUCUCAUU
1460


5077
ACCGUUAGCUGUCUCCUUG
1137
5077
ACCGUUAGCUGUCUCCUUG
1137
5095
CAAGGAGACAGCUAACGGU
1461


5095
GCCAAGCCCCAGGAAGAAA
1138
5095
GCCAAGCCCCAGGAAGAAA
1138
5113
UUUCUUCCUGGGGCUUGGC
1462


5113
AAUGAUGCAGCUCUGGCUC
1139
5113
AAUGAUGCAGCUCUGGCUC
1139
5131
GAGCCAGAGCUGCAUCAUU
1463


5131
CCUUGUCUCCCAGGCUGAU
1140
5131
CCUUGUCUCCCAGGCUGAU
1140
5149
AUCAGCCUGGGAGACAAGG
1464


5149
UCCUUUAUUCAGAAUACCA
1141
5149
UCCUUUAUUCAGAAUACCA
1141
5167
UGGUAUUCUGAAUAAAGGA
1465


5167
ACAAAGAAAGGACAUUCAG
1142
5167
ACAAAGAAAGGACAUUCAG
1142
5185
CUGAAUGUCCUUUCUUUGU
1466


5185
GCUCAAGGCUCCCUGCCGU
1143
5185
GCUCAAGGCUCCCUGCCGU
1143
5203
ACGGCAGGGAGCCUUGAGC
1467


5203
UGUUGAAGAGUUCUGACUG
1144
5203
UGUUGAAGAGUUCUGACUG
1144
5221
CAGUCAGAACUCUUCAACA
1468


5221
GCACAAACCAGCUUCUGGU
1145
5221
GCACAAACCAGCUUCUGGU
1145
5239
ACCAGAAGCUGGUUUGUGC
1469


5239
UUUCUUCUGGAAUGAAUAC
1146
5239
UUUCUUCUGGAAUGAAUAC
1146
5257
GUAUUCAUUCCAGAAGAAA
1470


5257
CCCUCAUAUCUGUCCUGAU
1147
5257
CCCUCAUAUCUGUCCUGAU
1147
5275
AUCAGGACAGAUAUGAGGG
1471


5275
UGUGAUAUGUCUGAGACUG
1148
5275
UGUGAUAUGUCUGAGACUG
1148
5293
CAGUCUCAGACAUAUCACA
1472


5293
GAAUGCGGGAGGUUCAAUG
1149
5293
GAAUGCGGGAGGUUCAAUG
1149
5311
CAUUGAACCUCCCGCAUUC
1473


5311
GUGAAGCUGUGUGUGGUGU
1150
5311
GUGAAGCUGUGUGUGGUGU
1150
5329
ACACCACACACAGCUUCAC
1474


5329
UCAAAGUUUCAGGAAGGAU
1151
5329
UCAAAGUUUCAGGAAGGAU
1151
5347
AUCCUUCCUGAAACUUUGA
1475


5347
UUUUACCCUUUUGUUCUUC
1152
5347
UUUUACCCUUUUGUUCUUC
1152
5365
GAAGAACAAAAGGGUAAAA
1476


5365
CCCCCUGUCCCCAACCCAC
1153
5365
CCCCCUGUCCCCAACCCAC
1153
5383
GUGGGUUGGGGACAGGGGG
1477


5383
CUCUCACCCCGCAACCCAU
1154
5383
CUCUCACCCCGCAACCCAU
1154
5401
AUGGGUUGCGGGGUGAGAG
1478


5401
UCAGUAUUUUAGUUAUUUG
1155
5401
UCAGUAUUUUAGUUAUUUG
1155
5419
CAAAUAACUAAAAUACUGA
1479


5419
GGCCUCUACUCCAGUAAAC
1156
5419
GGCCUCUACUCCAGUAAAC
1156
5437
GUUUACUGGAGUAGAGGCC
1480


5437
CCUGAUUGGGUUUGUUCAC
1157
5437
CCUGAUUGGGUUUGUUCAC
1157
5455
GUGAACAAACCCAAUCAGG
1481


5455
CUCUCUGAAUGAUUAUUAG
1158
5455
CUCUCUGAAUGAUUAUUAG
1158
5473
CUAAUAAUCAUUCAGAGAG
1482


5473
GCCAGACUUCAAAAUUAUU
1159
5473
GCCAGACUUCAAAAUUAUU
1159
5491
AAUAAUUUUGAAGUCUGGC
1483


5491
UUUAUAGCCCAAAUUAUAA
1160
5491
UUUAUAGCCCAAAUUAUAA
1160
5509
UUAUAAUUUGGGCUAUAAA
1484


5509
ACAUCUAUUGUAUUAUUUA
1161
5509
ACAUCUAUUGUAUUAUUUA
1161
5527
UAAAUAAUACAAUAGAUGU
1485


5527
AGACUUUUAACAUAUAGAG
1162
5527
AGACUUUUAACAUAUAGAG
1162
5545
CUCUAUAUGUUAAAAGUCU
1486


5545
GCUAUUUCUACUGAUUUUU
1163
5545
GCUAUUUCUACUGAUUUUU
1163
5563
AAAAAUCAGUAGAAAUAGC
1487


5563
UGCCCUUGUUCUGUCCUUU
1164
5563
UGCCCUUGUUCUGUCCUUU
1164
5581
AAAGGACAGAACAAGGGCA
1488


5581
UUUUUCAAAAAAGAAAAUG
1165
5581
UUUUUCAAAAAAGAAAAUG
1165
5599
CAUUUUCUUUUUUGAAAAA
1489


5599
GUGUUUUUUGUUUGGUACC
1166
5599
GUGUUUUUUGUUUGGUACC
1166
5617
GGUACCAAACAAAAAACAC
1490


5617
CAUAGUGUGAAAUGCUGGG
1167
5617
CAUAGUGUGAAAUGCUGGG
1167
5635
CCCAGCAUUUCACACUAUG
1491


5635
GAACAAUGACUAUAAGACA
1168
5635
GAACAAUGACUAUAAGACA
1168
5653
UGUCUUAUAGUCAUUGUUC
1492


5653
AUGCUAUGGCACAUAUAUU
1169
5653
AUGCUAUGGCACAUAUAUU
1169
5671
AAUAUAUGUGCCAUAGCAU
1493


5671
UUAUAGUCUGUUUAUGUAG
1170
5671
UUAUAGUCUGUUUAUGUAG
1170
5689
CUACAUAAACAGACUAUAA
1494


5689
GAAACAAAUGUAAUAUAUU
1171
5689
GAAACAAAUGUAAUAUAUU
1171
5707
AAUAUAUUACAUUUGUUUC
1495


5707
UAAAGCCUUAUAUAUAAUG
1172
5707
UAAAGCCUUAUAUAUAAUG
1172
5725
CAUUAUAUAUAAGGCUUUA
1496


5725
GAACUUUGUACUAUUCACA
1173
5725
GAACUUUGUACUAUUCACA
1173
5743
UGUGAAUAGUACAAAGUUC
1497


5743
AUUUUGUAUCAGUAUUAUG
1174
5743
AUUUUGUAUCAGUAUUAUG
1174
5761
CAUAAUACUGAUACAAAAU
1498


5761
GUAGCAUAACAAAGGUCAU
1175
5761
GUAGCAUAACAAAGGUCAU
1175
5779
AUGACCUUUGUUAUGCUAC
1499


5779
UAAUGCUUUCAGCAAUUGA
1176
5779
UAAUGCUUUCAGCAAUUGA
1176
5797
UCAAUUGCUGAAAGCAUUA
1500


5797
AUGUCAUUUUAUUAAAGAA
1177
5797
AUGUCAUUUUAUUAAAGAA
1177
5815
UUCUUUAAUAAAAUGACAU
1501


5812
AGAACAUUGAAAAACUUGA
1178
5812
AGAACAUUGAAAAACUUGA
1178
5830
UCAAGUUUUUCAAUGUUCU
1502


 







VEGFR3/FLT4 NM_002020.1
















1
ACCCACGCGCAGCGGCCGG
1503
1
ACCCACGCGCAGCGGCCGG
1503
19
CCGGCCGCUGCGCGUGGGU
1750



19
GAGAUGCAGCGGGGCGCCG
1504
19
GAGAUGCAGCGGGGCGCCG
1504
37
CGGCGCCCCGCUGCAUCUC
1751


37
GCGCUGUGCCUGCGACUGU
1505
37
GCGCUGUGCCUGCGACUGU
1505
55
ACAGUCGCAGGCACAGCGC
1752


55
UGGCUCUGCCUGGGACUCC
1506
55
UGGCUCUGCCUGGGACUCC
1506
73
GGAGUCCCAGGCAGAGCCA
1753


73
CUGGACGGCCUGGUGAGUG
1507
73
CUGGACGGCCUGGUGAGUG
1507
91
CACUCACCAGGCCGUCCAG
1754


91
GACUACUCCAUGACCCCCC
1508
91
GACUACUCCAUGACCCCCC
1508
109
GGGGGGUCAUGGAGUAGUC
1755


109
CCGACCUUGAACAUCACGG
1509
109
CCGACCUUGAACAUCACGG
1509
127
CCGUGAUGUUCAAGGUCGG
1756


127
GAGGAGUCACACGUCAUCG
1510
127
GAGGAGUCACACGUCAUCG
1510
145
CGAUGACGUGUGACUCCUC
1757


145
GACACCGGUGACAGCCUGU
1511
145
GACACCGGUGACAGCCUGU
1511
163
ACAGGCUGUCACCGGUGUC
1758


163
UCCAUCUCCUGCAGGGGAC
1512
163
UCCAUCUCCUGCAGGGGAC
1512
181
GUCCCCUGCAGGAGAUGGA
1759


181
CAGCACCCCCUCGAGUGGG
1513
181
CAGCACCCCCUCGAGUGGG
1513
199
CCCACUCGAGGGGGUGCUG
1760


199
GCUUGGCCAGGAGCUCAGG
1514
199
GCUUGGCCAGGAGCUCAGG
1514
217
CCUGAGCUCCUGGCCAAGC
1761


217
GAGGCGCCAGCCACCGGAG
1515
217
GAGGCGCCAGCCACCGGAG
1515
235
CUCCGGUGGCUGGCGCCUC
1762


235
GACAAGGACAGCGAGGACA
1516
235
GACAAGGACAGCGAGGACA
1516
253
UGUCCUCGCUGUCCUUGUC
1763


253
ACGGGGGUGGUGCGAGACU
1517
253
ACGGGGGUGGUGCGAGACU
1517
271
AGUCUCGCACCACCCCCGU
1764


271
UGCGAGGGCACAGACGCCA
1518
271
UGCGAGGGCACAGACGCCA
1518
289
UGGCGUCUGUGCCCUCGCA
1765


289
AGGCCCUACUGCAAGGUGU
1519
289
AGGCCCUACUGCAAGGUGU
1519
307
ACACCUUGCAGUAGGGCCU
1766


307
UUGCUGCUGCACGAGGUAC
1520
307
UUGCUGCUGCACGAGGUAC
1520
325
GUACCUCGUGCAGCAGCAA
1767


325
CAUGCCAACGACACAGGCA
1521
325
CAUGCCAACGACACAGGCA
1521
343
UGCCUGUGUCGUUGGCAUG
1768


343
AGCUACGUCUGCUACUACA
1522
343
AGCUACGUCUGCUACUACA
1522
361
UGUAGUAGCAGACGUAGCU
1769


361
AAGUACAUCAAGGCACGCA
1523
361
AAGUACAUCAAGGCACGCA
1523
379
UGCGUGCCUUGAUGUACUU
1770


379
AUCGAGGGCACCACGGCCG
1524
379
AUCGAGGGCACCACGGCCG
1524
397
CGGCCGUGGUGCCCUCGAU
1771


397
GCCAGCUCCUACGUGUUCG
1525
397
GCCAGCUCCUACGUGUUCG
1525
415
CGAACACGUAGGAGCUGGC
1772


415
GUGAGAGACUUUGAGCAGC
1526
415
GUGAGAGACUUUGAGCAGC
1526
433
GCUGCUCAAAGUCUCUCAC
1773


433
CCAUUCAUCAACAAGCCUG
1527
433
CCAUUCAUCAACAAGCCUG
1527
451
CAGGCUUGUUGAUGAAUGG
1774


451
GACACGCUCUUGGUCAACA
1528
451
GACACGCUCUUGGUCAACA
1528
469
UGUUGACCAAGAGCGUGUC
1775


469
AGGAAGGACGCCAUGUGGG
1529
469
AGGAAGGACGCCAUGUGGG
1529
487
CCCACAUGGCGUCCUUCCU
1776


487
GUGCCCUGUCUGGUGUCCA
1530
487
GUGCCCUGUCUGGUGUCCA
1530
505
UGGACACCAGACAGGGCAC
1777


505
AUCCCCGGCCUCAAUGUCA
1531
505
AUCCCCGGCCUCAAUGUCA
1531
523
UGACAUUGAGGCCGGGGAU
1778


523
ACGCUGCGCUCGCAAAGCU
1532
523
ACGCUGCGCUCGCAAAGCU
1532
541
AGCUUUGCGAGCGCAGCGU
1779


541
UCGGUGCUGUGGCCAGACG
1533
541
UCGGUGCUGUGGCCAGACG
1533
559
CGUCUGGCCACAGCACCGA
1780


559
GGGCAGGAGGUGGUGUGGG
1534
559
GGGCAGGAGGUGGUGUGGG
1534
577
CCCACACCACCUCCUGCCC
1781


577
GAUGACCGGCGGGGCAUGC
1535
577
GAUGACCGGCGGGGCAUGC
1535
595
GCAUGCCCCGCCGGUCAUC
1782


595
CUCGUGUCCACGCCACUGC
1536
595
CUCGUGUCCACGCCACUGC
1536
613
GCAGUGGCGUGGACACGAG
1783


613
CUGCACGAUGCCCUGUACC
1537
613
CUGCACGAUGCCCUGUACC
1537
631
GGUACAGGGCAUCGUGCAG
1784


631
CUGCAGUGCGAGACCACCU
1538
631
CUGCAGUGCGAGACCACCU
1538
649
AGGUGGUCUCGCACUGCAG
1785


649
UGGGGAGACCAGGACUUCC
1539
649
UGGGGAGACCAGGACUUCC
1539
667
GGAAGUCCUGGUCUCCCCA
1786


667
CUUUCCAACCCCUUCCUGG
1540
667
CUUUCCAACCCCUUCCUGG
1540
685
CCAGGAAGGGGUUGGAAAG
1787


685
GUGCACAUCACAGGCAACG
1541
685
GUGCACAUCACAGGCAACG
1541
703
CGUUGCCUGUGAUGUGCAC
1788


703
GAGCUCUAUGACAUCCAGC
1542
703
GAGCUCUAUGACAUCCAGC
1542
721
GCUGGAUGUCAUAGAGCUC
1789


721
CUGUUGCCCAGGAAGUCGC
1543
721
CUGUUGCCCAGGAAGUCGC
1543
739
GCGACUUCCUGGGCAACAG
1790


739
CUGGAGCUGCUGGUAGGGG
1544
739
CUGGAGCUGCUGGUAGGGG
1544
757
CCCCUACCAGCAGCUCCAG
1791


757
GAGAAGCUGGUCCUCAACU
1545
757
GAGAAGCUGGUCCUCAACU
1545
775
AGUUGAGGACCAGCUUCUC
1792


775
UGCACCGUGUGGGCUGAGU
1546
775
UGCACCGUGUGGGCUGAGU
1546
793
ACUCAGCCCACACGGUGCA
1793


793
UUUAACUCAGGUGUCACCU
1547
793
UUUAACUCAGGUGUCACCU
1547
811
AGGUGACACCUGAGUUAAA
1794


811
UUUGACUGGGACUACCCAG
1548
811
UUUGACUGGGACUACCCAG
1548
829
CUGGGUAGUCCCAGUCAAA
1795


829
GGGAAGCAGGCAGAGCGGG
1549
829
GGGAAGCAGGCAGAGCGGG
1549
847
CCCGCUCUGCCUGCUUCCC
1796


847
GGUAAGUGGGUGCCCGAGC
1550
847
GGUAAGUGGGUGCCCGAGC
1550
865
GCUCGGGCACCCACUUACC
1797


865
CGACGCUCCCAACAGACCC
1551
865
CGACGCUCCCAACAGACCC
1551
883
GGGUCUGUUGGGAGCGUCG
1798


883
CACACAGAACUCUCCAGCA
1552
883
CACACAGAACUCUCCAGCA
1552
901
UGCUGGAGAGUUCUGUGUG
1799


901
AUCCUGACCAUCCACAACG
1553
901
AUCCUGACCAUCCACAACG
1553
919
CGUUGUGGAUGGUCAGGAU
1800


919
GUCAGCCAGCACGACCUGG
1554
919
GUCAGCCAGCACGACCUGG
1554
937
CCAGGUCGUGCUGGCUGAC
1801


937
GGCUCGUAUGUGUGCAAGG
1555
937
GGCUCGUAUGUGUGCAAGG
1555
955
CCUUGCACACAUACGAGCC
1802


955
GCCAACAACGGCAUCCAGC
1556
955
GCCAACAACGGCAUCCAGC
1556
973
GCUGGAUGCCGUUGUUGGC
1803


973
CGAUUUCGGGAGAGCACCG
1557
973
CGAUUUCGGGAGAGCACCG
1557
991
CGGUGCUCUCCCGAAAUCG
1804


991
GAGGUCAUUGUGCAUGAAA
1558
991
GAGGUCAUUGUGCAUGAAA
1558
1009
UUUCAUGCACAAUGACCUC
1805


1009
AAUCCCUUCAUCAGCGUCG
1559
1009
AAUCCCUUCAUCAGCGUCG
1559
1027
CGACGCUGAUGAAGGGAUU
1806


1027
GAGUGGCUCAAAGGACCCA
1560
1027
GAGUGGCUCAAAGGACCCA
1560
1045
UGGGUCCUUUGAGCCACUC
1807


1045
AUCCUGGAGGCCACGGCAG
1561
1045
AUCCUGGAGGCCACGGCAG
1561
1063
CUGCCGUGGCCUCCAGGAU
1808


1063
GGAGACGAGCUGGUGAAGC
1562
1063
GGAGACGAGCUGGUGAAGC
1562
1081
GCUUCACCAGCUCGUCUCC
1809


1081
CUGCCCGUGAAGCUGGCAG
1563
1081
CUGCCCGUGAAGCUGGCAG
1563
1099
CUGCCAGCUUCACGGGCAG
1810


1099
GCGUACCCCCCGCCCGAGU
1564
1099
GCGUACCCCCCGCCCGAGU
1564
1117
ACUCGGGCGGGGGGUACGC
1811


1117
UUCCAGUGGUACAAGGAUG
1565
1117
UUCCAGUGGUACAAGGAUG
1565
1135
CAUCCUUGUACCACUGGAA
1812


1135
GGAAAGGCACUGUCCGGGC
1566
1135
GGAAAGGCACUGUCCGGGC
1566
1153
GCCCGGACAGUGCCUUUCC
1813


1153
CGCCACAGUCCACAUGCCC
1567
1153
CGCCACAGUCCACAUGCCC
1567
1171
GGGCAUGUGGACUGUGGCG
1814


1171
CUGGUGCUCAAGGAGGUGA
1568
1171
CUGGUGCUCAAGGAGGUGA
1568
1189
UCACCUCCUUGAGCACCAG
1815


1189
ACAGAGGCCAGCACAGGCA
1569
1189
ACAGAGGCCAGCACAGGCA
1569
1207
UGCCUGUGCUGGCCUCUGU
1816


1207
ACCUACACCCUCGCCCUGU
1570
1207
ACCUACACCCUCGCCCUGU
1570
1225
ACAGGGCGAGGGUGUAGGU
1817


1225
UGGAACUCCGCUGCUGGCC
1571
1225
UGGAACUCCGCUGCUGGCC
1571
1243
GGCCAGCAGCGGAGUUCCA
1818


1243
CUGAGGCGCAACAUCAGCC
1572
1243
CUGAGGCGCAACAUCAGCC
1572
1261
GGCUGAUGUUGCGCCUCAG
1819


1261
CUGGAGCUGGUGGUGAAUG
1573
1261
CUGGAGCUGGUGGUGAAUG
1573
1279
CAUUCACCACCAGCUCCAG
1820


1279
GUGCCCCCCCAGAUACAUG
1574
1279
GUGCCCCCCCAGAUACAUG
1574
1297
CAUGUAUCUGGGGGGGCAC
1821


1297
GAGAAGGAGGCCUCCUCCC
1575
1297
GAGAAGGAGGCCUCCUCCC
1575
1315
GGGAGGAGGCCUCCUUCUC
1822


1315
CCCAGCAUCUACUCGCGUC
1576
1315
CCCAGCAUCUACUCGCGUC
1576
1333
GACGCGAGUAGAUGCUGGG
1823


1333
CACAGCCGCCAGGCCCUCA
1577
1333
CACAGCCGCCAGGCCCUCA
1577
1351
UGAGGGCCUGGCGGCUGUG
1824


1351
ACCUGCACGGCCUACGGGG
1578
1351
ACCUGCACGGCCUACGGGG
1578
1369
CCCCGUAGGCCGUGCAGGU
1825


1369
GUGCCCCUGCCUCUCAGCA
1579
1369
GUGCCCCUGCCUCUCAGCA
1579
1387
UGCUGAGAGGCAGGGGCAC
1826


1387
AUCCAGUGGCACUGGCGGC
1580
1387
AUCCAGUGGCACUGGCGGC
1580
1405
GCCGCCAGUGCCACUGGAU
1827


1405
CCCUGGACACCCUGCAAGA
1581
1405
CCCUGGACACCCUGCAAGA
1581
1423
UCUUGCAGGGUGUCCAGGG
1828


1423
AUGUUUGCCCAGCGUAGUC
1582
1423
AUGUUUGCCCAGCGUAGUC
1582
1441
GACUACGCUGGGCAAACAU
1829


1441
CUCCGGCGGCGGCAGCAGC
1583
1441
CUCCGGCGGCGGCAGCAGC
1583
1459
GCUGCUGCCGCCGCCGGAG
1830


1459
CAAGACCUCAUGCCACAGU
1584
1459
CAAGACCUCAUGCCACAGU
1584
1477
ACUGUGGCAUGAGGUCUUG
1831


1477
UGCCGUGACUGGAGGGCGG
1585
1477
UGCCGUGACUGGAGGGCGG
1585
1495
CCGCCCUCCAGUCACGGCA
1832


1495
GUGACCACGCAGGAUGCCG
1586
1495
GUGACCACGCAGGAUGCCG
1586
1513
CGGCAUCCUGCGUGGUCAC
1833


1513
GUGAACCCCAUCGAGAGCC
1587
1513
GUGAACCCCAUCGAGAGCC
1587
1531
GGCUCUCGAUGGGGUUCAC
1834


1531
CUGGACACCUGGACCGAGU
1588
1531
CUGGACACCUGGACCGAGU
1588
1549
ACUCGGUCCAGGUGUCCAG
1835


1549
UUUGUGGAGGGAAAGAAUA
1589
1549
UUUGUGGAGGGAAAGAAUA
1589
1567
UAUUCUUUCCCUCCACAAA
1836


1567
AAGACUGUGAGCAAGCUGG
1590
1567
AAGACUGUGAGCAAGCUGG
1590
1585
CCAGCUUGCUCACAGUCUU
1837


1585
GUGAUCCAGAAUGCCAACG
1591
1585
GUGAUCCAGAAUGCCAACG
1591
1603
CGUUGGCAUUCUGGAUCAC
1838


1603
GUGUCUGCCAUGUACAAGU
1592
1603
GUGUCUGCCAUGUACAAGU
1592
1621
ACUUGUACAUGGCAGACAC
1839


1621
UGUGUGGUCUCCAACAAGG
1593
1621
UGUGUGGUCUCCAACAAGG
1593
1639
CCUUGUUGGAGACCACACA
1840


1639
GUGGGCCAGGAUGAGCGGC
1594
1639
GUGGGCCAGGAUGAGCGGC
1594
1657
GCCGCUCAUCCUGGCCCAC
1841


1657
CUCAUCUACUUCUAUGUGA
1595
1657
CUCAUCUACUUCUAUGUGA
1595
1675
UCACAUAGAAGUAGAUGAG
1842


1675
ACCACCAUCCCCGACGGCU
1596
1675
ACCACCAUCCCCGACGGCU
1596
1693
AGCCGUCGGGGAUGGUGGU
1843


1693
UUCACCAUCGAAUCCAAGC
1597
1693
UUCACCAUCGAAUCCAAGC
1597
1711
GCUUGGAUUCGAUGGUGAA
1844


1711
CCAUCCGAGGAGCUACUAG
1598
1711
CCAUCCGAGGAGCUACUAG
1598
1729
CUAGUAGCUCCUCGGAUGG
1845


1729
GAGGGCCAGCCGGUGCUCC
1599
1729
GAGGGCCAGCCGGUGCUCC
1599
1747
GGAGCACCGGCUGGCCCUC
1846


1747
CUGAGCUGCCAAGCCGACA
1600
1747
CUGAGCUGCCAAGCCGACA
1600
1765
UGUCGGCUUGGCAGCUCAG
1847


1765
AGCUACAAGUACGAGCAUC
1601
1765
AGCUACAAGUACGAGCAUC
1601
1783
GAUGCUCGUACUUGUAGCU
1848


1783
CUGCGCUGGUACCGCCUCA
1602
1783
CUGCGCUGGUACCGCCUCA
1602
1801
UGAGGCGGUACCAGCGCAG
1849


1801
AACCUGUCCACGCUGCACG
1603
1801
AACCUGUCCACGCUGCACG
1603
1819
CGUGCAGCGUGGACAGGUU
1850


1819
GAUGCGCACGGGAACCCGC
1604
1819
GAUGCGCACGGGAACCCGC
1604
1837
GCGGGUUCCCGUGCGCAUC
1851


1837
CUUCUGCUCGACUGCAAGA
1605
1837
CUUCUGCUCGACUGCAAGA
1605
1855
UCUUGCAGUCGAGCAGAAG
1852


1855
AACGUGCAUCUGUUCGCCA
1606
1855
AACGUGCAUCUGUUCGCCA
1606
1873
UGGCGAACAGAUGCACGUU
1853


1873
ACCCCUCUGGCCGCCAGCC
1607
1873
ACCCCUCUGGCCGCCAGCC
1607
1891
GGCUGGCGGCCAGAGGGGU
1854


1891
CUGGAGGAGGUGGCACCUG
1608
1891
CUGGAGGAGGUGGCACCUG
1608
1909
CAGGUGCCACCUCCUCCAG
1855


1909
GGGGCGCGCCACGCCACGC
1609
1909
GGGGCGCGCCACGCCACGC
1609
1927
GCGUGGCGUGGCGCGCCCC
1856


1927
CUCAGCCUGAGUAUCCCCC
1610
1927
CUCAGCCUGAGUAUCCCCC
1610
1945
GGGGGAUACUCAGGCUGAG
1857


1945
CGCGUCGCGCCCGAGCACG
1611
1945
CGCGUCGCGCCCGAGCACG
1611
1963
CGUGCUCGGGCGCGACGCG
1858


1963
GAGGGCCACUAUGUGUGCG
1612
1963
GAGGGCCACUAUGUGUGCG
1612
1981
CGCACACAUAGUGGCCCUC
1859


1981
GAAGUGCAAGACCGGCGCA
1613
1981
GAAGUGCAAGACCGGCGCA
1613
1999
UGCGCCGGUCUUGCACUUC
1860


1999
AGCCAUGACAAGCACUGCC
1614
1999
AGCCAUGACAAGCACUGCC
1614
2017
GGCAGUGCUUGUCAUGGCU
1861


2017
CACAAGAAGUACCUGUCGG
1615
2017
CACAAGAAGUACCUGUCGG
1615
2035
CCGACAGGUACUUCUUGUG
1862


2035
GUGCAGGCCCUGGAAGCCC
1616
2035
GUGCAGGCCCUGGAAGCCC
1616
2053
GGGCUUCCAGGGCCUGCAC
1863


2053
CCUCGGCUCACGCAGAACU
1617
2053
CCUCGGCUCACGCAGAACU
1617
2071
AGUUCUGCGUGAGCCGAGG
1864


2071
UUGACCGACCUCCUGGUGA
1618
2071
UUGACCGACCUCCUGGUGA
1618
2089
UCACCAGGAGGUCGGUCAA
1865


2089
AACGUGAGCGACUCGCUGG
1619
2089
AACGUGAGCGACUCGCUGG
1619
2107
CCAGCGAGUCGCUCACGUU
1866


2107
GAGAUGCAGUGCUUGGUGG
1620
2107
GAGAUGCAGUGCUUGGUGG
1620
2125
CCACCAAGCACUGCAUCUC
1867


2125
GCCGGAGCGCACGCGCCCA
1621
2125
GCCGGAGCGCACGCGCCCA
1621
2143
UGGGCGCGUGCGCUCCGGC
1868


2143
AGCAUCGUGUGGUACAAAG
1622
2143
AGCAUCGUGUGGUACAAAG
1622
2161
CUUUGUACCACACGAUGCU
1869


2161
GACGAGAGGCUGCUGGAGG
1623
2161
GACGAGAGGCUGCUGGAGG
1623
2179
CCUCCAGCAGCCUCUCGUC
1870


2179
GAAAAGUCUGGAGUCGACU
1624
2179
GAAAAGUCUGGAGUCGACU
1624
2197
AGUCGACUCCAGACUUUUC
1871


2197
UUGGCGGACUCCAACCAGA
1625
2197
UUGGCGGACUCCAACCAGA
1625
2215
UCUGGUUGGAGUCCGCCAA
1872


2215
AAGCUGAGCAUCCAGCGCG
1626
2215
AAGCUGAGCAUCCAGCGCG
1626
2233
CGCGCUGGAUGCUCAGCUU
1873


2233
GUGCGCGAGGAGGAUGCGG
1627
2233
GUGCGCGAGGAGGAUGCGG
1627
2251
CCGCAUCCUCCUCGCGCAC
1874


2251
GGACCGUAUCUGUGCAGCG
1628
2251
GGACCGUAUCUGUGCAGCG
1628
2269
CGCUGCACAGAUACGGUCC
1875


2269
GUGUGCAGACCCAAGGGCU
1629
2269
GUGUGCAGACCCAAGGGCU
1629
2287
AGCCCUUGGGUCUGCACAC
1876


2287
UGCGUCAACUCCUCCGCCA
1630
2287
UGCGUCAACUCCUCCGCCA
1630
2305
UGGCGGAGGAGUUGACGCA
1877


2305
AGCGUGGCCGUGGAAGGCU
1631
2305
AGCGUGGCCGUGGAAGGCU
1631
2323
AGCCUUCCACGGCCACGCU
1878


2323
UCCGAGGAUAAGGGCAGCA
1632
2323
UCCGAGGAUAAGGGCAGCA
1632
2341
UGCUGCCCUUAUCCUCGGA
1879


2341
AUGGAGAUCGUGAUCCUUG
1633
2341
AUGGAGAUCGUGAUCCUUG
1633
2359
CAAGGAUCACGAUCUCCAU
1880


2359
GUCGGUACCGGCGUCAUCG
1634
2359
GUCGGUACCGGCGUCAUCG
1634
2377
CGAUGACGCCGGUACCGAC
1881


2377
GCUGUCUUCUUCUGGGUCC
1635
2377
GCUGUCUUCUUCUGGGUCC
1635
2395
GGACCCAGAAGAAGACAGC
1882


2395
CUCCUCCUCCUCAUCUUCU
1636
2395
CUCCUCCUCCUCAUCUUCU
1636
2413
AGAAGAUGAGGAGGAGGAG
1883


2413
UGUAACAUGAGGAGGCCGG
1637
2413
UGUAACAUGAGGAGGCCGG
1637
2431
CCGGCCUCCUCAUGUUACA
1884


2431
GCCCACGCAGACAUCAAGA
1638
2431
GCCCACGCAGACAUCAAGA
1638
2449
UCUUGAUGUCUGCGUGGGC
1885


2449
ACGGGCUACCUGUCCAUCA
1639
2449
ACGGGCUACCUGUCCAUCA
1639
2467
UGAUGGACAGGUAGCCCGU
1886


2467
AUCAUGGACCCCGGGGAGG
1640
2467
AUCAUGGACCCCGGGGAGG
1640
2485
CCUCCCCGGGGUCCAUGAU
1887


2485
GUGCCUCUGGAGGAGCAAU
1641
2485
GUGCCUCUGGAGGAGCAAU
1641
2503
AUUGCUCCUCCAGAGGCAC
1888


2503
UGCGAAUACCUGUCCUACG
1642
2503
UGCGAAUACCUGUCCUACG
1642
2521
CGUAGGACAGGUAUUCGCA
1889


2521
GAUGCCAGCCAGUGGGAAU
1643
2521
GAUGCCAGCCAGUGGGAAU
1643
2539
AUUCCCACUGGCUGGCAUC
1890


2539
UUCCCCCGAGAGCGGCUGC
1644
2539
UUCCCCCGAGAGCGGCUGC
1644
2557
GCAGCCGCUCUCGGGGGAA
1891


2557
CACCUGGGGAGAGUGCUCG
1645
2557
CACCUGGGGAGAGUGCUCG
1645
2575
CGAGCACUCUCCCCAGGUG
1892


2575
GGCUACGGCGCCUUCGGGA
1646
2575
GGCUACGGCGCCUUCGGGA
1646
2593
UCCCGAAGGCGCCGUAGCC
1893


2593
AAGGUGGUGGAAGCCUCCG
1647
2593
AAGGUGGUGGAAGCCUCCG
1647
2611
CGGAGGCUUCCACCACCUU
1894


2611
GCUUUCGGCAUCCACAAGG
1648
2611
GCUUUCGGCAUCCACAAGG
1648
2629
CCUUGUGGAUGCCGAAAGC
1895


2629
GGCAGCAGCUGUGACACCG
1649
2629
GGCAGCAGCUGUGACACCG
1649
2647
CGGUGUCACAGCUGCUGCC
1896


2647
GUGGCCGUGAAAAUGCUGA
1650
2647
GUGGCCGUGAAAAUGCUGA
1650
2665
UCAGCAUUUUCACGGCCAC
1897


2665
AAAGAGGGCGCCACGGCCA
1651
2665
AAAGAGGGCGCCACGGCCA
1651
2683
UGGCCGUGGCGCCCUCUUU
1898


2683
AGCGAGCAGCGCGCGCUGA
1652
2683
AGCGAGCAGCGCGCGCUGA
1652
2701
UCAGCGCGCGCUGCUCGCU
1899


2701
AUGUCGGAGCUCAAGAUCC
1653
2701
AUGUCGGAGCUCAAGAUCC
1653
2719
GGAUCUUGAGCUCCGACAU
1900


2719
CUCAUUCACAUCGGCAACC
1654
2719
CUCAUUCACAUCGGCAACC
1654
2737
GGUUGCCGAUGUGAAUGAG
1901


2737
CACCUCAACGUGGUCAACC
1655
2737
CACCUCAACGUGGUCAACC
1655
2755
GGUUGACCACGUUGAGGUG
1902


2755
CUCCUCGGGGCGUGCACCA
1656
2755
CUCCUCGGGGCGUGCACCA
1656
2773
UGGUGCACGCCCCGAGGAG
1903


2773
AAGCCGCAGGGCCCCCUCA
1657
2773
AAGCCGCAGGGCCCCCUCA
1657
2791
UGAGGGGGCCCUGCGGCUU
1904


2791
AUGGUGAUCGUGGAGUUCU
1658
2791
AUGGUGAUCGUGGAGUUCU
1658
2809
AGAACUCCACGAUCACCAU
1905


2809
UGCAAGUACGGCAACCUCU
1659
2809
UGCAAGUACGGCAACCUCU
1659
2827
AGAGGUUGCCGUACUUGCA
1906


2827
UCCAACUUCCUGCGCGCCA
1660
2827
UCCAACUUCCUGCGCGCCA
1660
2845
UGGCGCGCAGGAAGUUGGA
1907


2845
AAGCGGGACGCCUUCAGCC
1661
2845
AAGCGGGACGCCUUCAGCC
1661
2863
GGCUGAAGGCGUCCCGCUU
1908


2863
CCCUGCGCGGAGAAGUCUC
1662
2863
CCCUGCGCGGAGAAGUCUC
1662
2881
GAGACUUCUCCGCGCAGGG
1909


2881
CCCGAGCAGCGCGGACGCU
1663
2881
CCCGAGCAGCGCGGACGCU
1663
2899
AGCGUCCGCGCUGCUCGGG
1910


2899
UUCCGCGCCAUGGUGGAGC
1664
2899
UUCCGCGCCAUGGUGGAGC
1664
2917
GCUCCACCAUGGCGCGGAA
1911


2917
CUCGCCAGGCUGGAUCGGA
1665
2917
CUCGCCAGGCUGGAUCGGA
1665
2935
UCCGAUCCAGCCUGGCGAG
1912


2935
AGGCGGCCGGGGAGCAGCG
1666
2935
AGGCGGCCGGGGAGCAGCG
1666
2953
CGCUGCUCCCCGGCCGCCU
1913


2953
GACAGGGUCCUCUUCGCGC
1667
2953
GACAGGGUCCUCUUCGCGC
1667
2971
GCGCGAAGAGGACCCUGUC
1914


2971
CGGUUCUCGAAGACCGAGG
1668
2971
CGGUUCUCGAAGACCGAGG
1668
2989
CCUCGGUCUUCGAGAACCG
1915


2989
GGCGGAGCGAGGCGGGCUU
1669
2989
GGCGGAGCGAGGCGGGCUU
1669
3007
AAGCCCGCCUCGCUCCGCC
1916


3007
UCUCCAGACCAAGAAGCUG
1670
3007
UCUCCAGACCAAGAAGCUG
1670
3025
CAGCUUCUUGGUCUGGAGA
1917


3025
GAGGACCUGUGGCUGAGCC
1671
3025
GAGGACCUGUGGCUGAGCC
1671
3043
GGCUCAGCCACAGGUCCUC
1918


3043
CCGCUGACCAUGGAAGAUC
1672
3043
CCGCUGACCAUGGAAGAUC
1672
3061
GAUCUUCCAUGGUCAGCGG
1919


3061
CUUGUCUGCUACAGCUUCC
1673
3061
CUUGUCUGCUACAGCUUCC
1673
3079
GGAAGCUGUAGCAGACAAG
1920


3079
CAGGUGGCCAGAGGGAUGG
1674
3079
CAGGUGGCCAGAGGGAUGG
1674
3097
CCAUCCCUCUGGCCACCUG
1921


3097
GAGUUCCUGGCUUCCCGAA
1675
3097
GAGUUCCUGGCUUCCCGAA
1675
3115
UUCGGGAAGCCAGGAACUC
1922


3115
AAGUGCAUCCACAGAGACC
1676
3115
AAGUGCAUCCACAGAGACC
1676
3133
GGUCUCUGUGGAUGCACUU
1923


3133
CUGGCUGCUCGGAACAUUC
1677
3133
CUGGCUGCUCGGAACAUUC
1677
3151
GAAUGUUCCGAGCAGCCAG
1924


3151
CUGCUGUCGGAAAGCGACG
1678
3151
CUGCUGUCGGAAAGCGACG
1678
3169
CGUCGCUUUCCGACAGCAG
1925


3169
GUGGUGAAGAUCUGUGACU
1679
3169
GUGGUGAAGAUCUGUGACU
1679
3187
AGUCACAGAUCUUCACCAC
1926


3187
UUUGGCCUUGCCCGGGACA
1680
3187
UUUGGCCUUGCCCGGGACA
1680
3205
UGUCCCGGGCAAGGCCAAA
1927


3205
AUCUACAAAGACCCCGACU
1681
3205
AUCUACAAAGACCCCGACU
1681
3223
AGUCGGGGUCUUUGUAGAU
1928


3223
UACGUCCGCAAGGGCAGUG
1682
3223
UACGUCCGCAAGGGCAGUG
1682
3241
CACUGCCCUUGCGGACGUA
1929


3241
GCCCGGCUGCCCCUGAAGU
1683
3241
GCCCGGCUGCCCCUGAAGU
1683
3259
ACUUCAGGGGCAGCCGGGC
1930


3259
UGGAUGGCCCCUGAAAGCA
1684
3259
UGGAUGGCCCCUGAAAGCA
1684
3277
UGCUUUCAGGGGCCAUCCA
1931


3277
AUCUUCGACAAGGUGUACA
1685
3277
AUCUUCGACAAGGUGUACA
1685
3295
UGUACACCUUGUCGAAGAU
1932


3295
ACCACGCAGAGUGACGUGU
1686
3295
ACCACGCAGAGUGACGUGU
1686
3313
ACACGUCACUCUGCGUGGU
1933


3313
UGGUCCUUUGGGGUGCUUC
1687
3313
UGGUCCUUUGGGGUGCUUC
1687
3331
GAAGCACCCCAAAGGACCA
1934


3331
CUCUGGGAGAUCUUCUCUC
1688
3331
CUCUGGGAGAUCUUCUCUC
1688
3349
GAGAGAAGAUCUCCCAGAG
1935


3349
CUGGGGGCCUCCCCGUACC
1689
3349
CUGGGGGCCUCCCCGUACC
1689
3367
GGUACGGGGAGGCCCCCAG
1936


3367
CCUGGGGUGCAGAUCAAUG
1690
3367
CCUGGGGUGCAGAUCAAUG
1690
3385
CAUUGAUCUGCACCCCAGG
1937


3385
GAGGAGUUCUGCCAGCGCG
1691
3385
GAGGAGUUCUGCCAGCGCG
1691
3403
CGCGCUGGCAGAACUCCUC
1938


3403
GUGAGAGACGGCACAAGGA
1692
3403
GUGAGAGACGGCACAAGGA
1692
3421
UCCUUGUGCCGUCUCUCAC
1939


3421
AUGAGGGCCCCGGAGCUGG
1693
3421
AUGAGGGCCCCGGAGCUGG
1693
3439
CCAGCUCCGGGGCCCUCAU
1940


3439
GCCACUCCCGCCAUACGCC
1694
3439
GCCACUCCCGCCAUACGCC
1694
3457
GGCGUAUGGCGGGAGUGGC
1941


3457
CACAUCAUGCUGAACUGCU
1695
3457
CACAUCAUGCUGAACUGCU
1695
3475
AGCAGUUCAGCAUGAUGUG
1942


3475
UGGUCCGGAGACCCCAAGG
1696
3475
UGGUCCGGAGACCCCAAGG
1696
3493
CCUUGGGGUCUCCGGACCA
1943


3493
GCGAGACCUGCAUUCUCGG
1697
3493
GCGAGACCUGCAUUCUCGG
1697
3511
CCGAGAAUGCAGGUCUCGC
1944


3511
GACCUGGUGGAGAUCCUGG
1698
3511
GACCUGGUGGAGAUCCUGG
1698
3529
CCAGGAUCUCCACCAGGUC
1945


3529
GGGGACCUGCUCCAGGGCA
1699
3529
GGGGACCUGCUCCAGGGCA
1699
3547
UGCCCUGGAGCAGGUCCCC
1946


3547
AGGGGCCUGCAAGAGGAAG
1700
3547
AGGGGCCUGCAAGAGGAAG
1700
3565
CUUCCUCUUGCAGGCCCCU
1947


3565
GAGGAGGUCUGCAUGGCCC
1701
3565
GAGGAGGUCUGCAUGGCCC
1701
3583
GGGCCAUGCAGACCUCCUC
1948


3583
CCGCGCAGCUCUCAGAGCU
1702
3583
CCGCGCAGCUCUCAGAGCU
1702
3601
AGCUCUGAGAGCUGCGCGG
1949


3601
UCAGAAGAGGGCAGCUUCU
1703
3601
UCAGAAGAGGGCAGCUUCU
1703
3619
AGAAGCUGCCCUCUUCUGA
1950


3619
UCGCAGGUGUCCACCAUGG
1704
3619
UCGCAGGUGUCCACCAUGG
1704
3637
CCAUGGUGGACACCUGCGA
1951


3637
GCCCUACACAUCGCCCAGG
1705
3637
GCCCUACACAUCGCCCAGG
1705
3655
CCUGGGCGAUGUGUAGGGC
1952


3655
GCUGACGCUGAGGACAGCC
1706
3655
GCUGACGCUGAGGACAGCC
1706
3673
GGCUGUCCUCAGCGUCAGC
1953


3673
CCGCCAAGCCUGCAGCGCC
1707
3673
CCGCCAAGCCUGCAGCGCC
1707
3691
GGCGCUGCAGGCUUGGCGG
1954


3691
CACAGCCUGGCCGCCAGGU
1708
3691
CACAGCCUGGCCGCCAGGU
1708
3709
ACCUGGCGGCCAGGCUGUG
1955


3709
UAUUACAACUGGGUGUCCU
1709
3709
UAUUACAACUGGGUGUCCU
1709
3727
AGGACACCCAGUUGUAAUA
1956


3727
UUUCCCGGGUGCCUGGCCA
1710
3727
UUUCCCGGGUGCCUGGCCA
1710
3745
UGGCCAGGCACCCGGGAAA
1957


3745
AGAGGGGCUGAGACCCGUG
1711
3745
AGAGGGGCUGAGACCCGUG
1711
3763
CACGGGUCUCAGCCCCUCU
1958


3763
GGUUCCUCCAGGAUGAAGA
1712
3763
GGUUCCUCCAGGAUGAAGA
1712
3781
UCUUCAUCCUGGAGGAACC
1959


3781
ACAUUUGAGGAAUUCCCCA
1713
3781
ACAUUUGAGGAAUUCCCCA
1713
3799
UGGGGAAUUCCUCAAAUGU
1960


3799
AUGACCCCAACGACCUACA
1714
3799
AUGACCCCAACGACCUACA
1714
3817
UGUAGGUCGUUGGGGUCAU
1961


3817
AAAGGCUCUGUGGACAACC
1715
3817
AAAGGCUCUGUGGACAACC
1715
3835
GGUUGUCCACAGAGCCUUU
1962


3835
CAGACAGACAGUGGGAUGG
1716
3835
CAGACAGACAGUGGGAUGG
1716
3853
CCAUCCCACUGUCUGUCUG
1963


3853
GUGCUGGCCUCGGAGGAGU
1717
3853
GUGCUGGCCUCGGAGGAGU
1717
3871
ACUCCUCCGAGGCCAGCAC
1964


3871
UUUGAGCAGAUAGAGAGCA
1718
3871
UUUGAGCAGAUAGAGAGCA
1718
3889
UGCUCUCUAUCUGCUCAAA
1965


3889
AGGCAUAGACAAGAAAGCG
1719
3889
AGGCAUAGACAAGAAAGCG
1719
3907
CGCUUUCUUGUCUAUGCCU
1966


3907
GGCUUCAGGUAGCUGAAGC
1720
3907
GGCUUCAGGUAGCUGAAGC
1720
3925
GCUUCAGCUACCUGAAGCC
1967


3925
CAGAGAGAGAGAAGGCAGC
1721
3925
CAGAGAGAGAGAAGGCAGC
1721
3943
GCUGCCUUCUCUCUCUCUG
1968


3943
CAUACGUCAGCAUUUUCUU
1722
3943
CAUACGUCAGCAUUUUCUU
1722
3961
AAGAAAAUGCUGACGUAUG
1969


3961
UCUCUGCACUUAUAAGAAA
1723
3961
UCUCUGCACUUAUAAGAAA
1723
3979
UUUCUUAUAAGUGCAGAGA
1970


3979
AGAUCAAAGACUUUAAGAC
1724
3979
AGAUCAAAGACUUUAAGAC
1724
3997
GUCUUAAAGUCUUUGAUCU
1971


3997
CUUUCGCUAUUUCUUCUAC
1725
3997
CUUUCGCUAUUUCUUCUAC
1725
4015
GUAGAAGAAAUAGCGAAAG
1972


4015
CUGCUAUCUACUACAAACU
1726
4015
CUGCUAUCUACUACAAACU
1726
4033
AGUUUGUAGUAGAUAGCAG
1973


4033
UUCAAAGAGGAACCAGGAG
1727
4033
UUCAAAGAGGAACCAGGAG
1727
4051
CUCCUGGUUCCUCUUUGAA
1974


4051
GGACAAGAGGAGCAUGAAA
1728
4051
GGACAAGAGGAGCAUGAAA
1728
4069
UUUCAUGCUCCUCUUGUCC
1975


4069
AGUGGACAAGGAGUGUGAC
1729
4069
AGUGGACAAGGAGUGUGAC
1729
4087
GUCACACUCCUUGUCCACU
1976


4087
CCACUGAAGCACCACAGGG
1730
4087
CCACUGAAGCACCACAGGG
1730
4105
CCCUGUGGUGCUUCAGUGG
1977


4105
GAGGGGUUAGGCCUCCGGA
1731
4105
GAGGGGUUAGGCCUCCGGA
1731
4123
UCCGGAGGCCUAACCCCUC
1978


4123
AUGACUGCGGGCAGGCCUG
1732
4123
AUGACUGCGGGCAGGCCUG
1732
4141
CAGGCCUGCCCGCAGUCAU
1979


4141
GGAUAAUAUCCAGCCUCCC
1733
4141
GGAUAAUAUCCAGCCUCCC
1733
4159
GGGAGGCUGGAUAUUAUCC
1980


4159
CACAAGAAGCUGGUGGAGC
1734
4159
CACAAGAAGCUGGUGGAGC
1734
4177
GCUCCACCAGCUUCUUGUG
1981


4177
CAGAGUGUUCCCUGACUCC
1735
4177
CAGAGUGUUCCCUGACUCC
1735
4195
GGAGUCAGGGAACACUCUG
1982


4195
CUCCAAGGAAAGGGAGACG
1736
4195
CUCCAAGGAAAGGGAGACG
1736
4213
CGUCUCCCUUUCCUUGGAG
1983


4213
GCCCUUUCAUGGUCUGCUG
1737
4213
GCCCUUUCAUGGUCUGCUG
1737
4231
CAGCAGACCAUGAAAGGGC
1984


4231
GAGUAACAGGUGCCUUCCC
1738
4231
GAGUAACAGGUGCCUUCCC
1738
4249
GGGAAGGCACCUGUUACUC
1985


4249
CAGACACUGGCGUUACUGC
1739
4249
CAGACACUGGCGUUACUGC
1739
4267
GCAGUAACGCCAGUGUCUG
1986


4267
CUUGACCAAAGAGCCCUCA
1740
4267
CUUGACCAAAGAGCCCUCA
1740
4285
UGAGGGCUCUUUGGUCAAG
1987


4285
AAGCGGCCCUUAUGCCAGC
1741
4285
AAGCGGCCCUUAUGCCAGC
1741
4303
GCUGGCAUAAGGGCCGCUU
1988


4303
CGUGACAGAGGGCUCACCU
1742
4303
CGUGACAGAGGGCUCACCU
1742
4321
AGGUGAGCCCUCUGUCACG
1989


4321
UCUUGCCUUCUAGGUCACU
1743
4321
UCUUGCCUUCUAGGUCACU
1743
4339
AGUGACCUAGAAGGCAAGA
1990


4339
UUCUCACAAUGUCCCUUCA
1744
4339
UUCUCACAAUGUCCCUUCA
1744
4357
UGAAGGGACAUUGUGAGAA
1991


4357
AGCACCUGACCCUGUGCCC
1745
4357
AGCACCUGACCCUGUGCCC
1745
4375
GGGCACAGGGUCAGGUGCU
1992


4375
CGCCGAUUAUUCCUUGGUA
1746
4375
CGCCGAUUAUUCCUUGGUA
1746
4393
UACCAAGGAAUAAUCGGCG
1993


4393
AAUAUGAGUAAUACAUCAA
1747
4393
AAUAUGAGUAAUACAUCAA
1747
4411
UUGAUGUAUUACUCAUAUU
1994


4411
AAGAGUAGUAUUAAAAGCU
1748
4411
AAGAGUAGUAUUAAAAGCU
1748
4429
AGCUUUUAAUACUACUCUU
1995


4429
UAAUUAAUCAUGUUUAUAA
1749
4429
UAAUUAAUCAUGUUUAUAA
1749
4447
UUAUAAACAUGAUUAAUUA
1996


 







