# Patent

## Title
RNA interference mediated inhibition of respiratory syncytial virus (RSV) expression using short interfering nucleic acid (siNA)

## Abstract

This invention relates to compounds, compositions, and methods useful for modulating sespiratory syncytial virus (RSV) 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 RSV 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 RSV genes, including cocktails of such small nucleic acid molecules and lipid nanoparticle formulations of such small nucleic acid molecules cocktails thereof. The application also relates to methods of treating diseases and conditions associated with RSV gene expression, such as RSV infection, respiratory failure, bronchiolitis and pneumonia, as well as providing dosing regimens and treatment protocols.

## Background
This application is a continuation of U.S. patent application Ser. No. 11/395,833, filed Mar. 31, 2006 (now abandoned), which is a continuation-in-part of U.S. patent application Ser. No. 11/369,108, filed Mar. 6, 2006 (now abandoned), which is a continuation-in-part of U.S. patent application Ser. No. 10/923,536, filed Aug. 20, 2004 (now abandoned), which is 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 (now abandoned), 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 (now abandoned), 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. The parent U.S. patent application Ser. No. 11/395,833 is also a continuation-in-part of International Patent Application No. PCT/US05/04270, filed Feb. 9, 2005 which 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.


SEQUENCE LISTING
The sequence listing submitted via EFS, in compliance with 37 CFR §1.52(e)(5), is incorporated herein by reference. The sequence listing text file submitted via EFS contains the file “SequenceListing54USCNT”, created on Oct. 8, 2008, which is 434,294 bytes in size.
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 respiratory syncytial virus (RSV) 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 respiratory syncytial virus (RSV) gene expression pathways or other cellular processes that mediate the maintenance or development of such traits, diseases and conditions. Specifically, the invention relates to double stranded nucleic acid molecules including 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 RNA interference (RNAi) against respiratory syncytial virus (RSV) gene expression. Such small nucleic acid molecules are useful, for example, in providing compositions to prevent, inhibit, or reduce RSV infection, liver failure, hepatocellular carcinoma, cirrhosis, and/or other disease states associated with RSV infection in a subject or organism.
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′-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.
McSwiggen et al., WO 03/070918 describe double stranded nucleic acid molecules, including short interfering nucleic acids, targeting RSV and conserved sequences within the RSV genome.
Bushman et al., US 2003/0203868 describe the inhibition of certain pathogens, including RSV, using certain RNA interference mediating ribonucleic acid molecules.
Vaillant et al., US 2004/0229828 describe certain antiviral single-stranded oligonucleotides targeting RSV.
Mohapatra et al., WO 05/056021 describe certain siRNA molecules targeting RSV.
Bitco et al., 2005, Nature Medicine, 11, 50-55, describes the use of certain nasally administered vector expressed siRNA constructs targeting RSV.
Zhang et al., 2005, Nature Medicine, 11, 56-62, describes the use of certain nasally administered vector expressed siRNA constructs targeting the Ni gene of RSV.
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 the development or maintenance of RSV infection and other disease states associated with RSV infection (e.g., respiratory distress, bronchiolitis and pneumonia), by RNA interference (RNAi) 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 RSV 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 RSV genes and/or other genes (e.g., cellular or host genes) involved in pathways of RSV gene expression and/or infection.