VEGF NM_003376.3
















3
GCGGAGGCUUGGGGCAGCC
1997
3
GCGGAGGCUUGGGGCAGCC
1997
21
GGCUGCCCCAAGCCUCCGC
2093



21
CGGGUAGCUCGGAGGUCGU
1998
21
CGGGUAGCUCGGAGGUCGU
1998
39
ACGACCUCCGAGCUACCCG
2094


39
UGGCGCUGGGGGCUAGCAC
1999
39
UGGCGCUGGGGGCUAGCAC
1999
57
GUGCUAGCCCCCAGCGCCA
2095


57
CCAGCGCUCUGUCGGGAGG
2000
57
CCAGCGCUCUGUCGGGAGG
2000
75
CCUCCCGACAGAGCGCUGG
2096


75
GCGCAGCGGUUAGGUGGAC
2001
75
GCGCAGCGGUUAGGUGGAC
2001
93
GUCCACCUAACCGCUGCGC
2097


93
CCGGUCAGCGGACUCACCG
2002
93
CCGGUCAGCGGACUCACCG
2002
111
CCGUGAGUCCGCUGACCCG
2098


111
GGCCAGGGCGCUCGGUGCU
2003
111
GGCCAGGGCGCUCGGUGCU
2003
129
AGCACCGAGCGCCCUGGCC
2099


129
UGGAAUUUGAUAUUCAUUG
2004
129
UGGAAUUUGAUAUUCAUUG
2004
147
CAAUGAAUAUCAAAUUCCA
2100


147
GAUCCGGGUUUUAUCCCUC
2005
147
GAUCCGGGUUUUAUCCCUC
2005
165
GAGGGAUAAAACCCGGAUC
2101


165
CUUCUUUUUUCUUAAACAU
2006
165
CUUCUUUUUUCUUAAACAU
2006
183
AUGUUUAAGAAAAAAGAAG
2102


183
UUUUUUUUUAAAACUGUAU
2007
183
UUUUUUUUUAAAACUGUAU
2007
201
AUACAGUUUUAAAAAAAAA
2103


201
UUGUUUCUCGUUUUAAUUU
2008
201
UUGUUUCUCGUUUUAAUUU
2008
219
AAAUUAAAACGAGAAACAA
2104


219
UAUUUUUGCUUGCCAUUCC
2009
219
UAUUUUUGCUUGCCAUUCC
2009
237
GGAAUGGCAAGCAAAAAUA
2105


237
CCCACUUGAAUCGGGCCGA
2010
237
CCCACUUGAAUCGGGCCGA
2010
255
UCGGCCCGAUUCAAGUGGG
2106


255
ACGGCUUGGGGAGAUUGCU
2011
255
ACGGCUUGGGGAGAUUGCU
2011
273
AGCAAUCUCCCCAAGCCGU
2107


273
UCUACUUCCCCAAAUCACU
2012
273
UCUACUUCCCCAAAUCACU
2012
291
AGUGAUUUGGGGAAGUAGA
2108


291
UGUGGAUUUUGGAAACCAG
2013
291
UGUGGAUUUUGGAAACCAG
2013
309
CUGGUUUCCAAAAUCCACA
2109


309
GCAGAAAGAGGAAAGAGGU
2014
309
GCAGAAAGAGGAAAGAGGU
2014
327
ACCUCUUUCCUCUUUCUGC
2110


327
UAGCAAGAGCUCCAGAGAG
2015
327
UAGCAAGAGCUCCAGAGAG
2015
345
CUCUCUGGAGCUCUUGCUA
2111


345
GAAGUCGAGGAAGAGAGAG
2016
345
GAAGUCGAGGAAGAGAGAG
2016
363
CUCUCUCUUCCUCGACUUC
2112


363
GACGGGGUCAGAGAGAGCG
2017
363
GACGGGGUCAGAGAGAGCG
2017
381
CGCUCUCUCUGACCCCGUC
2113


381
GCGCGGGCGUGCGAGCAGC
2018
381
GCGCGGGCGUGCGAGCAGC
2018
399
GCUGCUCGCACGCCCGCGC
2114


399
CGAAAGCGACAGGGGCAAA
2019
399
CGAAAGCGACAGGGGCAAA
2019
417
UUUGCCCCUGUCGCUUUCG
2115


417
AGUGAGUGACCUGCUUUUG
2020
417
AGUGAGUGACCUGCUUUUG
2020
435
CAAAAGCAGGUCACUCACU
2116


435
GGGGGUGACCGCCGGAGCG
2021
435
GGGGGUGACCGCCGGAGCG
2021
453
CGCUCCGGCGGUCACCCCC
2117


453
GCGGCGUGAGCCCUCCCCC
2022
453
GCGGCGUGAGCCCUCCCCC
2022
471
GGGGGAGGGCUCACGCCGC
2118


471
CUUGGGAUCCCGCAGCUGA
2023
471
CUUGGGAUCCCGCAGCUGA
2023
489
UCAGCUGCGGGAUCCCAAG
2119


489
ACCAGUCGCGCUGACGGAC
2024
489
ACCAGUCGCGCUGACGGAC
2024
507
GUCCGUCAGCGCGACUGGU
2120


507
CAGACAGACAGACACCGCC
2025
507
CAGACAGACAGACACCGCC
2025
525
GGCGGUGUCUGUCUGUCUG
2121


525
CCCCAGCCCCAGCUACCAC
2026
525
CCCCAGCCCCAGCUACCAC
2026
543
GUGGUAGCUGGGGCUGGGG
2122


543
CCUCCUCCCCGGCCGGCGG
2027
543
CCUCCUCCCCGGCCGGCGG
2027
561
CCGCCGGCCGGGGAGGAGG
2123


561
GCGGACAGUGGACGCGGCG
2028
561
GCGGACAGUGGACGCGGCG
2028
579
CGCCGCGUCCACUGUCCGC
2124


579
GGCGAGCCGCGGGCAGGGG
2029
579
GGCGAGCCGCGGGCAGGGG
2029
597
CCCCUGCCCGCGGCUCGCC
2125


597
GCCGGAGCCCGCGCCCGGA
2030
597
GCCGGAGCCCGCGCCCGGA
2030
615
UCCGGGCGCGGGCUCCGGC
2126


615
AGGCGGGGUGGAGGGGGUC
2031
615
AGGCGGGGUGGAGGGGGUC
2031
633
GACCCCCUCCACCCCGCCU
2127


633
CGGGGCUCGCGGCGUCGCA
2032
633
CGGGGCUCGCGGCGUCGCA
2032
651
UGCGACGCCGCGAGCCCCG
2128


651
ACUGAAACUUUUCGUCCAA
2033
651
ACUGAAACUUUUCGUCCAA
2033
669
UUGGACGAAAAGUUUCAGU
2129


669
ACUUCUGGGCUGUUCUCGC
2034
669
ACUUCUGGGCUGUUCUCGC
2034
687
GCGAGAACAGCCCAGAAGU
2130


687
CUUCGGAGGAGCCGUGGUC
2035
687
CUUCGGAGGAGCCGUGGUC
2035
705
GACCACGGCUCCUCCGAAG
2131


705
CCGCGCGGGGGAAGCCGAG
2036
705
CCGCGCGGGGGAAGCCGAG
2036
723
CUCGGCUUCCCCCGCGCGG
2132


723
GCCGAGCGGAGCCGCGAGA
2037
723
GCCGAGCGGAGCCGCGAGA
2037
741
UCUCGCGGCUCCGCUCGGC
2133


741
AAGUGCUAGCUCGGGCCGG
2038
741
AAGUGCUAGCUCGGGCCGG
2038
759
CCGGCCCGAGCUAGCACUU
2134


759
GGAGGAGCCGCAGCCGGAG
2039
759
GGAGGAGCCGCAGCCGGAG
2039
777
CUCCGGCUGCGGCUCCUCC
2135


777
GGAGGGGGAGGAGGAAGAA
2040
777
GGAGGGGGAGGAGGAAGAA
2040
795
UUCUUCCUCCUCCCCCUCC
2136


795
AGAGAAGGAAGAGGAGAGG
2041
795
AGAGAAGGAAGAGGAGAGG
2041
813
CCUCUCCUCUUCCUUCUCU
2137


813
GGGGCCGCAGUGGCGACUC
2042
813
GGGGCCGCAGUGGCGACUC
2042
831
GAGUCGCCACUGCGGCCCC
2138


831
CGGCGCUCGGAAGCCGGGC
2043
831
CGGCGCUCGGAAGCCGGGC
2043
849
GCCCGGCUUCCGAGCGCCG
2139


849
CUCAUGGACGGGUGAGGCG
2044
849
CUCAUGGACGGGUGAGGCG
2044
867
CGCCUCACCCGUCCAUGAG
2140


867
GGCGGUGUGCGCAGACAGU
2045
867
GGCGGUGUGCGCAGACAGU
2045
885
ACUGUCUGCGCACACCGCC
2141


885
UGCUCCAGCCGCGCGCGCU
2046
885
UGCUCCAGCCGCGCGCGCU
2046
903
AGCGCGCGCGGCUGGAGCA
2142


903
UCCCCAGGCCCUGGCCCGG
2047
903
UCCCCAGGCCCUGGCCCGG
2047
921
CCGGGCCAGGGCCUGGGGA
2143


921
GGCCUCGGGCCGGGGAGGA
2048
921
GGCCUCGGGCCGGGGAGGA
2048
939
UCCUCCCCGGCCCGAGGCC
2144


939
AAGAGUAGCUCGCCGAGGC
2049
939
AAGAGUAGCUCGCCGAGGC
2049
957
GCCUCGGCGAGCUACUCUU
2145


957
CGCCGAGGAGAGCGGGCCG
2050
957
CGCCGAGGAGAGCGGGCCG
2050
975
CGGCCCGCUCUCCUCGGCG
2146


975
GCCCCACAGCCCGAGCCGG
2051
975
GCCCCACAGCCCGAGCCGG
2051
993
CCGGCUCGGGCUGUGGGGC
2147


993
GAGAGGGAGCGCGAGCCGC
2052
993
GAGAGGGAGCGCGAGCCGC
2052
1011
GCGGCUCGCGCUCCCUCUC
2148


1011
CGCCGGCCCCGGUCGGGCC
2053
1011
CGCCGGCCCCGGUCGGGCC
2053
1029
GGCCCGACCGGGGCCGGCG
2149


1029
CUCCGAAACCAUGAACUUU
2054
1029
CUCCGAAACCAUGAACUUU
2054
1047
AAAGUUCAUGGUUUCGGAG
2150


1047
UCUGCUGUCUUGGGUGCAU
2055
1047
UCUGCUGUCUUGGGUGCAU
2055
1065
AUGCACCCAAGACAGCAGA
2151


1065
UUGGAGCCUUGCCUUGCUG
2056
1065
UUGGAGCCUUGCCUUGCUG
2056
1083
CAGCAAGGCAAGGCUCCAA
2152


1083
GCUCUACCUCCACCAUGCC
2057
1083
GCUCUACCUCCACCAUGCC
2057
1101
GGCAUGGUGGAGGUAGAGC
2153


1101
CAAGUGGUCCCAGGCUGCA
2058
1101
CAAGUGGUCCCAGGCUGCA
2058
1119
UGCAGCCUGGGACCACUUG
2154


1119
ACCCAUGGCAGAAGGAGGA
2059
1119
ACCCAUGGCAGAAGGAGGA
2059
1137
UCCUCCUUCUGCCAUGGGU
2155


1137
AGGGCAGAAUCAUCACGAA
2060
1137
AGGGCAGAAUCAUCACGAA
2060
1155
UUCGUGAUGAUUCUGCCCU
2156


1155
AGUGGUGAAGUUCAUGGAU
2061
1155
AGUGGUGAAGUUCAUGGAU
2061
1173
AUCCAUGAACUUCACCACU
2157


1173
UGUCUAUCAGCGCAGCUAC
2062
1173
UGUCUAUCAGCGCAGCUAC
2062
1191
GUAGCUGCGCUGAUAGACA
2158


1191
CUGCCAUCCAAUCGAGACC
2063
1191
CUGCCAUCCAAUCGAGACC
2063
1209
GGUCUCGAUUGGAUGGCAG
2159


1209
CCUGGUGGACAUCUUCCAG
2064
1209
CCUGGUGGACAUCUUCCAG
2064
1227
CUGGAAGAUGUCCACCAGG
2160


1227
GGAGUACCCUGAUGAGAUC
2065
1227
GGAGUACCCUGAUGAGAUC
2065
1245
GAUCUCAUCAGGGUACUCC
2161


1245
CGAGUACAUCUUCAAGCCA
2066
1245
CGAGUACAUCUUCAAGCCA
2066
1263
UGGCUUGAAGAUGUACUCG
2162


1263
AUCCUGUGUGCCCCUGAUG
2067
1263
AUCCUGUGUGCCCCUGAUG
2067
1281
CAUCAGGGGCACACAGGAU
2163


1281
GCGAUGCGGGGGCUGCUGC
2068
1281
GCGAUGCGGGGGCUGCUGC
2068
1299
GCAGCAGCCCCCGCAUCGC
2164


1299
CAAUGACGAGGGCCUGGAG
2069
1299
CAAUGACGAGGGCCUGGAG
2069
1317
CUCCAGGCCCUCGUCAUUG
2165


1317
GUGUGUGCCCACUGAGGAG
2070
1317
GUGUGUGCCCACUGAGGAG
2070
1335
CUCCUCAGUGGGCACACAC
2166


1335
GUCCAACAUCACCAUGCAG
2071
1335
GUCCAACAUCACCAUGCAG
2071
1353
CUGCAUGGUGAUGUUGGAC
2167


1353
GAUUAUGCGGAUCAAACCU
2072
1353
GAUUAUGCGGAUCAAACCU
2072
1371
AGGUUUGAUCCGCAUAAUC
2168


1371
UCACCAAGGCCAGCACAUA
2073
1371
UCACCAAGGCCAGCACAUA
2073
1389
UAUGUGCUGGCCUUGGUGA
2169


1389
AGGAGAGAUGAGCUUCCUA
2074
1389
AGGAGAGAUGAGCUUCCUA
2074
1407
UAGGAAGCUCAUCUCUCCU
2170


1407
ACAGCACAACAAAUGUGAA
2075
1407
ACAGCACAACAAAUGUGAA
2075
1425
UUCACAUUUGUUGUGCUGU
2171


1425
AUGCAGACCAAAGAAAGAU
2076
1425
AUGCAGACCAAAGAAAGAU
2076
1443
AUCUUUCUUUGGUCUGCAU
2172


1443
UAGAGCAAGACAAGAAAAA
2077
1443
UAGAGCAAGACAAGAAAAA
2077
1461
UUUUUCUUGUCUUGCUCUA
2173


1461
AAAAUCAGUUCGAGGAAAG
2078
1461
AAAAUCAGUUCGAGGAAAG
2078
1479
CUUUCCUCGAACUGAUUUU
2174


1479
GGGAAAGGGGCAAAAACGA
2079
1479
GGGAAAGGGGCAAAAACGA
2079
1497
UCGUUUUUGCCCCUUUCCC
2175


1497
AAAGCGCAAGAAAUCCCGG
2080
1497
AAAGCGCAAGAAAUCCCGG
2080
1515
CCGGGAUUUCUUGCGCUUU
2176


1515
GUAUAAGUCCUGGAGCGUU
2081
1515
GUAUAAGUCCUGGAGCGUU
2081
1533
AACGCUCCAGGACUUAUAC
2177


1533
UCCCUGUGGGCCUUGCUCA
2082
1533
UCCCUGUGGGCCUUGCUCA
2082
1551
UGAGCAAGGCCCACAGGGA
2178


1551
AGAGCGGAGAAAGCAUUUG
2083
1551
AGAGCGGAGAAAGCAUUUG
2083
1569
CAAAUGCUUUCUCCGCUCU
2179


1569
GUUUGUACAAGAUCCGCAG
2084
1569
GUUUGUACAAGAUCCGCAG
2084
1587
CUGCGGAUCUUGUACAAAC
2180


1587
GACGUGUAAAUGUUCCUGC
2085
1587
GACGUGUAAAUGUUCCUGC
2085
1605
GCAGGAACAUUUACACGUC
2181


1605
CAAAAACACAGACUCGCGU
2086
1605
CAAAAACACAGACUCGCGU
2086
1623
ACGCGAGUCUGUGUUUUUG
2182


1623
UUGCAAGGCGAGGCAGCUU
2087
1623
UUGCAAGGCGAGGCAGCUU
2087
1641
AAGCUGCCUCGCCUUGCAA
2183


1641
UGAGUUAAACGAACGUACU
2088
1641
UGAGUUAAACGAACGUACU
2088
1659
AGUACGUUCGUUUAACUCA
2184


1659
UUGCAGAUGUGACAAGCCG
2089
1659
UUGCAGAUGUGACAAGCCG
2089
1677
CGGCUUGUCACAUCUGCAA
2185


1677
GAGGCGGUGAGCCGGGCAG
2090
1677
GAGGCGGUGAGCCGGGCAG
2090
1695
CUGCCCGGCUCACCGCCUC
2186


1695
GGAGGAAGGAGCCUCCCUC
2091
1695
GGAGGAAGGAGCCUCCCUC
2091
1713
GAGGGAGGCUCCUUCCUCC
2187


1703
GAGCCUCCCUCAGGGUUUC
2092
1703
GAGCCUCCCUCAGGGUUUC
2092
1721
GAAACCCUGAGGGAGGCUC
2188


 







Sequence Alignments: Lower case shows mismatches
















SEQ



Gene
Pos
Sequence
Upper Case Seq
ID





hFLT1
3645
AUCAUGCUGGACUGCUGGCACAG
AUCAUGCUGGACUGCUGGCACAG
2189


hKDR
3717
AcCAUGCUGGACUGCUGGCACgG
ACCAUGCUGGACUGCUGGCACGG
2190


mFLT1
3422
AUCAUGUUGGAUUGCUGGCACAa
AUCAUGUUGGAUUGCUGGCACAA
2191


mKDR
3615
AcCAUGCUGGACUGCUGGCAUga
ACCAUGCUGGACUGCUGGCAUGA
2192


rFLT1
3632
AUCAUGCUGGAUUGCUGGCACAa
AUCAUGCUGGAUUGCUGGCACAA
2193


rKDR
3650
ACCAUGCUGGAUUGCUGGCAUga
ACCAUGCUGGAUUGCUGGCAUGA
2194


hFLT1
3646
UCAUGCUGGACUGCUGGCACAGA
UCAUGCUGGACUGCUGGCACAGA
2195


hKDR
3718
cCAUGCUGGACUGCUGGCACgGg
CCAUGCUGGACUGCUGGCACGGG
2196


mFLT1
3423
UCAUGUUGGAUUGCUGGCACAaA
UCAUGUUGGAUUGCUGGCACAAA
2197


mKDR
3616
cCAUGCUGGACUGCUGGCAUgag
CCAUGCUGGACUGCUGGCAUGAG
2198


rFLT1
3633
UCAUGCUGGAUUGCUGGCACAaA
UCAUGCUGGAUUGCUGGCACAAA
2199


rKDR
3651
CCAUGCUGGAUUGCUGGCAUgag
CCAUGCUGGAUUGCUGGCAUGAG
2200


hFLT1
3647
CAUGCUGGACUGCUGGCACAGAG
CAUGCUGGACUGCUGGCACAGAG
2201


hKDR
3719
CAUGCUGGACUGCUGGCACgGgG
CAUGCUGGACUGCUGGCACGGGG
2202


mFLT1
3424
CAUGUUGGAUUGCUGGCACAaAG
CAUGUUGGAUUGCUGGCACAAAG
2203


mKDR
3617
CAUGCUGGACUGCUGGCAUgagG
CAUGCUGGACUGCUGGCAUGAGG
2204


rFLT1
3634
CAUGCUGGAUUGCUGGCACAaAG
CAUGCUGGAUUGCUGGCACAAAG
2205


rKDR
3652
CAUGCUGGAUUGCUGGCAUgagG
CAUGCUGGAUUGCUGGCAUGAGG
2206


hKDR
2764
UGCCUUAUGAUGCCAGCAAAUGG
UGCCUUAUGAUGCCAGCAAAUGG
2207


hFLT1
2689
UcCCUUAUGAUGCCAGCAAgUGG
UCCCUUAUGAUGCCAGCAAGUGG
2208


mFLT1
2469
UGCCcUAUGAUGCCAGCAAgUGG
UGCCCUAUGAUGCCAGCAAGUGG
2209


mKDR
2662
UGCCUUAUGAUGCCAGCAAgUGG
UGCCUUAUGAUGCCAGCAAGUGG
2210


rFLT1
2676
UGCCcUAUGAUGCCAGCAAgUGG
UGCCCUAUGAUGCCAGCAAGUGG
2209


rKDR
2697
UGCCUUAUGAUGCCAGCAAgUGG
UGCCUUAUGAUGCCAGCAAGUGG
2210


hKDR
2765
GCCUUAUGAUGCCAGCAAAUGGG
GCCUUAUGAUGCCAGCAAAUGGG
2211


hFLT1
2690
cCCUUAUGAUGCCAGCAAgUGGG
CCCUUAUGAUGCCAGCAAGUGGG
2212


mFLT1
2470
GCCCUAUGAUGCCAGCAAgUGGG
GCCCUAUGAUGCCAGCAAGUGGG
2213


mKDR
2663
GCCUUAUGAUGCCAGCAAgUGGG
GCCUUAUGAUGCCAGCAAGUGGG
2214


rFLT1
2677
GCCCUAUGAUGCCAGCAAgUGGG
GCCCUAUGAUGCCAGCAAGUGGG
2213


rKDR
2698
GCCUUAUGAUGCCAGCAAgUGGG
GCCUUAUGAUGCCAGCAAGUGGG
2214


hKDR
2766
CCUUAUGAUGCCAGCAAAUGGGA
CCUUAUGAUGCCAGCAAAUGGGA
2215


hFLT1
2691
CCUUAUGAUGCCAGCAAgUGGGA
CCUUAUGAUGCCAGCAAGUGGGA
2216


mFLT1
2471
CCcUAUGAUGCCAGCAAgUGGGA
CCCUAUGAUGCCAGCAAGUGGGA
2217


mKDR
2664
CCUUAUGAUGCCAGCAAgUGGGA
CCUUAUGAUGCCAGCAAGUGGGA
2216


rFLT1
2678
CCcUAUGAUGCCAGCAAgUGGGA
CCCUAUGAUGCCAGCAAGUGGGA
2217


rKDR
2699
CCUUAUGAUGCCAGCAAgUGGGA
CCUUAUGAUGCCAGCAAGUGGGA
2216


hKDR
2767
CUUAUGAUGCCAGCAAAUGGGAA
CUUAUGAUGCCAGCAAAUGGGAA
2218


hFLT1
2692
CUUAUGAUGCCAGCAAgUGGGAg
CUUAUGAUGCCAGCAAGUGGGAG
2219


mFLT1
2472
CcUAUGAUGCCAGCAAgUGGGAg
CCUAUGAUGCCAGCAAGUGGGAG
2220


mKDR
2665
CUUAUGAUGCCAGCAAgUGGGAA
CUUAUGAUGCCAGCAAGUGGGAA
2221


rFLT1
2679
CcUAUGAUGCCAGCAAgUGGGAg
CCUAUGAUGCCAGCAAGUGGGAG
2220


rKDR
2700
CUUAUGAUGCCAGCAAgUGGGAg
CUUAUGAUGCCAGCAAGUGGGAG
2219


hKDR
2768
UUAUGAUGCCAGCAAAUGGGAAU
UUAUGAUGCCAGCAAAUGGGAAU
2222


hFLT1
2693
UUAUGAUGCCAGCAAgUGGGAgU
UUAUGAUGCCAGCAAGUGGGAGU
2223


mFLT1
2473
cUAUGAUGCCAGCAAgUGGGAgu
CUAUGAUGCCAGCAAGUGGGAGU
2224


mKDR
2666
UUAUGAUGCCAGCAAgUGGGAAU
UUAUGAUGCCAGCAAGUGGGAAU
2225


rFLT1
2680
cUAUGAUGCCAGCAAgUGGGAgU
CUAUGAUGCCAGCAAGUGGGAGU
2224


rKDR
2701
UUAUGAUGCCAGCAAQUGGGAQU
UUAUGAUGCCAGCAAGUGGGAGU
2223


hKDR
3712
ACCAGACCAUGCUGGACUGCUGG
ACCAGACCAUGCUGGACUGCUGG
2226


hFLT1
3640
AUCAGAUCAUGCUGGACUGCUGG
AUCAGAUCAUGCUGGACUGCUGG
2227


mFLT1
3417
ACCAaAUCAUGUUGGAUUGCUGG
ACCAAAUCAUGUUGGAUUGCUGG
2228


mKDR
3610
ACCAGACCAUGCUGGACUGCUGG
ACCAGACCAUGCUGGACUGCUGG
2226


rFLT1
3627
ACCAaAUCAUGCUGGAUUGCUGG
ACCAAAUCAUGCUGGAUUGCUGG
2229


rKDR
3645
ACCAaACCAUGCUGGAUUGCUGG
ACCAAACCAUGCUGGAUUGCUGG
2230


hKDR
3713
CCAGACCAUGCUGGACUGCUGGC
CCAGACCAUGCUGGACUGCUGGC
2231


hFLT1
3641
UCAGAUCAUGCUGGACUGCUGGC
UCAGAUCAUGCUGGACUGCUGGC
2232


mFLT1
3418
CCAaAUCAUGUUGGAUUGCUGGC
CCAAAUCAUGUUGGAUUGCUGGC
2233


mKDR
3611
CCAGACCAUGCUGGACUGCUGGC
CCAGACCAUGCUGGACUGCUGGC
2231


rFLT1
3628
CCAaAUCAUGCUGGAUUGCUGGC
CCAAAUCAUGCUGGAUUGCUGGC
2234


rKDR
3646
CCAaACCAUGCUGGAUUGCUGGC
CCAAACCAUGCUGGAUUGCUGGC
2235


hKDR
3714
CAGACCAUGCUGGACUGCUGGCA
CAGACCAUGCUGGACUGCUGGCA
2236


hFLT1
3642
CAGAUCAUGCUGGACUGCUGGCA
CAGAUCAUGCUGGACUGCUGGCA
2237


mFLT1
3419
CAaAUCAUGUUGGAUUGCUGGCA
CAAAUCAUGUUGGAUUGCUGGCA
2238


mKDR
3612
CAGACCAUGCUGGACUGCUGGCA
CAGACCAUGCUGGACUGCUGGCA
2236


rFLT1
3629
CAaAUCAUGCUGGAUUGCUGGCA
CAAAUCAUGCUGGAUUGCUGGCA
2239


rKDR
3647
CAaACCAUGCUGGAUUGCUGGCA
CAAACCAUGCUGGAUUGCUGGCA
2240


hKDR
3715
AGACCAUGCUGGACUGCUGGCAC
AGACCAUGCUGGACUGCUGGCAC
2241


hFLT1
3643
AGAUCAUGCUGGACUGCUGGCAC
AGAUCAUGCUGGACUGCUGGCAC
2242


mFLT1
3420
AaAUCAUGUUGGAUUGCUGGCAC
AAAUCAUGUUGGAUUGCUGGCAC
2243


mKDR
3613
AGACCAUGCUGGACUGCUGGCAU
AGACCAUGCUGGACUGCUGGCAU
2244


rFLT1
3630
AaAUCAUGCUGGAUUGCUGGCAC
AAAUCAUGCUGGAUUGCUGGCAC
2245


rKDR
3648
AaACCAUGCUGGAUUGCUGGCAU
AAACCAUGCUGGAUUGCUGGCAU
2246


hKDR
3716
GACCAUGCUGGACUGCUGGCACG
GACCAUGCUGGACUGCUGGCACG
2247


hFLT1
3644
GAUCAUGCUGGACUGCUGGCACa
GAUCAUGCUGGACUGCUGGCACA
2248


mFLT1
3421
aAUCAUGUUGGAUUGCUGGCACa
AAUCAUGUUGGAUUGCUGGCACA
2249


mKDR
3614
GACCAUGCUGGACUGCUGGCAUG
GACCAUGCUGGACUGCUGGCAUG
2250


rFLT1
3631
aAUCAUGCUGGAUUGCUGGCACa
AAUCAUGCUGGAUUGCUGGCACA
2251


rKDR
3649
aACCAUGCUGGAUUGCUGGCAUG
AACCAUGCUGGAUUGCUGGCAUG
2252


hKDR
3811
AGCAGGAUGGCAAAGACUACAUU
AGCAGGAUGGCAAAGACUACAUU
2253


hFLT1
3739
AaCAGGAUGGUAAAGACUACAUc
AACAGGAUGGUAAAGACUACAUC
2254


mFLT1
3516
AaCAGGAUGGgAAAGAUUACAUc
AACAGGAUGGGAAAGAUUACAUC
2255


mKDR
3709
AGCAGGAUGGCAAAGACUAUAUU
AGCAGGAUGGCAAAGACUAUAUU
2256


rFLT1
3726
AaCAGGAUGGUAAAGACUACAUc
AACAGGAUGGUAAAGACUACAUC
2254


rKDR
3744
AGCAGGAUGGCAAAGACUAUAUU
AGCAGGAUGGCAAAGACUAUAUU
2256


hKDR
3812
GCAGGAUGGCAAAGACUACAUUG
GCAGGAUGGCAAAGACUACAUUG
2257


hFLT1
3740
aCAGGAUGGUAAAGACUACAUcc
ACAGGAUGGUAAAGACUACAUCC
2258


mFLT1
3517
aGAGGAUGGgAAAGAUUACAUcc
ACAGGAUGGGAAAGAUUACAUCC
2259


mKDR
3710
GCAGGAUGGCAAAGACUAUAUUG
GCAGGAUGGCAAAGACUAUAUUG
2260


rFLT1
3727
aCAGGAUGGUAAAGACUACAUcc
ACAGGAUGGUAAAGACUACAUCC
2258


rKDR
3745
GCAGGAUGGCAAAGACUAUAUUG
GCAGGAUGGCAAAGACUAUAUUG
2260







Conserved Regions











Gene
Pos
Len
Sequence
SeqID










Fragments of >= 10 nt that are present in both human VEGF


(NM_003376.3) and human FLT1 (NM_002019.1)












FLT1
18
12
CUCCUCCCCGGC
2261



FLT1
125
12
GGAGCCGCGAGA
2262


FLT1
155
12
GGCCGGCGGCGG
2263


FLT1
160
10
GCGGCGGCGA
2264


FLT1
1051
11
UACCCUGAUGA
2265


FLT1
1803
10
GGCUAGCACC
2266


FLT1
2841
10
AGAGGGGGCC
2267


FLT1
3133
12
AGCAGCGAAAGC
2268


FLT1
3191
11
AGGAAGAGGAG
2269


FLT1
3550
10
CCAGGAGUAC
2270


FLT1
4216
10
CCGCCCCCAG
2271


FLT1
5711
10
GUGGGCCUUG
2272


FLT1
5811
10
GUGGGCCUUG
2272


FLT1
5938
10
CUUGGGGAGA
2273


FLT1
6236
10
CCUCUUCUUU
2274


 







Fragments of >= 10 nt that are present in both human VEGF


(NM_003376.3) and human KDR (NM_002253.1)












KDR
1463
10
AAGUGAGUGA
2275



KDR
1689
11
GGAGGAAGAGU
2276


KDR
1886
11
ACAAAUGUGAA
2277


KDR
1983
10
GOCCACUGAG
2278


KDR
2228
10
GCCUUGCUCA
2279


KDR
2484
10
GAGGAAGGAG
2280


KDR
3064
10
UUUGGAAACC
2281


KDR
3912
11
GGAGGAGGAAG
2282


KDR
4076
10
CGGACAGUGG
2283


KDR
5138
10
UCCCAGGCUG
2284





The 3′-ends of the Upper sequence and the Lower sequence of the siNA construct can include an overhang sequence, for example about 1, 2, 3, or 4 nucleotides in length, preferably 2 nucleotides in length, wherein the overhanging sequence of the lower sequence is optionally complementary to a portion of the target sequence. The upper and lower sequences in the Table can further comprise a chemical modification having Formulae I–VII, such as exemplary siNA constructs shownin FIGS. 4 and 5, or having modifications described in Table IV or any combination thereof.













TABLE III







VEGF and/or VEGFR Synthetic Modified siNA Constructs














Target

Seq
Cmpd


Seq



Pos
Target
ID
#
Aliases
Sequence
ID










VEGFR1














298
GCUGUCUGCUUCUCACAGGAUCU
2285

FLT1:298U21 sense siNA
UGUCUGCUUCUCACAGGAUTT
2709



1956
GAAGGAGAGGACCUGAAACUGUC
2286

FLT1:1956U21 sense siNA
AGGAGAGGACCUGAAACUGTT
2710


1957
AAGGAGAGGACCUGAAACUGUCU
2287

FLT1:1957U21 sense siNA
GGAGAGGACCUGAAACUGUTT
2711


2787
GCAUUUGGCAUUAAGAAAUCACC
2288

FLT1:2787U21 sense siNA
AUUUGGCAUUAAGAAAUCATT
2712


298
GCUGUCUGCUUCUCACAGGAUCU
2285

FLT1:316L21 antisense siNA (298 C.)
AUCCUGUGAGAAGCAGACATT
2713


1956
GAAGGAGAGGACCUGAAACUGUC
2286

FLT1:1974L21 antisense siNA (1956 C.)
CAGUUUCAGGUCCUCUCCUTT
2714


1957
AAGGAGAGGACCUGAAACUGUCU
2287

FLT1:1975L21 antisense siNA (1957 C.)
ACAGUUUCAGGUCCUCUCCTT
2715


2787
GCAUUUGGCAUUAAGAAAUCACC
2288

FLT1:2805L21 antisense siNA (2787 C.)
UGAUUUCUUAAUGCCAAAUTT
2716


298
GCUGUCUGCUUCUCACAGGAUCU
2285

FLT1:298U21 sense siNA stab04
B uGucuGcuucucAcAGGAuTT B
2717


1956
GAAGGAGAGGACCUGAAACUGUC
2286

FLT1:1956U21 sense siNA stab04
B AGGAGAGGAccuGAAAcuGTT B
2718


1957
AAGGAGAGGACCUGAAACUGUCU
2287

FLT1:1957U21 sense siNA stab04
B GGAGAGGAccuGAAAcuGuTT B
2719


2787
GCAUUUGGCAUUAAGAAAUCACC
2288

FLT1:2787U21 sense siNA stab04
B AuuuGGcAuuAAGAAAucATT B
2720


298
GCUGUCUGCUUCUCACAGGAUCU
2285

FLT1:316L21 antisense siNA (298 C.)
AuccuGuGAGAAGcAGAcATsT
2721






stab05


1956
GAAGGAGAGGACCUGAAACUGUC
2286

FLT1:1974L21 antisense siNA (1956 C.)
cAGuuucAGGuccucuccuTsT
2722






stab05


1957
AAGGAGAGGACCUGAAACUGUCU
2287

FLT1:1975L21 antisense siNA (1957 C.)
AcAGuuucAGGuccucuccTsT
2723






stab05


2787
GCAUUUGGCAUUAAGAAAUCACC
2288

FLT1:2805L21 antisense siNA (2787 C.)
uGAuuucuuAAuGccAAAuTsT
2724






stab05


298
GCUGUCUGCUUCUCACAGGAUCU
2285

FLT1:298U21 sense siNA stab07
B uGucuGcuucucAcAGGAuTT B
2725


1956
GAAGGAGAGGACCUGAAACUGUC
2286
37387
FLT1:1956U21 sense siNA stab07
B AGGAGAGGAccuGAAAcuGTT B
2726


1957
AAGGAGAGGACCUGAAACUGUCU
2287
37388
FLT1:1957U21 sense siNA stab07
B GGAGAGGAccuGAAAcuGuTT B
2727


2787
GCAUUUGGCAUUAAGAAAUCACC
2288
37404
FLT1:2787U21 sense siNA stab07
B AuuuGGcAuuAAGAAAucATT B
2728


298
GCUGUCUGCUUCUCACAGGAUCU
2285

FLT1:316L21 antisense siNA (298 C.)
AuccuGuGAGAAGcAGAcATsT
2729






stab11


1956
GAAGGAGAGGACCUGAAACUGUC
2286

FLT1:1974L21 antisense siNA (1956 C.)
cAGuuucAGGuccucuccuTsT
2730






stab11


1957
AAGGAGAGGACCUGAAACUGUCU
2287

FLT1:1975L21 antisense siNA (1957 C.)
AcAGuuucAGGuccucuccTsT
2731






stab11


2787
GCAUUUGGCAUUAAGAAAUCACC
2288

FLT1:2805L21 antisense siNA (2787 C.)
uGAuuucuuAAuGccAAAuTsT
2732






stab11


349
AACUGAGUUUAAAAGGCACCCAG
2289
31209
FLT1:367L21 antisense siNA (349 C.)
GAcucAAAuuuuccGuGGGTsT
2733






stab05 inv


2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31210
FLT1:2967L21 antisense siNA (2949 C.)
cGuuccucccGGAGAcuAcTsT
2734






stab05 inv


3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31211
FLT1:3930L21 antisense siNA (3912 C.)
GGAccuuucuuAGuuuuGGTsT
2735






stab05 inv


349
AACUGAGUUUAAAAGGCACCCAG
2289
31212
FLT1:349U21 sense siNA stab07 inv
B ccCACGGAAAAuuuGAGucTT B
2736


2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31213
FLT1:2949U21 sense siNA stab07 inv
B GuAGucuccGGGAGGMcGTT B
2737


3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31214
FLT1:3912U21 sense siNA stab07 inv
B ccAAAAcuAAGAAAGGuccTT B
2738


349
AACUGAGUUUAAAAGGCACCCAG
2289
31215
FLT1:367L21 antisense siNA (349 C.) GAcucAAAuuuuccGuGGGTsT
2739






stab08 inv


2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31216
FLT1:2967L21 antisense siNA (2949 C.) cGuuccucccGGAGAcuAcTsT
2740






stab08 inv


3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31217
FLT1:3930L21 antisense siNA (3912 C.) GGAccuuucuuAGuuuuGGTsT
2741






stab08 inv


349
AACUGAGUUUAAAAGGCACCCAG
2289
31270
FLT1:349U21 sense siNA stab09
B CUGAGUUUAAAAGGCACCCTT B
2742


2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31271
FLT1:2949U21 sense siNA stab09
B GCAAGGAGGGCCUCUGAUGTT B
2743


3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31272
FLT1:3912U21 sense siNA stab09
B CCUGGAAAGAAUCAAAACCTT B
2744


349
AACUGAGUUUAAAAGGCACCCAG
2289
31273
FLT1:367L21 antisense siNA (349 C.) GGGUGCCUUUUAAACUCAGTsT
2745






stab10


2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31274
FLT1:2967L21 antisense siNA (2949 C.) CAUCAGAGGCCCUCCUUGCTsT
2746






stab10


3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31275
FLT1:3930L21 antisense siNA (3912 C.) GGUUUUGAUUCUUUCCAGGTsT
2747






stab10


349
AACUGAGUUUAAAAGGCACCCAG
2289
31276
FLT1:349U21 sense siNA stab09 inv
B CCCACGGAAAAUUUGAGUCTT B
2748


2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31277
FLT1:2949U21 sense siNA stab09 inv
B GUAGUCUCCGGGAGGAACGTT B
2749


3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31278
FLT1:3912U21 sense siNA stab09 inv
B CCAAAACUAAGAAAGGUCCTT B
2750


349
AACUGAGUUUAAAAGGCACCCAG
2289
31279
FLT1:367L21 antisense siNA (349 C.)
GACUCAAAUUUUCCGUGGGTsT
2751






stab10 inv


2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31280
FLT1:2967L21 antisense siNA (2949 C.)
CGUUCCUCOCGGAGACUACTsT
2752






stab10 inv


3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31281
FLT1:3930L21 antisense siNA (3912 C.)
GGACCUUUCUUAGUUUUGGTsT
2753






stab10 inv


2340
AACAACCACAAAAUACAACAAGA
2292
31424
FLT1:2358L21 antisense siNA (2340 C.)
uuGuuGuAuuuuGuGGuuGXsX
2754






stab11 3′-BrdU


2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31425
FLT1:2967L21 antisense siNA (2949 C.)
cAucAGAGGcccuccuuGcXsX
2755






stab11 3′BrdU


2340
AACAACCACAAAAUACAACAAGA
2292
31442
FLT1:2358L21 antisense siNA (2340 C.)
uuGuuGuAuuuuGuGGuuGXsT
2756






stab11 3′-BrdU


2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31443
FLT1:2967L21 antisense siNA (2949 C.)
cAucAGAGGcccuccuuGcXsT
2757