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, including fully modified siNA, retains its RNAi activity. The siNA molecules of the instant invention provide useful reagents and methods for a variety of therapeutic, prophylactic, 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 gene(s) encoding RSV and/or cellular proteins associated with the maintenance or development of RSV infection, respiratory distress, bronchiolitis, and pneumonia, such as genes encoding sequences comprising those sequences referred to by GenBank Accession Nos. shown in Table I, referred to herein generally as RSV. The description below of the various aspects and embodiments of the invention is provided with reference to exemplary respiratory syncytial virus (RSV) genes (e.g., genes encoding RSV proteins such as nucleopretein (N), large (L) and phosphoproteins (P), matrix (M), fusion (F), glycoprotein (G), NS1 and 2 non-structural proteins, including small hydrophobic (SH) and M2 proteins), generally referred to herein as RSV. However, such reference is meant to be exemplary only and the various aspects and embodiments of the invention are also directed to other genes that express alternate RSV genes, such as mutant RSV genes, splice variants of RSV genes, and genes encoding different strains of RSV, as well as as cellular targets for RSV, such as those described herein and also referred to by GenBank Accession Nos. herein and in U.S. Ser. No. 10/923,536 and International Patent Application No. PCT/US03/05028, both incorporated by reference herein. The various aspects and embodiments are also directed to other genes involved in RSV pathways, including genes that encode cellular proteins involved in the maintenance and/or development of RSV infection, respiratory distress, bronchiolitis, and pneumonia, or other genes that express other proteins associated with RSV infection, such as cellular proteins that are utilized in the RSV life-cycle. Such additional genes can be analyzed for target sites using the methods described herein for RSV. Thus, the inhibition and the effects of such inhibition of the other genes can be performed as described herein. In other words, the term “RSV” as it is defined herein below and recited in the described embodiments, is meant to encompass genes associated with the development and/or maintenance of RSV infection, such as genes which encode RSV polypeptides, including polypeptides of different strains of RSV, mutant RSV genes, and splice variants of RSV genes, as well as cellular genes involved in RSV pathways of gene expression, replication, and/or RSV activity. Also, the term “RSV” as it is defined herein below and recited in the described embodiments, is meant to encompass RSV viral gene products and cellular gene products involved in RSV infection, such as those described herein. Thus, each of the embodiments described herein with reference to the term “RSV” are applicable to all of the virus, cellular and viral protein, peptide, polypeptide, and/or polynucleotide molecules covered by the term “RSV”, as that term is defined herein. Comprehensively, such gene targets are also referred to herein generally as “target” sequences.
In one embodiment, the invention features a composition comprising two or more different siNA molecules of the invention (e.g., siNA, duplex forming siNA, or multifunctional siNA or any combination thereof) targeting different polynucleotide targets, such as different regions of RSV RNA (e.g., two different target sites herein or any combination of RSV proteins such as nucleopretein (N), large (L) and phosphoproteins (P), matrix (M), fusion (F), glycoprotein (G), NS1 and 2 non-structural proteins, including small hydrophobic (SH) and M2 protein targets), different viral strains (e.g., RSV strains, or HIV and RSV, RSV and HCV etc.), or different viral and cellular targets (e.g., a RSV target and a cellular target as described herein). Such pools of siNA molecules can prevent or overcome viral resistance or otherwise provide increased therapeutic effect.
In one embodiment, the invention features a pool of two or more different siNA molecules of the invention (e.g., siNA, duplex forming siNA, or multifunctional siNA or any combination thereof) targeting different polynucleotide targets, such as different regions of RSV RNA (e.g., two different target sites herein or any combination of RSV proteins such as nucleopretein (N), large (L) and phosphoproteins (P), matrix (M), fusion (F), glycoprotein (G), NS1 and 2 non-structural proteins, including small hydrophobic (SH) and M2 protein targets), different viral strains (e.g., RSV strains), or different viruses (e.g., HIV and RSV, RSV and HCV etc.), or different viral and cellular targets (e.g., a RSV target and a cellular target), wherein the pool comprises siNA molecules targeting about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different RSV targets.
In one embodiment, a siNA molecule of the invention targets the RSV negative strand RNA or has RNAi specificity for the RSV negative strand RNA.
In one embodiment, the invention features one or more siNA molecules and methods that independently or in combination modulate the expression of genes representing cellular targets for RSV infection (see for example Ghildyal et al, 2005, J Gen Virol, 86:1879-84), such as cellular receptors, cell surface molecules, cellular enzymes, cellular transcription factors, and/or cytokines, second messengers, and cellular accessory molecules including, but not limited to, ICAM-1 (e.g., Genbank Accession Number NM—000201), RhoA (see for example Budge et al., 2004, Journal of Antimicrobial Chemotherapy, 54(2):299-302, e.g., Genbank Accession No. NM—044472); FAS (e.g., Genbank Accession No. NM—000043) or FAS ligand (e.g., Genbank Accession No. NM—000639); interferon regulatory factors (IRFs; e.g., Genbank Accession No. AF082503.1); cellular PKR protein kinase (e.g., Genbank Accession No. XM—002661.7); human eukaryotic initiation factors 2B (elF2Bgamma; e.g., Genbank Accession No. AF256223, and/or elF2gamma; e.g., Genbank Accession No. NM—006874.1); human DEAD Box protein (DDX3; e.g., Genbank Accession No. XM—018021.2); and polypyrimidine tract-binding protein (e.g., Genbank Accession Nos. NM—031991.1 and XM—042972.3). Such cellular targets are also referred to herein generally as RSV targets, and specifically as “host target” or “host targets”.