stab11 3′-BrdU


2340
AACAACCACAAAAUACAACAAGA
2292
31449
FLT1:2340U21 sense siNA stab09
B CAACCACAAAAUACAACAATT B
2758


2340
AACAACCACAAAAUACAACAAGA
2292
31450
FLT1:2340U21 sense siNA inv stab09
B AACAACAUAAAACACCAACTT B
2759


2340
AACAACCACAAAAUACAACAAGA
2292
31451
FLT1:2358L21 antisense siNA (2340 C.)
UUGUUGUAUUUUGUGGUUGTsT
2760






stab10


2340
AACAACCACAAAAUACAACAAGA
2292
31452
FLT1:2358L21 antisense siNA (2340 C.) inv
GUUGGUGUUUUAUGUUGUUTsT
2761






stab10


2340
AACAACCACAAAAUACAACAAGA
2292
31509
FLT1:2358L21 antisense siNA (2340 C.)
uuGuuGuAuuuuGuGGuuGTsT
2762






stab11


349
AACUGAGUUUAAAAGGCACCCAG
2289
31794
2 × cholesterol + R31194 FLT1:349U21
(H)2 ZTa B
2763






sense siNA stab07
cuGAGuuuAAAAGGcAcccTT B


349
AACUGAGUUUAAAAGGCACCCAG
2289
31795
2 × cholesterol + R31212 FLT1:349U21
(H)2 ZTa B
2764






sense siNA stab07 inv
cccAcGGAAAAuuuGAGucTT B


349
AACUGAGUUUAAAAGGCACCCAG
2289
31796
2 × cholesterol + 31270 FLT1:349U21
(H)2 ZTA B
2765






sense siNA stab09
CUGAGUUUAAAAGGCACCCTT B


349
AACUGAGUUUAAAAGGCACCCAG
2289
31797
2 × cholesterol + R31276 FLT1:349U21
(H)2 ZTA B
2766






sense siNA stab09 inv
CCCACGGAAAAUUUGAGUCTT B


349
AACUGAGUUUAAAAGGCACCCAG
2289
31798
2 × C18 phospholipid + R31194
(L)2 ZTa B
2767






FLT1:349U21 sense siNA stab07
cuGAGuuuAAAAGGcAcccTT B


349
AACUGAGUUUAAAAGGCACCCAG
2289
31799
2 × C18 phospholipid + R31212
(L)2 ZTa B
2768






FLT1:349U21 sense siNA stab07 inv
cccAcGGAAAAuuuGAGucTT B


349
AACUGAGUUUAAAAGGCACCCAG
2289
31800
2 × C18 phospholipid + R31270
(L)2 ZTA B
2769






FLT1:349U21 sense siNA stab09
CUGAGUUUAAAAGGCACCCTT B


349
AACUGAGUUUAAAAGGCACCCAG
2289
31801
2 × C18 phospholipid + R31276
(L)2 ZTA B
2770






FLT1:349U21 sense siNA stab09 inv
CCCACGGAAAAUUUGAGUCTT B


3645
CAUGCUGGACUGCUGGCAC
2293
32235
FLT1:3645U21 sense siNA
CAUGCUGGACUGCUGGCACTT
2771


3646
AUGCUGGACUGCUGGCACA
2294
32236
FLT1:3646U21 sense siNA
AUGCUGGACUGCUGGCACATT
2772


3647
UGCUGGACUGCUGGCACAG
2295
32237
FLT1:3647U21 sense siNA
UGCUGGACUGCUGGCACAGTT
2773


3645
CAUGCUGGACUGCUGGCAC
2293
32250
FLT1:3663L21 antisense siNA (3645 C.)
GUGCCAGCAGUCCAGCAUGTT
2774


3646
AUGCUGGACUGCUGGCACA
2294
32251
FLT1:3664L21 antisense siNA (3646 C.)
UGUGCCAGCAGUCCAGCAUTT
2775


3647
UGCUGGACUGCUGGCACAG
2295
32252
FLT1:3665L21 antisense siNA (3647 C.)
CUGUGCCAGCAGUCCAGCATT
2776


349
AACUGAGUUUAAAAGGCACCCAG
2289
32278
FLT1:349U21 sense siNA stab16
B CUGAGUUUAAAAGGCACCCTT B
2777


349
AACUGAGUUUAAAAGGCACCCAG
2289
32279
FLT1:349U21 sense siNA stab18
B cuGAGuuuAAAAGGcAcccTT B
2778


349
AACUGAGUUUAAAAGGCACCCAG
2289
32280
FLT1:349U21 sense siNA inv stab16
B CCCACGGAAAAUUUGAGUCTT B
2779


349
AACUGAGUUUAAAAGGCACCCAG
2289
32281
FLT1:349U21 sense siNA inv stab18
B cccAcGGAAAAuuuGAGucTT B
2780


346
CUGAACUGAGUUUAAAAGGCACC
2296
32282
FLT1:346U21 sense siNA stab09
B GAACUGAGUUUAAAAGGCATT B
2781


347
UGAACUGAGUUUAAAAGGCACCC
2297
32283
FLT1:347U21 sense siNA stab09
B AACUGAGUUUAAAAGGCACTT B
2782


348
GAACUGAGUUUAAAAGGCACCCA
2298
32284
FLT1:348U21 sense siNA stab09
B ACUGAGUUUAAAAGGCACCTT B
2783


350
ACUGAGUUUAAAAGGCACCCAGC
2299
32285
FLT1:350U21 sense siNA stab09
B UGAGUUUAAAAGGCACCCATT B
2784


351
CUGAGUUUAAAAGGCACCCAGCA
2300
32286
FLT1:351U21 sense siNA stab09
B GAGUUUAAAAGGCACCCAGTT B
2785


352
UGAGUUUAAAAGGCACCCAGCAC
2301
32287
FLT1:352U21 sense siNA stab09
B AGUUUAAAAGGCACCCAGCTT B
2786


353
GAGUUUAAAAGGCACCCAGCACA
2302
32288
FLT1:353U21 sense siNA stab09
B GUUUAAAAGGCACCCAGCATT B
2787


346
CUGAACUGAGUUUAAAAGGCACC
2296
32289
FLT1.:364L21 antisense siNA (346 C.)
UGCCUUUUAAACUCAGUUCTsT
2788






stab10


347
UGAACUGAGUUUAAAAGGCACCC
2297
32290
FLT1:365L21 antisense siNA (347 C.)
GUGCCUUUUAAACUCAGUUTsT
2789






stab10


348
GAACUGAGUUUAAAAGGCACCCA
2298
32291
FLT1:366L21 antisense siNA (348 C.)
GGUGCCUUUUAAACUCAGUTsT
2790






stab10


350
ACUGAGUUUAAAAGGCACCCAGC
2299
32292
FLT1:368L21 antisense siNA (350 C.)
UGGGUGCCUUUUAAACUCATsT
2791






stab10


351
CUGAGUUUAAAAGGCACCCAGCA
2300
32293
FLT1:369L21 antisense siNA (351 C.)
CUGGGUGCCUUUUAAACUCTsT
2792






stab10


352
UGAGUUUAAAAGGCACCCAGCAC
2301
32294
FLT1:370L21 antisense siNA (352 C.)
GCUGGGUGCCUUUUAAACUTsT
2793






stab10


353
GAGUUUAAAAGGCACCCAGCACA
2302
32295
FLT1:371L21 antisense siNA (353 C.)
UGCUGGGUGCCUUUUAAACTsT
2794






stab10


346
CUGAACUGAGUUUAAAAGGCACC
2296
32296
FLT1:346U21 sense siNA inv stab09
B ACGGAAAAUUUGAGUCAAGTT B
2795


347
UGAACUGAGUUUAAAAGGCACCC
2297
32297
FLT1:347U21 sense siNA inv stab09
B CACGGAAAAUUUGAGUCAATT B
2796


348
GAACUGAGUUUAAAAGGCACCCA
2298
32298
FLT1:348U21 sense siNA inv stab09
B CCACGGAAAAUUUGAGUCATT B
2797


350
ACUGAGUUUAAAAGGCACCCAGC
2299
32299
FLT1:350U21 sense siNA inv stab09
B ACCCACGGAAAAUUUGAGUTT B
2798


351
CUGAGUUUAAAAGGCACCCAGCA
2300
32300
FLT1:351U21 sense siNA inv stab09
B GACCCACGGAAAAUUUGAGTT B
2799


352
UGAGUUUAAAAGGCACCCAGCAC
2301
32301
FLT1:352U21 sense siNA inv stab09
B CGACCCACGGAAAAUUUGATT B
2800


353
GAGUUUAAAAGGCACCCAGCACA
2302
32302
FLT1:353U21 sense siNA inv stab09
B ACGACCCACGGAAAAUUUGTT B
2801


346
CUGAACUGAGUUUAAAAGGCACC
2296
32303
FLT1:364L21 antisense siNA (346 C.) inv
CUUGACUCAAAUUUUCCGUTsT
2802






stab10


347
UGAACUGAGUUUAAAAGGCACCC
2297
32304
FLT1:365121 antisense siNA (347 C.) inv
UUGACUCAAAUUUUCCGUGTsT
2803






stab10


348
GAACUGAGUUUAAAAGGCACCCA
2298
32305
FLT1:366L21 antisense siNA (348 C.) inv
UGACUCAAAUUUUCCGUGGTsT
2804






stab10


350
ACUGAGUUUAAAAGGCACCCAGC
2299
32306
FLT1:368121 antisense siNA (350 C.) inv
ACUCAAAUUUUCCGUGGGUTsT
2805






stab10


351
CUGAGUUUAAAAGGCACCCAGCA
2300
32307
FLT1:369L21 antisense siNA (351 C.) inv
CUCAAAUUUUCCGUGGGUCTsT
2806






stab10


352
UGAGUUUAAAAGGCACCCAGCAC
2301
32308
FLT1:370L21 antisense siNA (352 C.) inv
UCAAAUUUUCCGUGGGUCGTsT
2807






stab10


353
GAGUUUAAAAGGCACCCAGCACA
2302
32309
FLT1:371L21 antisense siNA (353 C.) inv
CAAAUUUUCCGUGGGUCGUTsT
2808






stab10


349
AACUGAGUUUAAAAGGCACCCAG
2289
32338
FLT1:367L21 antisense siNA (349 C.)
GGGUGCCUUUUAAACUCAGXsT
2809






stab10 3′-BrdU


349
AACUGAGUUUAAAAGGCACCCAG
2289
32718
FLT1:367L21 antisense siNA (349 C.) v1 5′p
pGGGUGCCUUUUAAACUC
2810







GAGUUUAAAAG B


349
AACUGAGUUUAAAAGGCACCCAG
2289
32719
FLT1:367L21 antisense siNA (349 C.) v2 5′p
pGGGUGCCUUUUAAACUCAG
2811







GAGUUUAAAAG B


2967
AAGCAAGGAGGGCCUCUGAUGGU
2290
32720
FLT1:2967L21 antisense siNA (2949 C.) v1
pCAUCAGAGGCCCUCCUUGC
2812






5′p
AAGGAGGGCCUCTT B


2967
AAGCAAGGAGGGCCUCUGAUGGU
2290
32721
FLT1:2967L21 antisense siNA (2949 C.) v2
pCAUCAGAGGCCCUCCUU
2813






5′p
AAGGAGGGCCUCUG B


2967
AAGCAAGGAGGGCCUCUGAUGGU
2290
32722
FLT1:2967L21 antisense siNA (2949 C.) v3
pCAUCAGAGGCCCUCCU
2814






5′p
AGGAGGGCCUCUG B


346
CUGAACUGAGUUUAAAAGGCACC
2296
32748
FLT1:346U21 sense siNA stab07
B GAAcuGAGuuuAAAAGGcATT B
2815


347
UGAACUGAGUUUAAAAGGCACCC
2297
32749
FLT1:347U21 sense siNA stab07
B AAcuGAGuuuAAAAGGcAcTT B
2816


348
GAACUGAGUUUAAAAGGCACCCA
2298
32750
FLT1:348U21 sense siNA stab07
B AcuGAGuuuAAAAGGcAccTT B
2817


350
ACUGAGUUUAAAAGGCACCCAGC
2299
32751
FLT1:350U21 sense siNA stab07
B uGAGuuuAAAAGGcAcccATT B
2818


351
CUGAGUUUAAAAGGCACCCAGCA
2300
32752
FLT1:351U21 sense siNA stab07
B GAGuuuAAAAGGcAcccAGTT B
2819


352
UGAGUUUAAAAGGCACCCAGCAC
2301
32753
FLT1:352U21 sense siNA stab07
B AGuuuAAAAGGcAcccAGcTT B
2820


353
GAGUUUAAAAGGCACCCAGCACA
2302
32754
FLT1:353U21 sense siNA stab07
B GuuuAAAAGGcAcccAGcATT B
2821


346
CUGAACUGAGUUUAAAAGGCACC
2296
32755
FLT1:364L21 antisense siNA (346 C.)
uGccuuuuAAAcucAGuucTsT
2822






stab08


347
UGAACUGAGUUUAAAAGGCACCC
2297
32756
FLT1:365L21 antisense siNA (347 C.)
GuGccuuuuAAAcucAGuuTsT
2823






stab08


348
GAACUGAGUUUAAAAGGCACCCA
2298
32757
FLT1:366L21 antisense siNA (348 C.)
GGuGccuuuuAAAcucAGuTsT
2824






stab08


350
ACUGAGUUUAAAAGGCACCCAGC
2299
32758
FLT1:368L21 antisense siNA (350 C.)
uGGGuGccuuuuAAAcucATsT
2825






stab08


351
CUGAGUUUAAAAGGCACCCAGCA
2300
32759
FLT1:369L21 antisense siNA (351 C.)
cuGGGuGccuuuuAAAcucTsT
2826






stab08


352
UGAGUUUAAAAGGCACCCAGCAC
2301
32760
FLT1:370L21 antisense siNA (352 C.)
GcuGGGuGccuuuuAAAcuTsT
2827






stab08


353
GAGUUUAAAAGGCACCCAGCACA
2302
32761
FLT1:371L21 antisense siNA (353 C.)
uGcuGGGuGccuuuuAAAcTsT
2828






stab08


346
CUGAACUGAGUUUAAAAGGCACC
2296
32772
FLT1:346U21 sense siNA inv stab07
B AcGGAAAAuuuGAGucAAGTT B
2829


347
UGAACUGAGUUUAAAAGGCACCC
2297
32773
FLT1:347U21 sense siNA inv stab07
B cAcGGAAAAuuuGAGucAATT B
2830


348
GAACUGAGUUUAAAAGGCACCCA
2298
32774
FLT1:348U21 sense siNA inv stab07
B ccAcGGAAAAuuuGAGucATT B
2831


350
ACUGAGUUUAAAAGGCACCCAGC
2299
32775
FLT1:350U21 sense siNA inv stab07
B AcccAcGGAAAAuuuGAGuTT B
2832


351
CUGAGUUUAAAAGGCACCCAGCA
2300
32776
FLT1:351U21 sense siNA inv stab07
B GAcccAcGGAAAAuuuGAGTT B
2833


352
UGAGUUUAAAAGGCACCCAGCAC
2301
32777
FLT1:352U21 sense siNA inv stab07
B cGAcccAcGGAAAAuuuGATT B
2834


353
GAGUUUAAAAGGCACCCAGCACA
2302
32778
FLT1:353U21 sense siNA inv stab07
B AcGAcccAcGGAAAAuuuGTT B
2835


346
CUGAACUGAGUUUAAAAGGCACC
2296
32779
FLT1:364L21 antisense siNA (346 C.) inv
cuuGAcucAAAuuuuccGuTsT
2836






stab08


347
UGAACUGAGUUUAAAAGGCACCC
2297
32780
FLT1:365L21 antisense siNA (347 C.) inv
uuGAcucAAAuuuuccGuGTsT
2837






stab08


348
GAACUGAGUUUAAAAGGCACCCA
2298
32781
FLT1:366L21 antisense siNA (348 C.) inv
uGAcucAAAuuuuccGuGGTsT
2838






stab08


350
ACUGAGUUUAAAAGGCACCCAGC
2299
32782
FLT1:368L21 antisense siNA (350 C.) inv
AcucAAAuuuuccGuGGGuTsT
2839






stab08


351
CUGAGUUUAAAAGGCACCCAGCA
2300
32783
FLT1:369L21 antisense siNA (351 C.) inv
cucAAAuuuuccGuGGGucTsT
2840






stab08


352
UGAGUUUAAAAGGCACCCAGCAC
2301
32784
FLT1:370L21 antisense siNA (352 C.) inv
ucAAAuuuuccGuGGGucGTsT
2841






stab08


353
GAGUUUAAAAGGCACCCAGCACA
2302
32785
FLT1:371L21 antisense siNA (353 C.) inv
cAAAuuuuccGuGGGucGuTsT
2842






stab08


349
AACUGAGUUUAAAAGGCACCCAG
2289
33121
FLT1:349U21 sense siNA stab22
CUGAGUUUAAAAGGCACCCTTB
2843


349
AACUGAGUUUAAAAGGCACCCAG
2289
33321
FLT1:367L21 antisense siNA (349 C.)
pGGGuGccuuuuAAAcucAGTsT
2844






stab08 + 5′ P


349
AACUGAGUUUAAAAGGCACCCAG
2289
33338
FLT1:367L21 antisense siNA (349 C.)
L GGGuGccuuuuAAAcucAGTsT
2845






stab08 + 5′ aminoL


349
AACUGAGUUUAAAAGGCACCCAG
2289
33553
FLT1:367L21 antisense siNA (349 C.)
L GGGuGccuuuuAAAcucAGTsT
2846






stab08 + 5′ aminoL


349
AACUGAGUUUAAAAGGCACCCAG
2289
33571
FLT1:367L21 antisense siNA (349 C.)
IGGUGCCUUUUAAACUCAGTT
2847






stab10 + 5′I


3645
AUCAUGCUGGACUGCUGGCACAG
2189
33725
FLT1:3645U21 sense siNA stab07
B cAuGcuGGAcuGcuGGcAcTT B
2848


3646
UCAUGCUGGACUGCUGGCACAGA
2195
33726
FLT1:3646U21 sense siNA stab07
B AuGcuGGAcuGcuGGcAcATT B
2849


3645
AUCAUGCUGGACUGCUGGCACAG
2189
33731
FLT1:3663L21 antisense siNA (3645 C.)
GuGccAGcAGuccAGcAuGTsT
2850






stab08


3646
UCAUGCUGGACUGCUGGCACAGA
2195
33732
FLT1:3664L21 antisense siNA (3646 C.)
uGuGccAGcAGuccAGcAuTsT
2851






stab08


3645
AUCAUGCUGGACUGCUGGCACAG
2189
33737
FLT1:3645U21 sense siNA stab09
B CAUGCUGGACUGCUGGCACTT B
2852


3646
UCAUGCUGGACUGCUGGCACAGA
2195
33738
FLT1:3646U21 sense siNA stab09
B AUGCUGGACUGCUGGCACATT B
2853


3645
AUCAUGCUGGACUGCUGGCACAG
2189
33743
FLT1:3663L21 antisense siNA (3645 C.)
GUGCCAGCAGUCCAGCAUGTsT
2854






stab10


3646
UCAUGCUGGACUGCUGGCACAGA
2195
33744
FLT1:3664L21 antisense siNA (3646 C.)
UGUGCCAGCAGUCCAGCAUTsT
2855






stab10


3645
AUCAUGCUGGACUGCUGGCACAG
2189
33749
FLT1:3645U21 sense siNA inv stab07
B cAcGGucGucAGGucGuAcTT B
2856


3646
UCAUGCUGGACUGCUGGCACAGA
2195
33750
FLT1:3646U21 sense siNA inv stab07
B AcAcGGucGucAGGucGuATT B
2857


3645
AUCAUGCUGGACUGCUGGCACAG
2189
33755
FLT1:3663L21 antisense siNA (3645 C.) inv
GuAcGAccuGAcGAccGuGTsT
2858






stab08


3646
UCAUGCUGGACUGCUGGCACAGA
2195
33756
FLT1:3664L21 antisense siNA (3646 C.) inv
uAcGAccuGAcGAccGuGuTsT
2859






stab08


3645
AUCAUGCUGGACUGCUGGCACAG
2189
33761
FLT1:3645U21 sense siNA inv stab09
B CACGGUCGUCAGGUCGUACTT B
2860


3646
UCAUGCUGGACUGCUGGCACAGA
2195
33762
FLT1:3646U21 sense siNA inv stab09
B ACACGGUCGUCAGGUCGUATT B
2861


3645
AUCAUGCUGGACUGCUGGCACAG
2189
33767
FLT1:3663L21 antisense siNA (3645 C.) inv
GUACGACCUGACGACCGUGTsT
2862






stab10


3646
UCAUGCUGGACUGCUGGCACAGA
2195
33768
FLT1:3664L21 antisense siNA (3646 C.) inv
UACGACCUGACGACCGUGUTsT
2863






stab10


349
AACUGAGUUUAAAAGGCACCCAG
2289
34487
FLT1:349U21 sense siNA stab09 w/block
B CsUsGAGUUUsAsAsAsAsGGCAC
2864






Ps
CsCsTsT B


349
AACUGAGUUUAAAAGGCACCCAG
2289
34488
FLT1:367L21 antisense siNA (349 C.)
GGGsUsGsCsCsUUUUAAsAsCsUs
2865






stab10 w/block PS
CsAGTsT


349
AACUGAGUUUAAAAGGCACCCAG
2289
34489
FLT1:349U21 sense siNA stab09 inv
B CsCsCACGGAsAsAsAsUsUUGAG
2866






w/block PS
UsCsTsT B


349
AACUGAGUUUAAAAGGCACCCAG
2289
34490
FLT1:367L21 antisense siNA (349 C.)
GACsUsCsAsAsAUUUUCsCsGsUs
2867






stab10 inv w/block PS
GsGGTsT


349
AACUGAGUUUAAAAGGCACCCAG
2289
29694
FLT1:349U21 sense siNA stab01
CsUsGsAsGsUUUAAAAGGCACCC
2868







TsT


2340
AACAACCACAAAAUACAACAAGA
2292
29695
FLT1:2340U21 sense siNA stab01
CsAsAsCsCsACAAAAUACAACAAT
2869







sT


3912
AGCCUGGAAAGAAUCAAAACCUU
2291
29696
FLT1:3912U21 sense siNA stab01
CsCsUsGsGsAAAGAAUCAAAACC
2870







TsT


2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
29697
FLT1:2949U21 sense siNA stab01
GsCsAsAsGsGAGGGCCUCUGAU
2871







GTsT


349
AACUGAGUUUAAAAGGCACCCAG
2289
29698
FLT1:367L21 antisense siNA (349 C.)
GsGsGsUsGsCCUUUUAAACUCA
2872






stab01
GTsT


2340
AACAACCACAAAAUACAACAAGA
2292
29699
FLT1:2358L21 antisense siNA (2340 C.)
UsUsGsUsUsGUAUUUUGUGGUU
2873






stab01
GTsT


3912
AGCCUGGAAAGAAUCAAAACCUU
2291
29700
FLT1:3930L21 antisense siNA (3912 C.)
GsGsUsUsUsUGAUUCUUUCCAG
2874






stab01
GTsT


2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
29701
FLT1:2967L21 antisense siNA (2949 C.)
CsAsUsCsAsGAGGCCCUCCUUG
2875






stab01
CTsT


349
AACUGAGUUUAAAAGGCACCCAG
2289
29702
FLT1:349U21 sense siNA stab03
csusGsAsGuuuAAAAGGcAcscscsTsT
2876


2340
AACAACCACAAAAUACAACAAGA
2292
29703
FLT1:2340U21 sense siNA stab03
csAsAscscAcAAAAuAcAAcsAsAsTsT
2877


3912
AGCCUGGAAAGAAUCAAAACCUU
2291
29704
FLT1:3912U21 sense siNA stab03
cscsusGsGAAAGAAucAAAAscscsT
2878







sT


2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
29705
FLT1:2949U21 sense siNA stab03
GscsAsAsGGAGGGccucuGAsusGs
2879







TsT


349
AACUGAGUUUAAAAGGCACCCAG
2289
29706
FLT1:367L21 antisense siNA (349 C.)
GsGsGsUsGsCsCsUsUsUsUsAsAs
2880






stab02
AsCsUsCsAsGsTsT


2340
AACAACCACAAAAUACAACAAGA
2292
29707
FLT1:2358L21 antisense siNA (2340 C.)
UsUsGsUsUsGsUsAsUsUsUsUsGs
2881






stab02
UsGsGsUsUsGsTsT


3912
AGCCUGGAAAGAAUCAAAACCUU
2291
29708
FLT1:3930L21 antisense siNA (3912 C.)
GsGsUsUsUsUsGsAsUsUsCsUsUs
2882






stab02
UsCsCsAsGsGsTsT


2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
29709
FLT1:2967L21 antisense siNA (2949 C.)
CsAsUsCsAsGsAsGsGsCsCsCsUs
2883






stab02
CsCsUsUsGsCsTsT


2340
AACAACCACAAAAUACAACAAGA
2292
29981
FLT1:2340U21 sense siNA Native
CAACCACAAAAUACAACAAGA
2884


2340
AACAACCACAAAAUACAACAAGA
2292
29982
FLT1:2358L21 antisense siNA (2340 C.)
UUGUUGUAUUUUGUGGUUGUU
2885






Native


2340
AACAACCACAAAAUACAACAAGA
2292
29983
FLT1:2340U21 sense siNA stab01 inv
AsAsCsAsAsCAUAAAACACCAACT
2886






FLT1:2358L21 antisense siNA (2340 C.)
sT


2340
AACAACCACAAAAUACAACAAGA
2292
29984
stab01 inv
GsUsUsGsGsUGUUUUAUGUUGU
2887







UTsT


2340
AACAACCACAAAAUACAACAAGA
2292
29985
FLT1:2340U21 sense siNA stab03 inv
AsAscsAsAcAuAAAAcAccAsAscsTsT
2888


2340
AACAACCACAAAAUACAACAAGA
2292
29986
FLT1:2358L21 antisense siNA (2340 C.)
GsUsUsGsGsUsGsUsUsUsUsAsUs
2889






stab02 inv
GsUsUsGsUsUsTsT


2340
AACAACCACAAAAUACAACAAGA
2292
29987
FLT1:2340U21 sense siNA inv Native
AGAACAACAUAAAACACCAAC
2890


2340
AACAACCACAAAAUACAACAAGA
2292
29988
FLT1:2358L21 antisense siNA (2340 C.) inv
UUGUUGGUGUUUUAUGUUGUU
2891






Native


2340
AACAACCACAAAAUACAACAAGA
2292
30075
FLT1:2340U21 sense siNA
CAACCACAAAAUACAACAATT
2892


2340
AACAACCACAAAAUACAACAAGA
2292
30076
FLT1:2358L21 antisense siNA (2340 C.)
UUGUUGUAUUUUGUGGUUGTT
2893


2342
AACAACCACAAAAUACAACAAGA
2292
30077
FLT1:2342U21 sense siNA inv
AGAACAACAUAAAACACCATT
2894


2340
AACAACCACAAAAUACAACAAGA
2292
30078
FLT1:2358L21 antisense siNA (2340 C.) inv
UUGUUGGUGUUUUAUGUUGTT
2895


2340
AACAACCACAAAAUACAACAAGA
2292
30187
FLT1:2358L21 antisense siNA (2340 C.) 2′-
uuGuuGuAuuuuGuGGuuGTT
2896






F U,C


2340
AACAACCACAAAAUACAACAAGA
2292
30190
FLT1:2358L21 antisense siNA (2340 C.)
uuGuuGuAuuuuGuGGuuGXX
2897






nitroindole


2340
AACAACCACAAAAUACAACAAGA
2292
30193
FLT1:2358L21 antisense siNA (2340 C.)
uuGuuGuAuuuuGuGGuuGZZ
2898






nitropyrole


2340
AACAACCACAAAAUACAACAAGA
2292
30196
FLT1:2340U21 sense siNA stab04
B cAAccAcAAAAuAcAAcAATT B
2899


2340
AACAACCACAAAAUACAACAAGA
2292
30199
FLT1:2340U21 sense siNA sense iB caps
cAAccAcAAAAuAcAAcAATT
2900


2340
AACAACCACAAAAUACAACAAGA
2292
30340
FLT1:2358L21 antisense siNA (2340 C.)
uuGuuGuAuuuuGuGGuuGTX
2901






3′dT


2340
AACAACCACAAAAUACAACAAGA
2292
30341
FLT1:2358L21 antisense siNA (2340 C.)
uuGuuGuAuuuuGuGGuuGTGly
2902






glyceryl


2340
AACAACCACAAAAUACAACAAGA
2292
30342
FLT1:2358L21 antisense siNA (2340 C.)
uuGuuGuAuuuuGuGGuuGTU
2903






3′OMeU


2340
AACAACCACAAAAUACAACAAGA
2292
30343
FLT1:2358L21 antisense siNA (234C C.) L-
uuGuuGuAuuuuGuGGuuGTt
2904






dT


2340
AACAACCACAAAAUACAACAAGA
2292
30344
FLT1:2358L21 antisense siNA (2340 C.) L-
uuGuuGuAuuuuGuGGuuGTu
2905






rU


2340
AACAACCACAAAAUACAACAAGA
2292
30345
FLT1:2358L21 antisense siNA (2340 C.) idT
uuGuuGuAuuuuGuGGuuGTD
2906


2340
AACAACCACAAAAUACAACAAGA
2292
30346
FLT1:2358L21 antisense siNA (2340 C.)
uuGuuGuAuuuuGuGGuuGXT
2907






3′dT


2340
AACAACCACAAAAUACAACAAGA
2292
30416
FLT1:2358L21 antisense siNA (2340 C.)
uuGuuGuAuuuuGuGGuuGTsT
2908






stab05


1184
UCGUGUAAGGAGUGGACCAUCAU
2303
30777
FLT1:1184U21 sense siNA stab04
B GuGuAAGGAGuGGAccAucTT B
2909


3503
UUACGGAGUAUUGCUGUGGGAAA
2304
30778
FLT1:3503U21 sense siNA stab04
B AcGGAGuAuuGcuGuGGGATT B
2910


4715
UAGCAGGCCUAAGACAUGUGAGG
2305
30779
FLT1:4715U21 sense siNA stab04
B GcAGGccuAAGAcAuGuGATT B
2911


4753
AGCAAAAAGCAAGGGAGAAAAGA
2306
30780
FLT1:4753U21 sense siNA stab04
B cAAAAAGcAAGGGAGAAAATT B
2912


1184
UCGUGUAAGGAGUGGACCAUCAU
2303
30781
FLT1:1202L21 antisense siNA (1184 C.)
GAuGGuccAcuccuuAcAcTsT
2913





stab05


3503
UUACGGAGUAUUGCUGUGGGAAA
2304
30782
FLT1:3521L21 antisense siNA (3503 C.)
ucccAcAGcAAuAcuccGuTsT
2914






stab05


4715
UAGCAGGCCUAAGACAUGUGAGG
2305
30783
FLT1:4733L21 antisense siNA (4715 C.)
ucAcAuGucuuAGGccuGcTsT
2915






stab05


4753
AGCAAAAAGCAAGGGAGAAAAGA
2306
30784
FLT1:4771L21 antisense siNA (4753 C.)
uuuucucccuuGcuuuuuGTsT
2916






stab05


2340
AACAACCACAAAAUACAACAAGA
2292
30955
FLT1:2340U21 sense siNA stab07
B cAAccAcAAAAuAcAAcAATT B
2917


2340
AACAACCACAAAAUACAACAAGA
2292
30956
FLT1:2358L21 antisense siNA (2340 C.)
uuGuuGuAuuuuGuGGuuGTsT
2918






stab08


2340
AACAACCACAAAAUACAACAAGA
2292
30963
FLT1:2340U21 sense siNA inv
AACAACAUAAAACACCAACTT
2919


2340
AACAACCACAAAAUACAACAAGA
2292
30964
FLT1:2358L21 antisense siNA (2340 C.) inv
GUUGGUGUUUUAUGUUGUUTT
2920


2340
AACAACCACAAAAUACAACAAGA
2292
30965
FLT1:2340U21 sense siNA stab04 inv
B AAcAAcAuAAAAcAccAAcTT B
2921


2340
AACAACCACAAAAUACAACAAGA
2292
30966
FLT1:2358L21 antisense siNA (2340 C.)
GuuGGuGuuuuAuGuuGuuTsT
2922






stab05 inv


2340
AACAACCACAAAAUACAACAAGA
2292
30967
FLT1:2340U21 sense siNA stab07 inv
B AAcAAcAuAAAAcAccAAcTT B
2923


2340
AACAACCACAAAAUACAACAAGA
2292
30968
FLT1:2358L21 antisense siNA (2340 C.)
GuuGGuGuuuuAuGuuGuuTsT
2924






stab08 inv


349
AACUGAGUUUAAAAGGCACCCAG
2289
31182
FLT1:349U21 sense siNA stab00
CUGAGUUUAAAAGGCACCCTT
2925


2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31183
FLT1:2949U21 sense siNA TT
GCAAGGAGGGCCUCUGAUGTT
2926


3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31184
FLT1:3912U21 sense siNA TT
CCUGGAAAGAAUCAAAACCTT
2927


349
AACUGAGUUUAAAAGGCACCCAG
2289
31185
FLT1:367L21 antisense siNA (349 C.)
GGGUGCCUUUUAAACUCAGTT
2928






stab00


2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31186
FLT1:2967L21 antisense siNA (2949 C.) TT
CAUCAGAGGCCCUCCUUGCTT
2929


3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31187
FLT1:3930L21 antisense siNA (3912 C.) TT
GGUUUUGAUUCUUUCCAGGTT
2930


349
AACUGAGUUUAAAAGGCACCCAG
2289
31188
FLT1:349U21 sense siNA stab04
B cuGAGuuuAAAAGGcAcccTT B
2931


2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31189
FLT1:2949U21 sense siNA stab04
B GcAAGGAGGGccucuGAuGTT B
2932


3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31190
FLT1:3912U21 sense siNA stab04
B ccuGGAAAGAAucAAAAccTT B
2933


349
AACUGAGUUUAAAAGGCACCCAG
2289
31191
FLT1:367L21 antisense siNA (349 C.)
GGGuGccuuuuAAAcucAGTsT
2934






stab05


2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31192
FLT1:2967L21 antisense siNA (2949 C.)
cAucAGAGGcccuccuuGcTsT
2935






stab05


3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31193
FLT1:3930L21 antisense siNA (3912 C.)
GGuuuuGAuucuuuccAGGTsT
2936






stab05


349
AACUGAGUUUAAAAGGCACCCAG
2289
31194
FLT1:349U21 sense siNA stab07
B cuGAGuuuAAAAGGcAcccTT B
2937


2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31195
FLT1:2949U21 sense siNA stab07
B GcAAGGAGGGccucuGAuGTT B
2938


3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31196
FLT1:3912U21 sense siNA stab07
B ccuGGAAAGAAucAAAAccTT B
2939


349
AACUGAGUUUAAAAGGCACCCAG
2289
31197
FLT1:367L21 antisense siNA (349 C.)
GGGuGccuuuuAAAcucAGTsT
2940






stab08


2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31198
FLT1:2967L21 antisense siNA (2949 C.)
cAucAGAGGcccuccuuGcTsT
2941






stab08


3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31199
FLT1:3930L21 antisense siNA (3912 C.)
GGuuuuGAuucuuuccAGGTsT
2942






stab08


349
AACUGAGUUUAAAAGGCACCCAG
2289
31200
FLT1:349U21 sense siNA inv TT
CCCACGGAAAAUUUGAGUCTT
2943


2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31201
FLT1:2949U21 sense siNA inv TT
GUAGUCUCCGGGAGGAACGTT
2944


3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31202
FLT1:3912U21 sense siNA inv TT
CCAAAACUAAGAAAGGUCCTT
2945


349
AACUGAGUUUAAAAGGCACCCAG
2289
31203
FLT1:367L21 antisense siNA (349 C.) inv
GACUCAAAUUUUCCGUGGGTT
2946






TV


2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31204
FLT1:2967L21 antisense siNA (2949 C.) inv
CGUUCCUCCCGGAGACUACTT
2947






TT


3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31205
FLT1:3930L21 antisense siNA (3912 C.) inv
GGACCUUUCUUAGUUUUGGTT
2948






TT


349
AACUGAGUUUAAAAGGCACCCAG
2289
31206
FLT1:349U21 sense siNA stab04 inv
B cccAcGGAAAAuuuGAGucTT B
2949


2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31207
FLT1:2949U21 sense siNA stab04 inv
B GuAGucuccGGGAGGAAcGTT B
2950


3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31208
FLT1:3912U21 sense siNA stab04 inv
B ccAAAAcuAAGAAAGGuccTT B
2951


2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31510
FLT1:2967L21 antisense siNA (2949 C.)
cAucAGAGGcccuccuuGcTsT
2952






stab11


349
AACUGAGUUUAAAAGGCACCCAG
2289
31511
FLT1:367L21 antisense siNA (349 C.)
GGGuGccuuuuAAAcucAGTsT
2953






stab11


3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31512
FLT1:3930L21 antisense siNA (3912 C.)
GGuuuuGAuucuuuccAGGTsT
2954






stab11


2340
AACAACCACAAAAUACAACAAGA
2292
31513
FLT1:2358L21 antisense siNA (2340 C.) inv
GuuGGuGuuuuAuGuuGuuTsT
2955






stab11


2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
31514
FLT1:2967L21 antisense siNA (2949 C.) inv
cGuuccucccGGAGAcuAcTsT
2956