Due to the high sequence variability of the RSV genome, selection of siNA molecules for broad therapeutic applications likely involve the conserved regions of the RSV genome.
In one embodiment, the present invention relates to siNA molecules that target the conserved regions of the RSV genome. Examples of conserved regions of the RSV genome include, but are not limited to, the attachment (G) glycoprotein (see for example Trento et al., 2006, J. Virology, 80, 975-984) and polyadenylation/termination signal sequences (see for example Harmon et al., 2001, J. Virology, 75, 36-44). siNA molecules designed to target conserved regions of various RSV isolates enable efficient inhibition of RSV replication in diverse patient populations and ensure the effectiveness of the siNA molecules against RSV quasi species which evolve due to mutations in the non-conserved regions of the RSV genome. As described, a single siNA molecule can be targeted against all isolates of RSV by designing the siNA molecule to interact with conserved nucleotide sequences of RSV (e.g., sequences that are expected to be present in the RNA of various RSV isolates).
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 RSV target nucleic acid molecule, or a portion thereof. In one embodiment, the predetermined nucleotide sequence is a nucleotide RSV target sequence described herein. In another embodiment, the predetermined nucleotide sequence is a RSV 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 RSV target gene or that directs cleavage of a RSV 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 RSV 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 RSV 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 RSV target RNA for the siNA molecule to direct cleavage of the RSV 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 RSV 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 RSV target RNA for the siNA molecule to direct cleavage of the RSV 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 RSV 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 RSV target RNA for the siNA molecule to direct cleavage of the RSV 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 RSV 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 RSV target RNA for the siNA molecule to direct cleavage of the RSV target RNA via RNA interference.
In one embodiment, the invention features a siNA molecule that down-regulates expression of a RSV target gene or that directs cleavage of a RSV target RNA, for example, wherein the RSV target gene or RNA comprises protein encoding sequence. In one embodiment, the invention features a siNA molecule that down-regulates expression of a RSV target gene or that directs cleavage of a RSV target RNA, for example, wherein the RSV target gene or RNA comprises non-coding sequence or regulatory elements involved in RSV target gene expression (e.g., non-coding RNA).
In one embodiment, a siNA of the invention is used to inhibit the expression of RSV target genes or a RSV target gene family (e.g., different RSV strains, such as subgroup A and B strains), 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 RSV 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 RSV 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 RSV targeting sequences for differing polynucleotide RSV 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 RSV 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 Table I herein, or is U.S. Ser. No. 10/923,536 and PCT/US03/05028, both incorporated by reference herein. In another embodiment, the invention features a siNA molecule having RNAi activity against RSV 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 Table 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 RSV target gene and thereby mediate silencing of RSV target gene expression, for example, wherein the siNA mediates regulation of RSV target gene expression by cellular processes that modulate the chromatin structure or methylation patterns of the RSV target gene and prevent transcription of the RSV target gene.
In one embodiment, siNA molecules of the invention are used to down regulate or inhibit the expression of proteins arising from haplotype polymorphisms that are associated with a trait, disease or condition in a subject or organism. 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. 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 target gene expression. As such, analysis of protein or RNA levels can be used to determine treatment type and the course of therapy in treating a subject. Monitoring of 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 proteins associated with a trait, disorder, condition, or disease.
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 RSV target protein. The siNA further comprises a sense strand, wherein said sense strand comprises a nucleotide sequence of a RSV target 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 RSV target protein or a portion thereof. The siNA molecule further comprises a sense region, wherein said sense region comprises a nucleotide sequence of a RSV target 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 RSV target 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 RSV target 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 RSV target 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 Table I and in U.S. Ser. No. 10/923,536 and PCT/US03/05028, both incorporated by reference herein. Chemical modifications in Tables III and IV and 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 RSV target 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 one embodiment, a siNA molecule of the invention (e.g., a double stranded nucleic acid molecule) comprises an antisense (guide) 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 that are complementary to a RNA sequence of RSV or a portion thereof. In one embodiment, at least 15 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) of a RSV RNA sequence are complementary to the antisense (guide) strand of a siNA molecule of the invention.