stab11


349
AACUGAGUUUAAAAGGCACCCAG
2289
31515
FLT1:367121 antisense siNA (349 C.) inv
GAcucAAAuuuuccGuGGGTsT
2957






stab11


3912
AGCCUGGAAAGAAUCAAAACCUU
2291
31516
FLT1:3930L21 antisense siNA (3912 C.) inv
GGAccuuucuuAGuuuuGGTsT
2958






stab11


349
AACUGAGUUUAAAAGGCACCCAG
2289
34426
5′ n-1 C31270 FLT1:349U21 sense siNA
CUGAGUUUAAAAGGCACCCTT B
2843






stab09


349
AACUGAGUUUAAAAGGCACCCAG
2289
34427
5′ n-2 C31270 FLT1:349U21 sense siNA
UGAGUUUAAAAGGCACCCTT B
2959






stab09


349
AACUGAGUUUAAAAGGCACCCAG
2289
34428
5′ n-3 C31270 FLT1:349U21 sense siNA
GAGUUUAAAAGGCACCCTT B
2960






stab09


349
AACUGAGUUUAAAAGGCACCCAG
2289
34429
5′ n-4 C31270 FLT1:349U21 sense siNA
AGUUUAAAAGGCACCCTT B
2961






stab09


349
AACUGAGUUUAAAAGGCACCCAG
2289
34430
5′ n-5 C31270 FLT1:349U21 sense siNA
GUUUAAAAGGCACCCTT B
2962






stab09


349
AACUGAGUUUAAAAGGCACCCAG
2289
34431
5′ n-7 C31270 FLT1:349U21 sense siNA
UUAAAAGGCACCCTT B
2963






stab09


349
AACUGAGUUUAAAAGGCACCCAG
2289
34432
5′ n-9 C31270 FLT1:349U21 sense siNA
AAAAGGCACCCTT B
2964






stab09


349
AACUGAGUUUAAAAGGCACCCAG
2289
34433
3′ n-1 C31270 FLT1:349U21 sense siNA
B CUGAGUUUAAAAGGCACCCTT
2965






stab09


349
AACUGAGUUUAAAAGGCACCCAG
2289
34434
3′ n-2 C31270 FLT1:349U21 sense siNA
B CUGAGUUUAAAAGGCACCCT
2966






stab09


349
AACUGAGUUUAAAAGGCACCCAG
2289
34435
3′ n-3 C31270 FLT1:349U21 sense siNA
     |
2967






stab09
B CUGAGUUUAAAAGGCACCC


349
AACUGAGUUUAAAAGGCACCCAG
2289
34436
3′ n-4 C31270 FLT1:349U21 sense siNA
B CUGAGUUUAAAAGGCACC
2968






stab09


349
AACUGAGUUUAAAAGGCACCCAG
2289
34437
3′ n-5 C31270 FLT1:349U21 sense siNA
B CUGAGUUUAAAAGGCAC
2969






stab09


349
AACUGAGUUUAAAAGGCACCCAG
2289
34438
3′ n-7 C31270 FLT1:349U21 sense siNA
B CUGAGUUUAAAAGGC
2970






stab09


349
AACUGAGUUUAAAAGGCACCCAG
2289
34439
5′ n-1 C31273 FLT1:367L21 antisense
GGUGCCUUUUAAACUCAGTsT
2971






siNA (349 C.) stab10


349
AACUGAGUUUAAAAGGCACCCAG
2289
34440
5′ n-2 C31273 FLT1:367L21 antisense
GUGCCUUUUAAACUCAGTsT
2972






siNA (349 C.) stab10


349
AACUGAGUUUAAAAGGCACCCAG
2289
34441
5′ n-3 C31273 FLT1:367L21 antisense
UGCCUUUUAAACUCAGTsT
2973






siNA (349 C.) stab10


349
AACUGAGUUUAAAAGGCACCCAG
2289
34442
5′ n-4 C31273 FLT1:367L21 antisense
GCCUUUUAAACUCAGTsT
2974






siNA (349 C.) stab10


349
AACUGAGUUUAAAAGGCACCCAG
2289
34443
5′ n-5 C31273 FLT1:367L21 antisense
CCUUUUAAACUCAGTsT
2975






siNA (349 C.) stab10


349
AACUGAGUUUAAAAGGCACCCAG
2289
34444
3′ n-1 C31273 FLT1:367L21 antisense
GGGUGCCUUUUAAACUCAGT
2976






siNA (349 C.) stab10


349
AACUGAGUUUAAAAGGCACCCAG
2289
34445
3′ n-2 C31273 FLT1:367L21 antisense
GGGUGCCUUUUAAACUCAG
2977






siNA (349 C.) stab10


349
AACUGAGUUUAAAAGGCACCCAG
2289
34446
3′ n-3 031273 FLT1:367L21 antisense
GGGUGCCUUUUAAACUCA
2978






siNA (349 C.) stab10


349
AACUGAGUUUAAAAGGCACCCAG
2289
34447
3′ n-4 C31273 FLT1:367L21 antisense
GGGUGCCUUUUAAACUC
2979






siNA (349 C.) stab10


349
AACUGAGUUUAAAAGGCACCCAG
2289
34448
3′ n-5 C31273 FLT1:367L21 antisense
GGGUGCCUUUUAAACU
2980






siNA (349 C.) stab10


349
AACUGAGUUUAAAAGGCACCCAG
2289
34449
3′ n-7 C31273 FLT1:367L21 antisense
GGGUGCCUUUUAAA
2981






siNA (349 C.) stab10


349
AACUGAGUUUAAAAGGCACCCAG
2289
34450
3′ n-9 031273 FLT1:367L21 antisense
GGGUGCCUUUUA
2982






siNA (349 C.) stab10


349
AACUGAGUUUAAAAGGCACCCAG
2289
34452
FLT1:367L21 antisense siNA (349 C.)
CUACCAGCGAGUUUGUAGUUUA
2983






scram1 + A15 all 2′OMe
AAAAAAAAAAAAAsA


349
AACUGAGUUUAAAAGGCACCCAG
2289
34453
FLT1:367L21 antisense siNA (349 C.)
CUACCAGCGAGUUUGUAGUUUA
2984






scram1 + A20 all 2′OMe
AAAAAAAAAAAAAAAAAAsA


349
AACUGAGUUUAAAAGGCACCCAG
2289
34454
FLT1:367L21 antisense siNA (349 C.)
CUACCAGCGAGUUUGUAGUUUA
2985






scram1 + A25 all 2′OMe
AAAAAAAAAAAAAAAAAAAAAAAsA


349
AACUGAGUUUAAAAGGCACCCAG
2289
34455
FLT1:367L21 antisense siNA (349 C.)
CUACCAGCGAGUUUGUAGUUUA
2986






scram1 + A30 all 2′OMe
AAAAAAAAAAAAAAAAAAAAAAAA







AAAAsA


1501
ACCUCACUGCCACUCUAAUUGUC
2307
34676
FLT1:1501U21 sense siNA stab00
CUCACUGCCACUCUAAUUGTT
2987


1502
CCUCACUGCCACUCUAAUUGUCA
2308
34677
FLT1:1502U21 sense siNA stab00
UCACUGCCACUCUAAUUGUTT
2988


1503
CUCACUGCCACUCUAAUUGUCAA
2309
34678
FLT1:1503U21 sense siNA stab00
CACUGCCACUCUAAUUGUCTT
2989


5353
AAGACCCCGUCUCUAUACCAACC
2310
34679
FLT1:5353U21 sense siNA stab00
GACCCCGUCUCUAUACCAATT
2990


1501
ACCUCACUGCCACUCUAAUUGUC
2307
34684
FLT1:1519L21 (1501 C.) siRNA stab00
CAAUUAGAGUGGCAGUGAGTT
2991


1502
CCUCACUGCCACUCUAAUUGUCA
2308
34685
FLT1:1520L21 (1502 C.) siRNA stab00
ACAAUUAGAGUGGCAGUGATT
2992


1503
CUCACUGCCACUCUAAUUGUCAA
2309
34686
FLT1:1521L21 (1503 C.) siRNA stab00
GACAAUUAGAGUGGCAGUGTT
2993


5353
AAGACCCCGUCUCUAUACCAACC
2310
34687
FLT1:5371L21 (5353 C.) siRNA stab00
UUGGUAUAGAGACGGGGUCTT
2994


349
AACUGAGUUUAAAAGGCACCCAG
2289
35117
FLT1:349U21 sense siNA stab07 N1
B cuGAGuuuAAAAGGCACCCTT B
2995


349
AACUGAGUUUAAAAGGCACCCAG
2289
35118
FLT1:367L21 antisense siNA (349 C.)
GGGUGCcuuuuAAAcucAGTsT
2996






stab08 N1


349
AACUGAGUUUAAAAGGCACCCAG
2289
35119
FLT1:367L21 antisense siNA (349 C.)
GGGUGccuuuuAAAcucAGTsT
2997






stab08 N2


349
AACUGAGUUUAAAAGGCACCCAG
2289
35120
FLT1:367L21 antisense siNA (349 C.)
GGGUGccuuuuAAAcucAGTsT
2998






stab08 N3


349
AACUGAGUUUAAAAGGCACCCAG
2289
35121
FLT1:367L21 antisense siNA (349 C.)
GGGuGccuuuuAAAcucAGTsT
2999






stab25


349
AACUGAGUUUAAAAGGCACCCAG
2289
35122
FLT1:367L21 antisense siNA (349 C.)
GGGuGccuuuuAAAcucAGTsT
3000






stab08 N5


349
AACUGAGUUUAAAAGGCACCCAG
2289
35123
FLT1:367L21 antisense siNA (349 C.)
GGGuGccuuuuAAAcucAGTsT
3001






stab24


346
CUGAACUGAGUUUAAAAGGCACC
2296
35814
FLT1:346U21 sense siNA stab23
B GAAcuGAGuuuAAAAGGcATT B
3002


346
CUGAACUGAGUUUAAAAGGCACC
2296
35815
FLT1:346U21 sense siNA stab07 N2
B GAAcuGAGuuuAAAGGCATT B
3003


346
CUGAACUGAGUUUAAAAGGCACC
2296
35816
FLT1:364L21 antisense siNA (346 C.)
UGccuuuuAAAcucAGuucTsT
3004






stab24


346
CUGAACUGAGUUUAAAAGGCACC
2296
35817
FLT1:364L21 antisense siNA (346 C.)
UGccuuuuAAAcucAGuucTsT
3005






stab08 N2


346
CUGAACUGAGUUUAAAAGGCACC
2296
35818
FLT1:364L21 antisense siNA (346 C.)
UGCcuuuuAAAcucAGuucTsT
3006






stab24


346
CUGAACUGAGUUUAAAAGGCACC
2296
35909
FLT1:346U21 sense siNA stab07 J1
GAAcuGAGuUuAAAAGGcATT
3007


346
CUGAACUGAGUUUAAAAGGCACC
2296
35910
FLT1:364L21 antisense siNA (346 C.)
UGccuuuUAAAcucAGUucTsT
3008






stab08 J1


47
GAGCGGGCUCCGGGGCUCGGGUG
2311
36152
FLT1:47U21 sense siNA stab00
GCGGGCUCCGGGGCUCGGGTT
3009


121
CUGGCUGGAGCCGCGAGACGGGC
2312
36153
FLT1:121U21 sense siNA stab00
GGCUGGAGCCGCGAGACGGTT
3010


122
UGGCUGGAGCCGCGAGACGGGCG
2313
36154
FLT1:122U21 sense siNA stab00
GCUGGAGCCGCGAGACGGGTT
3011


251
CAUGGUCAGCUACUGGGACACCG
2314
36155
FLT1:251U21 sense siNA stab00
UGGUCAGCUACUGGGACACTT
3012


252
AUGGUCAGCUACUGGGACACCGG
2315
36156
FLT1:252U21 sense siNA stab00
GGUCAGCUACUGGGACACCTT
3013


354
AGUUUAAAAGGCACCCAGCACAU
2316
36157
FLT1:354U21 sense siNA stab00
UUUAAAAGGCACCCAGCACTT
3014


419
AGCAGCCCAUAAAUGGUCUUUGC
2317
36158
FLT1:419U21 sense siNA stab00
CAGCCCAUAAAUGGUCUUUTT
3015


594
UCAAAGAAGAAGGAAACAGAAUC
2318
36159
FLT1:594U21 sense siNA stab00
AAAGAAGAAGGAAACAGAATT
3016


595
CAAAGAAGAAGGAAACAGAAUCU
2319
36160
FLT1:595U21 sense siNA stab00
AAGAAGAAGGAAACAGAAUTT
3017


709
AGCUCGUCAUUCCCUGCCGGGUU
2320
36161
FLT1:709U21 sense siNA stab00
CUCGUCAUUCCCUGCCGGGTT
3018


710
GCUCGUCAUUCCCUGCCGGGUUA
2321
36162
FLT1:710U21 sense siNA stab00
UCGUCAUUCCCUGCCGGGUTT
3019


758
AAAAAAGUUUCCACUUGACACUU
2322
36163
FLT1:758U21 sense siNA stab00
AAAAGUUUCCACUUGACACTT
3020


759
AAAAAGUUUCCACUUGACACUUU
2323
36164
FLT1:759U21 sense siNA stab00
AAAGUUUCCACUUGACACUTT
3021


796
AACGCAUAAUCUGGGACAGUAGA
2324
36165
FLT1:796U21 sense siNA stab00
CGCAUAAUCUGGGACAGUATT
3022


797
ACGCAUAAUCUGGGACAGUAGAA
2325
36166
FLT1:797U21 sense siNA stab00
GCAUAAUCUGGGACAGUAGTT
3023


798
CGCAUAAUCUGGGACAGUAGAAA
2326
36167
FLT1:798U21 sense siNA stab00
CAUAAUCUGGGACAGUAGATT
3024


799
GCAUAAUCUGGGACAGUAGAAAG
2327
36168
FLT1:799U21 sense siNA stab00
AUAAUCUGGGACAGUAGAATT
3025


1220
CACCUCAGUGCAUAUAUAUGAUA
2328
36169
FLT1:1220U21 sense siNA stab00
CCUCAGUGCAUAUAUAUGATT
3026


1438
CUGAAGAGGAUGCAGGGAAUUAU
2329
36170
FLT1:1438U21 sense siNA stab00
GAAGAGGAUGCAGGGAAUUTT
3027


1541
UUACGAAAAGGCCGUGUCAUCGU
2330
36171
FLT1:1541U21 sense siNA stab00
ACGAAAAGGCCGUGUCAUCTT
3028


1640
AAUCAAGUGGUUCUGGCACCCCU
2331
36172
FLT1:1640U21 sense siNA stab00
UCAAGUGGUUCUGGCACCCTT
3029


1666
ACCAUAAUCAUUCCGAAGCAAGG
2332
36173
FLT1:1666U21 sense siNA stab00
CAUAAUCAUUCCGAAGCAATT
3030


1877
GACUGUGGGAAGAAACAUAAGCU
2333
36174
FLT1:1877U21 sense siNA stab00
CUGUGGGAAGAAACAUAAGTT
3031


2247
AACCUCAGUGAUCACACAGUGGC
2334
36175
FLT1:2247U21 sense siNA stab00
CCUCAGUGAUCACACAGUGTT
3032


2248
ACCUCAGUGAUCACACAGUGGCC
2335
36176
FLT1:2248U21 sense siNA stab00
CUCAGUGAUCACACAGUGGTT
3033


2360
AGAGCCUGGAAUUAUUUUAGGAC
2336
36177
FLT1:2360U21 sense siNA stab00
AGCCUGGAAUUAUUUUAGGTT
3034


2415
ACAGAAGAGGAUGAAGGUGUCUA
2337
36178
FLT1:2415U21 sense siNA stab00
AGAAGAGGAUGAAGGUGUCTT
3035


2514
UCUAAUCUGGAGCUGAUCACUCU
2338
36179
FLT1:2514U21 sense siNA stab00
UAAUCUGGAGCUGAUCACUTT
3036


2518
AUCUGGAGCUGAUCACUCUAACA
2339
36180
FLT1:2518U21 sense siNA stab00
CUGGAGCUGAUCACUCUAATT
3037


2703
AGCAAGUGGGAGUUUGCCCGGGA
2340
36181
FLT1:2703U21 sense siNA stab00
CAAGUGGGAGUUUGCCCGGTT
3038


2795
CAUUAAGAAAUCACCUACGUGCC
2341
36182
FLT1:2795U21 sense siNA stab00
UUAAGAAAUCACCUACGUGTT
3039


2965
UGAUGGUGAUUGUUGAAUACUGC
2342
36183
FLT1:2965U21 sense siNA stab00
AUGGUGAUUGUUGAAUACUTT
3040


3074
GAAAGAAAAAAUGGAGCCAGGCC
2343
36184
FLT1:3074U21 sense siNA stab00
AAGAAAAAAUGGAGCCAGGTT
3041


3100
AACAAGGCAAGAAACCAAGACUA
2344
36185
FLT1:3100U21 sense siNA stab00
CAAGGCAAGAAACCAAGACTT
3042


3101
ACAAGGCAAGAAACCAAGACUAG
2345
36186
FLT1:3101U21 sense siNA stab00
AAGGCAAGAAACCAAGACUTT
3043


3182
GAGUGAUGUUGAGGAAGAGGAGG
2346
36187
FLT1:3182U21 sense siNA stab00
GUGAUGUUGAGGAAGAGGATT
3044


3183
AGUGAUGUUGAGGAAGAGGAGGA
2347
36188
FLT1:3183U21 sense siNA stab00
UGAUGUUGAGGAAGAGGAGTT
3045


3253
CUUACAGUUUUCAAGUGGCCAGA
2348
36189
FLT1:3253U21 sense siNA stab00
UACAGUUUUCAAGUGGCCATT
3046


3254
UUACAGUUUUCAAGUGGCCAGAG
2349
36190
FLT1:3254U21 sense siNA stab00
ACAGUUUUCAAGUGGCCAGTT
3047


3260
UUUUCAAGUGGCCAGAGGCAUGG
2350
36191
FLT1:3260U21 sense siNA stab00
UUCAAGUGGCCAGAGGCAUTT
3048


3261
UUUCAAGUGGCCAGAGGCAUGGA
2351
36192
FLT1:3261U21 sense siNA stab00
UCAAGUGGCCAGAGGCAUGTT
3049


3294
UCCAGAAAGUGCAUUCAUCGGGA
2352
36193
FLT1:3294U21 sense siNA stab00
CAGAAAGUGCAUUCAUCGGTT
3050


3323
AGCGAGAAACAUUCUUUUAUCUG
2353
36194
FLT1:3323U21 sense siNA stab00
CGAGAAACAUUCUUUUAUCTT
3051


3324
GCGAGAAACAUUCUUUUAUCUGA
2354
36195
FLT1:3324U21 sense siNA stab00
GAGAAACAUUCUUUUAUCUTT
3052


3325
CGAGAAACAUUCUUUUAUCUGAG
2355
36196
FLT1:3325U21 sense siNA stab00
AGAAACAUUCUUUUAUCUGTT
3053


3513
UUGCUGUGGGAAAUCUUCUCCUU
2356
36197
FLT1:3513U21 sense siNA stab00
GCUGUGGGAAAUCUUCUCCTT
3054


3812
UGCCUUCUCUGAGGACUUCUUCA
2357
36198
FLT1:3812U21 sense siNA stab00
CCUUCUCUGAGGACUUCUUTT
3055


3864
UCAGGAAGCUCUGAUGAUGUCAG
2358
36199
FLT1:3864U21 sense siNA stab00
AGGAAGCUCUGAUGAUGUCTT
3056


3865
CAGGAAGCUCUGAUGAUGUCAGA
2359
36200
FLT1:3865U21 sense siNA stab00
GGAAGCUCUGAUGAUGUCATT
3057


3901
UCAAGUUCAUGAGCCUGGAAAGA
2360
36201
FLT1:3901U21 sense siNA stab00
AAGUUCAUGAGCCUGGAAATT
3058


3902
CAAGUUCAUGAGCCUGGAAAGAA
2361
36202
FLT1:3902U21 sense siNA stab00
AGUUCAUGAGCCUGGAAAGTT
3059


3910
UGAGCCUGGAAAGAAUCAAAACC
2362
36203
FLT1:3910U21 sense siNA stab00
AGCCUGGAAAGAAUCAAAATT
3060


4136
CAGCUGUGGGCACGUCAGCGAAG
2363
36204
FLT1:4136U21 sense siNA stab00
GCUGUGGGCACGUCAGCGATT
3061


4154
CGAAGGCAAGCGCAGGUUCACCU
2364
36205
FLT1:4154U21 sense siNA stab00
AAGGCAAGCGCAGGUUCACTT
3062


4635
UGCAGCCCAAAACCCAGGGCAAC
2365
36206
FLT1:4635U21 sense siNA stab00
CAGCCCAAAACCCAGGGCATT
3063


4945
GAGGCAAGAAAAGGACAAAUAUC
2366
36207
FLT1:4945U21 sense siNA stab00
GGCAAGAAAAGGACAAAUATT
3064


5090
UUGGCUCCUCUAGUAAGAUGCAC
2367
36208
FLT1:5090U21 sense siNA stab00
GGCUCCUCUAGUAAGAUGCTT
3065


5137
GUCUCCAGGCCAUGAUGGCCUuA
2368
36209
FLT1:5137U21 sense siNA stab00
CUCCAGGCCAUGAUGGCCUTT
3066


5138
UCUCCAGGCCAUGAUGGCCUUAC
2369
36210
FLT1:5138U21 sense siNA stab00
UCCAGGCCAUGAUGGCCUUTT
3067


5354
AGACCCCGUCUCUAUACCAACCA
2370
36211
FLT1:5354U21 sense siNA stab00
ACCCCGUCUCUAUACCAACTT
3068


5356
ACCCCGUCUCUAUACCAACCAAA
2371
36212
FLT1:5356U21 sense siNA stab00
CCCGUCUCUAUACCAACCATT
3069


5357
CCCCGUCUCUAUACCAACCAAAC
2372
36213
FLT1:5357U21 sense siNA stab00
CCGUCUCUAUACCAACCAATT
3070


5707
GAUCAAGUGGGCCUUGGAUCGCU
2373
36214
FLT1:5707U21 sense siNA stab00
UCAAGUGGGCCUUGGAUCGTT
3071


5708
AUCAAGUGGGCCUUGGAUCGCUA
2374
36215
FLT1:5708U21 sense siNA stab00
CAAGUGGGCCUUGGAUCGCTT
3072


47
GAGCGGGCUCCGGGGCUCGGGUG
2311
36216
FLT1:65L21 antisense siNA (47 C.) stab00
CCCCGAGCCCCGGAGCCCGCTT
3073


121
CUGGCUGGAGCCGCGAGACGGGC
2312
36217
FLT1:139L21 antisense siNA (121 C.)
CCGUCUCGCGGCUCCAGCCTT
3074






stab00


122
UGGCUGGAGCCGCGAGACGGGCG
2313
36218
FLT1:140L21 antisense siNA (122 C.)
CCCGUCUCGCGGCUCCAGCTT
3075






stab00


251
CAUGGUCAGCUACUGGGACACCG
2314
36219
FLT1:269L21 antisense siNA (251 C.)
GUGUCCCAGUAGCUGACCATT
3076






stab00


252
AUGGUCAGCUACUGGGACACCGG
2315
36220
FLT1:270121 antisense siNA (252 C.)
GGUGUCCCAGUAGCUGACCTT
3077






stab00


354
AGUUUAAAAGGCACCCAGCACAU
2316
36221
FLT1:372121 antisense siNA (354 C.)
GUGCUGGGUGCCUUUUAAATT
3078






stab00


419
AGCAGCCCAUAAAUGGUCUUUGC
2317
36222
FLT1:437L21 antisense siNA (419 C.)
AAAGACCAUUUAUGGGCUGTT
3079






stab00


594
UCAAAGAAGAAGGAAACAGAAUC
2318
36223
FLT1:612L21 antisense siNA (594 C.)
UUCUGUUUCCUUCUUCUUUTT
3080






stab00


595
CAAAGAAGAAGGAAACAGAAUCU
2319
36224
FLT1:613L21 antisense siNA (595 C.)
AUUCUGUUUCCUUCUUCUUTT
3081






stab00


709
AGCUCGUCAUUCCCUGCCGGGUU
2320
36225
FLT1:727L21 antisense siNA (709 C.)
CCCGGCAGGGAAUGACGAGTT
3082






stab00


710
GCUCGUCAUUCCCUGCCGGGUUA
2321
36226
FLT1:728L21 antisense siNA (710 C.)
ACCCGGCAGGGAAUGACGATT
3083






stab00


758
AAAAAAGUUUCCACUUGACACUU
2322
36227
FLT1:776121 anhisense siNA (758 C.)
GUGUCAAGUGGAAACUUUUTT
3084






stab00


759
AAAAAGUUUCCACUUGACACUUU
2323
36228
FLT1:777L21 antisense siNA (759 C.)
AGUGUCAAGUGGAAACUUUTT
3085






stab00


796
AACGCAUAAUCUGGGACAGUAGA
2324
36229
FLT1:814L21 antisense siNA (796 C.)
UACUGUCCCAGAUUAUGCGTT
3086






stab00


797
ACGCAUAAUCUGGGACAGUAGAA
2325
36230
FLT1:815L21 antisense siNA (797 C.)
CUACUGUCCCAGAUUAUGCTT
3087






stab00


798
CGCAUAAUCUGGGACAGUAGAAA
2326
36231
FLT1:816L21 antisense siNA (798 C.)
UCUACUGUCCCAGAUUAUGTT
3088






stab00


799
GCAUAAUCUGGGACAGUAGAAAG
2327
36232
FLT1:817L21 antisense siNA (799 C.)
UUCUACUGUCCCAGAUUAUTT
3089






stab00


1220
CACCUCAGUGCAUAUAUAUGAUA
2328
36233
FLT1:1238L21 antisense siNA (1220 C.)
UCAUAUAUAUGCACUGAGGTT
3090






stab00


1438
CUGAAGAGGAUGCAGGGAAUUAU
2329
36234
FLT1:1456L21 antisense siNA (1438 C.)
AAUUCCCUGCAUCCUCUUCTT
3091






stab0G


1541
UUACGAAAAGGCCGUGUCAUCGU
2330
36235
FLT1:1559L21 antisense siNA (1541 C.)
GAUGACACGGCCUUUUCGUTT
3092






stab00


1640
AAUCAAGUGGUUCUGGCACCCCU
2331
36236
FLT1:1658L21 antisense siNA (1640 C.)
GGGUGCCAGAACCACUUGATT
3093






stab00


1666
ACCAUAAUCAUUCCGAAGCAAGG
2332
36237
FLT1:1684L21 antisense siNA (1666 C.)
UUGCUUCGGAAUGAUUAUGTT
3094






stab00


1877
GACUGUGGGAAGAAACAUAAGCU
2333
36238
FLT1:1895L21 antisense siNA (1877 C.)
CUUAUGUUUCUUCCCACAGTT
3095






stab00


2247
AACCUCAGUGAUCACACAGUGGC
2334
36239
FLT1:2265L21 antisense siNA (2247 C.)
CACUGUGUGAUCACUGAGGTT
3096






stab00


2248
ACCUCAGUGAUCACACAGUGGCC
2335
36240
FLT1:2266L21 antisense siNA (2248 C.)
CCACUGUGUGAUCACUGAGTT
3097


2360
AGAGCCUGGAAUUAUUUUAGGAC
2336
36241
FLT1:2378L21 antisense siNA (2360 C.)
CCUAAAAUAAUUCCAGGCUTT
3098






stab00


2415
ACAGAAGAGGAUGAAGGUGUCUA
2337
36242
FLT1:2433L21 antisense siNA (2415 C.)
GACACCUUCAUCCUCUUCUTT
3099






stab00


2514
UCUAAUCUGGAGCUGAUCACUCU
2338
36243
FLT1:2532L21 antisense siNA (2514 C.)
AGUGAUCAGCUCCAGAUUATT
3100






stab00


2518
AUCUGGAGCUGAUCACUCUAACA
2339
36244
FLT1:2536L21 antisense siNA (2518 C.)
UUAGAGUGAUCAGCUCCAGTT
3101






stab00


2703
AGCAAGUGGGAGUUUGCCCGGGA
2340
36245
FLT1:2721L21 antisense siNA (2703 C.)
CCGGGCAAACUCCCACUUGTT
3102






stab00


2795
CAUUAAGAAAUCACCUACGUGCC
2341
36246
FLT1:2813L21 antisense siNA (2795 C.)
CACGUAGGUGAUUUCUUAATT
3103






stab00


2965
UGAUGGUGAUUGUUGAAUACUGC
2342
36247
FLT1:2983L21 antisense siNA (2965 C.)
AGUAUUCAACAAUCACCAUTT
3104






stab00


3074
GAAAGAAAAAAUGGAGCCAGGCC
2343
36248
FLT1:3092L21 antisense siNA (3074 C.)
CCUGGCUCCAUUUUUUCUUTT
3105






stab00


3100
AACAAGGCAAGAAACCAAGACUA
2344
36249
FLT1:3118L21 antisense siNA (3100 C.)
GUCUUGGUUUCUUGCCUUGTT
3106






stab00


3101
ACAAGGCAAGAAACCAAGACUAG
2345
36250
FLT1:3119L21 antisense siNA (3101 C.)
AGUCUUGGUUUCUUGCCUUTT
3107






stab00


3182
GAGUGAUGUUGAGGAAGAGGAGG
2346
36251
FLT1:3200L21 antisense siNA (3182 C.)
UCCUCUUCCUCAACAUCACTT
3108






stab00


3183
AGUGAUGUUGAGGAAGAGGAGGA
2347
36252
FLT1:3201L21 antisense siNA (3183 C.)
CUCCUCUUCCUCAACAUCATT
3109






stab00


3253
CUUACAGUUUUCAAGUGGCCAGA
2348
36253
FLT1:3271L21 antisense siNA (3253 C.)
UGGCCACUUGAAAACUGUATT
3110






stab00


3254
UUACAGUUUUCAAGUGGCCAGAG
2349
36254
FLT1:3272L21 antisense siNA (3254 C.)
CUGGCCACUUGAAAACUGUTT
3111






stab00


3260
UUUUCAAGUGGCCAGAGGCAUGG
2350
36255
FLT1:3278L21 antisense siNA (3260 C.)
AUGCCUCUGGCCACUUGAATT
3112






stab00


3261
UUUCAAGUGGCCAGAGGCAUGGA
2351
36256
FLT1:3279L21 antisense siNA (3261 C.)
CAUGCCUCUGGCCACUUGATT
3113






stab00


3294
UCCAGAAAGUGCAUUCAUCGGGA
2352
36257
FLT1:3312L21 antisense siNA (3294 C.)
CCGAUGAAUGCACUUUCUGTT
3114






stab00


3323
AGCGAGAAACAUUCUUUUAUCUG
2353
36258
FLT1:3341L21 antisense siNA (3323 C.)
GAUAAAAGAAUGUUUCUCGTT
3115






stab00


3324
GCGAGAAACAUUCUUUUAUCUGA
2354
36259
FLT1:3342L21 antisense siNA (3324 C.)
AGAUAAAAGAAUGUUUCUCTT
3116






stab00


3325
CGAGAAACAUUCUUUUAUCUGAG
2355
36260
FLT1:3343L21 antisense siNA (3325 C.)
CAGAUAAAAGAAUGUUUCUTT
3117






stab00


3513
UUGCUGUGGGAAAUCUUCUCCUU
2356
36261
FLT1:3531L21 antisense siNA (3513 C.)
GGAGAAGAUUUCCCACAGCTT
3118






stab00


3812
UGCCUUCUCUGAGGACUUCUUCA
2357
36262
FLT1:3830L21 antisense siNA (3812 C.)
AAGAAGUCCUCAGAGAAGGTT
3119






stab00


3864
UCAGGAAGCUCUGAUGAUGUCAG
2358
36263
FLT1:3882L21 antisense siNA (3864 C.)
GACAUCAUCAGAGCUUCCUTT
3120






stab00


3865
CAGGAAGCUCUGAUGAUGUCAGA
2359
36264
FLT1:3883L21 antisense siNA (3865 C.)
UGACAUCAUCAGAGCUUCCTT
3121






stab00


3901
UCAAGUUCAUGAGCCUGGAAAGA
2360
36265
FLT1:3919L21 antisense siNA (3901 C.)
UUUCCAGGCUCAUGAACUUTT
3122






stab00


3902
CAAGUUCAUGAGCCUGGAAAGAA
2361
36266
FLT1:3920L21 antisense siNA (3902 C.)
CUUUCCAGGCUCAUGAACUTT
3123






stab0G


3910
UGAGCCUGGAAAGAAUCAAAACC
2362
36267
FLT1:3928L21 antisense siNA (3910 C.)
UUUUGAUUCUUUCCAGGCUTT
3124






stab00


4136
CAGCUGUGGGCACGUCAGCGAAG
2363
36268
FLT1:4154L21 antisense siNA (4136 C.)
UCGCUGACGUGCCCACAGCTT
3125






stab00


4154
CGAAGGCAAGCGCAGGUUCACCU
2364
36269
FLT1:4172L21 antisense siNA (4154 C.)
GUGAACCUGCGCUUGCCUUTT
3126






stab00


4635
UGCAGCCCAAAACCCAGGGCAAC
2365
36270
FLT1:4653L21 antisense siNA (4635 C.)
UGCCCUGGGUUUUGGGCUGTT
3127






stab00


4945
GAGGCAAGAAAAGGACAAAUAUC
2366
36271
FLT1:4963L21 antisense siNA (4945 C.)
UAUUUGUCCUUUUCUUGCCTT
3128






stab00


5090
UUGGCUCCUCUAGUAAGAUGCAC
2367
36272
FLT1:5108L21 antisense siNA (5090 C.)
GCAUCUUACUAGAGGAGCCTT
3129






stab00


5137
GU0U0CAGGCCAUGAUGGCCUUA
2368
36273
FLT1:5155L21 antisense siNA (5137 C.)
AGGCCAUCAUGGCCUGGAGTT
3130






stab00


5138
UCUCCAGGCCAUGAUGGCCUUAC
2369
36274
FLT1:5156L21 antisense siNA (5138 C.)
AAGGCCAUCAUGGCCUGGATT
3131






stab00


5354
AGACCCCGUCUCUAUACCAACCA
2370
36275
FLT1:5372L21 antisense siNA (5354 C.)
GUUGGUAUAGAGACGGGGUTT
3132






stab00


5356
ACCCCGUCUCUAUACCAACCAAA
2371
36276
FLT1:5374L21 antisense siNA (5356 C.)
UGGUUGGUAUAGAGACGGGTT
3133






stab00


5357
CCCCGUCUCUAUACCAACCAAAC
2372
36277
FLT1:5375L21 antisense siNA (5357 C.)
UUGGUUGGUAUAGAGACGGTT
3134






stab00


5707
GAUCAAGUGGGCCUUGGAUCGCU
2373
36278
FLT1:5725L21 antisense siNA (5707 C.)
CGAUCCAAGGCCCACUUGATT
3135






stab00


5708
AUCAAGUGGGCCUUGGAUCGCUA
2374
36279
FLT1:5726L21 antisense siNA (5708 C.)
GCGAUCCAAGGCCCACUUGTT
3136






stab00


346
CUGAACUGAGUUUAAAAGGCACC
2296
36431
FLT1:346U21 sense siNA stab00
GAACUGAGUUUAAAAGGCATT
3137


346
CUGAACUGAGUUUAAAAGGCACC
2296
36439
FLT1:364L21 antisense siNA (346 C.)
UGCCUUUUAAACUCAGUUCTT
3138






stab00


349
AACUGAGUUUAAAAGGCACCCAG
2289
36457
FLT1:349U19 sense siNA stab00 -3′ TT
CUGAGUUUAAAAGGCACCC
3139


349
AACUGAGUUUAAAAGGCACCCAG
2289
36458
FLT1:367L21 antisense siNA (349 C.)
B GGGUGCCUUUUAAACUCAGTsT B
3140






stab10 +5′ &3′ iB


349
AACUGAGUUUAAAAGGCACCCAG
2289
36459
FLT1:367L19 siRNA (349 C.) stab00 +5′ iB-
B GGGUGCCUUUUAAACUCAG
3141






3′ TT


349
AACUGAGUUUAAAAGGCACCCAG
2289
36460
FLT1:349U21 sense siNA stab07 −5′ &3′
cuGAGuuuAAAAGGcAcccTT
3142






iB


349
AACUGAGUUUAAAAGGCACCCAG
2289
36461
FLT1:349U21 sense siNA stab07 −5′ iB −3′
cuGAGuuuAAAAGGcAccc
3143






TTB


349
AACUGAGUUUAAAAGGCACCCAG
2289
36462
FLT1:367L19 siRNA (349 C.) stab08 −3′ TsT
GGGuGccuuuuAAAcucAG
3144


2338
AAAACAACCACAAAAUACAACAA
2375
37389
FLT1:2338U21 sense siNA stab07
B AAcAAccAcAAAAuAcAAcTT B
3145


2342
CAACCACAAAAUACAACAAGAGC
2376
37390
FLT1:2342U21 sense siNA stab07
B AccAcAAAAuAcAAcAAGATT B
3146


2365
CUGGAAUUAUUUUAGGACCAGGA
2377
37391
FLT1:2365U21 sense siNA stab07
B GGAAuuAuuuuAGGAccAGTT B
3147


2391
AGCACGCUGUUUAUUGAAAGAGU
2378
37392
FLT1:2391U21 sense siNA stab07
B cAcGcuGuuuAuuGAAAGATT B
3148


2392
GCACGCUGUUUAUUGAAAGAGUC
2379
37393
FLT1:2392U21 sense siNA stab07
B AcGcuGuuuAuuGAAAGAGTT B
3149


2393
CACGCUGUUUAUUGAAAGAGUCA
2380
37394
FLT1:2393U21 sense siNA stab07
B cGcuGuuuAuuGAAAGAGuTT B
3150


2394
ACGCUGUUUAUUGAAAGAGUCAC
2381
37395
FLT1:2394U21 sense siNA stab07
B GcuGuuuAuuGAAAGAGucTT B
3151


2395
CGCUGUUUAUUGAAAGAGUCACA
2382
37396
FLT1:2395U21 sense siNA stab07
B cuGuuuAuuGAAAGAGucATT B
3152


2396
GCUGUUUAUUGAAAGAGUCACAG
2383
37397
FLT1:2396U21 sense siNA stab07
B uGuuuAuuGAAAGAGucAcTT B
3153


2397
CUGUUUAUUGAAAGAGUCACAGA
2384
37398
FLT1:2397U21 sense siNA stab07
B GuuuAuuGAAAGAGucAcATT B
3154


2398
UGUUUAUUGAAAGAGUCACAGAA
2385
37399
FLT1:2398U21 sense siNA stab07
B uuuAuuGAAAGAGucAcAGTT B
3155


2697
GAUGCCAGCAAGUGGGAGUUUGC
2386
37400
FLT1:2697U21 sense siNA stab07
B uGccAGcAAGuGGGAGuuuTT B
3156


2699
UGCCAGCAAGUGGGAGUUUGCCC
2387
37401
FLT1:2699U21 sense siNA stab07
B ccAGcAAGuGGGAGuuuGcTT B
3157


2785
CAGCAUUUGGCAUUAAGAAAUCA
2388
37402
FLT1:2785U21 sense siNA stab07
B GcAuuuGGcAuuAAGAAAuTT B
3158


2786
AGCAUUUGGCAUUAAGAAAUCAC
2389
37403
FLT1:2786U21 sense siNA stab07
B cAuuuGGcAuuAAGAAAucTT B
3159


2788
CAUUUGGCAUUAAGAAAUCACCU
2390
37405
FLT1:2788U21 sense siNA stab07
B uuuGGcAuuAAGAAAucAcTT B
3160


2789
AUUUGGCAUUAAGAAAUCACCUA
2391
37406
FLT1:2789U21 sense siNA stab07
B uuGGcAuuAAGAAAucAccTT B
3161


2812
CGUGCCGGACUGUGGCUGUGAAA
2392
37407
FLT1:2812U21 sense siNA stab07
B uGccGGAcuGuGGcuGuGATT B
3162


2860
GCGAGUACAAAGCUCUGAUGACU
2393
37408
FLT1:2860U21 sense siNA stab07
B GAGuAcAAAGcucuGAuGATT B
3163


2861
CGAGUACAAAGCUCUGAUGACUG
2394
37409
FLT1:2861U21 sense siNA stab07
B AGuAcAAAGcucuGAuGAcTT B
3164


2947
CCAAGCAAGGAGGGCCUCUGAUG
2395
37410
FLT1:2947U21 sense siNA stab07
B AAGcAAGGAGGGccucuGATT B
3165


2950
AGCAAGGAGGGCCUCUGAUGGUG
2396
37411
FLT1:2950U21 sense siNA stab07
B cAAGGAGGGccucuGAuGGTT B
3166


2952
CAAGGAGGGCCUCUGAUGGUGAU
2397
37412
FLT1:2952U21 sense siNA stab07
B AGGAGGGccucuGAuGGuGTT B
3167


2953
AAGGAGGGCCUCUGAUGGUGAUU
2398
37413
FLT1:2953U21 sense siNA stab07
B GGAGGGccucuGAuGGuGATT B
3168


2954
AGGAGGGCCUCUGAUGGUGAUUG
2399
37414
FLT1:2954U21 sense siNA stab07
B GAGGGccucuGAuGGuGAuTT B
3169


3262
UUCAAGUGGCCAGAGGCAUGGAG
2400
37415
FLT1:3262U21 sense siNA stab07
B cAAGuGGccAGAGGcAuGGTT B
3170


3263
UCAAGUGGCCAGAGGCAUGGAGU
2401
37416
FLT1:3263U21 sense siNA stab07
B AAGuGGccAGAGGcAuGGATT B
3171


3266
AGUGGCCAGAGGCAUGGAGUUCC
2402
37417
FLT1:3266U21 sense siNA stab07
B uGGccAGAGGcAuGGAGuuTT B
3172


3911
GAGCCUGGAAAGAAUCAAAACCU
2403
37418
FLT1:3911U21 sense siNA stab07
B GccuGGAAAGAAucAAAAcTT B
3173


4419
UUUUUUGACUAACAAGAAUGUAA
2404
37419
FLT1:4419U21 sense siNA stab07
B uuuuGAcuAAcAAGAAuGuTT B
3174


346
CUGAACUGAGUUUAAAAGGCACC
2296
37420
FLT1:364L21 antisense siNA (346 C.)
UGCcuuuuAAAcucAGuucTT
3175






stab26


347
UGAACUGAGUUUAAAAGGCACCC
2297
37421
FLT1:365L21 antisense siNA (347 C.)
GUGccuuuuAAAcucAGuuTT
3176






stab26


349
AACUGAGUUUAAAAGGCACCCAG
2289
37422
FLT1:367L21 antisense siNA (349 C.)
GGGuGccuuuuAAAcucAGTT
3177






stab26


351
CUGAGUUUAAAAGGCACCCAGCA
2300
37423
FLT1:369L21 antisense siNA (351 C.)
CUGGGuGccuuuuAAAcucTT
3178






stab26


353
GAGUUUAAAAGGCACCCAGCACA
2302
37424
FLT1:371L21 antisense siNA (353 C.)
UGCuGGGuGccuuuuAAAcTT
3179






stab26


1956
GAAGGAGAGGACCUGAAACUGUC
2286
37425
FLT1:1974L21 antisense siNA (1956 C.)
CAGuuucAGGuccucuccuTT
3180






stab26


1957
AAGGAGAGGACCUGAAACUGUCU
2287
37426
FLT1:1975L21 antisense siNA (1957 C.)
ACAGuuucAGGuccucuccTT
3181






stab26


2338
AAAACAACCACAAAAUACAACAA
2375
37427
FLT1:2356L21 antisense siNA (2338 C.)
GUUGuAuuuuGuGGuuGuuTT
3182






stab26


2340
AACAACCACAAAAUACAACAAGA
2292
37428
FLT1:2358L21 antisense siNA (2340 C.)
UUGuuGuAuuuuGuGGuuGTT
3183






stab26


2342
CAACCACAAAAUACAACAAGAGC
2376
37429
FLT1:2360L21 antisense siNA (2342 C.)
UCUuGuuGuAuuuuGuGGuTT
3184






stab26


2365
CUGGAAUUAUUUUAGGACCAGGA
2377
37430
FLT1:2383L21 antisense siNA (2365 C.)
CUGGuccuAAAAuAAuuccTT
3185






stab26


2391
AGCACGCUGUUUAUUGAAAGAGU
2378
37431
FLT1:2409L21 antisense siNA (2391 C.)
UCUuucAAuAAAcAGcGuGTT
3186






stab26


2392
GCACGCUGUUUAUUGAAAGAGUC
2379
37432
FLT1:2410L21 antisense siNA (2392 C.)
CUCuuucAAuAAAcAGcGuTT
3187






stab26


2393
CACGCUGUUUAUUGAAAGAGUCA
2380
37433
FLT1:2411L21 antisense siNA (2393 C.)
ACUcuuucAAuAAAcAGcGTT
3188






stab26


2394
ACGCUGUUUAUUGAAAGAGUCAC
2381
37434
FLT1:2412L21 antisense siNA (2394 C.)
GACucuuucAAuAAAcAGcTT
3189






stab26


2395
CGCUGUUUAUUGAAAGAGUCACA
2382
37435
FLT1:2413L21 antisense siNA (2395 C.)
UGAcucuuucAAuAAAcAGTT
3190






stab26


2396
GCUGUUUAUUGAAAGAGUCACAG
2383
37436
FLT1:2414L21 antisense siNA (2396 C.)
GUGAcucuuucAAuAAAcATT
3191






stab26


2397
CUGUUUAUUGAAAGAGUCACAGA
2384
37437
FLT1:2415L21 antisense siNA (2397 C.)
UGUGAcucuuucAAuAAAcTT
3192






stab26


2398
UGUUUAUUGAAAGAGUCACAGAA
2385
37438
FLT1:2416L21 antisense siNA (2398 C.)
CUGuGAcucuuucAAuAAATT
3193






stab26


2697
GAUGCCAGCAAGUGGGAGUUUGC
2386
37439
FLT1:2715L21 antisense siNA (2697 C.)
AAAcucccAcuuGcuGGcATT
3194






stab26


2699
UGCCAGCAAGUGGGAGUUUGCCC
2387
37440
FLT1:2717L21 antisense siNA (2699 C.)
GCAAAcucccAcuuGcuGGTT
3195






stab26


2785
CAGCAUUUGGCAUUAAGAAAUCA
2388
37441
FLT1:2803L21 antisense siNA (2785 C.)
AUUucuuAAuGccAAAuGcTT
3196






stab26


2786
AGCAUUUGGCAUUAAGAAAUCAC
2389
37442
FLT1:2804L21 antisense siNA (2786 C.)
GAUuuCuuAAuGccAAAuGTT
3197






stab26


2787
GCAUUUGGCAUUAAGAAAUCACC
2288
37443
FLT1:2805L21 antisense siNA (2787 C.)
UGAuuucuuAAuGccAAAuTT
3198






stab26


2788
CAUUUGGCAUUAAGAAAUCACCU
2390
37444
FLT1:2806L21 antisense siNA (2788 C.)
GUGAuuucuuAAuGccAAATT
3199






stab26


2789
AUUUGGCAUUAAGAAAUCACCUA
2391
37445
FLT1:2807L21 antisense siNA (2789 C.)
GGUGAuuucuuAAuGccAATT
3200






stab26


2812
CGUGCCGGACUGUGGCUGUGAAA
2392
37446
FLT1:2830L21 antisense siNA (2812 C.)
UCAcAGccAcAGuccGGcATT
3201






stab26


2860
GCGAGUACAAAGCUCUGAUGACU
2393
37447
FLT1:2878L21 antisense siNA (2860 C.)
UCAucAGAGcuuuGuAcucTT
3202






stab26


2861
CGAGUACAAAGCUCUGAUGACUG
2394
37448
FLT1:2879L21 antisense siNA (2861 C.)
GUCAucAGAGcuuuGuAcuTT
3203






stab26


2947
CCAAGCAAGGAGGGCCUCUGAUG
2395
37449
FLT1:2965L21 antisense siNA (2947 C.)
UCAGAGGcccuccuuGcuuTT
3204






stab26


2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
37450
FLT1:2967L21 antisense siNA (2949 C.)
CAUcAGAGGcccuccuuGcTT
3205






stab26


2950
AGCAAGGAGGGCCUCUGAUGGUG
2396
37451
FLT1:2968L21 antisense siNA (2950 C.)
CCAucAGAGGcccuccuuGTT
3206






stab26


2952
CAAGGAGGGCCUCUGAUGGUGAU
2397
37452
FLT1:2970L21 antisense siNA (2952 C.)
CACcAucAGAGGcccuccuTT
3207






stab26


2953
AAGGAGGGCCUCUGAUGGUGAUU
2398
37453
FLT1:2971L21 antisense siNA (2953 C.)
UCAccAucAGAGGcccuccTT
3208






stab26


2954
AGGAGGGCCUCUGAUGGUGAUUG
2399
37454
FLT1:2972L21 antisense siNA (2954 C.)
AUCAccAucAGAGGcccucTT
3209






stab26


3262
UUCAAGUGGCCAGAGGCAUGGAG
2400
37455
FLT1:3280L21 antisense siNA (3262 C.)
CCAuGccucuGGccAcuuGTT
3210






stab26


3263
UCAAGUGGCCAGAGGCAUGGAGU
2401
37456
FLT1:3281L21 antisense siNA (3263 C.)
UCCAuGccucuGGccAcuuTT
3211






stab26


3266
AGUGGCCAGAGGCAUGGAGUUCC
2402
37457
FLT1:3284L21 antisense siNA (3266 C.)
AACuccAuGccucuGGccATT
3212






stab26


3911
GAGCCUGGAAAGAAUCAAAACCU
2403
37458
FLT1:3929L21 antisense siNA (3911 C.)
GUUuuGAuucuuuccAGGcTT
3213






stab26


4419
UUUUUUGACUAACAAGAAUGUAA
2404
37459
FLT1:4437L21 antisense siNA (4419 C.)
ACAuucuuGuuAGucAAAATT
3214






stab26


3646
UCAUGCUGGACUGCUGGCACAGA
2195
37576
FLT1:3664L21 antisense siNA (3646 C.)
UGUGCcAGcAGuccAGcAuTT
3215






stab26


349
AACUGAGUUUAAAAGGCACCCAG
2289
38285
5′CB 31270 FLT1:349U21 sense siNA
CBUGAGUUUAAAAGGCACCCTT B
3216






stab09


 