In one embodiment, a siNA molecule of the invention (e.g., a double stranded nucleic acid molecule) comprises a sense (passenger) 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 that comprise sequence of a RSV RNA or a portion thereof. In one embodiment, at least 15 nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides of a RSV RNA sequence comprise the sense (passenger) strand of a siNA molecule of the invention.
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 RSV target 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 a RSV gene. Because RSV genes can share some degree of sequence homology with each other, siNA molecules can be designed to target a class of RSV genes (e.g., a class of different RSV strains or subtypes) or alternately specific RSV genes (e.g., escape mutants, resistant strains, or other polymorphic variants) by selecting sequences that are either shared amongst different RSV targets or alternatively that are unique for a specific RSV target (e.g., unique for any of the NS1, NS2, N, P, M, SH, G, F, M2, or L genes/proteins). Therefore, in one embodiment, the siNA molecule can be designed to target conserved regions of RSV RNA sequences having homology among several RSV gene variants so as to target a class of RSV genes with one siNA molecule. Accordingly, in one embodiment, the siNA molecule of the invention modulates the expression of one or more RSV stains in a subject or organism. In another embodiment, the siNA molecule can be designed to target a sequence that is unique to a specific RSV RNA sequence (e.g., a single RSV strain or RSV 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, 2′-deoxy-2′-fluoroarabino (FANA), 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 RSV target 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 RSV target 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 RSV target 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 RSV target 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 RSV target 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 RSV target polynucleotide sequence, i.e. those nucleotide positions in the RSV target 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 RSV target polynucleotide sequence, i.e. those nucleotide positions in the RSV target 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 RSV target 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 RSV target polynucleotide sequence. In another embodiment, the overhang nucleotides of a siNA molecule of the invention are 2′-O-methyl nucleotides, 2′-deoxy-2′-fluoroarabino, 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 or 2′-deoxy-2′-fluoroarabino 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 are 2′-deoxy-2′-fluoro or 2′-deoxy-2′-fluoroarabino nucleotides.
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 RSV target 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 RSV target polynucleotide sequence. In another embodiment, the nucleotides comprising the overhanging portion of a siNA molecule of the invention are 2′-O-methyl nucleotides, 2′-deoxy-2′-fluoroarabino 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 ain 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 RSV target nucleic acid molecules, such as DNA, or RNA encoding a protein or non-coding RNA associated with the expression of RSV target 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, 2′-deoxy-2′-fluoroarabino (FANA, see for example Dowler et al., 2006, Nucleic Acids Research, 34, 1669-1675) 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.
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, 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′-deoxy-2′-fluoroarabino, 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 RSV target 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 RSV target gene or that directs cleavage of a RSV target 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 RSV target gene, and the second strand of the double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence of the RSV target 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 RSV target gene or that directs cleavage of a RSV target RNA, comprising an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of the RSV target 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 RSV target 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 RSV target gene or that directs cleavage of a RSV target 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 RSV target 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 RSV target gene or that directs cleavage of a RSV target 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, (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 one 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 RSV target gene or that directs cleavage of a RSV target 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 RSV target 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 RSV target 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 RSV target 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 RSV target 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 RSV target gene can comprise, for example, sequences referred to herein or incorporated herein by reference. The RSV gene can comprise, for example, sequences referred to in Table I.
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 and FIGS. 4 and 5.