VEGFR2














3304
UGACCUUGGAGCAUCUCAUCUGU
2405

KDR:3304U21 sense siNA stab04
B AccuuGGAGcAucucAucuTT B
3217



3894
UCACCUGUUUCCUGUAUGGAGGA
2406

KDR:3894U21 sense siNA stab04
B AccuGuuuccuGuAuGGAGTT B
3218


3304
UGACCUUGGAGCAUCUCAUCUGU
2405

KDR:3322L21 antisense siNA (3304 C.)
AGAuGAGAuGcuccAAGGuTsT
3219






stab05


3894
UCACCUGUUUCCUGUAUGGAGGA
2406

KDR:3912L21 antisense siNA (3894 C.)
cuccAuAcAGGAAAcAGGuTsT
3220






stab05


3304
UGACCUUGGAGCAUCUCAUCUGU
2405

KDR:3304U21 sense siNA stab07
B AccuuGGAGcAucucAucuTT B
3221


3894
UCACCUGUUUCCUGUAUGGAGGA
2406
32766
KDR:3894U21 sense siNA stab07
B AccuGuuuccuGuAuGGAGTT B
3222


3304
UGACCUUGGAGCAUCUCAUCUGU
2405

KDR:3322L21 antisense siNA (3304 C.)
AGAuGAGAuGCuccAAGGuTsT
3223






stab11


3854
UUUGAGCAUGGAAGAGGAUUCUG
2407

KDR:3872L21 antisense siNA (3854 C.)
GAAuccucuuccAuGcucATsT
3224






stab11


3894
UCACCUGUUUCCUGUAUGGAGGA
2406

KDR:3912L21 antisense siNA (3894 C.)
cuccAuAcAGGAAAcAGGuTsT
3225






stab11


3948
GACAACACAGCAGGAAUCAGUCA
2408

KDR:3966L21 antisense siNA (3948 C.)
AcuGAuuccuGcuGuGuuGTsT
3226






stab11


3076
UGUCCACUUACCUGAGGAGCAAG
2409
30785
KDR:3076U21 sense siNA stab04
B uccAcuuAccuGAGGAGcATT B
3227


3854
UUUGAGCAUGGAAGAGGAUUCUG
2407
30786
KDR:3854U21 sense siNA stab04
B uGAGcAuGGAAGAGGAuucTT B
3228


4089
AUGGUUCUUGCCUCAGAAGAGCU
2410
30787
KDR:4089U21 sense siNA stab04
B GGuucuuGccucAGAAGAGTT B
3229


4191
UCUGAAGGCUCAAACCAGACAAG
2411
30788
KDR:4191U21 sense siNA stab04
B uGAAGGcucAAAccAGAcATT B
3230


3076
UGUCCACUUACCUGAGGAGCAAG
2409
30789
KDR:3094L21 antisense siNA (3076 C.)
uGcuccucAGGuAAGuGGATsT
3231






stab05


3854
UUUGAGCAUGGAAGAGGAUUCUG
2407
30790
KDR:3872L21 antisense siNA (3854 C.)
GAAuccucuuccAuGcucATsT
3232






stab05


4089
AUGGUUCUUGCCUCAGAAGAGCU
2410
30791
KDR:4107L21 antisense siNA (4089 C.)
cucuucuGAGGcAAGAAccTsT
3233






stab05


4191
UCUGAAGGCUCAAACCAGACAAG
2411
30792
KDR:4209L21 antisense siNA (4191 C.)
uGucuGGuuuGAGccuucATsT
3234






stab05


3076
UGUCCACUUACCUGAGGAGCAAG
2409
31426
KDR:3076U21 sense siNA
UCCACUUACCUGAGGAGCATT
3235


3854
UUUGAGCAUGGAAGAGGAUUCUG
2407
31435
KDR:3854U21 sense siNA
UGAGCAUGGAAGAGGAUUCTT
3236


4089
AUGGUUCUUGCCUCAGAAGAGCU
2410
31428
KDR:4089U21 sense siNA
GGUUCUUGCCUCAGAAGAGTT
3237


4191
UCUGAAGGCUCAAACCAGACAAG
2411
31429
KDR:4191U21 sense siNA
UGAAGGCUCAAACCAGACATT
3238


3076
UGUCCACUUACCUGAGGAGCAAG
2409
31430
KDR:3094L21 antisense siNA (3076 C.)
UGCUCCUCAGGUAAGUGGATT
3239


3854
UUUGAGCAUGGAAGAGGAUUCUG
2407
31439
KDR:3872L21 antisense siNA (3854 C.)
GAAUCCUCUUCCAUGCUCATT
3240


4089
AUGGUUCUUGCCUCAGAAGAGCU
2410
31432
KDR:4107L21 antisense siNA (4089 C.)
CUCUUCUGAGGCAAGAACCTT
3241


4191
UCUGAAGGCUCAAACCAGACAAG
2411
31433
KDR:4209L21 antisense siNA (4191 C.)
UGUCUGGUUUGAGCCUUCATT
3242


3304
UGACCUUGGAGCAUCUCAUCUGU
2405
31434
KDR:3304U21 sense siNA
ACCUUGGAGCAUCUCAUCUTT
3243


3894
UCACCUGUUUCCUGUAUGGAGGA
2406
31436
KDR:3894U21 sense siNA
ACCUGUUUCCUGUAUGGAGTT
3244


3948
GACAACACAGCAGGAAUCAGUCA
2408
31437
KDR:3948U21 sense siNA
CAACACAGCAGGAAUCAGUTT
3245


3304
UGACCUUGGAGCAUCUCAUCUGU
2405
31438
KDR:3322L21 antisense siNA (3304 C.)
AGAUGAGAUGCUCCAAGGUTT
3246


3894
UCACCUGUUUCCUGUAUGGAGGA
2406
31440
KDR:3912L21 antisense siNA (3894 C.)
CUCCAUACAGGAAACAGGUTT
3247


3948
GACAACACAGCAGGAAUCAGUCA
2408
31441
KDR:3966L21 antisense siNA (3948 C.)
ACUGAUUCCUGCUGUGUUGTT 3248


3948
GACAACACAGCAGGAAUCAGUCA
2408
31856
KDR:3948U21 sense siNA stab04
B cAAcAcAGcAGGAAucAGuTT B 3249


3948
GACAACACAGCAGGAAUCAGUCA
2408
31857
KDR:3966L21 antisense siNA (3948 C.)
AcuGAuuccuGcuGuGuuGTsT
3250






stab05


3854
UUUGAGCAUGGAAGAGGAUUCUG
2407
31858
KDR:3854U21 sense siNA stab07
B uGAGcAuGGAAGAGGAuucTT B
3251


3948
GACAACACAGCAGGAAUCAGUCA
2408
31859
KDR:3948U21 sense siNA stab07
B cAAcAcAGcAGGAAucAGuTT B 3252


3854
UUUGAGCAUGGAAGAGGAUUCUG
2407
31860
KDR:3872L21 antisense siNA (3854 C.)
GAAuccucuuccAuGcucATsT
3253






stab08


3948
GACAACACAGCAGGAAUCAGUCA
2408
31861
KDR:3966L21 antisense siNA (3948 C.)
AcuGAuuccuGcuGuGuuGTsT
3254






stab08


3854
UUUGAGCAUGGAAGAGGAUUCUG
2407
31862
KDR:3854U21 sense siNA stab09
B UGAGCAUGGAAGAGGAUUCTT B
3255


3948
GACAACACAGCAGGAAUCAGUCA
2408
31863
KDR:3948U21 sense siNA stab09
B CAACACAGCAGGAAUCAGUTT B
3256


3854
UUUGAGCAUGGAAGAGGAUUCUG
2407
31864
KDR:3872L21 antisense siNA (3854 C.)
GAAUCCUCUUCCAUGCUCATsT
3257






stab10


3948
GACAACACAGCAGGAAUCAGUCA
2408
31865
KDR:3966L21 antisense siNA (3948 C.)
ACUGAUUCCUGCUGUGUUGTsT
3258






stab10


3854
UUUGAGCAUGGAAGAGGAUUCUG
2407
31878
KDR:3854U21 sense siNA inv stab04
B cuuAGGAGAAGGuAcGAGuTT B
3259


3948
GACAACACAGCAGGAAUCAGUCA
2408
31879
KDR:3948U21 sense siNA inv stab04
B uGAcuAAGGAcGAcAcAAcTT B
3260


3854
UUUGAGCAUGGAAGAGGAUUCUG
2407
31880
KDR:3872L21 antisense siNA (3854 C.) inv
AcucGuAccuucuccuAAGTsT
3261






stab05


3948
GACAACACAGCAGGAAUCAGUCA
2408
31881
KDR:3966L21 antisense siNA (3948 C.) inv
GuuGuGucGuccuuAGucATsT
3262






stab05


3854
UUUGAGCAUGGAAGAGGAUUCUG
2407
31882
KDR:3854U21 sense siNA inv stab07
B cuuAGGAGAAGGuAcGAGuTT B
3263


3948
GACAACACAGCAGGAAUCAGUCA
2408
31883
KDR:3948U21 sense siNA inv stab07
B uGAcuAAGGAcGAcAcAAcTT B
3264


3854
UUUGAGCAUGGAAGAGGAUUCUG
2407
31884
KDR:3872L21 antisense siNA (3854 C.) inv
AcucGuAccuucuccuAAGTsT
3265






stab08


3948
GACAACACAGCAGGAAUCAGUCA
2408
31885
KDR:3966L21 antisense siNA (3948 C.) inv
GuuGuGucGuccuuAGucATsT
3266






stab08


3854
UUUGAGCAUGGAAGAGGAUUCUG
2407
31886
KDR:3854U21 sense siNA inv stab09
B CUUAGGAGAAGGUACGAGUTT B
3267


3948
GACAACACAGCAGGAAUCAGUCA
2408
31887
KDR:3948U21 sense siNA inv stab09
B UGACUAAGGACGACACAACTT B
3268


3854
UUUGAGCAUGGAAGAGGAUUCUG
2407
31888
KDR:3872L21 antisense siNA (3854 C.) inv
ACUCGUACCUUCUCCUAAGTsT
3269






stab10


3948
GACAACACAGCAGGAAUCAGUCA
2408
31889
KDR:3966L21 antisense siNA (3948 C.) inv
GUUGUGUCGUCCUUAGUCATsT
3270






stab10


2764
CCUUAUGAUGCCAGCAAAU
2412
32238
KDR:2764U21 sense siNA
CCUUAUGAUGCCAGCAAAUTT
3271


2765
CUUAUGAUGCCAGCAAAUG
2413
32239
KDR:2765U21 sense siNA
CUUAUGAUGCCAGCAAAUGTT
3272


2766
UUAUGAUGCCAGCAAAUGG
2414
32240
KDR:2766U21 sense siNA
UUAUGAUGCCAGCAAAUGGTT
3273


2767
UAUGAUGCCAGCAAAUGGG
2415
32241
KDR:2767U21 sense siNA
UAUGAUGCCAGCAAAUGGGTT
3274


2768
AUGAUGCCAGCAAAUGGGA
2416
32242
KDR:2768U21 sense siNA
AUGAUGCCAGCAAAUGGGATT
3275


3712
CAGACCAUGCUGGACUGCU
2417
32243
KDR:3712U21 sense siNA
CAGACCAUGCUGGACUGCUTT
3276


3713
AGACCAUGCUGGACUGCUG
2418
32244
KDR:3713U21 sense siNA
AGACCAUGCUGGACUGCUGTT
3277


3714
GACCAUGCUGGACUGCUGG
2419
32245
KDR:3714U21 sense siNA
GACCAUGCUGGACUGCUGGTT
3278


3715
ACCAUGCUGGACUGCUGGC
2420
32246
KDR:3715U21 sense siNA
ACCAUGCUGGACUGCUGGCTT
3279


3716
CCAUGCUGGACUGCUGGCA
2421
32247
KDR:3716U21 sense siNA
CCAUGCUGGACUGCUGGCATT
3280


3811
CAGGAUGGCAAAGACUACA
2422
32248
KDR:3811U21 sense siNA
CAGGAUGGCAAAGACUACATT
3281


3812
AGGAUGGCAAAGACUACAU
2423
32249
KDR:3812U21 sense siNA
AGGAUGGCAAAGACUACAUTT
3282


2764
CCUUAUGAUGCCAGCAAAU
2412
32253
KDR:2782L21 antisense siNA (2764 C.)
AUUUGCUGGCAUCAUAAGGTT
3283


2765
CUUAUGAUGCCAGCAAAUG
2413
32254
KDR:2783L21 antisense siNA (2765 C.)
CAUUUGCUGGCAUCAUAAGTT
3284


2766
UUAUGAUGCCAGCAAAUGG
2414
32255
KDR:2784L21 antisense siNA (2766 C.)
CCAUUUGCUGGCAUCAUAATT
3285


2767
UAUGAUGCCAGCAAAUGGG
2415
32256
KDR:2785L21 antisense siNA (2767 C.)
CCCAUUUGCUGGCAUCAUATT
3286


2768
AUGAUGCCAGCAAAUGGGA
2416
32257
KDR:2786L21 antisense siNA (2768 C.)
UCCCAUUUGCUGGCAUCAUTT
3287


3712
CAGACCAUGCUGGACUGCU
2417
32258
KDR:3730L21 antisense siNA (3712 C.)
AGCAGUCCAGCAUGGUCUGTT
3288


3713
AGACCAUGCUGGACUGCUG
2418
32259
KDR:3731L21 antisense siNA (3713 C.)
CAGCAGUCCAGCAUGGUCUTT
3289


3714
GACCAUGCUGGACUGCUGG
2419
32260
KDR:3732L21 antisense siNA (3714 C.)
CCAGCAGUCCAGCAUGGUCTT
3290


3715
ACCAUGCUGGACUGCUGGC
2420
32261
KDR:3733L21 antisense siNA (3715 C.)
GCCAGCAGUCCAGCAUGGUTT
3291


3716
CCAUGCUGGACUGCUGGCA
2421
32262
KDR:3734L21 antisense siNA (3716 C.)
UGCCAGCAGUCCAGCAUGGTT
3292


3811
CAGGAUGGCAAAGACUACA
2422
32263
KDR:3829L21 antisense siNA (3811 C.)
UGUAGUCUUUGCCAUCCUGTT
3293


3812
AGGAUGGCAAAGACUACAU
2423
32264
KDR:3830L21 antisense siNA (3812 C.)
AUGUAGUCUUUGCCAUCCUTT
3294


3304
UGACCUUGGAGCAUCUCAUCUGU
2405
32310
KDR:3304U21 sense siNA stab09
B ACCUUGGAGCAUCUCAUCUTT B
3295


3894
UCACCUGUUUCCUGUAUGGAGGA
2406
32311
KDR:3894U21 sense siNA stab09
B ACCUGUUUCCUGUAUGGAGTT B
3296


3304
UGACCUUGGAGCAUCUCAUCUGU
2405
32312
KDR:3322L21 antisense siNA (3304 C.)
AGAUGAGAUGCUCCAAGGUTsT
3297






stab10


3894
UCACCUGUUUCCUGUAUGGAGGA
2406
32313
KDR:3912L21 antisense siNA (3894 C.)
CUCCAUACAGGAAACAGGUTsT
3298






stab10


3304
UGACCUUGGAGCAUCUCAUCUGU
2405
32314
KDR:3304U21 sense siNA inv stab09
B UCUACUCUACGAGGUUCCATT B
3299


3894
UCACCUGUUUCCUGUAUGGAGGA
2406
32315
KDR:3894U21 sense siNA inv stab09
B GAGGUAUGUCCUUUGUCCATT B
3300


3304
UGACCUUGGAGCAUCUCAUCUGU
2405
32316
KDR:3322L21 antisense siNA (3304 C.)
inv
UGGAACCUCGUAGAGUAGATsT
3301






stab10


3894
UCACCUGUUUCCUGUAUGGAGGA
2406
32317
KDR:3912L21 antisense siNA (3894 C.)
inv
UGGACAAAGGACAUACCUCTsT
3302






stab10


828
AACAGAAUUUCCUGGGACAGCAA
2424
32762
KDR:828U21 sense siNA stab07
B cAGAAuuuccuGGGAcAGcTT B
3303


3310
UGGAGCAUCUCAUCUGUUACAGC
2425
32763
KDR:3310U21 sense siNA stab07
B GAGcAucucAucuGuuAcATT B
3304


3758
CACGUUUUCAGAGUUGGUGGAAC
2426
32764
KDR:3758U21 sense siNA stab07
B cGuuuucAGAGuuGGuGGATT B
3305


3893
CUCACCUGUUUCCUGUAUGGAGG
2427
32765
KDR:3893U21 sense siNA stab07
B cAccuGuuuccuGuAuGGATT B
3306


828
AACAGAAUUUCCUGGGACAGCAA
2424
32767
KDR:846L21 antisense siNA (828 C.) stab08
GcuGucccAGGAAAuucuGTsT
3307


3310
UGGAGCAUCUCAUCUGUUACAGC
2425
32768
KDR:3328L21 antisense siNA (3310 C.)
uGuAAcAGAuGAGAuGcucTsT
3308






stab08


3758
CACGUUUUCAGAGUUGGUGGAAC
2426
32769
KDR:3776L21 antisense siNA (3758 C.)
uccAccAAcucuGAAAAcGTsT
3309






stab08


3893
CUCACCUGUUUCCUGUAUGGAGG
2427
32770
KDR:3911L21 antisense siNA (3893 C.)
uccAuAcAGGAAAcAGGuGTsT
3310






stab08


3894
UCACCUGUUUCCUGUAUGGAGGA
2406
32771
KDR:3912L21 antisense siNA (3894 C.)
cucCAuAcAGGAAAcAGGuTsT
3311






stab08


828
AACAGAAUUUCCUGGGACAGCAA
2424
32786
KDR:828U21 sense siNA inv stab07
B cGAcAGGGuccuuuAAGAcTT B
3312


3310
UGGAGCAUCUCAUCUGUUACAGC
2425
32787
KDR:3310U21 sense siNA inv stab07
B AcAuuGucuAcucuAcGAGTT B
3313


3758
CACGUUUUCAGAGUUGGUGGAAC
2426
32788
KDR:3758U21 sense siNA inv stab07
B AGGuGGuuGAGAcuuuuGcTT B
3314


3893
CUCACCUGUUUCCUGUAUGGAGG
2427
32789
KDR:3893U21 sense siNA inv stab07
B AGGuAuGuccuuuGuccAcTT B
3315


3894
UCACCUGUUUCCUGUAUGGAGGA
2406
32790
KDR:3894U21 sense siNA inv stab07
B GAGGuAuGuccuuuGuccATT B
3316


828
AACAGAAUUUCCUGGGACAGCAA
2424
32791
KDR:846L21 antisense siNA (828 C.) inv
GucuuAAAGGAcccuGucGTsT
3317






stab08


3310
UGGAGCAUCUCAUCUGUUACAGC
2425
32792
KDR:3328L21 antisense siNA (3310 C.) inv
CucGuAGAGuAGAcAAuGuTsT
3318






stab08


3758
CACGUUUUCAGAGUUGGUGGAAC
2426
32793
KDR:3776L21 antisense siNA (3758 C.) inv
GcAAAAGucucAAccAccuTsT
3319






stab08


3893
CUCACCUGUUUCCUGUAUGGAGG
2427
32794
KDR:3911L21 antisense siNA (3893 C.) inv
GuGGAcAAAGGAcAuAccuTsT
3320






stab08


3894
UCACCUGUUUCCUGUAUGGAGGA
2406
32795
KDR:3912L21 antisense siNA (3894 C.) inv
uGGAcAAAGGAcAuAccucTsT
3321






stab08


828
AACAGAAUUUCCUGGGACAGCAA
2424
32958
KDR:828U21 sense siNA stab09
B CAGAAUUUCCUGGGACAGCTT B
3322


3310
UGGAGCAUCUCAUCUGUUACAGC
2425
32959
KDR:3310U21 sense siNA stab09
B GAGCAUCUCAUCUGUUACATT B
3323


3758
CACGUUUUCAGAGUUGGUGGAAC
2426
32960
KDR:3758U21 sense siNA stab09
B CGUUUUCAGAGUUGGUGGATT B
3324


3893
CUCACCUGUUUCCUGUAUGGAGG
2427
32961
KDR:3893U21 sense siNA stab09
B CACCUGUUUCCUGUAUGGATT B
3325


828
AACAGAAUUUCCUGGGACAGCAA
2424
32963
KDR:846L21 antisense siNA (828 C.) stab10
GCUGUCCCAGGAAAUUCUGTsT
3326


3310
UGGAGCAUCUCAUCUGUUACAGC
2425
32964
KDR:3328L21 antisense siNA (3310 C.)
UGUAACAGAUGAGAUGCUCTsT
3327






stab10


3758
CACGUUUUCAGAGUUGGUGGAAC
2426
32965
KDR:3776L21 antisense siNA (3758 C.)
UCCACCAACUCUGAAAACGTsT
3328






stab10


3893
CUCACCUGUUUCCUGUAUGGAGG
2427
32966
KDR:3911L21 antisense siNA (3893 C.)
UCCAUACAGGAAACAGGUGTsT
3329






stab10


828
AACAGAAUUUCCUGGGACAGCAA
2424
32988
KDR:828U21 sense siNA inv stab09
B CGACAGGGUCCUUUAAGACTT B
3330


3310
UGGAGCAUCUCAUCUGUUACAGC
2425
32989
KDR:3310U21 sense siNA inv stab09
B ACAUUGUCUACUCUACGAGTT B
3331


3758
CACGUUUUCAGAGUUGGUGGAAC
2426
32990
KDR:3758U21 sense siNA inv stab09
B AGGUGGUUGAGACUUUUGCTT B
3332


3893
CUCACCUGUUUCCUGUAUGGAGG
2427
32991
KDR:3893U21 sense siNA inv stab09
B AGGUAUGUCCUUUGUCCACTT B
3333


828
AACAGAAUUUCCUGGGACAGCAA
2424
32993
KDR:846L21 antisense siNA (828 C.) inv
GUCUUAAAGGACCCUGUCGTsT
3334






stab10


3310
UGGAGCAUCUCAUCUGUUACAGC
2425
32994
KDR:3328L21 antisense siNA (3310 C.) inv
CUCGUAGAGUAGACAAUGUTsT
3335






stab10


3758
CACGUUUUCAGAGUUGGUGGAAC
2426
32995
KDR:3776L21 antisense siNA (3758 C.) inv
GCAAAAGUCUCAACCACCUTsT
3336






stab10


3893
CUCACCUGUUUCCUGUAUGGAGG
2427
32996
KDR:3911L21 antisense siNA (3893 C.) inv
GUGGACAAAGGACAUACCUTsT
3337






stab10


2767
CUUAUGAUGCCAGCAAAUGGGAA
2218
33727
KDR:2767U21 sense siNA stab07
B uAuGAuGccAGcAAAuGGGTT B
3338


2768
UUAUGAUGCCAGCAAAUGGGAAU
2222
33728
KDR:2768U21 sense siNA stab07
B AuGAuGccAGcAAAuGGGATT B
3339


3715
AGACCAUGCUGGACUGCUGGCAC
2241
33729
KDR:3715U21 sense siNA stab07
B AccAuGcuGGAcuGcuGGcTT B
3340


3716
GACCAUGCUGGACUGCUGGCACG
2247
33730
KDR:3716U21 sense siNA stab07
B ccAuGcuGGAcuGcuGGcATT B
3341


2767
CUUAUGAUGCCAGCAAAUGGGAA
2218
33733
KDR:2785L21 antisense siNA (2767 C.)
cccAuuuGcuGGcAucAuATsT
3342






stab08


2768
UUAUGAUGCCAGCAAAUGGGAAU
2222
33734
KDR:2786L21 antisense siNA (2768 C.)
ucccAuuuGcuGGcAucAuTsT
3343






stab08


3715
AGACCAUGCUGGACUGCUGGCAC
2241
33735
KDR:3733L21 antisense siNA (3715 C.)
GccAGcAGuccAGcAuGGuTsT
3344






stab08


3716
GACCAUGCUGGACUGCUGGCACG
2247
33736
KDR:3734L21 antisense siNA (3716 C.)
uGccAGcAGuccAGcAuGGTsT
3345






stab08


2767
CUUAUGAUGCCAGCAAAUGGGAA
2218
33739
KDR:2767U21 sense siNA stab09
B UAUGAUGCCAGCAAAUGGGTT B
3346


2768
UUAUGAUGCCAGCAAAUGGGAAU
2222
33740
KDR:2768U21 sense siNA stab09
B AUGAUGCCAGCAAAUGGGATT B
3347


3715
AGACCAUGCUGGACUGGUGGCAC
2241
33741
KDR:3715U21 sense siNA stab09
B ACCAUGCUGGACUGCUGGCTT B
3348


3716
GACCAUGCUGGACUGCUGGCACG
2247
33742
KDR:3716U21 sense siNA stab09
B CCAUGCUGGACUGCUGGCATT B
3349


2767
CUUAUGAUGCCAGCAAAUGGGAA
2218
33745
KDR:2785L21 antisense siNA (2767 C.)
CCCAUUUGCUGGCAUCAUATsT
3350






stab10


2768
UUAUGAUGCCAGCAAAUGGGAAU
2222
33746
KDR:2786L21 antisense siNA (2768 C.)
UCCCAUUUGCUGGCAUCAUTsT
3351






stab10


3715
AGACCAUGCUGGACUGCUGGCAC
2241
33747
KDR:3733L21 antisense siNA (3715 C.)
GCCAGCAGUCCAGCAUGGUTsT
3352






stab10


3716
GACCAUGCUGGACUGCUGGCACG
2247
33748
KDR:3734L21 antisense siNA (3716 C.)
UGCCAGCAGUCCAGCAUGGTsT
3353






stab10


2767
CUUAUGAUGCCAGCAAAUGGGAA
2218
33751
KDR:2767U21 sense siNA inv stab07
B GGGuAAAcGAccGuAGuAuTT B
3354


2768
UUAUGAUGCCAGCAAAUGGGAAU
2222
33752
KDR:2768U21 sense siNA inv stab07
B AGGGuAAAcGAccGuAGuATT B
3355


3715
AGACCAUGCUGGACUGCUGGCAC
2241
33753
KDR:3715U21 sense siNA inv stab07
B cGGucGucAGGucGuAccATT B
3356


3716
GACCAUGCUGGACUGCUGGCACG
2247
33754
KDR:3716U21 sense siNA inv stab07
B AcGGucGucAGGucGuAccTT B
3357


2767
CUUAUGAUGCCAGCAAAUGGGAA
2218
33757
KDR:2785L21 antisense siNA (2767 C.) inv
AuAcuAcGGucGuuuAcccTsT
3358






stab08


2768
UUAUGAUGCCAGCAAAUGGGAAU
2222
33758
KDR:2786L21 antisense siNA (2768 C.) inv
uAcuAcGGucGuuuAcccuTsT
3359






stab08


3715
AGACCAUGCUGGACUGCUGGCAC
2241
33759
KDR:3733L21 antisense siNA (3715 C.) inv
uGGuAcGAccuGAcGAccGTsT
3360






stab08


3716
GACCAUGCUGGACUGCUGGCACG
2247
33760
KDR:3734L21 antisense siNA (3716 C.) inv
GGuAcGAccuGAcGAccGuTsT
3361






stab08


2767
CUUAUGAUGCCAGCAAAUGGGAA
2218
33763
KDR:2767U21 sense siNA inv stab09
B GGGUAAACGACCGUAGUAUTT B
3362


2768
UUAUGAUGCCAGCAAAUGGGAAU
2222
33764
KDR:2768U21 sense siNA inv stab09
B AGGGUAAACGACCGUAGUATT B
3363


3715
AGACCAUGCUGGACUGCUGGCAC
2241
33765
KDR:3715U21 sense siNA inv stab09
B CGGUCGUCAGGUCGUACCATT B
3364


3716
GACCAUGCUGGACUGCUGGCACG
2247
33766
KDR:3716U21 sense siNA inv stab09
B ACGGUCGUCAGGUCGUACCTT B
3365


2767
CUUAUGAUGCCAGCAAAUGGGAA
2218
33769
KDR:2785L21 antisense siNA (2767 C.) inv
AUACUACGGUCGUUUACCCTsT
3366






stab10


2768
UUAUGAUGCCAGCAAAUGGGAAU
2222
33770
KDR:2786L21 antisense siNA (2768 C.) inv
UACUACGGUCGUUUACCCUTsT
3367






stab10


3715
AGACCAUGCUGGACUGCUGGCAC
2241
33771
KDR:3733L21 antisense siNA (3715 C.) inv
UGGUACGACCUGACGACCGTsT
3368






stab10


3716
GACCAUGCUGGACUGCUGGCACG
2247
33772
KDR:3734L21 antisense siNA (3716 C.) inv
GGUACGACCUGACGACCGUTsT
3369






stab10


3715
AGACCAUGCUGGACUGCUGGCAC
2241
34502
KDR:3733L21 antisense siNA (3715 C.)
GccAGcAGuccAGcAuGGuTTB
3370






stab19


3715
AGACCAUGCUGGACUGCUGGCAC
2241
34503
KDR:3733L21 antisense siNA (3715 C.)
GccAGcAGuccAGcAuGGU
3371






stab08 Blunt


3715
AGACCAUGCUGGACUGCUGGCAC
2241
34504
KDR:3733L21 antisense siNA (3715 C.)
inv
uGGuAcGAccuGAcGAccGTTB
3372






stab19


3715
AGACCAUGCUGGACUGCUGGCAC
2241
34505
KDR:3733L21 anhisense siNA (3715 C.)
inv
uGGuAcGAccuGAcGAccG
3373






stab08 Blunt


503
UCAGAGUGGCAGUGAGCAAAGGG
2428
34680
KDR:503U21 sense siNA stab00
AGAGUGGCAGUGAGCAAAGTT
3374


503
UCAGAGUGGCAGUGAGCAAAGGG
2428
34688
KDR:521L21 (503 C.) siRNA stab00
CUUUGCUCACUGCCACUCUTT
3375


3715
AGACCAUGCUGGACUGCUGGCAC
2241
35124
KDR:3715U21 sense siNA stab04
B AccAuGcuGGAcuGcuGGcTT B
3376


3715
AGACCAUGCUGGACUGCUGGCAC
2241
35125
KDR:3715U21 sense siNA stab07 N1
B AccAuGcuGGAcuGCUGGCTT B
3377


3715
AGACCAUGCUGGACUGCUGGCAC
2241
35126
KDR:3733L21 antisense siNA (3715 C.)
GCCAGCAGuccAGcAuGGuTsT
3378






stab08 N1


3715
AGACCAUGCUGGACUGCUGGCAC
2241
35127
KDR:3733L21 antisense siNA (3715 C.)
GCCAGcAGuccAGcAuGGuTsT
3379






stab08 N2


3715
AGACCAUGCUGGACUGCUGGCAC
2241
35128
KDR:3733L21 antisense siNA (3715 C.)
GCCAGcAGuccAGcAuGGuTsT
3380






stab08 N3


3715
AGACCAUGCUGGACUGCUGGCAC
2241
35129
KDR:3733L21 antisense siNA (3715 C.)
GCCAGcAGuccAGcAuGGuTsT
3381






stab25


3715
AGACCAUGCUGGACUGCUGGCAC
2241
35130
KDR:3733L21 antisense siNA (3715 C.)
GCcAGcAGuccAGcAuGGuTsT
3382






stab08 N5


3715
AGACCAUGCUGGACUGCUGGCAC
2241
35131
KDR:3733L21 antisense siNA (3715 C.)
GccAGcAGuccAGcAuGGuTsT
3383






stab24


83
CCGCAGAAAGUCCGUCUGGCAGC
2429
36280
KDR:83U21 sense siNA stab00
GCAGAAAGUCCGUCUGGCATT
3384


84
CGCAGAAAGUCCGUCUGGCAGCC
2430
36281
KDR:84U21 sense siNA stab00
CAGAAAGUCCGUCUGGCAGTT
3385


85
GCAGAAAGUCCGUCUGGCAGCCU
2431
36282
KDR:85U21 sense siNA stab00
AGAAAGUCCGUCUGGCAGCTT
3386


99
UGGCAGCCUGGAUAUCCUCUCCU
2432
36283
KDR:99U21 sense siNA stab00
GCAGCCUGGAUAUCCUCUCTT
3387


100
GGCAGCCUGGAUAUCCUCUCCUA
2433
36284
KDR:100U21 sense siNA stab00
CAGCCUGGAUAUCCUCUCCTT
3388


161
CCCGGGCUCCCUAGCCCUGUGCG
2434
36285
KDR:161U21 sense siNA stab00
CGGGCUCCCUAGCCCUGUGTT
3389


162
CCGGGCUCCCUAGCCCUGUGCGC
2435
36286
KDR:162U21 sense siNA stab00
GGGCUCCCUAGCCCUGUGCTT
3390


229
CCUCCUUCUCUAGACAGGCGCUG
2436
36287
KDR:229U21 sense siNA stab00
UCCUUCUCUAGACAGGCGCTT
3391


230
CUCCUUCUCUAGACAGGCGCUGG
2437
36288
KDR:230U21 sense siNA stab00
CCUUCUCUAGACAGGCGCUTT
3392


231
UCCUUCUCUAGACAGGCGCUGGG
2438
36289
KDR:231U21 sense siNA stab00
CUUCUCUAGACAGGCGCUGTT
3393


522
AGGGUGGAGGUGACUGAGUGCAG
2439
36290
KDR:522U21 sense siNA stab00
GGUGGAGGUGACUGAGUGCTT
3394


523
GGGUGGAGGUGACUGAGUGCAGC
2440
36291
KDR:523U21 sense siNA stab00
GUGGAGGUGACUGAGUGCATT
3395


888
GCUGGCAUGGUCUUCUGUGAAGC
2441
36292
KDR:888U21 sense siNA stab00
UGGCAUGGUCUUCUGUGAATT
3396


889
CUGGCAUGGUCUUCUGUGAAGCA
2442
36293
KDR:889U21 sense siNA stab00
GGCAUGGUCUUCUGUGAAGTT
3397


905
UGAAGCAAAAAUUAAUGAUGAAA
2443
36294
KDR:905U21 sense siNA stab00
AAGCAAAAAUUAAUGAUGATT
3398


906
GAAGCAAAAAUUAAUGAUGAAAG
2444
36295
KDR:906U21 sense siNA stab00
AGCAAAAAUUAAUGAUGAATT
3399


1249
CCAAGAAGAACAGCACAUUUGUC
2445
36296
KDR:1249U21 sense siNA stab00
AAGAAGAACAGCACAUUUGTT
3400


1260
AGCACAUUUGUCAGGGUCCAUGA
2446
36297
KDR:1260U21 sense siNA stab00
CACAUUUGUCAGGGUCCAUTT
3401


1305
AGUGGCAUGGAAUCUCUGGUGGA
2447
36298
KDR:1305U21 sense siNA stab00
UGGCAUGGAAUCUCUGGUGTT
3402


1315
AAUCUCUGGUGGAAGCCACGGUG
2448
36299
KDR:1315U21 sense siNA stab00
UCUCUGGUGGAAGCCACGGTT
3403


1541
GGUCUCUCUGGUUGUGUAUGUCC
2449
36300
KDR:1541U21 sense siNA stab00
UCUCUCUGGUUGUGUAUGUTT
3404


1542
GUCUCUCUGGUUGUGUAUGUCCC
2450
36301
KDR:1542U21 sense siNA stab00
CUCUCUGGUUGUGUAUGUCTT
3405


1588
UAAUCUCUCCUGUGGAUUCCUAC
2451
36302
KDR:1588U21 sense siNA stab00
AUCUCUCCUGUGGAUUCCUTT
3406


1589
AAUCUCUCCUGUGGAUUCCUACC
2452
36303
KDR:1589U21 sense siNA stab00
UCUCUCCUGUGGAUUCCUATT
3407


1875
GUGUCAGCUUUGUACAAAUGUGA
2453
36304
KDR:1875U21 sense siNA stab00
GUCAGCUUUGUACAAAUGUTT
3408


2874
GACAAGACAGCAACUUGCAGGAC
2454
36305
KDR:2874U21 sense siNA stab00
CAAGACAGCAACUUGCAGGTT
3409


2875
ACAAGACAGCAACUUGCAGGACA
2455
36306
KDR:2875U21 sense siNA stab00
AAGACAGCAACUUGCAGGATT
3410


2876
CAAGACAGCAACUUGCAGGACAG
2456
36307
KDR:2876U21 sense siNA stab00
AGACAGCAACUUGCAGGACTT
3411


3039
CUCAUGGUGAUUGUGGAAUUCUG
2457
36308
KDR:3039U21 sense siNA stab00
CAUGGUGAUUGUGGAAUUCTT
3412


3040
UCAUGGUGAUUGUGGAAUUCUGC
2458
36309
KDR:3040U21 sense siNA stab00
AUGGUGAUUGUGGAAUUCUTT
3413


3249
UCCCUCAGUGAUGUAGAAGAAGA
2459
36310
KDR:3249U21 sense siNA stab00
CCUCAGUGAUGUAGAAGAATT
3414


3263
AGAAGAAGAGGAAGCUCCUGAAG
2460
36311
KDR:3263U21 sense siNA stab00
AAGAAGAGGAAGCUCCUGATT
3415


3264
GAAGAAGAGGAAGCUCCUGAAGA
2461
36312
KDR:3264U21 sense siNA stab00
AGAAGAGGAAGCUCCUGAATT
3416


3269
AGAGGAAGCUCCUGAAGAUCUGU
2462
36313
KDR:3269U21 sense siNA stab00
AGGAAGCUCCUGAAGAUCUTT
3417


3270
GAGGAAGCUCCUGAAGAUCUGUA
2463
36314
KDR:3270U21 sense siNA stab00
GGAAGCUCCUGAAGAUCUGTT
3418


3346
AGGGCAUGGAGUUCUUGGCAUCG
2464
36315
KDR:3346U21 sense siNA stab00
GGCAUGGAGUUCUUGGCAUTT
3419


3585
UUGCUGUGGGAAAUAUUUUCCUU
2465
36316
KDR:3585U21 sense siNA stab00
GCUGUGGGAAAUAUUUUCCTT
3420


3586
UGCUGUGGGAAAUAUUUUCCUUA
2466
36317
KDR:3586U21 sense siNA stab00
CUGUGGGAAAUAUUUUCCUTT
3421


3860
CAUGGAAGAGGAUUCUGGACUCU
2467
36318
KDR:3860U21 sense siNA stab00
UGGAAGAGGAUUCUGGACUTT
3422


3877
GACUCUCUCUGCCUACCUCACCU
2468
36319
KDR:3877U21 sense siNA stab00
CUCUCUCUGCCUACCUCACTT
3423


3878
ACUCUCUCUGCCUACCUCACCUG
2469
36320
KDR:3878U21 sense siNA stab00
UCUCUCUGCCUACCUCACCTT
3424


4287
AAGCUGAUAGAGAUUGGAGUGCA
2470
36321
KDR:4287U21 sense siNA stab00
GCUGAUAGAGAUUGGAGUGTT
3425


4288
AGCUGAUAGAGAUUGGAGUGCAA
2471
36322
KDR:4288U21 sense siNA stab00
CUGAUAGAGAUUGGAGUGCTT
3426


4318
GCACAGCCCAGAUUCUCCAGCCU
2472
36323
KDR:4318U21 sense siNA stab00
ACAGCCCAGAUUCUCCAGCTT
3427


4319
CACAGCCCAGAUUCUCCAGCCUG
2473
36324
KDR:4319U21 sense siNA stab00
CAGCCCAGAUUCUCCAGCCTT
3428


4320
ACAGCCCAGAUUCUCCAGCCUGA
2474
36325
KDR:4320U21 sense siNA stab00
AGCCCAGAUUCUCCAGCCUTT
3429


4321
CAGCCCAGAUUCUCCAGCCUGAC
2475
36326
KDR:4321U21 sense siNA stab00
GCCCAGAUUCUCCAGCCUGTT
3430


4359
AGCUCUCCUCCUGUUUAAAAGGA
2476
36327
KDR:4359U21 sense siNA stab00
CUCUCCUCCUGUUUAAAAGTT
3431


4534
UAUCCUGGAAGAGGCUUGUGACC
2477
36328
KDR:4534U21 sense siNA stab00
UCCUGGAAGAGGCUUGUGATT
3432


4535
AUCCUGGAAGAGGCUUGUGACCC
2478
36329
KDR:4535U21 sense siNA stab00
CCUGGAAGAGGCUUGUGACTT
3433


4536
UCCUGGAAGAGGCUUGUGACCCA
2479
36330
KDR:4536U21 sense siNA stab00
CUGGAAGAGGCUUGUGACCTT
3434


4539
UGGAAGAGGCUUGUGACCCAAGA
2480
36331
KDR:4539U21 sense siNA stab00
GAAGAGGCUUGUGACCCAATT
3435


4769
UGUUGAAGAUGGGAAGGAUUUGC
2481
36332
KDR:4769U21 sense siNA stab00
UUGAAGAUGGGAAGGAUUUTT
3436


4934
UCUGGUGGAGGUGGGCAUGGGGU
2482
36333
KDR:4934U21 sense siNA stab00
UGGUGGAGGUGGGCAUGGGTT
3437


5038
UCGUUGUGCUGUUUCUGACUCCU
2483
36334
KDR:5038U21 sense siNA stab00
GUUGUGCUGUUUCUGACUCTT
3438


5039
CGUUGUGCUGUUUCUGACUCCUA
2484
36335
KDR:5039U21 sense siNA stab00
UUGUGCUGUUUCUGACUCCTT
3439


5040
GUUGUGCUGUUUCUGACUCCUAA
2485
36336
KDR:5040U21 sense siNA stab00
UGUGCUGUUUCUGACUCCUTT
3440


5331
UCAAAGUUUCAGGAAGGAUUUUA
2486
36337
KDR:5331U21 sense siNA stab00
AAAGUUUCAGGAAGGAUUUTT
3441


5332
CAAAGUUUCAGGAAGGAUUUUAC
2487
36338
KDR:5332U21 sense siNA stab00
AAGUUUCAGGAAGGAUUUUTT
3442


5333
AAAGUUUCAGGAAGGAUUUUACC
2488
36339
KDR:5333U21 sense siNA stab00
AGUUUCAGGAAGGAUUUUATT
3443


5587
UCAAAAAAGAAAAUGUGUUUUUU
2489
36340
KDR:5587U21 sense siNA stab00
AAAAAAGAAAAUGUGUUUUTT
3444


5737
CUAUUCACAUUUUGUAUCAGUAU
2490
36341
KDR:5737U21 sense siNA stab00
AUUCACAUUUUGUAUCAGUTT
3445


5738
UAUUCACAUUUUGUAUCAGUAUU
2491
36342
KDR:5738U21 sense siNA stab00
UUCACAUUUUGUAUCAGUATT
3446


5739
AUUCACAUUUUGUAUCAGUAUUA
2492
36343
KDR:5739U21 sense siNA stab00
UCACAUUUUGUAUCAGUAUTT
3447


83
CCGCAGAAAGUCCGUCUGGCAGC
2429
36344
KDR:101L21 antisense siNA (83 C.) stab00
UGCCAGACGGACUUUCUGCTT
3448


84
CGCAGAAAGUCCGUCUGGCAGCC
2430
36345
KDR:102L21 antisense siNA (84 C.) stab00
CUGCCAGACGGACUUUCUGTT
3449


85
GCAGAAAGUCCGUCUGGCAGCCU
2431
36346
KDR:103L21 antisense siNA (85 C.) stab00
GCUGCCAGACGGACUUUCUTT
3450


99
UGGCAGCCUGGAUAUCCUCUCCU
2432
36347
KDR:117L21 antisense siNA (99 C.) stab00
GAGAGGAUAUCCAGGCUGCTT
3451


100
GGCAGCCUGGAUAUCCUCUCCUA
2433
36348
KDR:118L21 antisense siNA (10O C.) stab00
GGAGAGGAUAUCCAGGCUGTT
3452


161
CCCGGGCUCCCUAGCCCUGUGCG
2434
36349
KDR:179L21 antisense siNA (161 C.) stab00
CACAGGGCUAGGGAGCCCGTT
3453


162
CCGGGCUCCCUAGCCCUGUGCGC
2435
36350
KDR:180L21 antisense siNA (162 C.) stab00
GCACAGGGCUAGGGAGCCCTT
3454


229
CCUCCUUCUCUAGACAGGCGCUG
2436
36351
KDR:247L21 antisense siNA (229 C.) stab00
GCGCCUGUCUAGAGAAGGATT
3455


230
CUCCUUCUCUAGACAGGCGCUGG
2437
36352
KDR:248L21 antisense siNA (230 C.) stab00
AGCGCCUGUCUAGAGAAGGTT
3456


231
UCCUUCUCUAGACAGGCGCUGGG
2438
36353
KDR:249L21 antisense siNA (231 C.) stab00
CAGCGCCUGUCUAGAGAAGTT
3457


522
AGGGUGGAGGUGACUGAGUGCAG
2439
36354
KDR:540L21 antisense siNA (522 C.) stab00
GCACUCAGUCACCUCCACCTT
3458


523
GGGUGGAGGUGACUGAGUGCAGC
2440
36355
KDR:541L21 antisense siNA (523 C.) stab00
UGCACUCAGUCACCUCCACTT
3459


888
GCUGGCAUGGUCUUCUGUGAAGC
2441
36356
KDR:906L21 antisense siNA (888 C.) stab00
UUCACAGAAGACCAUGCCATT
3460


889
CUGGCAUGGUCUUCUGUGAAGCA
2442
36357
KDR:907L21 antisense siNA (889 C.) stab00
CUUCACAGAAGACCAUGCCTT
3461


905
UGAAGCAAAAAUUAAUGAUGAAA
2443
36358
KDR:923L21 antisense siNA (905 C.) stab00
UCAUCAUUAAUUUUUGCUUTT
3462


906
GAAGCAAAAAUUAAUGAUGAAAG
2444
36359
KDR:924L21 antisense siNA (906 C.) stab00
UUCAUCAUUAAUUUUUGCUTT
3463


1249
CCAAGAAGAACAGCACAUUUGUC
2445
36360
KDR:1267L21 antisense siNA (1249 C.)
CAAAUGUGCUGUUCUUCUUTT
3464






stab00


126
CAGCACAUUUGUCAGGGUCCAUGA
2446
36361
KDR:1278L21 antisense siNA (1260 C.)
AUGGACCCUGACAAAUGUGTT
3465






stab00


1305
AGUGGCAUGGAAUCUCUGGUGGA
2447
36362
KDR:1323L21 antisense siNA (1305 C.)
CACCAGAGAUUCCAUGCCATT
3466






stab00


1315
AAUCUCUGGUGGAAGCCACGGUG
2448
36363
KDR:1333L21 antisense siNA (1315 C.)
CCGUGGCUUCCACCAGAGATT
3467






stab00


1541
GGUCUCUCUGGUUGUGUAUGUCC
2449
36364
KDR:1559L21 antisense siNA (1541 C.)
ACAUACACAACCAGAGAGATT
3468






stab00


1542
GUCUCUCUGGUUGUGUAUGUCCC
2450
36365
KDR:1560L21 antisense siNA (1542 C.)
GACAUACACAACCAGAGAGTT
3469






stab00


1588
UAAUCUCUCCUGUGGAUUCCUAC
2451
36366
KDR:1606L21 antisense siNA (1588 C.)
AGGAAUCCACAGGAGAGAUTT
3470






stab00


1589
AAUCUCUCCUGUGGAUUCCUACC
2452
36367
KDR:1607L21 antisense siNA (1589 C.)
UAGGAAUCCACAGGAGAGATT
3471






stab00


1875
GUGUCAGCUUUGUACAAAUGUGA
2453
36368
KDR:1893L21 antisense siNA (1875 C.)
ACAUUUGUACAAAGCUGACTT
3472






stab00


2874
GACAAGACAGCAACUUGCAGGAC
2454
36369
KDR:2892L21 antisense siNA (2874 C.)
CCUGCAAGUUGCUGUCUUGTT
3473






stab00


2875
ACAAGACAGCAACUUGCAGGACA
2455
36370
KDR:2893L21 antisense siNA (2875 C.)
UCCUGCAAGUUGCUGUCUUTT
3474






stab00


2876
CAAGACAGCAACUUGCAGGACAG
2456
36371
KDR:2894L21 antisense siNA (2876 C.)
GUCCUGCAAGUUGCUGUCUTT
3475






stab00


3039
CUCAUGGUGAUUGUGGAAUUCUG
2457
36372
KDR:3057L21 antisense siNA (3039 C.)
GAAUUCCACAAUCACCAUGTT
3476






stab00


3040
UCAUGGUGAUUGUGGAAUUCUGC
2458
36373
KDR:3058L21 antisense siNA (3040 C.)
AGAAUUCCACAAUCACCAUTT
3477






stab00


3249
UCCCUCAGUGAUGUAGAAGAAGA
2459
36374
KDR:3267L21 antisense siNA (3249 C.)
UUCUUCUACAUCACUGAGGTT
3478






stab00


3263
AGAAGAAGAGGAAGCUCCUGAAG
2460
36375
KDR:3281L21 antisense siNA (3263 C.)
UCAGGAGCUUCCUCUUCUUTT
3479






stab00


3264
GAAGAAGAGGAAGCUCCUGAAGA
2461
36376
KDR:3282L21 antisense siNA (3264 C.)
UUCAGGAGCUUCCUCUUCUTT
3480






stab00


3269
AGAGGAAGCUCCUGAAGAUCUGU
2462
36377
KDR:3287L21 antisense siNA (3269 C.)
AGAUCUUCAGGAGCUUCCUTT
3481






stab00


3270
GAGGAAGCUCCUGAAGAUCUGUA
2463
36378
KDR:3288L21 antisense siNA (3270 C.)
CAGAUCUUCAGGAGCUUCCTT
3482






stab00


3346
AGGGCAUGGAGUUCUUGGCAUCG
2464
36379
KDR:3364L21 antisense siNA (3346 C.)
AUGCCAAGAACUCCAUGCCTT
3483






stab00


3585
UUGCUGUGGGAAAUAUUUUCCUU
2465
36380
KDR:3603L21 antisense siNA (3585 C.)
GGAAAAUAUUUCCCACAGCTT
3484






stab00


3586
UGCUGUGGGAAAUAUUUUCCUUA
2466
36381
KDR:3604L21 antisense siNA (3586 C.)
AGGAAAAUAUUUCCCACAGTT
3485






stab00


3860
CAUGGAAGAGGAUUCUGGACUCU
2467
36382
KDR:3878L21 antisense siNA (3860 C.)
AGUCCAGAAUCCUCUUCCATT
3486






stab00


3877
GACUCUCUCUGCCUACCUCACCU
2468
36383
KDR:3895L21 antisense siNA (3877 C.)
GUGAGGUAGGCAGAGAGAGTT
3487






stab00


3878
ACUCUCUCUGCCUACCUCACCUG
2469
36384
KDR:3896L21 antisense siNA (3878 C.)
GGUGAGGUAGGCAGAGAGATT
3488






stab00


4287
AAGCUGAUAGAGAUUGGAGUGCA
2470
36385
KDR:4305L21 antisense siNA (4287 C.)
CACUCCAAUCUCUAUCAGCTT
3489






stab00


4288
AGCUGAUAGAGAUUGGAGUGCAA
2471
36386
KDR:4306L21 antisense siNA (4288 C.)
GCACUCCAAUCUCUAUCAGTT
3490






stab00


4318
GCACAGCCCAGAUUCUCCAGCCU
2472
36387
KDR:4336L21 antisense siNA (4318 C.)
GCUGGAGAAUCUGGGCUGUTT
3491






stab00


4319
CACAGCCCAGAUUCUCCAGCCUG
2473
36388
KDR:4337L21 antisense siNA (4319 C.)
GGCUGGAGAAUCUGGGCUGTT
3492






stab00


4320
ACAGCCCAGAUUCUCCAGCCUGA
2474
36389
KDR:4338L21 antisense siNA (4320 C.)
AGGCUGGAGAAUCUGGGCUTT
3493






stab00


4321
CAGCCCAGAUUCUCCAGCCUGAC
2475
36390
KDR:4339L21 antisense siNA (4321 C.)
CAGGCUGGAGAAUCUGGGCTT
3494






stab00


4359
AGCUCUCCUCCUGUUUAAAAGGA
2476
36391
KDR:4377L21 antisense siNA (4359 C.)
CUUUUAAACAGGAGGAGAGTT
3495






stab00


4534
UAUCCUGGAAGAGGCUUGUGACC
2477
36392
KDR:4552L21 antisense siNA (4534 C.)
UCACAAGCCUCUUCCAGGATT
3496






stab00


4535
AUCCUGGAAGAGGCUUGUGACCC
2478
36393
KDR:4553L21 antisense siNA (4535 C.)
GUCACAAGCCUCUUCCAGGTT
3497






stab00


4536
UCCUGGAAGAGGCUUGUGACCCA
2479
36394
KDR:4554L21 antisense siNA (4536 C.)
GGUCACAAGCCUCUUCCAGTT
3498






stab00


4539
UGGAAGAGGCUUGUGACCCAAGA
2480
36395
KDR:4557L21 antisense siNA (4539 C.)
UUGGGUCACAAGCCUCUUCTT
3499






stab00


4769
UGUUGAAGAUGGGAAGGAUUUGC
2481
36396
KDR:4787L21 antisense siNA (4769 C.)
AAAUCCUUCCCAUCUUCAATT
3500






stab00


4934
UCUGGUGGAGGUGGGCAUGGGGU
2482
36397
KDR:4952L21 antisense siNA (4934 C.)
CCCAUGCCCACCUCCACCATT
3501






stab00


5038
UCGUUGUGCUGUUUCUGACUCCU
2483
36398
KDR:5056L21 antisense siNA (5038 C.)
GAGUCAGAAACAGCACAACTT
3502






stab00


5039
CGUUGUGCUGUUUCUGACUCCUA
2484
36399
KDR:5057L21 antisense siNA (5039 C.)
GGAGUCAGAAACAGCACAATT
3503






stab00


5040
GUUGUGCUGUUUCUGACUCCUAA
2485
36400
KDR:5058L21 antisense siNA (5040 C.)
AGGAGUCAGAAACAGCACATT
3504






stab00


5331
UCAAAGUUUCAGGAAGGAUUUUA
2486
36401
KDR:5349L21 antisense siNA (5331 C.)
AAAUCCUUCCUGAAACUUUTT
3505






stab00


5332
CAAAGUUUCAGGAAGGAUUUUAC
2487
36402
KDR:5350L21 antisense siNA (5332 C.)
AAAAUCCUUCCUGAAACUUTT
3506






stab00


5333
AAAGUUUCAGGAAGGAUUUUACC
2488
36403
KDR:5351L21 antisense siNA (5333 C.)
UAAAAUCCUUCCUGAAACUTT
3507






stab00


5587
UCAAAAAAGAAAAUGUGUUUUUU
2489
36404
KDR:5605L21 antisense siNA (5587 C.)
AAAACACAUUUUCUUUUUUTT
3508






stab00


5737
CUAUUCACAUUUUGUAUCAGUAU
2490
36405
KDR:5755L21 antisense siNA (5737 C.)
ACUGAUACAAAAUGUGAAUTT
3509






stab00


5738
UAUUCACAUUUUGUAUCAGUAUU
2491
36406
KDR:5756L21 antisense siNA (5738 C.)
UACUGAUACAAAAUGUGAATT
3510






stab00


5739
AUUCACAUUUUGUAUCAGUAUUA
2492
36407
KDR:5757L21 antisense siNA (5739 C.)
AUACUGAUACAAAAUGUGATT
3511






stab00


359
GGCCGCCUCUGUGGGUUUGCCUA
2493
37460
KDR:359U21 sense siNA stab07
B ccGccucuGuGGGuuuGccTT B
3512


360
GCCGCCUCUGUGGGUUUGCCUAG
2494
37461
KDR:360U21 sense siNA stab07
B cGccucuGuGGGuuuGccuTT B
3513


799
ACCCAGAAAAGAGAUUUGUUCCU
2495
37462
KDR:799U21 sense siNA stab07
B ccAGAAAAGAGAuuuGuucTT B
3514


826
GUAACAGAAUUUCCUGGGACAGC
2496
37463
KDR:826U21 sense siNA stab07
B AAcAGAAuuuccuGGGAcATT B
3515


1027
AGCUUGUCUUAAAUUGUACAGCA
2497
37464
KDR:1027U21 sense siNA stab07
B cuuGucuuAAAuuGuAcAGTT B
3516


1827
GAAGGAAAAAACAAAACUGUAAG
2498
37465
KDR:1827U21 sense siNA stab07
B AGGAAAAAAcAAAAcuGuATT B
3517


1828
AAGGAAAAAACAAAACUGUAAGU
2499
37466
KDR:1828U21 sense siNA stab07
B GGAAAAAAcAAAAcuGuAATT B
3518


1947
ACCAGGGGUCCUGAAAUUACUUU
2500
37467
KDR:1947U21 sense siNA stab07
B cAGGGGuccuGAAAuuAcuTT B
3519


2247
AAGACCAAGAAAAGACAUUGCGU
2501
37468
KDR:2247U21 sense siNA stab07
B GAccAAGAAAAGAcAuuGcTT B
3520


2501
AGGCCUCUACACCUGCCAGGCAU
2502
37469
KDR:2501U21 sense siNA stab07
B GccucuAcAccuGccAGGcTT B
3521


2624
GAUUGCCAUGUUCUUCUGGCUAC
2503
37470
KDR:2624U21 sense siNA stab07
B uuGccAuGuucuucuGGcuTT B
3522


2685
GGAGGGGAACUGAAGACAGGCUA
2504
37471
KDR:2685U21 sense siNA stab07
B AGGGGAAcuGAAGAcAGGcTT B
3523


2688
GGGGAACUGAAGACAGGCUACUU
2505
37472
KDR:2688U21 sense siNA stab07
B GGAAcuGAAGAcAGGcuAcTT B
3524


2689
GGGAACUGAAGACAGGCUACUUG
2506
37473
KDR:2689U21 sense siNA stab07
B GAAcuGAAGAcAGGcuAcuTT B
3525


2690
GGAACUGAAGACAGGCUACUUGU
2507
37474
KDR:2690U21 sense siNA stab07
B AAcuGAAGAcAGGcuAcuuTT B
3526


2692
AACUGAAGACAGGCUACUUGUCC
2508
37475
KDR:2692U21 sense siNA stab07
B cuGAAGAcAGGcuAcuuGuTT B
3527


2762
ACUGCCUUAUGAUGCCAGCAAAU
2509
37476
KDR:2762U21 sense siNA stab07
B uGccuuAuGAuGccAGcAATT B
3528


3187
GGCGCUUGGACAGCAUCACCAGU
2510
37477
KDR:3187U21 sense siNA stab07
B cGcuuGGAcAGcAucAccATT B
3529


3293
UAAGGACUUCCUGACCUUGGAGC
2511
37478
KDR:3293U21 sense siNA stab07
B AGGAcuuccuGAccuuGGATT B
3530


3306
ACCUUGGAGCAUCUCAUCUGUUA
2512
37479
KDR:3306U21 sense siNA stab07
B cuuGGAGcAucucAucuGuTT B
3531


3308
CUUGGAGCAUCUCAUCUGUUACA
2513
37480
KDR:3308U21 sense siNA stab07
B uGGAGcAucucAucuGuuATT B
3532


3309
UUGGAGCAUCUCAUCUGUUACAG
2514
37481
KDR:3309U21 sense siNA stab07
B GGAGcAucucAucuGuuAcTT B
3533


3312
GAGCAUCUCAUCUGUUACAGCUU
2515
37482
KDR:3312U21 sense siNA stab07
B GcAucucAucuGuuAcAGcTT B
3534


3320
CAUCUGUUACAGCUUCCAAGUGG
2516
37483
KDR:3320U21 sense siNA stab07
B ucuGuuAcAGcuuccAAGuTT B
3535


3324
UGUUACAGCUUCCAAGUGGCUAA
2517
37484
KDR:3324U21 sense siNA stab07
B uuAcAGcuuccAAGuGGcuTT B
3536


3334
UCCAAGUGGCUAAGGGCAUGGAG
2518
37485
KDR:3334U21 sense siNA stab07
B cAAGuGGcuAAGGGcAuGGTT B
3537


3346
AGGGCAUGGAGUUCUUGGCAUCG
2464
37486
KDR:3346U21 sense siNA stab07
B GGcAuGGAGuucuuGGcAuTT B
3538


3347
GGGCAUGGAGUUCUUGGCAUCGC
2519
37487
KDR:3347U21 sense siNA stab07
B GcAuGGAGuucuuGGcAucTT B
3539


3857
GAGCAUGGAAGAGGAUUCUGGAC
2520
37488
KDR:3857U21 sense siNA stab07
B GcAuGGAAGAGGAuucuGGTT B
3540


3858
AGCAUGGAAGAGGAUUCUGGACU
2521
37489
KDR:3858U21 sense siNA stab07
B CAuGGAAGAGGAuucuGGATT B
3541


3860
CAUGGAAGAGGAUUCUGGACUCU
2467
37490
KDR:3860U21 sense siNA stab07
B uGGAAGAGGAuucuGGAcuTT B
3542


3883
CUCUGCCUACCUCACCUGUUUCC
2522
37491
KDR:3883U21 sense siNA stab07
B cuGccuAccucAccuGuuuTT B
3543


3884
UCUGCCUACCUCACCUGUUUCCU
2523
37492
KDR:3884U21 sense siNA stab07
B uGccuAccucAccuGuuucTT B
3544


3885
CUGCCUACCUCACCUGUUUCCUG
2524
37493
KDR:3885U21 sense siNA stab07
B GccuAccucAccuGuuuccTT B
3545


3892
CCUCACCUGUUUCCUGUAUGGAG
2525
37494
KDR:3892U21 sense siNA stab07
B ucAccuGuuuccuGuAuGGTT B
3546


3936
AAAUUCCAUUAUGACAACACAGC
2526
37495
KDR:3936U21 sense siNA stab07
B AuuccAuuAuGAcAAcAcATT B
3547


3940
UCCAUUAUGACAACACAGCAGGA
2527
37496
KDR:3940U21 sense siNA stab07
B cAuuAuGAcAAcAcAGcAGTT B
3548


359
GGCCGCCUCUGUGGGUUUGCCUA
2493
37497
KDR:377L21 antisense siNA (359 C.) stab26
GGCAAAcccAcAGAGGcGGTT
3549


360
GCCGCCUCUGUGGGUUUGCCUAG
2494
37498
KDR:378L21 antisense siNA (360 C.) stab26
AGGcAAAcccAcAGAGGcGTT
3550


799
ACCCAGAAAAGAGAUUUGUUCCU
2495
37499
KDR:817L21 antisense siNA (799 C.) stab26
GAAcAAAucucuuuucuGGTT
3551


826
GUAACAGAAUUUCCUGGGACAGC
2496
37500
KDR:844L21 antisense siNA (826 C.) stab26
UGUcccAGGAAAuucuGuuTT
3552


1027
AGCUUGUCUUAAAUUGUACAGCA
2497
37501
KDR:1045L21 antisense siNA (1027 C.)
CUGuAcAAuuuAAGAcAAGTT
3553






stab26


1827
GAAGGAAAAAACAAAACUGUAAG
2498
37502
KDR:1845L21 antisense siNA (1827 C.)
UACAGuuuuGuuuuuuccuTT
3554






stab26


1828
AAGGAAAAAACAAAACUGUAAGU
2499
37503
KDR:1846L21 antisense siNA (1828 C.)
UUAcAGuuuuGuuuuuuccTT
3555






stab26


1947
ACCAGGGGUCCUGAAAUUACUUU
2500
37504
KDR:1965L21 antisense siNA (1947 C.)
AGUAAuuucAGGAccccuGTT
3556






stab26


2247
AAGACCAAGAAAAGACAUUGCGU
2501
37505
KDR:2265L21 antisense siNA (2247 C.)
GCAAuGucuuuucuuGGucTT
3557






stab26


2501
AGGCCUCUACACCUGCCAGGCAU
2502
37506
KDR:2519L21 antisense siNA (2501 C.)
GCCuGGcAGGuGuAGAGGcTT
3558






stab26


2624
GAUUGCCAUGUUCUUCUGGCUAC
2503
37507
KDR:2642L21 antisense siNA (2624 C.)
AGCcAGAAGAAcAuGGcAATT
3559






stab26


2685
GGAGGGGAACUGAAGACAGGCUA
2504
37508
KDR:2703L21 antisense siNA (2685 C.)
GCCuGucuucAGuuccccuTT
3560






stab26


2688
GGGGAACUGAAGACAGGCUACUU
2505
37509
KDR:2706L21 antisense siNA (2688 C.)
GUAGccuGucuucAGuuccTT
3561






stab26


2689
GGGAACUGAAGACAGGCUACUUG
2506
37510
KDR:2707L21 antisense siNA (2689 C.)
AGUAGccuGucuucAGuucTT
3562






stab26


2690
GGAACUGAAGACAGGCUACUUGU
2507
37511
KDR:2708L21 antisense siNA (2690 C.)
AAGuAGccuGucuucAGuuTT
3563






stab26


2692
AACUGAAGACAGGCUACUUGUCC
2508
37512
KDR:2710L21 antisense siNA (2692 C.)
ACAAGuAGccuGucuucAGTT
3564






stab26


2762
ACUGCCUUAUGAUGCCAGCAAAU
2509
37513
KDR:2780L21 antisense siNA (2762 C.)
UUGcuGGcAucAuAAGGcATT
3565






stab26


3187
GGCGCUUGGACAGCAUCACCAGU
2510
37514
KDR:3205L21 antisense siNA (3187 C.)
UGGuGAuGcuGuccAAGcGTT
3566






stab26


3293
UAAGGACUUCCUGACCUUGGAGC
2511
37515
KDR:3311L21 antisense siNA (3293 C.)
UCCAAGGucAGGAAGuccuTT
3567






stab26


3306
ACCUUGGAGCAUCUCAUCUGUUA
2512
37516
KDR:3324L21 anhisense siNA (3306 C.)
ACAGAuGAGAuGcuccAAGTT
3568






stab26


3308
CUUGGAGCAUCUCAUCUGUUACA
2513
37517
KDR:3326L21 antisense siNA (3308 C.)
UAAcAGAuGAGAuGcuccATT
3569






stab26


3309
UUGGAGCAUCUCAUCUGUUACAG
2514
37518
KDR:3327L21 antisense siNA (3309 C.)
GUAAcAGAuGAGAuGcuccTT
3570






stab26


3312
GAGCAUCUCAUCUGUUACAGCUU
2515
37519
KDR:3330L21 antisense siNA (3312 C.)
GCUGuAAcAGAuGAGAuGcTT
3571






stab26


3320
CAUCUGUUACAGCUUCCAAGUGG
2516
37520
KDR:3338L21 antisense siNA (3320 C.)
ACUuGGAAGcuGuAAcAGATT
3572






stab26


3324
UGUUACAGCUUCCAAGUGGCUAA
2517
37521
KDR:3342L21 antisense siNA (3324 C.)
AGCcAcuuGGAAGcuGuAATT
3573






stab26


3334
UCCAAGUGGCUAAGGGCAUGGAG
2518
37522
KDR:3352L21 antisense siNA (3334 C.)
CCAuGcccuuAGccAcuuGTT
3574






stab26


3346
AGGGCAUGGAGUUCUUGGCAUCG
2464
37523
KDR:3364L21 antisense siNA (3346 C.)
AUGccAAGAAcuccAuGccTT
3575






stab26


3347
GGGCAUGGAGUUCUUGGCAUCGC
2519
37524
KDR:3365L21 antisense siNA (3347 C.)
GAUGccAAGAAcuccAuGcTT
3576






stab26


3758
CACGUUUUCAGAGUUGGUGGAAC
2426
37525
KDR:3776L21 antisense siNA (3758 C.)
UCCAccAAcucuGAAAAcGTT
3577






stab26


3857
GAGCAUGGAAGAGGAUUCUGGAC
2520
37526
KDR:3875L21 antisense siNA (3857 C.)
CCAGAAuccucuuccAuGcTT
3578






stab26


3858
AGCAUGGAAGAGGAUUCUGGACU
2521
37527
KDR:3876L21 antisense siNA (3858 C.)
UCCAGAAuccucuuccAuGTT
3579






stab26


3860
CAUGGAAGAGGAUUCUGGACUCU
2467
37528
KDR:3878L21 antisense siNA (3860 C.)
AGUccAGAAuccucuuccATT
3580






stab26


3883
CUCUGCCUACCUCACCUGUUUCC
2522
37529
KDR:3901L21 antisense siNA (3883 C.)
AAAcAGGuGAGGuAGGcAGTT
3581






stab26


3884
UCUGCCUACCUCACCUGUUUCCU
2523
37530
KDR:3902L21 antisense siNA (3884 C.)
GAAAcAGGuGAGGuAGGcATT
3582






stab26


3885
CUGCCUACCUCACCUGUUUCCUG
2524
37531
KDR:3903L21 antisense siNA (3885 C.)
GGAAAcAGGuGAGGuAGGcTT
3583






stab26


3892
CCUCACCUGUUUCCUGUAUGGAG
2525
37532
KDR:3910L21 antisense siNA (3892 C.)
CCAuAcAGGAAAcAGGuGATT
3584






stab26


3893
CUCACCUGUUUCCUGUAUGGAGG
2427
37533
KDR:3911L21 antisense siNA (3893 C.)
UCCAuAcAGGAAAcAGGuGTT
3585






stab26


3936
AAAUUCCAUUAUGACAACACAGC
2526
37534
KDR:3954L21 antisense siNA (3936 C.)
UGUGuuGucAuAAuGGAAuTT
3586






stab26


3940
UCCAUUAUGACAACACAGCAGGA
2527
37535
KDR:3958L21 antisense siNA (3940 C.)
CUGcuGuGuuGucAuAAuGTT
3587






stab26


3948
GACAACACAGCAGGAAUCAGUCA
2408
37536
KDR:3966L21 antisense siNA (3948 C.)
ACUGAuuccuGcuGuGuuGTT
3588






stab26


 







VEGFR3














2011
AGCACUGCCACAAGAAGUACCUG
2528
31904
FLT4:2011U21 sense siNA
CACUGCCACAAGAAGUACCTT
3589



3921
CUGAAGCAGAGAGAGAGAAGGCA
2529

FLT4:3921U21 sense siNA
GAAGCAGAGAGAGAGAAGGTT
3590


4038
AAAGAGGAACCAGGAGGACAAGA
2530

FLT4:4038U21 sense siNA
AGAGGAACCAGGAGGACAATT
3591


4054
GACAAGAGGAGCAUGAAAGUGGA
2531

FLT4:4054U21 sense siNA
CAAGAGGAGCAUGAAAGUGTT
3592


2011
AGCACUGCCACAAGAAGUACCUG
2528
31908
FLT4:2029L21 antisense siNA
GGUACUUCUUGUGGCAGUGTT
3593






(2011 C.)


3921
CUGAAGCAGAGAGAGAGAAGGCA
2529

FLT4:3939L21 antisense siNA
CCUUCUCUCUCUCUGCUUCTT
3594






(3921 C.)


4038
AAAGAGGAACCAGGAGGACAAGA
2530

FLT4:4056L21 antisense siNA
UUGUCCUCCUGGUUCCUCUTT
3595






(4038 C.)


4054
GACAAGAGGAGCAUGAAAGUGGA
2531

FLT4:4072L21 antisense siNA
CACUUUCAUGCUCCUCUUGTT
3596






(4054 C.)


2011
AGCACUGCCACAAGAAGUACCUG
2528

FLT4:2011U21 sense siNA stab04
B cAcuGccAcAAGAAGuAccTT B
3597


3921
CUGAAGCAGAGAGAGAGAAGGCA
2529

FLT4:3921U21 sense siNA stab04
B GAAGcAGAGAGAGAGAAGGTT B 3598


4038
AAAGAGGAACCAGGAGGACAAGA
2530

FLT4:4038U21 sense siNA stab04
B AGAGGAAccAGGAGGAcAATT B 3599


4054
GACAAGAGGAGCAUGAAAGUGGA
2531

FLT4:4054U21 sense siNA stab04
B cAAGAGGAGcAuGAAAGuGTT B 3600


2011
AGCACUGCCACAAGAAGUACCUG
2528

FLT4:2029L21 antisense siNA
GGuAcuucuuGuGGcAGuGTsT
3601






(2011 C.) stab05


3921
CUGAAGCAGAGAGAGAGAAGGCA
2529

FLT4:3939L21 antisense siNA
ccuucucucucucuGcuucTsT
3602






(3921 C.) stab05


4038
AAAGAGGAACCAGGAGGACAAGA
2530

FLT4:4056L21 antisense siNA
uuGuccuccuGGuuccucuTsT
3603






(4038 C.) stab05


4054
GACAAGAGGAGCAUGAAAGUGGA
2531

FLT4:4072L21 antisense siNA
cAcuuucAuGcuccucuuGTsT
3604






(4054 C.) stab05


2011
AGCACUGCCACAAGAAGUACCUG
2528

FLT4:2011U21 sense siNA stab07
B cAcuGccAcAAGAAGuAccTT B
3605


3921
CUGAAGCAGAGAGAGAGAAGGCA
2529

FLT4:3921U21 sense siNA stab07
B GAAGcAGAGAGAGAGAAGGTT B
3606


4038
AAAGAGGAACCAGGAGGACAAGA
2530

FLT4:4038U21 sense siNA stab07
B AGAGGAAccAGGAGGAcAATT B
3607


4054
GACAAGAGGAGCAUGAAAGUGGA
2531

FLT4:4054U21 sense siNA stab07
B cAAGAGGAGcAuGAAAGuGTT B
3608


2011
AGCACUGCCACAAGAAGUACCUG
2528

FLT4:2029L21 antisense siNA
GGuAcuucuuGuGGcAGuGTsT
3609






(2011 C.) stab11


3921
CUGAAGCAGAGAGAGAGAAGGCA
2529

FLT4:3939L21 antisense siNA
ccuucucucucucuGcuucTsT
3610






(3921 C.) stab11


4038
AAAGAGGAACCAGGAGGACAAGA
2530

FLT4:4056L21 antisense siNA
uuGuccuccuGGuuccucuTsT
3611






(4038 C.) stab11


4054
GACAAGAGGAGCAUGAAAGUGGA
2531

FLT4:4072L21 antisense siNA
cAcuuucAuGcuccucuuGTsT
3612






(4054 C.) stab11


1666
ACUUCUAUGUGACCACCAUCCCC
2532
31902
FLT4:1666U21 sense siNA
UUCUAUGUGACCACCAUCCTT
3613


2009
CAAGCACUGCCACAAGAAGUACC
2533
31903
FLT4:2009U21 sense siNA
AGCACUGCCACAAGAAGUATT
3614


2815
AGUACGGCAACCUCUCCAACUUC
2534
31905
FLT4:2815U21 sense siNA
UACGGCAACCUCUCCAACUTT
3615


1666
ACUUCUAUGUGACCACCAUCCCC
2532
31906
FLT4:1684L21 antisense siNA
GGAUGGUGGUCACAUAGAATT
3616






(1666 C.)


2009
CAAGCACUGCCACAAGAAGUACC
2533
31907
FLT4:2027L21 antisense siNA
UACUUCUUGUGGCAGUGCUTT
3617






(2009 C.)


2815
AGUACGGCAACCUCUCCAACUUC
2534
31909
FLT4:2833L21 antisense siNA
AGUUGGAGAGGUUGCCGUATT
3618






(2815 C.)


1609
CUGCCAUGUACAAGUGUGUGGUC
2535
34383
FLT4:1609U21 sense siNA stab09
B GCCAUGUACAAGUGUGUGGTT B
3619


1666
ACUUCUAUGUGACCACCAUCCCC
2532
34384
FLT4:1666U21 sense siNA stab09
B UUCUAUGUGACCACCAUCCTT B
3620


2009
CAAGCACUGCCACAAGAAGUACC
2533
34385
FLT4:2009U21 sense siNA stab09
B AGCACUGCCACAAGAAGUATT B
3621


2011
AGCACUGCCACAAGAAGUACCUG
2528
34386
FLT4:2011U21 sense siNA stab09
B CACUGCCACAAGAAGUACCTT B
3622


2014
ACUGCCACAAGAAGUACCUGUCG
2536
34387
FLT4:2014U21 sense siNA stab09
B UGCCACAAGAAGUACCUGUTT B
3623


2815
AGUACGGCAACCUCUCCAACUUC
2534
34388
FLT4:2815U21 sense siNA stab09
B UACGGCAACCUCUCCAACUTT B
3624


3172
UGGUGAAGAUCUGUGACUUUGGC
2537
34389
FLT4:3172U21 sense siNA stab09
B GUGAAGAUCUGUGACUUUGTT B
3625


3176
GAAGAUCUGUGACUUUGGCCUUG
2538
34390
FLT4:3176U21 sense siNA stab09
B AGAUCUGUGACUUUGGCCUTT B
3626


1609
CUGCCAUGUACAAGUGUGUGGUC
2535
34391
FLT4:1627L21 antisense siNA
CCACACACUUGUACAUGGCTsT
3627






(1609 C.) stab10


1666
ACUUCUAUGUGACCACCAUCCCC
2532
34392
FLT4:1684L21 antisense siNA
GGAUGGUGGUCACAUAGAATsT
3628






(1666 C.) stab10


2009
CAAGCACUGCCACAAGAAGUACC
2533
34393
FLT4:2027L21 antisense siNA
UACUUCUUGUGGCAGUGCUTsT
3629






(2009 C.) stab10


2011
AGCACUGCCACAAGAAGUACCUG
2528
34394
FLT4:2029L21 antisense siNA
GGUACUUCUUGUGGCAGUGTsT
3630






(2011 C.) stab10


2014
ACUGCCACAAGAAGUACCUGUCG
2536
34395
FLT4:2032L21 antisense siNA
ACAGGUACUUCUUGUGGCATsT
3631






(2014 C.) stab10


2815
AGUACGGCAACCUCUCCAACUUC
2534
34396
FLT4:2833L21 antisense siNA
AGUUGGAGAGGUUGCCGUATsT
3632






(2815 C.) stab10


3172
UGGUGAAGAUCUGUGACUUUGGC
2537
34397
FLT4:3190L21 antisense siNA
CAAAGUCACAGAUCUUCACTsT
3633






(3172 C.) stab10


3176
GAAGAUCUGUGACUUUGGCCUUG
2538
34398
FLT4:3194L21 antisensesiNA
AGGCCAAAGUCACAGAUCUTsT
3634






(3176 C.) stab10


1609
CUGCCAUGUACAAGUGUGUGGUC
2535
34399
FLT4:1627L21 antisense siNA
ccAcAcAcuuGuAcAuGGcTsT
3635






(1609 C.) stab08


1666
ACUUCUAUGUGACCACCAUCCCC
2532
34400
FLT4:1684L21 antisense siNA
GGAuGGuGGucAcAuAGAATsT
3636






(1666 C.) stab08


2009
CAAGCACUGCCACAAGAAGUACC
2533
34401
FLT4:2027L21 antisense siNA
uAcuucuuGuGGcAGuGcuTsT
3637






(2009 C.) stab08


2011
AGCACUGCCACAAGAAGUACCUG
2528
34402
FLT4:2029L21 antisense siNA
GGuAcuucuuGuGGcAGuGTsT
3638






(2011 C.) stab08


2014
ACUGCCACAAGAAGUACCUGUCG
2536
34403
FLT4:2032L21 antisense siNA
AcAGGuAcuucuuGuGGcATsT
3639






(2014 C.) stab08


2815
AGUACGGCAACCUCUCCAACUUC
2534
34404
FLT4:2833L21 antisense siNA
AGuuGGAGAGGuuGccGuATsT
3640






(2815 C.) stab08


3172
UGGUGAAGAUCUGUGACUUUGGC
2537
34405
FLT4:3190L21 antisense siNA
cAAAGucAcAGAucuucAcTsT
3641






(3172 C.) stab08


3176
GAAGAUCUGUGACUUUGGCCUUG
2538
34406
FLT4:3194L21 antisense siNA
AGGccAAAGucAcAGAucuTsT
3642






(3176 C.) stab08


 







VEGF














329
GCAAGAGCUCCAGAGAGAAGUCG
2539
32166
VEGF:331U21 sense siNA
AAGAGCUCCAGAGAGAAGUTT
3643



414
CAAAGUGAGUGACCUGCUUUUGG
2540
32167
VEGF:416U21 sense siNA
AAGUGAGUGACCUGCUUUUTT
3644


1151
ACGAAGUGGUGAAGUUCAUGGAU
2541
32168
VEGF:1153U21 sense siNA
GAAGUGGUGAAGUUCAUGGTT
3645


1212
GGUGGACAUCUUCCAGGAGUACC
2542
32525
VEGF:1214U21 sense siNA
UGGACAUCUUCCAGGAGUATT
3646


1213
GUGGACAUCUUCCAGGAGUACCC
2543
32526
VEGF:1215U21 sense siNA
GGACAUCUUCCAGGAGUACTT
3647


1215
GGACAUCUUCCAGGAGUACCCUG
2544
32527
VEGF:1217U21 sense siNA
ACAUCUUCCAGGAGUACCCTT
3648


1334
AGUCCAACAUCACCAUGCAGAUU
2545
32169
VEGF:1336U21 sense siNA
UCCAACAUCACCAUGCAGATT
3649


1650
CGAACGUACUUGCAGAUGUGACA
2546
32540
VEGF:1652U21 sense siNA
AACGUACUUGCAGAUGUGATT
3650


329
GCAAGAGCUCCAGAGAGAAGUCG
2539
32170
VEGF:349L21 antisense siNA (331 C.)
ACUUCUCUCUGGAGCUCUUTT
3651


414
CAAAGUGAGUGACCUGCUUUUGG
2540
32171
VEGF:434L21 antisense siNA (416 C.)
AAAAGCAGGUCACUCACUUTT
3652


1151
ACGAAGUGGUGAAGUUCAUGGAU
2541
32172
VEGF:1171L21 antisense siNA (1153 C.)
CCAUGAACUUCACCACUUCTT
3653


1212
GGUGGACAUCUUCCAGGAGUACC
2542
32543
VEGF:1232L21 antisense siNA (1214 C.)
UACUCCUGGAAGAUGUCCATT
3654


1213
GUGGACAUCUUCCAGGAGUACCC
2543
32544
VEGF:1233L21 antisense siNA (1215 C.)
GUACUCCUGGAAGAUGUCCTT
3655


1215
GGACAUCUUCCAGGAGUACCCUG
2544
32545
VEGF:1235L21 antisense siNA (1217 C.)
GGGUACUCCUGGAAGAUGUTT
3656


1334
AGUCCAACAUCACCAUGCAGAUU
2545
32173
VEGF:1354L21 antisense siNA (1336 C.)
UCUGCAUGGUGAUGUUGGATT
3657


1650
CGAACGUACUUGCAGAUGUGACA
2546
32558
VEGF:1670L21 antisense siNA (1652 C.)
UCACAUCUGCAAGUACGUUTT
3658


329
GCAAGAGCUCCAGAGAGAAGUCG
2539

VEGF:331U21 sense siNA stab04
B AAGAGcuccAGAGAGAAGuTT B
3659


414
CAAAGUGAGUGACCUGCUUUUGG
2540

VEGF:416U21 sense siNA stab04
B AAGuGAGuGAccuGcuuuuTT B
3660


1151
ACGAAGUGGUGAAGUUCAUGGAU
2541

VEGF:1153U21 sense siNA stab04
B GAAGuGGuGAAGuucAuGGTT B
3661


1212
GGUGGACAUCUUCCAGGAGUACC
2542

VEGF:1214U21 sense siNA stab04
B uGGAcAucuuccAGGAGuATT B
3662


1213
GUGGACAUCUUCCAGGAGUACCC
2543

VEGF:1215U21 sense siNA stab04
B GGAcAucuuccAGGAGuAcTT B
3663


1215
GGACAUCUUCCAGGAGUACCCUG
2544

VEGF:1217U21 sense siNA stab04
B AcAucuuccAGGAGuAcccTT B
3664


1334
AGUCCAACAUCACCAUGCAGAUU
2545

VEGF:1336U21 sense siNA stab04
B uccAAcAucAccAuGcAGATT B
3665


1650
CGAACGUACUUGCAGAUGUGACA
2546

VEGF:1652U21 sense siNA stab04
B AAcGuAcuuGcAGAuGuGATT B
3666


329
GCAAGAGCUCCAGAGAGAAGUCG
2539

VEGF:349L21 antisense siNA (331 C.) stab05
AcuucucucuGGAGcucuuTsT
3667


414
CAAAGUGAGUGACCUGCUUUUGG
2540

VEGF:434L21 antisense siNA (416 C.) stab05
AAAAGcAGGucAcucAcuuTsT
3668


1151
ACGAAGUGGUGAAGUUCAUGGAU
2541

VEGF:1171L21 antisense siNA (1153 C.) stab05
ccAuGAAcuucAccAcuucTsT
3669


1212
GGUGGACAUCUUCCAGGAGUACC
2542

VEGF:1232L21 antisense siNA (1214 C.) stab05
uAcuccuGGAAGAuGuccATsT
3670


1213
GUGGACAUCUUCCAGGAGUACCC
2543

VEGF:1233L21 antisense siNA (1215 C.) stab05
GuAcuccuGGAAGAuGuccTsT
3671


1215
GGACAUCUUCCAGGAGUACCCUG
2544

VEGF:1235L21 antisense siNA (1217 C.) stab05
GGGuAcuccuGGAAGAuGuTsT
3672


1334
AGUCCAACAUCACCAUGCAGAUU
2545

VEGF:1354L21 antisense siNA (1336 C.) stab05
ucuGcAuGGuGAuGuuGGATsT
3673


1650
CGAACGUACUUGCAGAUGUGACA
2546

VEGF:1670L21 antisense siNA (1652 C.) stab05
ucAcAucuGcAAGuAcGuuTsT
3674


329G
CAAGAGCUCCAGAGAGAAGUCG
2539

VEGF:331U21 sense siNA stab07
B AAGAGcuccAGAGAGAAGuTT B
3675


414C
AAAGUGAGUGACCUGCUUUUGG
2540

VEGF:416U21 sense siNA stab07
B AAGuGAGuGAccuGcuuuuTT B
3676


1151
ACGAAGUGGUGAAGUUCAUGGAU
2541

VEGF:1153U21 sense siNA stab07
B GAAGuGGuGAAGuucAuGGTT B
3677


1212
GGUGGACAUCUUCCAGGAGUACC
2542
33977
VEGF:1214U21 sense siNA stab07
B uGGAcAucuuccAGGAGuATT B
3678


1213
GUGGACAUCUUCCAGGAGUACCC
2543
33978
VEGF:1215U21 sense siNA stab07
B GGAcAucuuccAGGAGuAcTT B
3679


1215
GGACAUCUUCCAGGAGUACCCUG
2544

VEGF:1217U21 sense siNA stab07
B AcAucuuccAGGAGuAcccTT B
3680


1334
AGUCCAACAUCACCAUGCAGAUU
2545

VEGF:1336U21 sense siNA stab07
B uccAAcAucAccAuGcAGATT B
3681


1650
CGAACGUACUUGCAGAUGUGACA
2546

VEGF:1652U21 sense siNA stab07
B AAcGuAcuuGcAGAuGuGATT B
3682


329
GCAAGAGCUCCAGAGAGAAGUCG
2539

VEGF:349L21 antisense siNA (331 C.) stab11
AcuucucucuGGAGcucuuTsT
3683


414
CAAAGUGAGUGACCUGCUUUUGG
2540

VEGF:434L21 antisense siNA (416 C.) stab11
AAAAGcAGGucAcucAcuuTsT
3684


1151
ACGAAGUGGUGAAGUUCAUGGAU
2541

VEGF:1171L21 antisense siNA (1153 C.) stab11
ccAuGAAcuucAccAcuucTsT
3685


1212
GGUGGACAUCUUCCAGGAGUACC
2542

VEGF:1232L21 antisense siNA (1214 C.) stab11
uAcuccuGGAAGAuGuccATsT
3686


1213
GUGGACAUCUUCCAGGAGUACCC
2543

VEGF:1233L21 antisense siNA (1215 C.) stab11
GuAcuccuGGAAGAuGuccTsT
3687


1215
GGACAUCUUCCAGGAGUACCCUG
2544

VEGF:1235L21 antisense siNA (1217 C.) stab11
GGGuAcuccuGGAAGAuGuTsT
3688


1334
AGUCCAACAUCACCAUGCAGAUU
2545

VEGF:1354L21 antisense siNA (1336 C.) stab11
ucuGcAuGGuGAuGuuGGATsT
3689


1650
CGAACGUACUUGCAGAUGUGACA
2546

VEGF:1670L21 antisense siNA (1652 C.) stab11
ucAcAucuGcAAGuAcGuuTsT
3690


329
GCAAGAGCUCCAGAGAGAAGUCG
2539

VEGF:331U21 sense siNA stab18
B AAGAGcuCcAGAGAGAAGuTT B
3691


414
CAAAGUGAGUGACCUGCUUUUGG
2540

VEGF:416U21 sense siNA stab18
B AAGuGAGuGAccuGcuuuuTT B
3692


1151
ACGAAGUGGUGAAGUUCAUGGAU
2541

VEGF:1153U21 sense siNA stab18
B GAAGuGGuGAAGuucAuGGTT B
3693


1212
GGUGGACAUCUUCCAGGAGUACC
2542

VEGF:1214U21 sense siNA stab18
B uGGACAuCuuccAGGAGuATT B
3694


1213
GUGGACAUCUUCCAGGAGUACCC
2543

VEGF:1215U21 sense siNA stab18
B GGAcAucuuccAGGAGuAcTT B
3695


1215
GGACAUCUUCCAGGAGUACCCUG
2544

VEGF:1217U21 sense siNA stab18
B AcAucuuccAGGAGuAcccTT B
3696


1334
AGUCCAACAUCACCAUGCAGAUU
2545

VEGF:1336U21 sense siNA stab18
B uccAAcAucAccAuGcAGATT B
3697


1650
CGAACGUACUUGCAGAUGUGACA
2546

VEGF:1652U21 sense siNA stab18
B AAcGuAcuuGcAGAuGuGATT B
3698


329
GCAAGAGCUCCAGAGAGAAGUCG
2539

VEGF:349L21 antisense siNA (331 C.) stab08
AcuucucucuGGAGcucuuTsT
3699


414
CAAAGUGAGUGACCUGCUUUUGG
2540

VEGF:434L21 antisense siNA (416 C.) stab08
AAAAGcAGGucAcucAcuuTsT
3700


115
1ACGAAGUGGUGAAGUUCAUGGAU
2541

VEGF:1171L21 antisense siNA (1153 C.) stab08
ccAuGAAcuucAccAcuucTsT
3701


1212
GGUGGACAUCUUCCAGGAGUACC
2542
33983
VEGF:1232L21 antisense siNA (1214 C.) stab08
uAcuccuGGAAGAuGuccATsT
3702


1213
GUGGACAUCUUCCAGGAGUACCC
2543
33984
VEGF:1233L21 antisense siNA (1215 C.) stab08
GuAcuccuGGAAGAuGuccTsT
3703


1215
GGACAUCUUCCAGGAGUACCCUG
2544

VEGF:1235L21 antisense siNA (1217 C.) stab08
GGGuAcuccuGGAAGAuGuTsT
3704


1334
AGUCCAACAUCACCAUGCAGAUU
2545

VEGF:1354L21 antisense siNA (1336 C.) stab08
ucuGCAuGGuGAuGuuGGATsT
3705


1650
CGAACGUACUUGCAGAUGUGACA
2546

VEGF:1670L21 antisense siNA (1652 C.) stab08
ucAcAucuGcAAGuAcGuuTsT
3706


329
GCAAGAGCUCCAGAGAGAAGUCG
2539

VEGF:331U21 sense siNA stab09
B AAGAGCUCCAGAGAGAAGUTT B
3707


414
CAAAGUGAGUGACCUGCUUUUGG
2540

VEGF:416U21 sense siNA stab09
B AAGUGAGUGACCUGCUUUUTT B
3708


1151
ACGAAGUGGUGAAGUUCAUGGAU
2541

VEGF:1153U21 sense siNA stab09
B GAAGUGGUGAAGUUCAUGGTT B
3709


1212
GGUGGACAUCUUCCAGGAGUACC
2542
33965
VEGF:1214U21 sense siNA stab09
B UGGACAUCUUCCAGGAGUATT B
3710


1213
GUGGACAUCUUCCAGGAGUACCC
2543
33966
VEGF:1215U21 sense siNA stab09
B GGACAUCUUCCAGGAGUACTT B
3711


1215
GGACAUCUUCCAGGAGUACCCUG
2544

VEGF:1217U21 sense siNA stab09
B ACAUCUUCCAGGAGUACCCTT B
3712


1334
AGUCCAACAUCACCAUGCAGAUU
2545

VEGF:1336U21 sense siNA stab09
B UCCAACAUCACCAUGCAGATT B
3713


1650
CGAACGUACUUGCAGAUGUGACA
2546

VEGF:1652U21 sense siNA stab09
B AACGUACUUGCAGAUGUGATT B
3714


329
GCAAGAGCUCCAGAGAGAAGUCG
2539

VEGF:349L21 antisense siNA (331 C.) stab10
ACUUCUCUCUGGAGCUCUUTsT
3715


414
CAAAGUGAGUGACCUGCUUUUGG
2540

VEGF:434L21 antisense siNA (416 C.) stab10
AAAAGCAGGUCACUCACUUTsT
3716


1151
ACGAAGUGGUGAAGUUCAUGGAU
2541

VEGF:1171L21 antisense siNA (1153 C.) stab10
CCAUGAACUUCACCACUUCTsT
3717


1212
GGUGGACAUCUUCCAGGAGUACC
2542
33971
VEGF:1232L21 antisense siNA (1214 C.) stab10
UACUCCUGGAAGAUGUCCATsT
3718


1213
GUGGACAUCUUCCAGGAGUACCC
2543
33972
VEGF:1233L21 antisense siNA (1215 C.) stab10
GUACUCCUGGAAGAUGUCCTsT
3719


1215
GGACAUCUUCCAGGAGUACCCUG
2544

VEGF:1235L21 antisense siNA (1217 C.) stab10
GGGUACUCCUGGAAGAUGUTsT
3720


1334
AGUCCAACAUCACCAUGCAGAUU
2545

VEGF:1354L21 antisense siNA (1336 C.) stab10
UCUGCAUGGUGAUGUUGGATsT
3721


1650
CGAACGUACUUGCAGAUGUGACA
2546

VEGF:1670L21 antisense siNA (1652 C.) stab10
UCACAUCUGCAAGUACGUUTsT
3722


329
GCAAGAGCUCCAGAGAGAAGUCG
2539

VEGF:349L21 antisense siNA (331 C.) stab19
AcuucucucuGGAGcucuuTT B
3723


414
CAAAGUGAGUGACCUGCUUUUGG
2540

VEGF:434L21 antisense siNA (416 C.) stab19
AAAAGcAGGucAcucAcuuTT B
3724


1151
ACGAAGUGGUGAAGUUCAUGGAU
2541

VEGF:1171L21 antisense siNA (1153 C.) stab19
ccAuGAAcuucAccAcuucTT B
3725


1212
GGUGGACAUCUUCCAGGAGUACC
2542

VEGF:1232L21 antisense siNA (1214 C.) stab19
uAcuccuGGAAGAuGuccATT B
3726


1213
GUGGACAUCUUCCAGGAGUACCC
2543

VEGF:1233L21 antisense siNA (1215 C.) stab19
GuAcuccuGGAAGAuGuccTT B
3727


1215
GGACAUCUUCCAGGAGUACCCUG
2544

VEGF:1235L21 antisense siNA (1217 C.) stab19
GGGuAcuccuGGAAGAuGuTT B
3728


1334
AGUCCAACAUCACCAUGCAGAUU
2545

VEGF:1354L21 antisense siNA (1336 C.) stab19
ucuGcAuGGuGAuGuuGGATT B
3729


1650
CGAACGUACUUGCAGAUGUGACA
2546

VEGF:1670L21 antisense siNA (1652 C.) stab19
ucAcAucuGcAAGuAcGuuTT B
3730


329
GCAAGAGCUCCAGAGAGAAGUCG
2539

VEGF:349L21 antisense siNA (331 C.) stab22
ACUUCUCUCUGGAGCUCUUTT B
3731


414
CAAAGUGAGUGACCUGCUUUUGG
2540

VEGF:434L21 antisense siNA (416 C.) stab22
AAAAGCAGGUCACUCACUUTT B
3732


1151
ACGAAGUGGUGAAGUUCAUGGAU
2541

VEGF:1171L21 antisense siNA (1153 C.) stab22
CCAUGAACUUCACCACUUCTT B
3733


1212
GGUGGACAUCUUCCAGGAGUACC
2542

VEGF:1232L21 antisense siNA (1214 C.) stab22
UACUCCUGGAAGAUGUCCATT B
3734


1213
GUGGACAUCUUCCAGGAGUACCC
2543

VEGF:1233L21 antisense siNA (1215 C.) stab22
GUACUCCUGGAAGAUGUCCTT B
3735


1215
GGACAUCUUCCAGGAGUACCCUG
2544

VEGF:1235L21 antisense siNA (1217 C.) stab22
GGGUACUCCUGGAAGAUGUTT B
3736


1334
AGUCCAACAUCACCAUGCAGAUU
2545

VEGF:1354L21 antisense siNA (1336 C.) stab22
UCUGCAUGGUGAUGUUGGATT B
3737


1650
CGAACGUACUUGCAGAUGUGACA
2546

VEGF:1670L21 antisense siNA (1652 C.) stab22
UCACAUCUGCAAGUACGUUTT B
3738


1207
AGACCCUGGUGGACAUCUUCCAG
2547
32524
VEGF:1207U21 sense siNA stab00
ACCCUGGUGGACAUCUUCCTT
3739


1358
UAUGCGGAUCAAACCUCACCAAG
2548
32528
VEGF:1358U21 sense siNA stab00
UGCGGAUCAAACCUCACCATT
3740


1419
AAAUGUGAAUGCAGACCAAAGAA
2549
32529
VEGF:1419U21 sense siNA stab00
AUGUGAAUGCAGACCAAAGTT
3741


1420
AAUGUGAAUGCAGACCAAAGAAA
2550
32530
VEGF:1420U21 sense siNA stab00
UGUGAAUGCAGACCAAAGATT
3742


1421
AUGUGAAUGCAGACCAAAGAAAG
2551
32531
VEGF:1421U21 sense siNA stab00
GUGAAUGCAGACCAAAGAATT
3743


1423
GUGAAUGCAGACCAAAGAAAGAU
2552
32532
VEGF:1423U21 sense siNA stab00
GAAUGCAGACCAAAGAAAGTT
3744


1587
CAGACGUGUAAAUGUUCCUGCAA
2553
32533
VEGF:1587U21 sense siNA stab00
GACGUGUAAAUGUUCCUGCTT
3745


1591
CGUGUAAAUGUUCCUGCAAAAAC
2554
32534
VEGF:1591U21 sense siNA stab00
UGUAAAUGUUCCUGCAAAATT
3746


1592
GUGUAAAUGUUCCUGCAAAAACA
2555
32535
VEGF:1592U21 sense siNA stab00
GUAAAUGUUCCUGCAAAAATT
3747


1593
UGUAAAUGUUCCUGCAAAAACAC
2556
32536
VEGF:1593U21 sense siNA stab00
UAAAUGUUCCUGCAAAAACTT
3748


1594
GUAAAUGUUCCUGCAAAAACACA
2557
32537
VEGF:1594U21 sense siNA stab00
AAAUGUUCCUGCAAAAACATT
3749


1604
CUGCAAAAACACAGACUCGCGUU
2558
32538
VEGF:1604U21 sense siNA stab00
GCAAAAACACAGACUCGCGTT
3750


1637
GCAGCUUGAGUUAAACGAACGUA
2559
32539
VEGF:1637U21 sense siNA stab00
AGCUUGAGUUAAACGAACGTT
3751


1656
CGUACUUGCAGAUGUGACAAGCC
2560
32541
VEGF:1656U21 sense siNA stab00
UACUUGCAGAUGUGACAAGTT
3752


1207
AGACCCUGGUGGACAUCUUCCAG
2547
32542
VEGF:1225L21 antisense siNA (1207 C.) stab00
GGAAGAUGUCCACCAGGGUTT
3753


1358
UAUGCGGAUCAAACCUCACCAAG
2548
32546
VEGF:1376L21 antisense siNA (1358 C.) stab00
UGGUGAGGUUUGAUCCGCATT
3754


1419
AAAUGUGAAUGCAGACCAAAGAA
2549
32547
VEGF:1437L21 antisense siNA (1419 C.) stab00
CUUUGGUCUGCAUUCACAUTT
3755


1420
AAUGUGAAUGCAGACCAAAGAAA
2550
32548
VEGF:1438L21 antisense siNA (1420 C.) stab00
UCUUUGGUCUGCAUUCACATT
3756


1421
AUGUGAAUGCAGACCAAAGAAAG
2551
32549
VEGF:1439L21 antisense siNA (1421 C.) stab00
UUCUUUGGUCUGCAUUCACTT
3757


1423
GUGAAUGCAGACCAAAGAAAGAU
2552
32550
VEGF:1441L21 antisense siNA (1423 C.) stab00
CUUUCUUUGGUCUGCAUUCTT
3758


1587
CAGACGUGUAAAUGUUCCUGCAA
2553
32551
VEGF:1605L21 antisense siNA (1587 C.) stab00
GCAGGAACAUUUACACGUCTT
3759


1591
CGUGUAAAUGUUCCUGCAAAAAC
2554
32552
VEGF:1609L21 antisense siNA (1591 C.) stab00
UUUUGCAGGAACAUUUACATT
3760


1592
GUGUAAAUGUUCCUGCAAAAACA
2555
32553
VEGF:1610L21 antisense siNA (1592 C.) stab00
UUUUUGCAGGAACAUUUACTT
3761


1593
UGUAAAUGUUCCUGCAAAAACAC
2556
32554
VEGF:1611L21 antisense siNA (1593 C.) stab00
GUUUUUGCAGGAACAUUUATT
3762


1594
GUAAAUGUUCCUGCAAAAACACA
2557
32555
VEGF:1612L21 antisense siNA (1594 C.) stab00
UGUUUUUGCAGGAACAUUUTT
3763


1604
CUGCAAAAACACAGACUCGCGUU
2558
32556
VEGF:1622L21 antisense siNA (1604 C.) stab00
CGCGAGUCUGUGUUUUUGCTT
3764


1637
GCAGCUUGAGUUAAACGAACGUA
2559
32557
VEGF:1655L21 antisense siNA (1637 C.) stab00
CGUUCGUUUAACUCAAGCUTT
3765


1656
CGUACUUGCAGAUGUGACAAGCC
2560
32559
VEGF:1674L21 antisense siNA (1656 C.) stab00
CUUGUCACAUCUGCAAGUATT
3766


1206
GAGACCCUGGUGGACAUCUUCCA
2561
32560
VEGF:1206U21 sense siNA stab00
GACCCUGGUGGACAUCUUCTT
3767


1208
GACCCUGGUGGACAUCUUCCAGG
2562
32561
VEGF:1208U21 sense siNA stab00
CCCUGGUGGACAUCUUCCATT
3768


1551
UCAGAGCGGAGAAAGCAUUUGUU
2563
32562
VEGF:1551U21 sense siNA stab00
AGAGCGGAGAAAGCAUUUGTT
3769


1582
AUCCGCAGACGUGUAAAUGUUCC
2564
32563
VEGF:1582U21 sense siNA stab00
CCGCAGACGUGUAAAUGUUTT
3770


1584
CCGCAGACGUGUAAAUGUUCCUG
2565
32564
VEGF:1584U21 sense siNA stab00
GCAGACGUGUAAAUGUUCCTT
3771


1585
CGCAGACGUGUAAAUGUUCCUGC
2566
32565
VEGF:1585U21 sense siNA stab00
CAGACGUGUAAAUGUUCCUTT
3772


1589
GACGUGUAAAUGUUCCUGCAAAA
2567
32566
VEGF:1589U21 sense siNA stab00
CGUGUAAAUGUUCCUGCAATT
3773


1595
UAAAUGUUCCUGCAAAAACACAG
2568
32567
VEGF:1595U21 sense siNA stab00
AAUGUUCCUGCAAAAACACTT
3774


1596
AAAUGUUCCUGCAAAAACACAGA
2569
32568
VEGF:1596U21 sense siNA stab00
AUGUUCCUGCAAAAACACATT
3775


1602
UCCUGCAAAAACACAGACUCGCG
2570
32569
VEGF:1602U21 sense siNA stab00
CUGCAAAAACACAGACUCGTT
3776


1603
CCUGCAAAAACACAGACUCGCGU
2571
32570
VEGF:1603U21 sense siNA stab00
UGCAAAAACACAGACUCGCTT
3777


1630
AGGCGAGGCAGCUUGAGUUAAAC
2572
32571
VEGF:1630U21 sense siNA stab00
GCGAGGCAGCUUGAGUUAATT
3778


1633
CGAGGCAGCUUGAGUUAAACGAA
2573
32572
VEGF:1633U21 sense siNA stab00
AGGCAGCUUGAGUUAAACGTT
3779


1634
GAGGCAGCUUGAGUUAAACGAAC
2574
32573
VEGF:1634U21 sense siNA stab00
GGCAGCUUGAGUUAAACGATT
3780


1635
AGGCAGCUUGAGUUAAACGAACG
2575
32574
VEGF:1635U21 sense siNA stab00
GCAGCUUGAGUUAAACGAATT
3781


1636
GGCAGCUUGAGUUAAACGAACGU
2576
32575
VEGF:1636U21 sense siNA stab00
CAGCUUGAGUUAAACGAACTT
3782


1648
UAAACGAACGUACUUGCAGAUGU
2577
32576
VEGF:1648U21 sense siNA stab00
AACGAACGUACUUGCAGAUTT
3783


1649
AAACGAACGUACUUGCAGAUGUG
2578
32577
VEGF:1649U21 sense siNA stab00
ACGAACGUACUUGCAGAUGTT
3784


1206
GAGACCCUGGUGGACAUCUUCCA
2561
32578
VEGF:1224L21 antisense siNA (1206 C.) stab00
GAAGAUGUCCACCAGGGUCTT
3785


1208
GACCCUGGUGGACAUCUUCCAGG
2562
32579
VEGF:1226L21 antisense siNA (1208 C.) stab00
UGGAAGAUGUCCACCAGGGTT
3786


1551
UCAGAGCGGAGAAAGCAUUUGUU
2563
32580
VEGF:1569L21 antisense siNA (1551 C.) stab00
CAAAUGCUUUCUCCGCUCUTT
3787


1582
AUCCGCAGACGUGUAAAUGUUCC
2564
32581
VEGF:1600L21 antisense siNA (1582 C.) stab00
AACAUUUACACGUCUGCGGTT
3788


1584
CCGCAGACGUGUAAAUGUUCCUG
2565
32582
VEGF:1602L21 antisense siNA (1584 C.) stab00
GGAACAUUUACACGUCUGCTT
3789


1585
CGCAGACGUGUAAAUGUUCCUGC
2566
32583
VEGF:1603L21 antisense siNA (1585 C.) stab00
AGGAACAUUUACACGUCUGTT
3790


1589
GACGUGUAAAUGUUCCUGCAAAA
2567
32584
VEGF:1607L21 antisense siNA (1589 C.) stab00
UUGCAGGAACAUUUACACGTT
3791


1595
UAAAUGUUCCUGCAAAAACACAG
2568
32585
VEGF:1613L21 antisense siNA (1595 C.) stab00
GUGUUUUUGCAGGAACAUUTT
3792


1596
AAAUGUUCCUGCAAAAACACAGA
2569
32586
VEGF:1614L21 antisense siNA (1596 C.) stab00
UGUGUUUUUGCAGGAACAUTT
3793


1602
UCCUGCAAAAACACAGACUCGCG
2570
32587
VEGF:1620L21 antisense siNA (1602 C.) stab00
CGAGUCUGUGUUUUUGCAGTT
3794


1603
CCUGCAAAAACACAGACUCGCGU
2571
32588
VEGF:1621L21 antisense siNA (1603 C.) stab00
GCGAGUCUGUGUUUUUGCATT
3795


1630
AGGCGAGGCAGCUUGAGUUAAAC
2572
32589
VEGF:1648L21 antisense siNA (1630 C.) stab00
UUAACUCAAGCUGCCUCGCTT
3796


1633
CGAGGCAGCUUGAGUUAAACGAA
2573
32590
VEGF:1651L21 antisense siNA (1633 C.) stab00
CGUUUAACUCAAGCUGCCUTT
3797


1634
GAGGCAGCUUGAGUUAAACGAAC
2574
32591
VEGF:1652L21 antisense siNA (1634 C.) stab00
UCGUUUAACUCAAGCUGCCTT
3798


1635
AGGCAGCUUGAGUUAAACGAACG
2575
32592
VEGF:1653L21 antisense siNA (1635 C.) stab00
UUCGUUUAACUCAAGCUGCTT
3799


1636
GGCAGCUUGAGUUAAACGAACGU
2576
32593
VEGF:1654L21 antisense siNA (1636 C.) stab00
GUUCGUUUAACUCAAGCUGTT
3800


1648
UAAACGAACGUACUUGCAGAUGU
2577
32594
VEGF:1666L21 antisense siNA (1648 C.) stab00
AUCUGCAAGUACGUUCGUUTT
3801


1649
AAACGAACGUACUUGCAGAUGUG
2578
32595
VEGF:1667L21 antisense siNA (1649 C.) stab00
CAUCUGCAAGUACGUUCGUTT
3802


1358
UAUGCGGAUCAAACCUCACCAAG
2548
32968
VEGF:1358U21 sense siNA stab07
B uGcGGAucAAAccucAccATT B
3803


1419
AAAUGUGAAUGCAGACCAAAGAA
2549
32969
VEGF:1419U21 sense siNA stab07
B AuGuGAAuGcAGAccAAAGTT B
3804


1421
AUGUGAAUGCAGACCAAAGAAAG
2551
32970
VEGF:1421U21 sense siNA stab07
B GuGAAuGcAGAccAAAGAATT B
3805


1596
AAAUGUUCCUGCAAAAACACAGA
2569
32971
VEGF:1596U21 sense siNA stab07
B AuGuuccuGcAAAAAcAcATT B
3806


1636
GGCAGCUUGAGUUAAACGAACGU
2576
32972
VEGF:1636U21 sense siNA stab07
B cAGcuuGAGuuAAAcGAAcTT B
3807


1358
UAUGCGGAUCAAACCUCACCAAG
2548
32973
VEGF:1376L21 antisense siNA (1358 C.) stab08
uGGuGAGGuuuGAuccGcATsT
3808


1419
AAAUGUGAAUGCAGACCAAAGAA
2549
32974
VEGF:1437L21 antisense siNA (1419 C.) stab08
cuuuGGucuGcAuucAcAuTsT
3809


1421
AUGUGAAUGCAGACCAAAGAAAG
2551
32975
VEGF:1439L21 antisense siNA (1421 C.) stab08
uucuuuGGucuGcAuucAcTsT
3810


1596
AAAUGUUCCUGCAAAAACACAGA
2569
32976
VEGF:1614L21 antisense siNA (1596 C.) stab08
uGuGuuuuuGcAGGAAcAuTsT
3811


1636
GGCAGCUUGAGUUAAACGAACGU
2576
32977
VEGF:1654L21 antisense siNA (1636 C.) stab08
GuucGuuuAAcucAAGcuGTsT
3812


1358
UAUGCGGAUCAAACCUCACCAAG
2548
32978
VEGF:1358U21 sense siNA stab09
B UGCGGAUCAAACCUCACCATT B
3813


1419
AAAUGUGAAUGCAGACCAAAGAA
2549
32979
VEGF:1419U21 sense siNA stab09
B AUGUGAAUGCAGACCAAAGTT B
3814


1421
AUGUGAAUGCAGACCAAAGAAAG
2551
32980
VEGF:1421U21 sense siNA stab09
B GUGAAUGCAGACCAAAGAATT B
3815


1596
AAAUGUUCCUGCAAAAACACAGA
2569
32981
VEGF:1596U21 sense siNA stab09
B AUGUUCCUGCAAAAACACATT B
3816


1636
GGCAGCUUGAGUUAAACGAACGU
2576
32982
VEGF:1636U21 sense siNA stab09
B CAGCUUGAGUUAAACGAACTT B
3817


1358
UAUGCGGAUCAAACCUCACCAAG
2548
32983
VEGF:1376L21 antisense siNA (1358 C.) stab10
UGGUGAGGUUUGAUCCGCATsT
3818


1419
AAAUGUGAAUGCAGACCAAAGAA
2549
32984
VEGF:1437L21 antisense siNA (1419 C.) stab10
CUUUGGUCUGCAUUCACAUTsT
3819


1421
AUGUGAAUGCAGACCAAAGAAAG
2551
32985
VEGF:1439L21 anhisense siNA (1421 C.) stab10
UUCUUUGGUCUGCAUUCACTsT
3820


1596
AAAUGUUCCUGCAAAAACACAGA
2569
32986
VEGF:1614L21 antisense siNA (1596 C.) stab10
UGUGUUUUUGCAGGAACAUTsT
3821


1636
GGCAGCUUGAGUUAAACGAACGU
2576
32987
VEGF:1654L21 antisense siNA (1636 C.) stab10
GUUCGUUUAACUCAAGCUGTsT
3822


1358
UAUGCGGAUCAAACCUCACCAAG
2548
32998
VEGF:1358U21 sense siNA inv stab07
B AccAcuccAAAcuAGGcGuTT B
3823


1419
AAAUGUGAAUGCAGACCAAAGAA
2549
32999
VEGF:1419U21 sense siNA inv stab07
B GAAAccAGAcGuAAGuGuATT B
3824


1421
AUGUGAAUGCAGACCAAAGAAAG
2551
33000
VEGF:1421U21 sense siNA inv stab07
B AAGAAAccAGAcGuAAGuGTT B
3825


1596
AAAUGUUCCUGCAAAAACACAGA
2569
33001
VEGF:1596U21 sense siNA inv stab07
B AcAcAAAAAcGuccuuGuATT B
3826


1636
GGCAGCUUGAGUUAAACGAACGU
2576
33002
VEGF:1636U21 sense siNA inv stab07
B cAAGcAAAuuGAGuucGAcTT B
3827


1358
UAUGCGGAUCAAACCUCACCAAG
2548
33003
VEGF:1376L21 antisense siNA (1358 C.) inv stab08
AcGccuAGuuuGGAGuGGuTsT
3828


1419
AAAUGUGAAUGCAGACCAAAGAA
2549
33004
VEGF:1437L21 antisense siNA (1419 C.) inv stab08
uAcAcuuAcGucuGGuuucTsT
3829


1421
AUGUGAAUGCAGACCAAAGAAAG
2551
33005
VEGF:1439L21 antisense siNA (1421 C.) inv stab08
cAcuuAcGucuGGuuucuuTsT
3830


1596
AAAUGUUCCUGCAAAAACACAGA
2569
33006
VEGF:1614L21 antisense siNA (1596 C.) inv stab08
uAcAAGGAcGuuuuuGuGuTsT
3831


1636
GGCAGCUUGAGUUAAACGAACGU
2576
33007
VEGF:1654L21 antisense siNA (1636 C.) inv stab08
GucGAAcucAAuuuGcuuGTsT
3832


1358
UAUGCGGAUCAAACCUCACCAAG
2548
33008
VEGF:1358U21 sense siNA inv stab09
B ACCACUCCAAACUAGGCGUTT B
3833


1419
AAAUGUGAAUGCAGACCAAAGAA
2549
33009
VEGF:1419U21 sense siNA inv stab09
B GAAACCAGACGUAAGUGUATT B
3834


1421
AUGUGAAUGCAGACCAAAGAAAG
2551
33010
VEGF:1421U21 sense siNA inv stab09
B AAGAAACCAGACGUAAGUGTT B
3835


1596
AAAUGUUCCUGCAAAAACACAGA
2569
33011
VEGF:1596U21 sense siNA inv stab09
B ACACAAAAACGUCCUUGUATT B
3836


1636
GGCAGCUUGAGUUAAACGAACGU
2576
33012
VEGF:1636U21 sense siNA inv stab09
B CAAGCAAAUUGAGUUCGACTT B
3837


1358
UAUGCGGAUCAAACCUCACCAAG
2548
33013
VEGF:1376L21 antisense siNA (1358 C.) inv stab10
ACGCCUAGUUUGGAGUGGUTsT
3838


1419
AAAUGUGAAUGCAGACCAAAGAA
2549
33014
VEGF:1437L21 antisense siNA (1419 C.) inv stab10
UACACUUACGUCUGGUUUCTsT
3839


1421
AUGUGAAUGCAGACCAAAGAAAG
2551
33015
VEGF:1439L21 antisense siNA (1421 C.) inv stab10
CACUUACGUCUGGUUUCUUTsT
3840


1596
AAAUGUUCCUGCAAAAACACAGA
2569
33016
VEGF:1614L21 antisense siNA (1596 C.) inv stab10
UACAAGGACGUUUUUGUGUTsT
3841


1636
GGCAGCUUGAGUUAAACGAACGU
2576
33017
VEGF:1654L21 antisense siNA (1636 C.) inv stab10
GUCGAACUCAAUUUGCUUGTsT
3842


1420
AAUGUGAAUGCAGACCAAAGAAA
2550
33968
VEGF:1420U21 sense siNA stab09
B UGUGAAUGCAGACCAAAGATT B
3843


1423
GUGAAUGCAGACCAAAGAAAGAU
2552
33970
VEGF:1423U21 sense siNA stab09
B GAAUGCAGACCAAAGAAAGTT B
3844


1420
AAUGUGAAUGCAGACCAAAGAAA
2550
33974
VEGF:1438L21 antisense siNA (1420 C.) stab10
UCUUUGGUCUGCAUUCACATsT
3845


1423
GUGAAUGCAGACCAAAGAAAGAU
2552
33976
VEGF:1441L21 antisense siNA (1423 C.) stab10
CUUUCUUUGGUCUGCAUUCTsT
3846


1420
AAUGUGAAUGCAGACCAAAGAAA
2550
33980
VEGF:1420U21 sense siNA stab07
B uGuGAAuGcAGAccAAAGATT B
3847


1423
GUGAAUGCAGACCAAAGAAAGAU
2552
33982
VEGF:1423U21 sense siNA stab07
B GAAuGcAGAccAAAGAAAGTT B
3848


1420
AAUGUGAAUGCAGACCAAAGAAA
2550
33986
VEGF:1438L21 antisense siNA (142C C.) stab08
ucuuuGGucuGcAuucAcATsT
3849


1423
GUGAAUGCAGACCAAAGAAAGAU
2552
33988
VEGF:1441L21 antisense siNA (1423 C.) stab08
cuuucuuuGGucuGcAuucTsT
3850


1214
GGUGGACAUCUUCCAGGAGUACC
2542
33989
VEGF:1214U21 sense siNA inv stab09
B AUGAGGACCUUCUACAGGUTT B
3851


1215
GUGGACAUCUUCCAGGAGUACCC
2543
33990
VEGF:1215U21 sense siNA inv stab09
B CAUGAGGACCUUCUACAGGTT B
3852


1420
AAUGUGAAUGCAGACCAAAGAAA
2550
33992
VEGF:1420U21 sense siNA inv stab09
B AGAAACCAGACGUAAGUGUTT B
3853


1423
GUGAAUGCAGACCAAAGAAAGAU
2552
33994
VEGF:1423U21 sense siNA inv stab09
B GAAAGAAACCAGACGUAAGTT B
3854


1214
GGUGGACAUCUUCCAGGAGUACC
2542
33995
VEGF:1232L21 antisense siNA (1214 C.) inv stab10
ACCUGUAGAAGGUCCUCAUTsT
3855


1215
GUGGACAUCUUCCAGGAGUACCC
2543
33996
VEGF:1233L21 antisense siNA (1215 C.) inv stab10
CCUGUAGAAGGUCCUCAUGTsT
3856


1420
AAUGUGAAUGCAGACCAAAGAAA
2550
33998
VEGF:1438L21 antisense siNA (142C C.) inv stab10
ACACUUACGUCUGGUUUCUTsT
3857


1423
GUGAAUGCAGACCAAAGAAAGAU
2552
34000
VEGF:1441L21 antisense siNA (1423 C.) inv stab10
CUUACGUCUGGUUUCUUUCTsT
3858


1214
GGUGGACAUCUUCCAGGAGUACC
2542
34001
VEGF:1214U21 sense siNA inv stab07
B AuGAGGAccuucuAcAGGuTT B
3859


1215
GUGGACAUCUUCCAGGAGUACCC
2543
34002
VEGF:1215U21 sense siNA inv stab07
B cAuGAGGAccuucuAcAGGTT B
3860


1420
AAUGUGAAUGCAGACCAAAGAAA
2550
34004
VEGF:1420U21 sense siNA inv stab07
B AGAAAccAGAcGuAAGuGuTT B
3861


1423
GUGAAUGCAGACCAAAGAAAGAU
2552
34006
VEGF:1423U21 sense siNA inv stab07
B GAAAGAAAccAGAcGuAAGTT B
3862


1214
GGUGGACAUCUUCCAGGAGUACC
2542
34007
VEGF:1232L21 antisense siNA (1214 C.) inv stab08
AccuGuAGAAGGuccucAuTsT
3863


1215
GUGGACAUCUUCCAGGAGUACCC
2543
34008
VEGF:1233L21 antisense siNA (1215 C.) inv stab08
ccuGuAGAAGGuccucAuGTsT
3864


1420
AAUGUGAAUGCAGACCAAAGAAA
2550
34010
VEGF:1438L21 antisense siNA (1420 C.) inv stab08
AcAcuuAcGucuGGuuucuTsT
3865


1423
GUGAAUGCAGACCAAAGAAAGAU
2552
34012
VEGF:1441L21 antisense siNA (1423 C.) inv stab08
cuuAcGucuGGuuucuuucTsT
3866


1366
AAACCUCACCAAGGCCAGCACAU
2579
34062
VEGF:1366U21 sense siNA stab00 (HVEGF5)
ACCUCACCAAGGCCAGCACTT
3867


1366
AAACCUCACCAAGGCCAGCACAU
2579
34064
VEGF:1384L21 antisense siNA (1366 C.) stab00
GUGCUGGCCUUGGUGAGGUTT
3868






(HVEGF5)


1366
AAACCUCACCAAGGCCAGCACAU
2579
34066
VEGF:1366U21 sense siNA stab07 (HVEGF5)
B AccucAccAAGGccAGcAcTT B
3869


1366
AAACCUCACCAAGGCCAGCACAU
2579
34068
VEGF:1384L21 antisense siNA (1366 C.) stab08
GuGcuGGccuuGGuGAGGuTsT
3870






(HVEGF5)


1366
AAACCUCACCAAGGCCAGCACAU
2579
34070
VEGF:1366U21 sense siNA stab09 (HVEGF5)
B ACCUCACCAAGGCCAGCACTT B
3871


1366
AAACCUCACCAAGGCCAGCACAU
2579
34072
VEGF:1384L21 antisense siNA (1366 C.) stab10
GUGCUGGCCUUGGUGAGGUTsT
3872






(HVEGF5)


1366
AAACCUCACCAAGGCCAGCACAU
2579
34074
VEGF:1366U21 sense siNA inv stab00 (HVEGF5)
CACGACCGGAACCACUCCATT
3873


1366
AAACCUCACCAAGGCCAGCACAU
2579
34076
VEGF:1384L21 antisense siNA (1366 C.) inv stab00
UGGAGUGGUUCCGGUCGUGTT
3874






(HVEGF5)


1366
AAACCUCACCAAGGCCAGCACAU
2579
34078
VEGF:1366U21 sense siNA inv stab07 (HVEGF5)
B cAcGAccGGAAccAcuccATT B
3875


1366
AAACCUCACCAAGGCCAGCACAU
2579
34080
VEGF:1384L21 antisense siNA (1366 C.) inv stab08
uGGAGuGGuuccGGucGuGTsT
3876






(HVEGF5)


1366
AAACCUCACCAAGGCCAGCACAU
2579
34082
VEGF:1366U21 sense siNA inv stab09 (HVEGF5)
B CACGACCGGAACCACUCCATT B
3877


1366
AAACCUCACCAAGGCCAGCACAU
2579
34084
VEGF:1384L21 antisense siNA (1366 C.) inv stab10
UGGAGUGGUUCCGGUCGUGTsT
3878






(HVEGF5)


360
AGAGAGACGGGGUCAGAGAGAGC
2580
34681
VEGF:360U21 sense siNA stab00
AGAGACGGGGUCAGAGAGATT
3879


1562
AAAGCAUUUGUUUGUACAAGAUC
2581
34682
VEGF:1562U21 sense siNA stab00
AGCAUUUGUUUGUACAAGATT
3880


360
AGAGAGACGGGGUCAGAGAGAGC
2580
34689
VEGF:378L21 (360 C.) siRNA stab00
UCUCUCUGACCCCGUCUCUTT
3881


1562
AAAGCAUUUGUUUGUACAAGAUC
2581
34690
VEGF:1580L21 (1562 C.) siRNA stab00
UCUUGUACAAACAAAUGCUTT
3882


162
UCCCUCUUCUUUUUUCUUAAACA
2582
36002
VEGF:162U21 sense siNA stab00
CCUCUUCUUUUUUCUUAAATT
3883


163
CCCUCUUCUUUUUUCUUAAACAU
2583
36003
VEGF:163U21 sense siNA stab00
CUCUUCUUUUUUCUUAAACTT
3884


164
CCUCUUCUUUUUUCUUAAACAUU
2584
36004
VEGF:164U21 sense siNA stab00
UCUUCUUUUUUCUUAAACATT
3885


166
UCUUCUUUUUUCUUAAACAUUUU
2585
36005
VEGF:166U21 sense siNA stab00
UUCUUUUUUCUUAAACAUUTT
3886


169
UCUUUUUUCUUAAACAUUUUUUU
2586
36006
VEGF:169U21 sense siNA stab00
UUUUUUCUUAAACAUUUUUTT
3887


171
UUUUUUCUUAAACAUUUUUUUUU
2587
36007
VEGF:171U21 sense siNA stab00
UUUUCUUAAACAUUUUUUUTT
3888


172
UUUUUCUUAAACAUUUUUUUUUA
2588
36008
VEGF:172U21 sense siNA stab00
UUUCUUAAACAUUUUUUUUTT
3889


181
AACAUUUUUUUUUAAAACUGUAU
2589
36009
VEGF:181U21 sense siNA stab00
CAUUUUUUUUUAAAACUGUTT
3890


187
UUUUUUUAAAACUGUAUUGUUUC
2590
36010
VEGF:187U21 sense siNA stab00
UUUUUAAAACUGUAUUGUUTT
3891


188
UUUUUUAAAACUGUAUUGUUUCU
2591
36011
VEGF:188U21 sense siNA stab00
UUUUAAAACUGUAUUGUUUTT
3892


192
UUAAAACUGUAUUGUUUCUCGUU
2592
36012
VEGF:192U21 sense siNA stab00
AAAACUGUAUUGUUUCUCGTT
3893


202
AUUGUUUCUCGUUUUAAUUUAUU
2593
36013
VEGF:202U21 sense siNA stab00
UGUUUCUCGUUUUAAUUUATT
3894


220
UUAUUUUUGCUUGCCAUUCCCCA
2594
36014
VEGF:220U21 sense siNA stab00
AUUUUUGCUUGCCAUUCCCTT
3895


237
UCCCCACUUGAAUCGGGCCGACG
2595
36015
VEGF:237U21 sense siNA stab00
CCCACUUGAAUCGGGCCGATT
3896


238
CCCCACUUGAAUCGGGCCGACGG
2596
36016
VEGF:238U21 sense siNA stab00
CCACUUGAAUCGGGCCGACTT
3897


338
CUCCAGAGAGAAGUCGAGGAAGA
2597
36017
VEGF:338U21 sense siNA stab00
CCAGAGAGAAGUCGAGGAATT
3898


339
UCCAGAGAGAAGUCGAGGAAGAG
2598
36018
VEGF:339U21 sense siNA stab00
CAGAGAGAAGUCGAGGAAGTT
3899


371
GUCAGAGAGAGCGCGCGGGCGUG
2599
36019
VEGF:371U21 sense siNA stab00
CAGAGAGAGCGCGCGGGCGTT
3900


484
GCAGCUGACCAGUCGCGCUGACG
2600
36020
VEGF:484U21 sense siNA stab00
AGCUGACCAGUCGCGCUGATT
3901


598
GGCCGGAGCCCGCGCCCGGAGGC
2601
36021
VEGF:598U21 sense siNA stab00
CCGGAGCCCGCGCCCGGAGTT
3902


599
GCCGGAGCCCGCGCCCGGAGGCG
2602
36022
VEGF:599U21 sense siNA stab00
CGGAGCCCGCGCCCGGAGGTT
3903


600
CCGGAGCCCGCGCCCGGAGGCGG
2603
36023
VEGF:600U21 sense siNA stab00
GGAGCCCGCGCCCGGAGGCTT
3904


652
CACUGAAACUUUUCGUCCAACUU
2604
36024
VEGF:652U21 sense siNA stab00
CUGAAACUUUUCGUCCAACTT
3905


653
ACUGAAACUUUUCGUCCAACUUC
2605
36025
VEGF:653U21 sense siNA stab00
UGAAACUUUUCGUCCAACUTT
3906


654
CUGAAACUUUUCGUCCAACUUCU
2606
36026
VEGF:654U21 sense siNA stab00
GAAACUUUUCGUCCAACUUTT
3907


658
AACUUUUCGUCCAACUUCUGGGC
2607
36027
VEGF:658U21 sense siNA stab00
CUUUUCGUCCAACUUCUGGTT
3908


672
CUUCUGGGCUGUUCUCGCUUCGG
2608
36028
VEGF:672U21 sense siNA stab00
UCUGGGCUGUUCUCGCUUCTT
3909


674
UCUGGGCUGUUCUCGCUUCGGAG
2609
36029
VEGF:674U21 sense siNA stab00
UGGGCUGUUCUCGCUUCGGTT
3910


691
UCGGAGGAGCCGUGGUCCGCGCG
2610
36030
VEGF:691U21 sense siNA stab00
GGAGGAGCCGUGGUCCGCGTT
3911


692
CGGAGGAGCCGUGGUCCGCGCGG
2611
36031
VEGF:692U21 sense siNA stab00
GAGGAGCCGUGGUCCGCGCTT
3912


758
CCGGGAGGAGCCGCAGCCGGAGG
2612
36032
VEGF:758U21 sense siNA stab00
GGGAGGAGCCGCAGCCGGATT
3913


759
CGGGAGGAGCCGCAGCCGGAGGA
2613
36033
VEGF:759U21 sense siNA stab00
GGAGGAGCCGCAGCCGGAGTT
3914


760
GGGAGGAGCCGCAGCCGGAGGAG
2614
36034
VEGF:760U21 sense siNA stab00
GAGGAGCCGCAGCCGGAGGTT
3915


795
GAAGAGAAGGAAGAGGAGAGGGG
2615
36035
VEGF:795U21 sense siNA stab00
AGAGAAGGAAGAGGAGAGGTT
3916


886
GUGCUCCAGCCGCGCGCGCUCCC
2616
36036
VEGF:886U21 sense siNA stab00
GCUCCAGCCGCGCGCGCUCTT
3917


977
GCCCCACAGCCCGAGCCGGAGAG
2617
36037
VEGF:977U21 sense siNA stab00
CCCACAGCCCGAGCCGGAGTT
3918


978
CCCCACAGCCCGAGCCGGAGAGG
2618
36038
VEGF:978U21 sense siNA stab00
CCACAGCCCGAGCCGGAGATT
3919


1038
ACCAUGAACUUUCUGCUGUCUUG
2619
36039
VEGF:1038U21 sense siNA stab00
CAUGAACUUUCUGCUGUCUTT
3920


1043
GAACUUUCUGCUGUCUUGGGUGC
2620
36040
VEGF:1043U21 sense siNA stab00
ACUUUCUGCUGUCUUGGGUTT
3921


1049
UCUGCUGUCUUGGGUGCAUUGGA
2621
36041
VEGF:1049U21 sense siNA stab00
UGCUGUCUUGGGUGCAUUGTT
3922


1061
GGUGCAUUGGAGCCUUGCCUUGC
2622
36042
VEGF:1061U21 sense siNA stab00
UGCAUUGGAGCCUUGCCUUTT
3923


1072
GCCUUGCCUUGCUGCUCUACCUC
2623
36043
VEGF:1072U21 sense siNA stab00
CUUGCCUUGCUGCUCUACCTT
3924


1088
UCACCUCCACCAUGCCAAGUGGU
2624
36044
VEGF:1088U21 sense siNA stab00
ACCUCCACCAUGCCAAGUGTT
3925


1089
CUCCUCCACCAUGCCAAGUGGUC
2625
36045
VEGF:1089U21 sense siNA stab00
CCUCCACCAUGCCAAGUGGTT
3926


1095
CACCAUGCCAAGUGGUCCCAGGC
2626
36046
VEGF:1095U21 sense siNA stab00
CCAUGCCAAGUGGUCCCAGTT
3927


1110
UCCCAGGCUGCACCCAUGGCAGA
2627
36047
VEGF:1110U21 sense siNA stab00
CCAGGCUGCACCCAUGGCATT
3928


1175
AUUCUAUCAGCGCAGCUACUGCC
2628
36048
VEGF:1175U21 sense siNA stab00
UCUAUCAGCGCAGCUACUGTT
3929


1220
CAUCUUCCAGGAGUACCCUGAUG
2629
36049
VEGF:1220U21 sense siNA stab00
UCUUCCAGGAGUACCCUGATT
3930


1253
CAUCUUCAAGCCAUCCUGUGUGC
2630
36050
VEGF:1253U21 sense siNA stab00
UCUUCAAGCCAUCCUGUGUTT
3931


1300
CUAAUGACGAGGGCCUGGAGUGU
2631
36051
VEGF:1300U21 sense siNA stab00
AAUGACGAGGGCCUGGAGUTT
3932


1309
CGGGCCUGGAGUGUGUGCCCACU
2632
36052
VEGF:1309U21 sense siNA stab00
GGCCUGGAGUGUGUGCCCATT
3933


1326
CCCACUGAGGAGUCCAACAUCAC
2633
36053
VEGF:1326U21 sense siNA stab00
CACUGAGGAGUCCAACAUCTT
3934


1338
UCCAACAUCACCAUGCAGAUUAU
2634
36054
VEGF:1338U21 sense siNA stab00
CAACAUCACCAUGCAGAUUTT
3935


1342
ACAUCACCAUGCAGAUUAUGCGG
2635
36055
VEGF:1342U21 sense siNA stab00
AUCACCAUGCAGAUUAUGCTT
3936


1351
UGCAGAUUAUGCGGAUCAAACCU
2636
36056
VEGF:1351U21 sense siNA stab00
CAGAUUAUGCGGAUCAAACTT
3937


1352
GCAGAUUAUGCGGAUCAAACCUC
2637
36057
VEGF:1352U21 sense siNA stab00
AGAUUAUGCGGAUCAAACCTT
3938


1353
CAGAUUAUGCGGAUCAAACCUCA
2638
36058
VEGF:1353U21 sense siNA stab00
GAUUAUGCGGAUCAAACCUTT
3939


1389
AUAGGAGAGAUGAGCUUCCUACA
2639
36059
VEGF:1389U21 sense siNA stab00
AGGAGAGAUGAGCUUCCUATT
3940


1398
GAGAGCUUCCUACAGCACAACAA
2640
36060
VEGF:1398U21 sense siNA stab00
GAGCUUCCUACAGCACAACTT
3941


1401
AGCUUCCUACAGCACAACAAAUG
2641
36061
VEGF:1401U21 sense siNA stab00
CUUCCUACAGCACAACAAATT
3942


1407
CCACAGCACAACAAAUGUGAAUG
2642
36062
VEGF:1407U21 sense siNA stab00
ACAGCACAACAAAUGUGAATT
3943


1408
UACAGCACAACAAAUGUGAAUGC
2643
36063
VEGF:1408U21 sense siNA stab00
CAGCACAACAAAUGUGAAUTT
3944


1417
ACAAAUGUGAAUGCAGACCAAAG
2644
36064
VEGF:1417U21 sense siNA stab00
AAAUGUGAAUGCAGACCAATT
3945


162
UCCCUCUUCUUUUUUCUUAAACA
2582
36065
VEGF:180L21 antisense siNA (162 C.) stab00
UUUAAGAAAAAAGAAGAGGTT
3946


163
CCCUCUUCUUUUUUCUUAAACAU
2583
36066
VEGF:181L21 antisense siNA (163 C.) stab00
GUUUAAGAAAAAAGAAGAGTT
3947


164
CCUCUUCUUUUUUCUUAAACAUU
2584
36067
VEGF:182L21 antisense siNA (164 C.) stab00
UGUUUAAGAAAAAAGAAGATT
3948


166
UCUUCUUUUUUCUUAAACAUUUU
2585
36068
VEGF:184L21 antisense siNA (166 C.) stab00
AAUGUUUAAGAAAAAAGAATT
3949


169
UCUUUUUUCUUAAACAUUUUUUU
2586
36069
VEGF:187L21 antisense siNA (169 C.) stab00
AAAAAUGUUUAAGAAAAAATT
3950


171
UUUUUUCUUAAACAUUUUUUUUU
2587
36070
VEGF:189L21 antisense siNA (171 C.) stab00
AAAAAAAUGUUUAAGAAAATT
3951


172
UUUUUCUUAAACAUUUUUUUUUA
2588
36071
VEGF:190L21 antisense siNA (172 C.) stab00
AAAAAAAAUGUUUAAGAAATT
3952


181
AACAUUUUUUUUUAAAACUGUAU
2589
36072
VEGF:199L21 antisense siNA (181 C.) stab00
ACAGUUUUAAAAAAAAAUGTT
3953


187
UUUUUUUAAAACUGUAUUGUUUC
2590
36073
VEGF:205L21 antisense siNA (187 C.) stab00
AACAAUACAGUUUUAAAAATT
3954


188
UUUUUUAAAACUGUAUUGUUUCU
2591
36074
VEGF:206L21 antisense siNA (188 C.) stab00
AAACAAUACAGUUUUAAAATT
3955


192
UUAAAACUGUAUUGUUUCUCGUU
2592
36075
VEGF:210L21 antisense siNA (192 C.) stab00
CGAGAAACAAUACAGUUUUTT
3956


202
AUUGUUUCUCGUUUUAAUUUAUU
2593
36076
VEGF:220L21 antisense siNA (202 C.) stab00
UAAAUUAAAACGAGAAACATT
3957


220
UUAUUUUUGCUUGCCAUUCCCCA
2594
36077
VEGF:238L21 antisense siNA (220 C.) stab00
GGGAAUGGCAAGCAAAAAUTT
3958


237
UCCCCACUUGAAUCGGGCCGACG
2595
36078
VEGF:255L21 antisense siNA (237 C.) stab00
UCGGCCCGAUUCAAGUGGGTT
3959


238
CCCCACUUGAAUCGGGCCGACGG
2596
36079
VEGF:256L21 antisense siNA (238 C.) stab00
GUCGGCCCGAUUCAAGUGGTT
3960


338
CUCCAGAGAGAAGUCGAGGAAGA
2597
36080
VEGF:356L21 antisense siNA (338 C.) stab00
UUCCUCGACUUCUCUCUGGTT
3961


339
UCCAGAGAGAAGUCGAGGAAGAG
2598
36081
VEGF:357L21 antisense siNA (339 C.) stab00
CUUCCUCGACUUCUCUCUGTT
3962


371
GUCAGAGAGAGCGCGCGGGCGUG
2599
36082
VEGF:389L21 antisense siNA (371 C.) stab00
CGCCCGCGCGCUCUCUCUGTT
3963


484
GCAGCUGACCAGUCGCGCUGACG
2600
36083
VEGF:502L21 antisense siNA (484 C.) stab00
UCAGCGCGACUGGUCAGCUTT
3964


598
GGCCGGAGCCCGCGCCCGGAGGC
2601
36084
VEGF:616L21 antisense siNA (598 C.) stab00
CUCCGGGCGCGGGCUCCGGTT
3965


599
GCCGGAGCCCGCGCCCGGAGGCG
2602
36085
VEGF:617L21 antisense siNA (599 C.) stab00
CCUCCGGGCGCGGGCUCCGTT
3966


600
CCGGAGCCCGCGCCCGGAGGCGG
2603
36086
VEGF:618L21 antisense siNA (600 C.) stab00
GCCUCCGGGCGCGGGCUCCTT
3967


652
CACUGAAACUUUUCGUCCAACUU
2604
36087
VEGF:670L21 antisense siNA (652 C.) stab00
GUUGGACGAAAAGUUUCAGTT
3968


653
ACUGAAACUUUUCGUCCAACUUC
2605
36088
VEGF:671L21 antisense siNA (653 C.) stab00
AGUUGGACGAAAAGUUUCATT
3969


654
CUGAAACUUUUCGUCCAACUUCU
2606
36089
VEGF:672L21 antisense siNA (654 C.) stab00
AAGUUGGACGAAAAGUUUCTT
3970


658
AACUUUUCGUCCAACUUCUGGGC
2607
36090
VEGF:676L21 antisense siNA (658 C.) stab00
CCAGAAGUUGGACGAAAAGTT
3971


672
CUUCUGGGCUGUUCUCGCUUCGG
2608
36091
VEGF:690L21 antisense siNA (672 C.) stab00
GAAGCGAGAACAGCCCAGATT
3972


674
UCUGGGCUGUUCUCGCUUCGGAG
2609
36092
VEGF:692L21 antisense siNA (674 C.) stab00
CCGAAGCGAGAACAGCCCATT
3973


691
UCGGAGGAGCCGUGGUCCGCGCG
2610
36093
VEGF:709L21 antisense siNA (691 C.) stab00
CGCGGACCACGGCUCCUCCTT
3974


692
CGGAGGAGCCGUGGUCCGCGCGG
2611
36094
VEGF:710L21 antisense siNA (692 C.) stab00
GCGCGGACCACGGCUCCUCTT
3975


758
CCGGGAGGAGCCGCAGCCGGAGG
2612
36095
VEGF:776L21 antisense siNA (758 C.) stab00
UCCGGCUGCGGCUCCUCCCTT
3976


759
CGGGAGGAGCCGCAGCCGGAGGA
2613
36096
VEGF:777L21 antisense siNA (759 C.) stab00
CUCCGGCUGCGGCUCCUCCTT
3977


760
GGGAGGAGCCGCAGCCGGAGGAG
2614
36097
VEGF:778L21 antisense siNA (760 C.) stab00
CCUCCGGCUGCGGCUCCUCTT
3978


795
GAAGAGAAGGAAGAGGAGAGGGG
2615
36098
VEGF:813L21 antisense siNA (795 C.) stab00
CCUCUCCUCUUCCUUCUCUTT
3979


886
GUGCUCCAGCCGCGCGCGCUCCC
2616
36099
VEGF:904L21 antisense siNA (886 C.) stab00
GAGCGCGCGCGGCUGGAGCTT
3980


977
GCCCCACAGCCCGAGCCGGAGAG
2617
36100
VEGF:995L21 antisense siNA.(977 C.) stab00
CUCCGGCUCGGGCUGUGGGTT
3981


978
CCCCACAGCCCGAGCCGGAGAGG
2618
36101
VEGF:996L21 antisense siNA (978 C.) stab00
UCUCCGGCUCGGGCUGUGGTT
3982


1038
ACCAUGAACUUUCUGCUGUCUUG
2619
36102
VEGF:1056L21 antisense siNA (1038 C.) stab00
AGACAGCAGAAAGUUCAUGTT
3983


1043
GAACUUUCUGCUGUCUUGGGUGC
2620
36103
VEGF:1061L21 antisense siNA (1043 C.) stab00
ACCCAAGACAGCAGAAAGUTT
3984


1049
UCUGCUGUCUUGGGUGCAUUGGA
2621
36104
VEGF:1067L21 antisense siNA (1049 C.) stab00
CAAUGCACCCAAGACAGCATT
3985


1061
GGUGCAUUGGAGCCUUGCCUUGC
2622
36105
VEGF:1079L21 antisense siNA (1061 C.) stab00
AAGGCAAGGCUCCAAUGCATT
3986


1072
GCCUUGCCUUGCUGCUCUACCUC
2623
36106
VEGF:1090L21 antisense siNA (1072 C.) stab00
GGUAGAGCAGCAAGGCAAGTT
3987


1088
UCACCUCCACCAUGCCAAGUGGU
2624
36107
VEGF:1106L21 antisense siNA (1088 C.) stab00
CACUUGGCAUGGUGGAGGUTT
3988


1089
CUCCUCCACCAUGCCAAGUGGUC
2625
36108
VEGF:1107L21 antisense siNA (1089 C.) stab00
CCACUUGGCAUGGUGGAGGTT
3989


1095
CACCAUGCCAAGUGGUCCCAGGC
2626
36109
VEGF:1113L21 antisense siNA (1095 C.) stab00
CUGGGACCACUUGGCAUGGTT
3990


1110
UCCCAGGCUGCACCCAUGGCAGA
2627
36110
VEGF:1128L21 antisense siNA (1110 C.) stab00
UGCCAUGGGUGCAGCCUGGTT
3991


1175
AUUCUAUCAGCGCAGCUACUGCC
2628
36111
VEGF:1193L21 antisense siNA (1175 C.) stab00
CAGUAGCUGCGCUGAUAGATT
3992


1220
CAUCUUCCAGGAGUACCCUGAUG
2629
36112
VEGF:1238L21 anhisense siNA (1220 C.) stab00
UCAGGGUACUCCUGGAAGATT
3993


1253
CAUCUUCAAGCCAUCCUGUGUGC
2630
36113
VEGF:1271L21 antisense siNA (1253 C.) stab00
ACACAGGAUGGCUUGAAGATT
3994


1300
CUAAUGACGAGGGCCUGGAGUGU
2631
36114
VEGF:1318L21 antisense siNA (1300 C.) stab00
ACUCCAGGCCCUCGUCAUUTT
3995


1309
CGGGCCUGGAGUGUGUGCCCACU
2632
36115
VEGF:1327L21 antisense siNA (1309 C.) stab00
UGGGCACACACUCCAGGCCTT
3996


1326
CCCACUGAGGAGUCCAACAUCAC
2633
36116
VEGF:1344L21 antisense siNA (1326 C.) stab00
GAUGUUGGACUCCUCAGUGTT
3997


1338
UCCAACAUCACCAUGCAGAUUAU
2634
36117
VEGF:1356L21 antisense siNA (1338 C.) stab00
AAUCUGCAUGGUGAUGUUGTT
3998


1342
ACAUCACCAUGCAGAUUAUGCGG
2635
36118
VEGF:1360L21 antisense siNA (1342 C.) stab00
GCAUAAUCUGCAUGGUGAUTT
3999


1351
UGCAGAUUAUGCGGAUCAAACCU
2636
36119
VEGF:1369L21 antisense siNA (1351 C.) stab00
GUUUGAUCCGCAUAAUCUGTT
4000


1352
GCAGAUUAUGCGGAUCAAACCUC
2637
36120
VEGF:1370L21 antisense siNA (1352 C.) stab00
GGUUUGAUCCGCAUAAUCUTT
4001


1353
CAGAUUAUGCGGAUCAAACCUCA
2638
36121
VEGF:1371L21 antisense siNA (1353 C.) stab00
AGGUUUGAUCCGCAUAAUCTT
4002


1389
AUAGGAGAGAUGAGCUUCCUACA
2639
36122
VEGF:1407L21 antisense siNA (1389 C.) stab00
UAGGAAGCUCAUCUCUCCUTT
4003


1398
GAGAGCUUCCUACAGCACAACAA
2640
36123
VEGF:1416L21 antisense siNA (1398 C.) stab00
GUUGUGCUGUAGGAAGCUCTT
4004


1401
AGCUUCCUACAGCACAACAAAUG
2641
36124
VEGF:1419L21 antisense siNA (1401 C.) stab00
UUUGUUGUGCUGUAGGAAGTT
4005


1407
CCACAGCACAACAAAUGUGAAUG
2642
36125
VEGF:1425L21 antisense siNA (1407 C.) stab00
UUCACAUUUGUUGUGCUGUTT
4006


1408
UACAGCACAACAAAUGUGAAUGC
2643
36126
VEGF:1426L21 antisense siNA (1408 C.) stab00
AUUCACAUUUGUUGUGCUGTT
4007


1417
ACAAAUGUGAAUGCAGACCAAAG
2644
36127
VEGF:1435L21 antisense siNA (1417 C.) stab00
UUGGUCUGCAUUCACAUUUTT
4008


1089
UACCUCCACCAUGCCAAGUGGUC
2645
37293
VEGF:1089U21 sense siNA stab07
B ccuccAccAuGccAAGuGGTT B
4009


1090
ACCUCCACCAUGCCAAGUGGUCC
2646
37294
VEGF:1090U21 sense siNA stab07
B cuccAccAuGccAAGuGGuTT B
4010


1095
CACCAUGCCAAGUGGUCCCAGGC
2626
37295
VEGF:1095U21 sense siNA stab07
B ccAuGccAAGuGGucccAGTT B
4011


1096
ACCAUGCCAAGUGGUCCCAGGCU
2647
37296
VEGF:1096U21 sense siNA stab07
B cAuGccAAGuGGucccAGGTT B
4012


1097
CCAUGCCAAGUGGUCCCAGGCUG
2648
37297
VEGF:1097U21 sense siNA stab07
B AuGccAAGuGGucccAGGcTT B
4013


1098
CAUGCCAAGUGGUCCCAGGCUGC
2649
37298
VEGF:1098U21 sense siNA stab07
B uGccAAGuGGucccAGGcuTT B
4014


1099
AUGCCAAGUGGUCCCAGGCUGCA
2650
37299
VEGF:1099U21 sense siNA stab07
B GccAAGuGGucccAGGcuGTT B
4015


1100
UGCCAAGUGGUCCCAGGCUGCAC
2651
37300
VEGF:1100U21 sense siNA stab07
B ccAAGuGGucccAGGcuGcTT B
4016


1104
AAGUGGUCCCAGGCUGCACCCAU
2652
37301
VEGF:1104U21 sense siNA stab07
B GuGGucccAGGcuGcAcccTT B
4017


1105
AGUGGUCCCAGGCUGCACCCAUG
2653
37302
VEGF:1105U21 sense siNA stab07
B uGGucccAGGcuGcAcccATT B
4018


1208
GACCCUGGUGGACAUCUUCCAGG
2562
37303
VEGF:1208U21 sense siNA stab07
B cccuGGuGGAcAucuuccATT B
4019


1424
UGAAUGCAGACCAAAGAAAGAUA
2654
37304
VEGF:1424U21 sense siNA stab07
B AAuGcAGAccAAAGAAAGATT B
4020


1549
GCUCAGAGCGGAGAAAGCAUUUG
2655
37305
VEGF:1549U21 sense siNA stab07
B ucAGAGcGGAGAAAGcAuuTT B
4021


1584
CCGCAGACGUGUAAAUGUUCCUG
2565
37306
VEGF:1584U21 sense siNA stab07
B GcAGAcGuGuAAAuGuuccTT B
4022


1585
CGCAGACGUGUAAAUGUUCCUGC
2566
37307
VEGF:1585U21 sense siNA stab07
B cAGAcGuGuAAAuGuuccuTT B
4023


1589
GACGUGUAAAUGUUCCUGCAAAA
2567
37308
VEGF:1589U21 sense siNA stab07
B cGuGuAAAuGuuccuGcAATT B
4024


1591
CGUGUAAAUGUUCCUGCAAAAAC
2554
37309
VEGF:1591U21 sense siNA stab07
B uGuAAAuGuuccuGcAAAATT B
4025


1592
GUGUAAAUGUUCCUGCAAAAACA
2555
37310
VEGF:1592U21 sense siNA stab07
B GuAAAuGuuccuGcAAAAATT B
4026


1593
UGUAAAUGUUCCUGCAAAAACAC
2556
37311
VEGF:1593U21 sense siNA stab07
B uAAAuGuuccuGcAAAAAcTT B
4027


1594
GUAAAUGUUCCUGCAAAAACACA
2557
37312
VEGF:1594U21 sense siNA stab07
B AAAuGuuccuGcAAAAAcATT B
4028


1595
UAAAUGUUCCUGCAAAAACACAG
2568
37313
VEGF:1595U21 sense siNA stab07
B AAuGuuccuGcAAAAAcAcTT B
4029


1597
AAUGUUCCUGCAAAAACACAGAC
2656
37314
VEGF:1597U21 sense siNA stab07
B uGuuccuGcAAAAAcAcAGTT B
4030


1598
AUGUUCCUGCAAAAACACAGACU
2657
37315
VEGF:1598U21 sense siNA stab07
B GuuccuGcAAAAAcAcAGATT B
4031


1599
UGUUCCUGCAAAAACACAGACUC
2658
37316
VEGF:1599U21 sense siNA stab07
B uuccuGcAAAAAcAcAGAcTT B
4032


1600
GUUCCUGCAAAAACACAGACUCG
2659
37317
VEGF:1600U21 sense siNA stab07
B uccuGcAAAAAcAcAGAcuTT B
4033


1604
CUGCAAAAACACAGACUCGCGUU
2558
37318
VEGF:1604U21 sense siNA stab07
B GcAAAAAcAcAGAcucGcGTT B
4034


1605
UGCAAAAACACAGACUCGCGUUG
2660
37319
VEGF:1605U21 sense siNA stab07
B cAAAAAcAcAGAcucGcGuTT B
4035


1608
AAAAACACAGACUCGCGUUGCAA
2661
37320
VEGF:1608U21 sense siNA stab07
B AAAcAcAGAcucGcGuuGcTT B
4036


1612
ACACAGACUCGCGUUGCAAGGCG
2662
37321
VEGF:1612U21 sense siNA stab07
B AcAGAcucGcGuuGcAAGGTT B
4037


1616
AGACUCGCGUUGCAAGGCGAGGC
2663
37322
VEGF:1616U21 sense siNA stab07
B AcucGcGuuGcAAGGcGAGTT B
4038


1622
GCGUUGCAAGGCGAGGCAGCUUG
2664
37323
VEGF:1622U21 sense siNA stab07
B GuuGcAAGGcGAGGcAGcuTT B
4039


1626
UGCAAGGCGAGGCAGCUUGAGUU
2665
37324
VEGF:1626U21 sense siNA stab07
B cAAGGcGAGGcAGcuuGAGTT B
4040


1628
CAAGGCGAGGCAGCUUGAGUUAA
2666
37325
VEGF:1628U21 sense siNA stab07
B AGGcGAGGcAGcuuGAGuuTT B
4041


1633
CGAGGCAGCUUGAGUUAAACGAA
2573
37326
VEGF:1633U21 sense siNA stab07
B AGGcAGcuuGAGuuAAAcGTT B
4042


1634
GAGGCAGCUUGAGUUAAACGAAC
2574
37327
VEGF:1634U21 sense siNA stab07
B GGcAGcuuGAGuuAAAcGATT B
4043


1635
AGGCAGCUUGAGUUAAACGAACG
2575
37328
VEGF:1635U21 sense siNA stab07
B GcAGcuuGAGuuAAAcGAATT B
4044


1637
GCAGCUUGAGUUAAACGAACGUA
2559
37329
VEGF:1637U21 sense siNA stab07
B AGcuuGAGuuAAAcGAAcGTT B
4045


1643
UGAGUUAAACGAACGUACUUGCA
2667
37330
VEGF:1643U21 sense siNA stab07
B AGuuAAAcGAAcGuAcuuGTT B
4046


1645
AGUUAAACGAACGUACUUGCAGA
2668
37331
VEGF:1645U21 sense siNA stab07
B uuAAAcGAAcGuAcuuGcATT B
4047


1646
GUUAAACGAACGUACUUGCAGAU
2669
37332
VEGF:1646U21 sense siNA stab07
B uAAAcGAAcGuAcuuGcAGTT B
4048


1647
UUAAACGAACGUACUUGCAGAUG
2670
37333
VEGF:1647U21 sense siNA stab07
B AAAcGAAcGuAcuuGcAGATT B
4049


1648
UAAACGAACGUACUUGCAGAUGU
2577
37334
VEGF:1648U21 sense siNA stab07
B AAcGAAcGuAcuuGcAGAuTT B
4050


1655
ACGUACUUGCAGAUGUGACAAGC
2671
37335
VEGF:1655U21 sense siNA stab07
B GuAcuuGcAGAuGuGAcAATT B
4051


1656
CGUACUUGCAGAUGUGACAAGCC
2560
37336
VEGF:1656U21 sense siNA stab07
B uAcuuGcAGAuGuGAcAAGTT B
4052


1657
GUACUUGCAGAUGUGACAAGCCG
2672
37337
VEGF:1657U21 sense siNA stab07
B AcuuGcAGAuGuGAcAAGcTT B
4053


1089
UACCUCCACCAUGCCAAGUGGUC
2645
37338
VEGF:1107L21 antisense siNA (1089 C.) stab26
CCAcuuGGcAuGGuGGAGGTT
4054


1090
ACCUCCACCAUGCCAAGUGGUCC
2646
37339
VEGF:1108L21 antisense siNA (1090 C.) stab26
ACCAcuuGGcAuGGuGGAGTT
4055


1095
CACCAUGCCAAGUGGUCCCAGGC
2626
37340
VEGF:1113L21 antisense siNA (1095 C.) stab26
CUGGGAccAcuuGGcAuGGTT
4056


1096
ACCAUGCCAAGUGGUCCCAGGCU
2647
37341
VEGF:1114L21 antisense siNA (1096 C.) stab26
CCUGGGAccAcuuGGcAuGTT
4057


1097
CCAUGCCAAGUGGUCCCAGGCUG
2648
37342
VEGF:1115L21 antisense siNA (1097 C.) stab26
GCCuGGGAccAcuuGGcAuTT
4058


1098
CAUGCCAAGUGGUCCCAGGCUGC
2649
37343
VEGF:1116L21 antisense siNA (1098 C.) stab26
AGCcuGGGAccAcuuGGcATT
4059


1099
AUGCCAAGUGGUCCCAGGCUGCA
2650
37344
VEGF:1117L21 antisense siNA (1099 C.) stab26
CAGccuGGGAccAcuuGGcTT
4060


1100
UGCCAAGUGGUCCCAGGCUGCAC
2651
37345
VEGF:1118L21 antisense siNA (i10O C.) stab26
GCAGccuGGGAccAcuuGGTT
4061


1104
AAGUGGUCCCAGGCUGCACCCAU
2652
37346
VEGF:1122L21 antisense siNA (1104 C.) stab26
GGGuGcAGccuGGGAccAcTT
4062


1105
AGUGGUCCCAGGCUGCACCCAUG
2653
37347
VEGF:1123L21 antisense siNA (1105 C.) stab26
UGGGuGcAGccuGGGAccATT
4063


1208
GACCCUGGUGGACAUCUUCCAGG
2562
37348
VEGF:1226L21 antisense siNA (1208 C.) stab26
UGGAAGAuGuccAccAGGGTT
4064


1214
GGUGGACAUCUUCCAGGAGUACC
2542
37349
VEGF:1232L21 antisense siNA (1214 C.) stab26
UACuccuGGAAGAuGuccATT
4065


1421
AUGUGAAUGCAGACCAAAGAAAG
2551
37350
VEGF:1439L21 antisense siNA (1421 C.) stab26
UUCuuuGGucuGcAuucAcTT
4066


1423
GUGAAUGCAGACCAAAGAAAGAU
2552
37351
VEGF:1441L21 antisense siNA (1423 C.) stab26
CUUucuuuGGucuGcAuucTT
4067


1424
UGAAUGCAGACCAAAGAAAGAUA
2654
37352
VEGF:1442L21 antisense siNA (1424 C.) stab26
UCUuucuuuGGucuGcAuuTT
4068


1549
GCUCAGAGCGGAGAAAGCAUUUG
2655
37353
VEGF:1567L21 antisense siNA (1549 C.) stab26
AAUGcuuucuccGcucuGATT
4069


1584
CCGCAGACGUGUAAAUGUUCCUG
2565
37354
VEGF:1602L21 antisense siNA (1584 C.) stab26
GGAAcAuuuAcAcGucuGcTT
4070


1585
CGCAGACGUGUAAAUGUUCCUGC
2566
37355
VEGF:1603L21 antisense siNA (1585 C.) stab26
AGGAAcAuuuAcAcGucuGTT
4071


1589
GACGUGUAAAUGUUCCUGCAAAA
2567
37356
VEGF:1607L21 antisense siNA (1589 C.) stab26
UUGcAGGAAcAuuuAcAcGTT
4072


1591
CGUGUAAAUGUUCCUGCAAAAAC
2554
37357
VEGF:1609L21 antisense siNA (1591 C.) stab26
UUUuGcAGGAAcAuuuAcATT
4073


1592
GUGUAAAUGUUCCUGCAAAAACA
2555
37358
VEGF:1610L21 antisense siNA (1592 C.) stab26
UUUuuGcAGGAAcAuuuAcTT
4074


1593
UGUAAAUGUUCCUGCAAAAACAC
2556
37359
VEGF:1611L21 antisense siNA (1593 C.) stab26
GUUuuuGcAGGAAcAuuuATT
4075


1594
GUAAAUGUUCCUGCAAAAACACA
2557
37360
VEGF:1612L21 antisense siNA (1594 C.) stab26
UGUuuuuGcAGGAAcAuuuTT
4076


1595
UAAAUGUUCCUGCAAAAACACAG
2568
37361
VEGF:1613L21 antisense siNA (1595 C.) stab26
GUGuuuuuGcAGGAAcAuuTT
4077


1597
AAUGUUCCUGCAAAAACACAGAC
2656
37362
VEGF:1615L21 antisense siNA (1597 C.) stab26
CUGuGuuuuuGcAGGAAcATT
4078


1598
AUGUUCCUGCAAAAACACAGACU
2657
37363
VEGF:1616L21 antisense siNA (1598 C.) stab26
UCUGuGuuuuuGcAGGAAcTT
4079


1599
UGUUCCUGCAAAAACACAGACUC
2658
37364
VEGF:1617L21 antisense siNA (1599 C.) stab26
GUCuGuGuuuuuGcAGGAATT
4080


1600
GUUCCUGCAAAAACACAGACUCG
2659
37365
VEGF:1618L21 antisense siNA (1600 C.) stab26
AGUcuGuGuuuuuGcAGGATT
4081


1604
CUGCAAAAACACAGACUCGCGUU
2558
37366
VEGF:1622L21 antisense siNA (1604 C.) stab26
CGCGAGucuGuGuuuuuGcTT
4082


1605
UGCAAAAACACAGACUCGCGUUG
2660
37367
VEGF:1623L21 antisense siNA (1605 C.) stab26
ACGcGAGucuGuGuuuuuGTT
4083


1608
AAAAACACAGACUCGCGUUGCAA
2661
37368
VEGF:1626L21 antisense siNA (1608 C.) stab26
GCAACGcGAGucuGuGuuuTT
4084


1612
ACACAGACUCGCGUUGCAAGGCG
2662
37369
VEGF:1630L21 antisense siNA (1612 C.) stab26
CCUuGcAAcGcGAGucuGuTT
4085


1616
AGACUCGCGUUGCAAGGCGAGGC
2663
37370
VEGF:1634L21 antisense siNA (1616 C.) stab26
CUCGccuuGcAAcGcGAGuTT
4086


1622
GCGUUGCAAGGCGAGGCAGCUUG
2664
37371
VEGF:1640L21 antisense siNA (1622 C.) stab26
AGCuGccucGccuuGcAAcTT
4087


1626
UGCAAGGCGAGGCAGCUUGAGUU
2665
37372
VEGF:1644L21 antisense siNA (1626 C.) stab26
CUCAAGcuGccucGccuuGTT
4088


1628
CAAGGCGAGGCAGCUUGAGUUAA
2666
37373
VEGF:1646L21 antisense siNA (1628 C.) stab26
AACucAAGcuGccucGccuTT
4089


1633
CGAGGCAGCUUGAGUUAAACGAA
2573
37374
VEGF:1651L21 antisense siNA (1633 C.) stab26
CGUuuAAcucAAGcuGccuTT
4090


1634
GAGGCAGCUUGAGUUAAACGAAC
2574
37375
VEGF:1652L21 antisense siNA (1634 C.) stab26
UCGuuuAAcucAAGcuGccTT
4091


1635
AGGCAGCUUGAGUUAAACGAACG
2575
37376
VEGF:1653L21 antisense siNA (1635 C.) stab26
UUCGuuuAAcucAAGcuGcTT
4092


1636
GGCAGCUUGAGUUAAACGAACGU
2576
37377
VEGF:1654L21 antisense siNA (1636 C.) stab26
GUUcGuuuAAcucAAGcuGTT
4093


1637
GCAGCUUGAGUUAAACGAACGUA
2559
37378
VEGF:1655L21 antisense siNA (1637 C.) stab26
CGUucGuuuAAcucAAGcuTT
4094


1643
UGAGUUAAACGAACGUACUUGCA
2667
37379
VEGF:1661L21 antisense siNA (1643 C.) stab26
CAAGuAcGuucGuuuAAcuTT
4095


1645
AGUUAAACGAACGUACUUGCAGA
2668
37380
VEGF:1663L21 antisense siNA (1645 C.) stab26
UGCAAGuAcGuucGuuuAATT
4096


1646
GUUAAACGAACGUACUUGCAGAU
2669
37381
VEGF:1664L21 antisense siNA (1646 C.) stab26
CUGcAAGuAcGuucGuuuATT
4097


1647
UUAAACGAACGUACUUGCAGAUG
2670
37382
VEGF:1665L21 antisense siNA (1647 C.) stab26
UCUGcAAGuAcGuucGuuuTT
4098


1648
UAAACGAACGUACUUGCAGAUGU
2577
37383
VEGF:1666L21 antisense siNA (1648 C.) stab26
AUCuGcAAGuAcGuucGuuTT
4099


1655
ACGUACUUGCAGAUGUGACAAGC
2671
37384
VEGF:1673L21 antisense siNA (1655 C.) stab26
UUGucAcAucuGcAAGuAcTT
4100


1656
CGUACUUGCAGAUGUGACAAGCC
2560
37385
VEGF:1674L21 antisense siNA (1656 C.) stab26
CUUGucAcAucuGcAAGuATT
4101


1657
GUACUUGCAGAUGUGACAAGCCG
2672
37386
VEGF:1675L21 antisense siNA (1657 C.) stab26
GCUuGucAcAucuGcAAGuTT
4102


1562
AAAGCAUUUGUUUGUACAAGAUC
2581
37575
VEGF:1562U21 sense siNA stab07
B AGcAuuuGuuuGuAcAAGATT B
4103


1562
AAAGCAUUUGUUUGUACAAGAUC
2581
37577
VEGF:1580L21 antisense siNA (1562 C.) stab26
UCUuGuAcAAAcAAAuGcuTT
4104


1215
GUGGACAUCUUCCAGGAGUACCC
2543
37789
VEGF:1233L21 antisense siNA (1215 C.) stab26
GUAcuccuGGAAGAuGuccTT
4105


 







VEGF/VEGFR multifunctional siNA














1501
ACCUCACUGCCACUCUAAUUGUC
2673
34692
F/K bf-1a siNA stab00
CAAUUAGAGUGGCAGUGAGCAAAGTT
4106




CCUCACUGCCACUCUAAUUGUCA


[FLT1:1519L21 (1501 C.) −14 +






KDR:503U21]


1502
CCUCACUGCCACUCUAAUUGUCA
2674
34693
F/K bf-2a siNA stab00
ACAAUUAGAGUGGCAGUGAGCAAAGTT
4107



CCUCACUGCCACUCUAAUUGUCA


[FLT1:1520L21 (1502 C.) −13 +






KDR:503U21]


1503
CUCACUGCCACUCUAAUUGUCAA
2675
34694
F/K bf-3a siNA stab00
GACAAUUAGAGUGGCAGUGAGCAAAGTT
4108



CCUCACUGCCACUCUAAUUGUCA


[FLT1:1521L21 (1503 C.) −12 +






KDR:503U21]


3646
AAAGCAUUUGUUUGUACAAGAUC
2676
34695
V/F bf-1a siNA stab00
UGUGCCAGCAGUCCAGCAUUUGUUUGUACAAGATT
4109



UCAUGCUGGACUGCUGGCACAGA


[FLT1:3664L19 (3646 C.) −5 +






VEGF:1562U21]


5353
AGAGAGACGGGGUCAGAGAGAGC
2677
34696
V/F bf-2a siNA stab00
UUGGUAUAGAGACGGGGUCAGAGAGATT
4110



AAGACCCCGUCUCUAUACCAACC


[FLT1:5371L19 (5353 C.) −12 +






VEGF:360U21]


1501
ACCUCACUGCCACUCUAAUUGUC
2678
34697
F/K bf-1b siNA stab00
CUUUGCUCACUGCCACUCUAAUUGTT
4111



UCAGAGUGGCAGUGAGCAAAGGG


[KDR:521L21 (503 C.) −14 +






FLT1:1501U21]


1502
CCUCACUGCCACUCUAAUUGUCA
2679
34698
F/K bf-2b siNA stab00
CUUUGCUCACUGCCACUCUAAUUGUTT
4112



UCAGAGUGGCAGUGAGCAAAGGG


[KDR:521L21 (503 C.) −13 +






FLT1:1502U21]


1503
CUCACUGCCACUCUAAUUGUCAA
2680
34699
F/K bf-3b siNA stab00
CUUUGCUCACUGCCACUCUAAUUGUCTT
4113



UCAGAGUGGCAGUGAGCAAAGGG


[KDR:521L21 (503 C.) −12 +






FLT1:1503U21]


3646
AAAGCAUUUGUUUGUACAAGAUC
2676
34700
V/F bf-1b siNA stab00
UCUUGUACAAACAAAUGCUGGACUGCUGGCACATT
4114



UCAUGCUGGACUGCUGGCACAGA


[VEGF:1580L19 (1562 C.) −5 +






FLT1:3646U21]


5353
AGAGAGACGGGGUCAGAGAGAGC
2677
34701
V/F bf-2b siNA stab00
UCUCUCUGACCCCGUCUCUAUACCAATT
4115



AAGACCCCGUCUCUAUACCAACC


[VEGF:378L21 (360 C.) −12 +






FLT1:5353U21]


3646
AAUGUGAAUGCAGACCAAAGAAA
2681
34702
V/F bf-3a siNA stab00
UGUGCCAGCAGUCCAGCAU
4116



UCAUGCUGGACUGCUGGCACAGA


[FLT1:3664L19 (3646 C.) +
UGUGAAUGCAGACCAAAGATT






[VEGF1420:U21]


3646
AAUGUGAAUGCAGACCAAAGAAA
2681
34703
V/F bf-3b siNA stab00
UCUUUGGUCUGCAUUCACA
4117



UCAUGCUGGACUGCUGGCACAGA


[VEGF1438:L19 (1420 C.) +
AUGCUGGACUGCUGGCACATT






FLT1:3646U21]


3648
AAUGUGAAUGCAGACCAAAGAAA
2681
34704
V/F bf-4a siNA stab00
UGUGCCAGCAGUCCAGC
4118



UCAUGCUGGACUGCUGGCACAGA


[FLT1:3664L17 (3648 C.) +
UGAAUGCAGACCAAAGATT






VEGF1422:U19]


3648
AAUGUGAAUGCAGACCAAAGAAA
2681
34705
V/F bf-4b siNA stab00
UCUUUGGUCUGCAUUCA
4119



UCAUGCUGGACUGGUGGCACAGA


[VEGF1438:L17 (1422 C.) +
GCUGGACUGCUGGCACATT






FLT1:3648U19]


3646
AAUGUGAAUGCAGACCAAAGAAA
2681
34706
V/F bf-5a siNA stab00
UGUGCCAGCAGUCCAGCAU
4120



UCAUGCUGGACUGCUGGCACAGA


[FLT1:3664L19 (3646 C.) +
GAAUGCAGACCAAAGAAAGTT






VEGF1423:U19]


3646
AAUGUGAAUGCAGACCAAAGAAA
2681
34707
V/F bf-5b siNA stab00
CUUUCUUUGGUCUGCAUUC
4121



UCAUGCUGGACUGCUGGCACAGA


[VEGF1441:L19 (1420 C.) +
AUGCUGGACUGCUGGCACATT






FLT1:3646U21]


3646
AUGUGAAUGCAGACCAAAGAAAG
2682
34708
V/F bf-6a siNA stab00
UGUGCCAGCAGUCCAGCAU
4122



UCAUGCUGGACUGCUGGCACAGA


[FLT1:3664L19 (3646 C.) +
GUGAAUGCAGACCAAAGAATT






VEGF1421:U21]


3646
AUGUGAAUGCAGACCAAAGAAAG
2682
34709
V/F bf-6b siNA stab00
UUCUUUGGUCUGCAUUCAC
4123



UCAUGCUGGACUGCUGGCACAGA


[VEGF1439:LI 9(1421 C.) +
AUGCUGGACUGCUGGCACATT






FLT1:3646U21]


1215
GUGGACAUCUUCCAGGAGUACCC
2683
36408
V/F bf-L-03 siNA stab00
GGACAUCUUCCAGGAGUACTT L
4124



CUGAACUGAGUUUAAAAGGCACC


[VEGF:1215U21 o18S
GAACUGAGUUUAAAAGGCATT






FLT1:346U21]


1421
AUGUGAAUGCAGACCAAAGAAAG
2684
36409
V/F bf-L-02 siNA stab00
GUGAAUGCAGACCAAAGAATT L
4125



CUGAACUGAGUUUAAAAGGCACC


[VEGF:1421U21 o18S
GAACUGAGUUUAAAAGGCATT






FLT1:346U21]


3854
UUUGAGCAUGGAAGAGGAUUCUG
2685
36411
F/K bf-L-04 siNA stab00
UGAGCAUGGAAGAGGAUUCTT L
4126



CUGAACUGAGUUUAAAAGGCACC


[KDR:3854U21 o18S
GAACUGAGUUUAAAAGGCATT






FLT1:346U21]


346
CUGAACUGAGUUUAAAAGGCACC
2686
36416
V/F bf-L-01 siNA stab00
GAACUGAGUUUAAAAGGCATT L
4127



AUGUGAAUGCAGACCAAAGAAAG


[FLT1:346U21 o18S
GUGAAUGCAGACCAAAGAATT






VEGF:1421U21]


3646
UCAUGCUGGACUGCUGGCACAGA
2687
36425
V/F bf-L-05 siNA stab00
AUGCUGGACUGCUGGCACATT L
4128



AUGUGAAUGCAGACCAAAGAAAG


[FLT1:3646U21 o18S
GUGAAUGCAGACCAAAGAATT






VEGF:1421U21]


3646
UCAUGGUGGACUGGUGGCACAGA
2687
36426
V/F bf-L-06 siNA stab00
AUGCUGGACUGCUGGCACATT W
4129



AUGUGAAUGCAGACCAAAGAAAG


[FLT1:3646U21 c12S
GUGAAUGCAGACCAAAGAATT






VEGF:1421U21]


3646
UCAUGCUGGACUGCUGGCACAGA
2687
36427
V/F bf-L-07 siNA stab00
AUGCUGGACUGCUGGCACATT Y
4130



AUGUGAAUGCAGACCAAAGAAAG


[FLT1:3646U21 o95
GUGAAUGCAGACCAAAGAATT






VEGF:1421U21]


3646
UCAUGCUGGACUGCUGGCACAGA
2687
36428
V/F bf-L-08 siNA stab00
AUGCUGGACUGCUGGCACATT Z
4131



AUGUGAAUGCAGACCAAAGAAAG


[FLT1:3646U21 c35
GUGAAUGCAGACCAAAGAATT






VEGF:1421U21]


3646
UCAUGCUGGACUGCUGGCACAGA
2687
36429
V/F bf-L-09 siNA stab00
AUGCUGGACUGCUGGCACATT LL
4132



AUGUGAAUGCAGACCAAAGAAAG


[FLT1:3646U21 2x o18S
GUGAAUGCAGACCAAAGAATT






VEGF:1421U21]


162
UCCCUCUUCUUUUUUCUUAAACA
2688
37537
V/K bf-1a siNA stab00
UUUAAGAAAAAAGAAGAGGAAGCUCCUGATT
4133



AGAAGAAGAGGAAGCUCCUGAAG


[VEGF:180L21 (162 C.) −9 +






KDR:3263U21]


164
CCUCUUCUUUUUUCUUAAACAUU
2689
37538
V/F bf-7a siNA stab00
UGUUUAAGAAAAAAGAAGAAGGAAACAGAATT
4134



UCAAAGAAGAAGGAAACAGAAUC


[VEGF:182L21 (164 C.) −8 +






FLT1:594U21]


202
AUUGUUUCUCGUUUUAAUUUAUU
2690
37539
V/F bf-8a siNA stab00
UAAAUUAAAACGAGAAACAUUCUUUUAUCTT
4135



AGCGAGAAACAUUCUUUUAUCUG


[VEGF:220L21 (202 C.) −9 +






FLT1:3323U21]


237
UCCCCACUUGAAUCGGGCCGACG
2691
37540
V/F bf-9a siNA stab00
UCGGCCCGAUUCAAGUGGGCCUUGGAUCGTT
4136



GAUCAAGUGGGCCUUGGAUCGCU


[VEGF:255L21 (237 C.) −9 +






FLT1:5707U21]


238
CCCCACUUGAAUCGGGCCGACGG
2692
37541
V/F bf-10a siNA stab00
GUCGGCCCGAUUCAAGUGGCCAGAGGCAUTT
4137



UUUUCAAGUGGCCAGAGGCAUGG


[VEGF:256L21 (238 C.) −9 +






FLT1:3260U21]


338
CUCCAGAGAGAAGUCGAGGAAGA
2693
37542
V/K bf-2a siNA stab00
UUCCUCGACUUCUCUCUGGUUGUGUAUGUTT
4138



GGUCUCUCUGGUUGUGUAUGUCC


[VEGF:356L21 (338 C.) −9 +






KDR:1541U21]


360
AGAGAGACGGGGUCAGAGAGAGC
2694
37543
V/F bf-11 a siNA stab00
UCUCUCUGACCCCGUCUCUAUACCAACTT
4139



AGACCCCGUCUCUAUACCAACCA


[VEGF:378L21 (360 C.) −11 +






FLT1:5354U21]


484
GCAGCUGACCAGUCGCGCUGACG
2695
37544
V/F bf-12a siNA stab00
UCAGCGCGACUGGUCAGCUACUGGGACACTT
4140



CAUGGUCAGCUACUGGGACACCG


[VEGF:502L21 (484 C.) −9 +






FLT1:251U21]


654
CUGAAACUUUUCGUCCAACUUCU
2696
37545
V/F bf-13a siNA stab00
AAGUUGGACGAAAAGUUUCCACUUGACACTT
4141



AAAAAAGUUUCCACUUGACACUU


[VEGF:672L21 (654 C.) −9 +






FLT1:758U21]


978
CCCCACAGCCCGAGCCGGAGAGG
2697
37546
V/F bf-14a siNA stab00
UCUCCGGCUCGGGCUGUGGGAAAUCUUCUCCTT
4142



UUGCUGUGGGAAAUCUUCUCCUU


[VEGF:996L21 (978 C.) −7 +






FLT1:3513U21]


1038
ACCAUGAACUUUCUGCUGUCUUG
2698
37547
V/F bf-15a siNA stab00
AGACAGCAGAAAGUUCAUGAGCCUGGAAATT
4143



UCAAGUUCAUGAGCCUGGAAAGA


[VEGF:1056L21 (1038 C.) −9 +






FLT1:3901U21]


1095
CACCAUGCCAAGUGGUCCCAGGC
2699
37548
V/K bf-3a siNA stab00
CUGGGACCACUUGGCAUGGAGUUCUUGGCAUTT
4144



AGGGCAUGGAGUUCUUGGCAUCG


[VEGF:1113L21 (1095 C.) −7 +






KDR:3346U21]


1253
CAUCUUCAAGCCAUCCUGUGUGC
2700
37549
V/K bf-4a siNA stab00
ACACAGGAUGGCUUGAAGAUGGGAAGGAUUUTT
4145



UGUUGAAGAUGGGAAGGAUUUGC


[VEGF:1271L21 (1253 C.) −7 +






KDR:4769U21]


1351
UGCAGAUUAUGCGGAUCAAACCU
2701
37550
V/F bf-16a siNA stab00
GUUUGAUCCGCAUAAUCUGGGACAGUATT
4146



AACGCAUAAUCUGGGACAGUAGA


[VEGF:1369L21 (1351 C.) −11 +






FLT1:796U21]


1352
GCAGAUUAUGCGGAUCAAACCUC
2702
37551
V/F bf-17a siNA stab00
GGUUUGAUCCGCAUAAUCUGGGACAGUATT
4147



AACGCAUAAUCUGGGACAGUAGA


[VEGF:1370L21 (1352 C.) −10 +






FLT1:796U21]


1389
AUAGGAGAGAUGAGCUUCCUACA
2703
37552
V/K bf-5a siNA stab00
UAGGAAGCUCAUCUCUCCUGUGGAUUCCUTT
4148



UAAUCUCUCCUGUGGAUUCCUAC


[VEGF:1407L21 (1389 C.) −9 +






KDR:1588U21]


1401
AGCUUCCUACAGCACAACAAAUG
2704
37553
V/F bf-18a siNA stab00
UUUGUUGUGCUGUAGGAAGCUCUGAUGAUGUCTT
4149



UCAGGAAGCUCUGAUGAUGUCAG


[VEGF:1419L21 (1401 C.) −6 +






FLT1:3864U21]


1408
UACAGCACAACAAAUGUGAAUGC
2705
37554
V/K bf-6a siNA stab00
AUUCACAUUUGUUGUGCUGUUUCUGACUCTT
4150



UCGUUGUGCUGUUUCUGACUCCU


[VEGF:1426L21 (1408 C.) −9 +






KDR:5038U21]


1417
ACAAAUGUGAAUGCAGACCAAAG
2706
37555
V/K bf-7a siNA stab00
UUGGUCUGCAUUCACAUUUUGUAUCAGUTT
4151



CUAUUCACAUUUUGUAUCAGUAU


[VEGF:1435L21 (1417 C.) −10 +






KDR:5737U21]


162
UCCCUCUUCUUUUUUCUUAAACA
2688
37556
V/K bf-1b siNA stab00
UCAGGAGCUUCCUCUUCUUUUUUCUUAAATT
4152



AGAAGAAGAGGAAGCUCCUGAAG


[KDR:3281L21 (3263 C.) −9 +






VEGF:162U21]


164
CCUCUUCUUUUUUCUUAAACAUU
2689
37557
V/F bf-7b siNA stab00
UUCUGUUUCCUUCUUCUUUUUUCUUAAACATT
4153



UCAAAGAAGAAGGAAACAGAAUC


[FLT1:612L21 (594 C.) −8 +






VEGF:164U21]


202
AUUGUUUCUCGUUUUAAUUUAUU
2690
37558
V/F bf-8b siNA stab00
GAUAAAAGAAUGUUUCUCGUUUUAAUUUATT
4154



AGCGAGAAACAUUCUUUUAUCUG


[FLT1:3341L21 (3323 C.) −9 +






VEGF:202U21]


237
UCCCCACUUGAAUCGGGCCGACG
2691
37559
V/F bf-9b siNA stab00
CGAUCCAAGGCCCACUUGAAUCGGGCCGATT
4155



GAUCAAGUGGGCCUUGGAUCGCU


[FLT1:5725L21 (5707 C.) −9 +






VEGF:237U21]


238
CCCCACUUGAAUCGGGCCGACGG
2692
37560
V/F bf-10b siNA stab00
AUGCCUCUGGCCACUUGAAUCGGGCCGACTT
4156



UUUUCAAGUGGCCAGAGGCAUGG


[FLT1:3278L21 (3260 C.) -9 +






VEGF:238U21]


338
CUCCAGAGAGAAGUCGAGGAAGA
2693
37561
V/K bf-2b siNA stab00
ACAUACACAACCAGAGAGAAGUCGAGGAATT
4157



GGUCUCUCUGGUUGUGUAUGUCC


[KDR:1559L21 (1541 C.) −9 +






VEGF:338U21]


360
AGAGAGACGGGGUCAGAGAGAGC
2694
37562
V/F bf-11b siNA stab00
GUUGGUAUAGAGACGGGGUCAGAGAGATT
4158



AGACCCCGUCUCUAUACCAACCA


[FLT1:5372L21 (5354 C.) −11 +






VEGF:360U21]


484
GCAGCUGACCAGUCGCGCUGACG
2695
37563
V/F bf-12b siNA stab00
GUGUCCCAGUAGCUGACCAGUCGCGCUGATT
4159



CAUGGUCAGCUACUGGGACACCG


[FLT1:269L21 (251 C.) −9 +






VEGF:484U21]


654
CUGAAACUUUUCGUCCAACUUCU
2696
37564
V/F bf-13b siNA stab00
GUGUCAAGUGGAAACUUUUCGUCCAACUUTT
4160



AAAAAAGUUUCCACUUGACACUU


[FLT1:776L21 (758 C.) −9 +






VEGF:654U21]


978
CCCCACAGCCCGAGCCGGAGAGG
2697
37565
V/F bf-14b siNA stab00
GGAGAAGAUUUCCCACAGCCCGAGCCGGAGATT
4161



UUGCUGUGGGAAAUCUUCUCCUU


[FLT1:3531L21 (3513 C.) −7 +






VEGF:978U21]


1038
ACCAUGAACUUUCUGCUGUCUUG
2698
37566
V/F bf-15b siNA stab00
UUUCCAGGCUCAUGAACUUUCUGCUGUCUTT
4162



UCAAGUUCAUGAGCCUGGAAAGA


[FLT1:3919L21 (3901 C.) −9 +






VEGF:1038U21]


1095
CACCAUGCCAAGUGGUCCCAGGC
2699
37567
V/K bf-3b siNA stab00
AUGCCAAGAACUCCAUGCCAAGUGGUCCCAGTT
4163



AGGGCAUGGAGUUCUUGGCAUCG


[KDR:3364L21 (3346 C.) −7 +






VEGF:1095U21]


1253
CAUCUUCAAGCCAUCCUGUGUGC
2700
37568
V/K bf-4b siNA stab00
AAAUCCUUCCCAUCUUCAAGCCAUCCUGUGUTT
4164



UGUUGAAGAUGGGAAGGAUUUGC


[KDR:4787L21 (4769 C.) −7 +






VEGF:1253U21]


1351
UGCAGAUUAUGCGGAUCAAACCU
2701
37569
V/F bf-16b siNA stab00
UACUGUCCCAGAUUAUGCGGAUCAAACTT
4165



AACGCAUAAUCUGGGACAGUAGA


[FLT1:814L21 (796 C.) −11 +






VEGF:1351U21]


1352
GCAGAUUAUGCGGAUCAAACCUC
2702
37570
V/F bf-17b siNA stab00
UACUGUCCCAGAUUAUGCGGAUCAAACCTT
4166



AACGCAUAAUCUGGGACAGUAGA


[FLT1:814L21 (796 C.) −10 +






VEGF:1352U21]


1389
AUAGGAGAGAUGAGCUUCCUACA
2703
37571
V/K bf-5b siNA stab00
AGGAAUCCACAGGAGAGAUGAGCUUCCUATT
4167



UAAUCUCUCCUGUGGAUUCCUAC


[KDR:1606L21 (1588 C.) −9 +






VEGF:1389U21]


1401
AGCUUCCUACAGCACAACAAAUG
2704
37572
V/F bf-18b siNA stab00
GACAUCAUCAGAGCUUCCUACAGCACAACAAATT
4168



UCAGGAAGCUCUGAUGAUGUCAG


[FLT1:3882L21 (3864 C.) −6 +






VEGF:1401U21]


1408
UACAGCACAACAAAUGUGAAUGC
2705
37573
V/K bf-6b siNA stab00
GAGUCAGAAACAGCACAACAAAUGUGAAUTT
4169



UCGUUGUGCUGUUUCUGACUCCU


[KDR:5056L21 (5038 C.) −9 +






VEGF:1408U21]


1417
ACAAAUGUGAAUGCAGACCAAAG
2706
37574
V/K bf-7b siNA stab00
ACUGAUACAAAAUGUGAAUGCAGACCAATT
4170



CUAUUCACAUUUUGUAUCAGUAU


[KDR:5755L21 (5737 C.) −10 +






VEGF:1417U21]


3646
AAAGCAUUUGUUUGUACAAGAUC
2676
37578
V/F bf-1a siNA stab07/26
UGUGccAGcAGuccAGcAu
4171



UCAUGCUGGACUGCUGGCACAGA


[FLT1:3664L19 (3646 C.) −5 +
AGcAuuuGuuuGuAcAAGATT B






VEGF:1562U21]


3646
AAAGCAUUUGUUUGUACAAGAUC
2676
37579
V/F bf-1b siNA stab07/26
UCUuGuAcAAAcAAAuGcu
4172



UCAUGGUGGACUGCUGCCACAGA


[VEGF:1580L19 (1562 C.) −5 +
AuGcuGGAcuGcuGGcAcATT B






FLT1:3646U21]


1215
GUGGACAUCUUCCAGGAGUACCC
2683
37777
V/F bf-L-03 siNA stab07
B GGAcAucuuccAGGAGuAcTT L
4173



CUGAACUGAGUUUAAAAGGCACC


[VEGF:1215U21 o18S
GAAcuGAGuuuAAAAGGcATT B






FLT1:346U21]


1421
AUGUGAAUGCAGACCAAAGAAAG
2684
37778
V/F bf-L-02 siNA stab07
B GuGAAuGcAGAccAAAGAATT L
4174



CUGAACUGAGUUUAAAAGGCACC


[VEGF:1421U21 o18S
GAAcuGAGuuuAAAAGGcATT B






FLT1:346U21]


1421
CUGAACUGAGUUUAAAAGGCACC
2686
37779
V/F bf-L-01 siNA stab07
B GAAcuGAGuuuAAAAGGcATT L
4175



AUGUGAAUGCAGACCAAAGAAAG


[FLT1:346U21 o18S
GuGAAuGcAGAccAAAGAATT B






VEGF:1421U21]


1421
UCAUGCUGGACUGCUGGCACAGA
2687
37780
V/F bf-L-05 siNA stab07
B AuGcuGGAcuGcuGGcAcATT L
4176



AUGUGAAUGCAGACCAAAGAAAG


[FLT1:3646U21 o18S
GuGAAuGcAGAccAAAGAATT B






VEGF:1421U21]


1421
UCAUGCUGGACUGCUGGCACAGA
2687
37783
V/F bf-L-05 siNA stab00
AUGCUGGACUGCUGGCACATT GAUCATCGTA
4177



AUGUGAAUGCAGACCAAAGAAAG


[FLT1:3646U21 10 nt
GUGAAUGCAGACCAAAGAATT






VEGF:1421U21]


1421
UCAUGCUGGACUGCUGGCACAGA
2687
37784
V/F bf-L-05 siNA stab00
AUGCUGGACUGCUGGCACATT GAUCAT
4178



AUGUGAAUGCAGACCAAAGAAAG


[FLT1:3646U21 6 nt
GUGAAUGCAGACCAAAGAATT






VEGF:1421U21]


1421
UCAUGCUGGACUGCUGGCACAGA
2687
37785
V/F bf-L-05 siNA stab00
AUGCUGGACUGCUGGCACATT GAU
4179



AUGUGAAUGCAGACCAAAGAAAG


[FLT1:3646U21 3 nt
GUGAAUGCAGACCAAAGAATT






VEGF:1421U21]


1421
UCAUGCUGGACUGCUGGCACAGA
2687
37786
V/F bf-L-05 siNA stab00
AUGCUGGACUGCUGGCACATT
4180



AUGUGAAUGCAGACCAAAGAAAG


[FLT1:3646U21 no linker
GUGAAUGCAGACCAAAGAATT






VEGF:1421U21]


1421
AUGUGAAUGCAGACCAAAGAAAG
2682
37787
V/F bf-6a siNA stab07/26
UGUGccAGcAGuccAGcAuTT
4181



UCAUGCUGGACUGCUGGCACAGA


[FLT1:3664L19 (3646 C.) +
GuGAAuGcAGAccAAAGAATT B






VEGF1421:U21]


1421
AUGUGAAUGCAGACCAAAGAAAG
2682
37788
V/F bf-6b siNA stab07/26
UUCuuuGGucuGcAuucAcTT
4182



UCAUGCUGGACUGCUGGCACAGA


[VEGF1439:L19 (1421 C.) +
AuGcuGGAcuGcuGGcAcATT B






FLT1:3646U21]


346
CUGAACUGAGUUUAAAAGGCACC
2686
38287
V/F bf-L-10a siNA stab09
B GAACUGAGUUUAAAAGGCATT L
4183



AUGUGAAUGCAGACCAAAGAAAG


[FLT1:346U21 o18S
GUGAAUGCAGACCAAAGAATT B






VEGF:1421U21]


346
CUGAACUGAGUUUAAAAGGCACC
2686
38288
V/F bf-L-11a siNA stab09
B GAACUGAGUUUAAAAGGCA
4184



AUGUGAAUGCAGACCAAAGAAAG


[FLT1:346U21 +
GUGAAUGCAGACCAAAGAA B






VEGF:1421U21]


346
CUGAACUGAGUUUAAAAGGCACC
2686
38289
V/F bf-L-11b siNA stab00
UUCUUUGGUCUGCAUUCAC
4185



AUGUGAAUGCAGACCAAAGAAAG


[VEGF:1439L21 (1421 C.) +
UGCCUUUUAAACUCAGUUC






FLT1:364L21 (346 C.)]


346
CUGAACUGAGUUUAAAAGGCACC
2686
38369
V/F bf-L-26a siNA stab22
UGCCUUUUAAACUCAGUUC
4186



AUGUGAAUGCAGACCAAAGAAAG


[FLT1:364L21 siNA (346 C.) +
GUGAAUGCAGACCAAAGAATT B






VEGF:1421U21]






V/F bf-L-26b siNA stab22


346
CUGAACUGAGUUUAAAAGGCACC
2686
38370
[VEGF:1439L21 siNA
UUCUUUGGUCUGCAUUCAC
4187



AUGUGAAUGCAGACCAAAGAAAG


(1421 C.) + FLT1:346U21
GAACUGAGUUUAAAAGGCATT B






siNA]


 







VEGF/VEGFR DFO siNA














349
AACUGAGUUUAAAAGGCACCCAG
2289
32718
FLT1:367L2i siRNA (349 C.) v1 5′p
pGGGUGCCUUUUAAACUC GAGUUUAAAAG B
2810







palindrome


349
AACUGAGUUUAAAAGGCACCCAG
2289
32719
FLT1:367L21 siRNA (349 C.) v2 5′p
pGGGUGCCUUUUAAACUCAG GAGUUUAAAAG B
2811






palindrome


2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
32720
FLT1:2967L21 siRNA (2949 C.) v1 5′p
pCAUCAGAGGCCCUCCUUGC
2812






palindrome
AAGGAGGGCCUCTT B


2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
32721
FLT1:2967L21 siRNA (2949 C.) v2 5′p
pCAUCAGAGGCCCUCCUU
2813






palindrome
AAGGAGGGCCUCUG B


2949
AAGCAAGGAGGGCCUCUGAUGGU
2290
32722
FLT1:2967L21 siRNA (2949 C.) v3 5′p
pCAUCAGAGGCCCUCCU AGGAGGGCCUCUG B
2814






palindrome


354
AGUUUAAAAGGCACCCAGCACAUC
2707
32805
FLT1:372L21 siRNA (354 C.) v1 5′p
pGUGCUGGGUGCCUUUUAAA AGGCACCCAGC B
4188






palindrome


354
AGUUUAAAAGGCACCCAGCACAUC
2707
32806
FLT1:372L21 siRNA (354 C.) v2 5′p
pGUGCUGGGUGCCUUUAAA GGCACCCAGC B
4189






palindrome


354
AGUUUAAAAGGCACCCAGCACAUC
2707
32807
FLT1:372L21 siRNA (354 C.) v3 5′p
pGUGCUGGGUGCCUUAAGGCACCCAGC B
4190






palindrome


1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
32808
FLT1:1247L21 siRNA (1229 C.) v1 5′p
pAAUGCUUUAUCAUAUAUAU GAUAAAGC B
4191






palindrome


1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
32809
FLT1:1247L21 siRNA (1229 C.) v2 5′p
pAAUGCUUUAUCAUAUAU GAUAAAGC B
4192






palindrome


1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
32810
FLT1:1247L21 siRNA (1229 C.) v3 5′p
pAAUGCUUUAUCAUAU GAUAAAGC B
4193






palindrome


1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
32811
FLT1:1247L21 siRNA (1229 C.) v4 5′p
pAAUGCUUUAUCAUAU GAUAAAGCA B 4194






palindrome


1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
32812
FLT1:1247L21 siRNA (1229 C.) v5 5′p
pAAUGCUUUAUCAUAUAU GAUAAAGCAUTT B
4195






palindrome


1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
32813
FLT1:1247L21 siRNA (1229 C.) v6 5′p
pAAUGCUUUAUCAUAU GAUAAAGCAUTT B4196






palindrome


349
AACUGAGUUUAAAAGGCACCCAG
2289
33056
FLT1:367L21 siRNA (349 C.) v3 5′p
pGGGUGCCUUUUAAACUCAG
4197






palindrome
GAGUUUAAAAGG B


349
AACUGAGUUUAAAAGGCACCCAG
2289
33057
FLT1:367L21 siRNA (349 C.) v4 5′p
pGGGUGCCUUUUAAACUC
4198






palindrome
GAGUUUAAAAGGCA B


349
AACUGAGUUUAAAAGGCACCCAG
2289
33058
FLT1:367L21 siRNA (349 C.) v5 5′p
pGGGUGCCUUUUAAACU AGUUUAAAAGG B
4199






palindrome


349
AACUGAGUUUAAAAGGCACCCAG
2289
33059
FLT1:367L21 siRNA (349 C.) v6 5′p
pGGGUGCCUUUUAAACU AGUUUAAAAGGC B
4200






palindrome


349
AACUGAGUUUAAAAGGCACCCAG
2289
33060
FLT1:367L21 siRNA (349 C.) v7 5′p
pGGGUGCCUUUUAAACU AGUUUAAAAGGCA B
4201






palindrome


349
AACUGAGUUUAAAAGGCACCCAG
2289
33061
FLT1:367L21 siRNA (349 C.) v8 5′p
pGGGUGCCUUUUAAACU AGUUUAAAAGGCAC B
4202






palindrome


349
AACUGAGUUUAAAAGGCACCCAG
2289
33062
FLT1:367L21 siRNA (349 C.) v9 5′p
pGGGUGCCUUUUAAAC GUUUAAAAGGC B
4203






palindrome


349
AACUGAGUUUAAAAGGCACCCAG
2289
33063
FLT1:367L21 siRNA (349 C.) v10 5′p
pGGGUGCCUUUUAAAC GUUUAAAAGGCA B
4204






palindrome


349
AACUGAGUUUAAAAGGCACCCAG
2289
33064
FLT1:367L21 siRNA (349 C.) v11 5′p
pGGGUGCCUUUUAAAC GUUUAAAAGGCAC B
4205






palindrome


354
AGUUUAAAAGGCACCCAGCACAU
2316
34092
FLT1:371L18 siRNA (354 C.) v4 5′p
pUGCUGGGUGCCUUUUAAA AGGCACCCAGC B
4206






palindrome


354
AGUUUAAAAGGCACCCAGCACAU
2316
34093
FLT1:370L17 siRNA (354 C.) v5 5′p
pGCUGGGUGCCUUUUAAA AGGCACCCAGC B
4207






palindrome


354
AGUUUAAAAGGCACCCAGCACAU
2316
34094
FLT1:370L17 siRNA (354 C.) v6 5′p
pGCUGGGUGCCUUUUAAA AGGCACCCAGCT B
4208






palindrome


354
AGUUUAAAAGGCACCCAGCACAU
2316
34095
FLT1:370L17 siRNA (354 C.) v7 5′p
pGCUGGGUGCCUUUUAAA AGGCACCCAG B
4209






palindrome


354
AGUUUAAAAGGCACCCAGCACAU
2316
34096
FLT1:369L16 siRNA (354 C.) v8 5′p
pCUGGGUGCCUUUUAAA AGGCACCCAG B
4210






palindrome


354
AGUUUAAAAGGCACCCAGCACAU
2316
34097
FLT1:369L16 siRNA (354 C.) v9 5′p
pCUGGGUGCCUUUUAAAAGGCACCCA B
4211






palindrome


354
AGUUUAAAAGGCACCCAGCACAU
2316
34098
FLT1:368L15 siRNA (354 C.) v10 5′p
pUGGGUGCCUUUUAAA AGGCACCCA B
4212






palindrome


354
AGUUUAAAAGGCACCCAGCACAU
2316
34099
FLT1:368L15 siRNA (354 C.) v11 5′p
pUGGGUGCCUUUUAAAAGGCACCCAT B
4213






palindrome


354
AGUUUAAAAGGCACCCAGCACAU
2316
34100
FLT1:368L15 siRNA (354 C.) v12 5′p
pUGGGUGCCUUUUAAA AGGCACCCATT B
4214






palindrome


1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34101
FLT1:1247L21 siRNA (1229 C.) v14
pUGCUUUAUCAUAUAUAU GAUAAAGCA B
4215






5′p palindrome


1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34102
FLT1:1247L21 siRNA (1229 C.) v15
pUGCUUUAUCAUAUAUAU GAUAAAGC B
4216






5′p palindrome


1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34103
FLT1:1247L21 siRNA (1229 C.) v16
pGCUUUAUCAUAUAUAU GAUAAAGC B
4217






5′p palindrome


1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34104
FLT1:1247L17 siRNA (1229 C.) v5
AAUGCUUUAUCAUAUAU GAUAAAGCAUTT B
4218






palindrome


1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34105
FLT1:1247L17 siRNA (1229 C.) v7 5′p
pAAUGCUUUAUCAUAUAU GAUAAAGCAUUT B
4219






palindrome


1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34106
FLT1:1247L17 siRNA (1229 C.) v8 5′p
pAAUGCU UUAUCAUAUAU GAUAAAGCAUUTT B
4220






palindrome


1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34107
FLT1:1247L17 siRNA (1229 C.) v9 5′p
pAAUGCUUUAUCAUAUAU GAUAAAGCATT B
4221






palindrome


1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34108
FLT1:1247L16 siRNA (1229 C.) v10
pAUGCUUUAUCAUAUAU GAUAAAGCATT B
4222






5′p palindrome


1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34109
FLT1:1247L16 siRNA (1229 C.) v11
pAUGCUUUAUCAUAUAU GAUAAAGCAUT B
4223






5′p palindrome


1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34110
FLT1:1247L16 siRNA (1229 C.) v12
pAUGCUUUAUCAUAUAU GAUAAAGCAUTT B
4224






5′p palindrome


1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34111
FLT1:1247L16 siRNA (1229 C.) v13
pAUGCUUUAUCAUAUAU GAUAAAGCA B
4225






5′p palindrome


1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34112
FLT1:1247L17 siRNA (1229 C.) v14
pAAUGCUUUAUCAUAUAU CUAUAAGCAUTT B
4226






5′p palindrome


1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34113
FLT1:1247L17 siRNA (1229 C.) v15
pAAUGCUUUUAGUUAUAU GAUAAAGCAUTT B
4227






5′p palindrome


1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34114
FLT1:1247L17 siRNA (1229 C.) v16
pAAUCCUUAAUCUUAUUU GAUAAAGCAUTT B
4228






5′p palindrome


1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34115
FLT1:1247L17 siRNA (1229 C.) v17
pAAuGcuuuAucAuAuAu GAuAAAGcAuu B
4229






5′p palindrome


1229
GCAUAUAUAUGAUAAAGCAUUCA
2708
34116
FLT1:1247L17 siRNA (1229 C.) v18
pAAuGcuuuAucAuAuAu GAuAAAGcAuu B
4230






5′p palindrome





Uppercase = ribonucleotide


u, c = 2′-deoxy-2′-fluoro U, C


T = thymidine


B = inverted deoxy abasic


s = phosphorothioate linkage


A = deoxy Adenosine


G = deoxy Guanosine


G = 2′-O-methyl Guanosine


A = 2′-O-methyl Adenosine


X = 3′-deoxy T


X = nitroindole


Z = nitropyrrole


T = thymidine


t = L-thymidine


u = L uridine


D = inverted thymidine


L = 5′amino mod-C5 TFA ( from W.W.)


L = hegS = hexethelyne glycol spacer; spacer-18 (Glen Research 10-1918-xx)


W = C12 spacer; spacer C12 (Glen Research 10-1928-xx)


Y = tetraethelyne glycol spacer; spacer 9 (Glen Research 10-1909-xx)


Z = C3 spacer; spacer C3 (Glen Research 10-1913-xx)


p = terminal phosphate


I = rI = rib0 inosine (Glen Res #10-3044-xx)


U = 3′-O-Methyl Uridine


Gyl = glyceryl













TABLE IV







Non-limiting examples of Stabilization Chemistries


for chemically modified siNA constructs












Chem-







istry
pyrimidine
Purine
cap
p = S
Strand





“Stab
Ribo
Ribo
TT at 3′-

S/AS


00”


ends


“Stab
Ribo
Ribo
—
5 at 5′-end
S/AS


1”



1 at 3′-end


“Stab
Ribo
Ribo
—
All linkages
Usually AS


2”


“Stab
2′-fluoro
Ribo
—
4 at 5′-end
Usually S


3”



4 at 3′-end


“Stab
2′-fluoro
Ribo
5′ and 3′-
—
Usually S


4”


ends


“Stab
2′-fluoro
Ribo
—
1 at 3′-end
Usually AS


5”


“Stab
2′-O-
Ribo
5′ and 3′-
—
Usually S


6”
Methyl

ends


“Stab
2′-fluoro
2′-deoxy
5′ and 3′-
—
Usually S


7”


ends


“Stab
2′-fluoro
2′-O-
—
1 at 3′-end
S/AS


8”

Methyl


“Stab
Ribo
Ribo
5′ and 3′-
—
Usually S


9”


ends


“Stab
Ribo
Ribo
—
1 at 3′-end
Usually AS


10”


“Stab
2′-fluoro
2′-deoxy
—
1 at 3′-end
Usually AS


11”


“Stab
2′-fluoro
LNA
5′ and 3′-

Usually S


12”


ends


“Stab
2′-fluoro
LNA

1 at 3′-end
Usually AS


13”


“Stab
2′-fluoro
2′-deoxy

2 at 5′-end
Usually AS


14”



1 at 3′-end


“Stab
2′-deoxy
2′-deoxy

2 at 5′-end
Usually AS


15”



1 at 3′-end


“Stab
Ribo
2′-O-
5′ and 3′-

Usually S


16”

Methyl
ends


“Stab
2′-O-
2′-O-
5′ and 3′-

Usually S


17”
Methyl
Methyl
ends


“Stab
2′-fluoro
2′-O-
5′ and 3′-

Usually S


18”

Methyl
ends


“Stab
2′-fluoro
2′-O-
3′-end

S/AS


19”

Methyl


“Stab
2′-fluoro
2′-deoxy
3′-end

Usually AS


20”


“Stab
2′-fluoro
Ribo
3′-end

Usually AS


21”


“Stab
Ribo
Ribo
3′-end

Usually AS


22”


“Stab
2′-fluoro*
2′-deoxy*
5′ and 3′-

Usually S


23”


ends


“Stab
2′-fluoro*
2′-O-
—
1 at 3′-end
S/AS


24”

Methyl*


“Stab
2′-fluoro*
2′-O-
—
1 at 3′-end
S/AS


25”

Methyl*


“Stab
2′-fluoro*
2′-O-
—

S/AS


26”

Methyl*


“Stab
2′-fluoro*
2′-O-
3′-end

S/AS


27”

Methyl*


“Stab
2′-fluoro*
2′-O-
3′-end

S/AS


28”

Methyl*


“Stab
2′-fluoro*
2′-O-

1 at 3′-end
S/AS


29”

Methyl*


“Stab
2′-fluoro*
2′-O-


S/AS


30”

Methyl*


“Stab
2′-fluoro*
2′-O-
3′-end

S/AS


31”

Methyl*


“Stab
2′-fluoro
2′-O-


S/AS


32”

Methyl


“Stab
2′-fluoro
2′-deoxy*
5′ and 3′-
—
Usually S


33”


ends


“Stab
2′-fluoro
2′-O-
5′ and 3′-

Usually S


34”

Methyl*
ends


“Stab
2′-OCF3
Ribo
—
4 at 5′-end
Usually S


3F”



4 at 3′-end


“Stab
2′-OCF3
Ribo
5′ and 3′-
—
Usually S


4F”


ends


“Stab
2′-OCF3
Ribo
—
1 at 3′-end
Usually AS


5F”


“Stab
2′-OCF3
2′-deoxy
5′ and 3′-
—
Usually S


7F”


ends


“Stab
2′-OCF3
2′-O-
—
1 at 3′-end
S/AS


8F”

Methyl


“Stab
2′-OCF3
2′-deoxy
—
1 at 3′-end
Usually AS


11F”


“Stab
2′-OCF3
LNA
5′ and 3′-

Usually S


12F”


ends


“Stab
2′-OCF3
LNA

1 at 3′-end
Usually AS


13F”


“Stab
2′-OCF3
2′-deoxy

2 at 5′-end
Usually AS


14F”



1 at 3′-end


“Stab
2′-OCF3
2′-deoxy

2 at 5′-end
Usually AS


15F”



1 at 3′-end


“Stab
2′-OCF3
2′-O-
5′ and 3′-

Usually S


18F”

Methyl
ends


“Stab
2′-OCF3
2′-O-
3′-end

S/AS


19F”

Methyl


“Stab
2′-OCF3
2′-deoxy
3′-end

Usually AS


20F”


“Stab
2′-OCF3
Ribo
3′-end

Usually AS


21F”


“Stab
2′-OCF3*
2′-deoxy*
5′ and 3′-

Usually S


23F”


ends


“Stab
2′-OCF3*
2′-O-
—
1 at 3′-end
S/AS


24F”

Methyl*


“Stab
2′-OCF3*
2′-O-
—
1 at 3′-end
S/AS


25F”

Methyl*


“Stab
2′-OCF3*
2′-O-
—

S/AS


26F”

Methyl*


“Stab
2′-OCF3*
2′-O-
3′-end

S/AS


27F”

Methyl*


“Stab
2′-OCF3*
2′-O-
3′-end

S/AS


28F”

Methyl*


“Stab
2′-OCF3*
2′-O-

1 at 3′-end
S/AS


29F”

Methyl*


“Stab
2′-OCF3*
2′-O-


S/AS


30F”

Methyl*


“Stab
2′-OCF3*
2′-O-
3′-end

S/AS


31F”

Methyl*


“Stab
2′-OCF3
2′-O-


S/AS


32F”

Methyl


“Stab
2′-OCF3
2′-deoxy*
5′ and 3′-
—
Usually S


33F”


ends


“Stab
2′-OCF3
2′-O-
5′ and 3′-

Usually S


34F”

Methyl*
ends





CAP = any terminal cap, see for example FIG. 10.


All Stab 00-34 chemistries can comprise 3′-terminal thymidine (TT) residues


All Stab 00-34 chemistries typically comprise about 21 nucleotides, but can vary as described herein.


All Stab 00-34 chemistries can also include a single ribonucleotide in the sense or passenger strand at the 11th base paired position of the double stranded nucleic acid duplex as determined from the 5′-end of the antisense or guide strand (see FIG. 6C)


S = sense strand


AS = antisense strand


*Stab 23 has a single ribonucleotide adjacent to 3′-CAP


*Stab 24 and Stab 28 have a single ribonucleotide at 5′-terminus


*Stab 25, Stab 26, and Stab 27 have three ribonucleotides at 5′-terminus


*Stab 29, Stab 30, Stab 31, Stab 33, and Stab 34 any purine at first three nucleotide positions from 5′-terminus are ribonucleotides


p = phosphorothioate linkage













TABLE V







A. 2.5 μmol Synthesis Cycle ABI 394 Instrument












Reagent
Equivalents
Amount
Wait Time* DNA
Wait Time* 2′-O-methyl
Wait Time* RNA


















Phosphoramidites
6.5
163
μL
45 sec
2.5
min
7.5
min


S-Ethyl Tetrazole
23.8
238
μL
45 sec
2.5
min
7.5
min


Acetic Anhydride
100
233
μL
 5 sec
5
sec
5
sec


N-Methyl
186
233
μL
 5 sec
5
sec
5
sec


Imidazole


TCA
176
2.3
mL
21 sec
21
sec
21
sec


Iodine
11.2
1.7
mL
45 sec
45
sec
45
sec


Beaucage
12.9
645
μL
100 sec 
300
sec
300
sec













Acetonitrile
NA
6.67
mL
NA
NA
NA










B. 0.2 μmol Synthesis Cycle ABI 394 Instrument












Reagent
Equivalents
Amount
Wait Time* DNA
Wait Time* 2′-O-methyl
Wait Time* RNA


















Phosphoramidites
15
31
μL
45 sec
233
sec
465
sec


S-Ethyl Tetrazole
38.7
31
μL
45 sec
233
min
465
sec


Acetic Anhydride
655
124
μL
 5 sec
5
sec
5
sec


N-Methyl
1245
124
μL
 5 sec
5
sec
5
sec


Imidazole


TCA
700
732
μL
10 sec
10
sec
10
sec


Iodine
20.6
244
μL
15 sec
15
sec
15
sec


Beaucage
7.7
232
μL
100 sec 
300
sec
300
sec













Acetonitrile
NA
2.64
mL
NA
NA
NA










C. 0.2 μmol Synthesis Cycle 96 well Instrument













Equivalents: DNA/
Amount: DNA/
Wait Time*
Wait Time* 2′-O-
Wait Time*


Reagent
2′-O-methyl/Ribo
2′-O-methyl/Ribo
DNA
methyl
Ribo

















Phosphoramidites
22/33/66
40/60/120
μL
60 sec
180
sec
360 sec 


S-Ethyl Tetrazole
 70/105/210
40/60/120
μL
60 sec
180
min
360 sec 


Acetic Anhydride
265/265/265
50/50/50
μL
10 sec
10
sec
10 sec


N-Methyl
502/502/502
50/50/50
μL
10 sec
10
sec
10 sec


Imidazole


TCA
238/475/475
250/500/500
μL
15 sec
15
sec
15 sec


Iodine
6.8/6.8/6.8
80/80/80
μL
30 sec
30
sec
30 sec


Beaucage
34/51/51
80/120/120

100 sec 
200
sec
200 sec 













Acetonitrile
NA
1150/1150/1150
μL
NA
NA
NA





Wait time does not include contact time during delivery.


Tandem synthesis utilizes double coupling of linker molecule

## Claims

1. A method for the treatment of an ocular disease in a subject comprising contacting the subject via intravitreal injection with a double stranded nucleic acid molecule 21-40 nucleotides in length comprising a first strand having SEQ ID NO: 2742 and a second strand having SEQ ID NO: 2745 under conditions suitable to inhibit the expression of VEGFR1 in the subject, wherein the ocular disease is an angiogenic disease.

2. The method of claim 1, wherein said ocular disease is age related macular degeneration (AMD).

3. The method of claim 1, wherein said ocular disease is diabetic retinopathy.

4. The method of claim 1, wherein said treatment comprises intravitreal injection of said double stranded nucleic acid molecule in a pharmaceutically acceptable carrier or diluent.

5. The method of claim 4, wherein said injection takes place every 4 weeks.

6. The method of claim 4, wherein said injection takes place every 6 weeks.

7. The method of claim 4, wherein said injection takes place every 8 weeks.

8. The method of claim 4, wherein said injection takes place every 10 weeks.

9. A method for the treatment of ocular disease in a subject comprising contacting the subject with a double stranded nucleic acid molecule 21-40 nucleotides in length comprising a first strand having SEQ ID NO: 2742 and a second strand having SEQ ID NO: 2745, wherein the treatment comprises administration of the double stranded nucleic acid molecule via periocular injection to an eye of the subject, wherein the ocular disease is an angiogenic disease.

10. The method of claim 9, wherein said ocular disease is age related macular degeneration (AMD).

11. The method of claim 9, wherein said ocular disease is diabetic retinopathy.

12. The method of claim 9, wherein the double stranded nucleic acid molecule is in a pharmaceutically acceptable carrier or diluent.

13. The method of claim 9, wherein said administration takes place every 4 weeks.

14. The method of claim 9, wherein said administration takes place every 6 weeks.

15. The method of claim 9, wherein said administration takes place every 8 weeks.

16. The method of claim 9, wherein said administration takes place every 10 weeks.