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 RSV target 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 RSV target 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 RSV target 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 RSV target 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 RSV target 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 RSV target 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 RSV target gene or that directs cleavage of a RSV target 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 RSV target 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-methylpyrimidine 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 RSV target gene or that directs cleavage of a RSV target 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′-deoxy-2′-fluoroarabino, 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′-deoxy-2′-fluoroarabino, 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′-deoxy-2′-fluoroarabino, 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′-deoxy-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 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′-fluoroarabino nucleotide. In one embodiment, all pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoroarabino pyrimidine nucleotides. In one embodiment, the modified nucleotides in the siNA include at least one 2′-deoxy-2′-fluoroarabino cytidine or 2′-deoxy-2′-fluoroarabino 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′-fluoroarabino uridine nucleotides. In one embodiment, all uridine nucleotides present in the siNA are 2′-deoxy-2′-fluoroarabino uridine nucleotides. In one embodiment, all cytidine nucleotides present in the siNA are 2′-deoxy-2′-fluoroarabino cytidine nucleotides. In one embodiment, all adenosine nucleotides present in the siNA are 2′-deoxy-2′-fluoroarabino adenosine nucleotides. In one embodiment, all guanosine nucleotides present in the siNA are 2′-deoxy-2′-fluoroarabino 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′-fluoroarabinonucleotides 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 RSV target gene or that directs cleavage of a RSV target 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 RSV target 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 viral strain, or disease or trait related allele in a subject or organism, such as sequence comprising a single nucleotide polymorphism (SNP) associated with the virus, or 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 RSV target gene or that directs cleavage of a RSV target 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 RSV target 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 RSV target 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 RSV target 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 IV 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 IV. 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 RSV target 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 RSV target RNA for the RNA molecule to direct cleavage of the RSV target 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′-deoxy-2′-fluoroarabino, 2′-O-methoxyethyl nucleotides, 4′-thio nucleotides, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides, etc. or any combination thereof.
In one embodiment, a RSV target RNA of the invention comprises sequence encoding a protein.
In one embodiment, RSV target RNA of the invention comprises non-coding RNA sequence (e.g., miRNA, snRNA, siRNA etc.), see for example Mattick, 2005, Science, 309, 1527-1528; Clayerie, 2005, Science, 309, 1529-1530; Sethupathy et al., 2006, RNA, 12, 192-197; and Czech, 2006 NEJM, 354, 11: 1194-1195.
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 RSV target 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 RSV target 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 RSV target 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 RSV target 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 RSV target 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 RSV target 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 RSV target 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 RSV target 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 RSV target 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 RSV target 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 RSV target 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 RSV target 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 RSV target 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 RSV target 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 RSV target 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 RSV target 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 RSV target 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 RSV target RNA.
In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a RSV target 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 RSV target 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 RSV target RNA or a portion thereof that is present in the RSV target RNA.
In one embodiment, the invention features a composition comprising a siNA molecule of the invention in a pharmaceutically acceptable carrier or diluent. In another embodiment, the invention features two or more differing siNA molecules of the invention (e.g. siNA molecules that target different regions of RSV target RNA or siNA molecules that target RSV RNA and cellular targets) 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 RSV targeting 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 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 RSV target 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-SH, 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 another 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-SH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-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 another 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 RSV target-complementary strand, for example, a strand complementary to a RSV target 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 RSV target-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 RSV target-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 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 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 sense 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-SH, 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, 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-SH, 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-SH, 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 I-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 0 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, a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprises a sense strand or sense region having one or more (e.g., 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 or more) 2′-O-alkyl (e.g. 2′-O-methyl), 2′-deoxy-2′-fluoro, 2′-deoxy, or abasic chemical modifications or any combination thereof.
In one embodiment, a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprises an antisense strand or antisense region having one or more (e.g., 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 or more) 2′-O-alkyl (e.g. 2′-O-methyl), 2′-deoxy-2′-fluoro, 2′-deoxy, or abasic chemical modifications or any combination thereof.
In one embodiment, a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprises a sense strand or sense region and an antisense strand or antisense region, each having one or more (e.g., 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 or more) 2′-O-alkyl (e.g. 2′-O-methyl), 2′-deoxy-2′-fluoro, 2′-deoxy, or abasic chemical modifications or any combination thereof.
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).
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 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).
In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region and an antisense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the sense region and the antisense 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).
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) 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 an antisense region, wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-O-methyl 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 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 III 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 RSV target molecule wherein the nucleic acid molecule has sequence that comprises a sequence recognized by the RSV target molecule in its natural setting. Alternately, an aptamer can be a nucleic acid molecule that binds to a RSV target molecule where the RSV target molecule does not naturally bind to a nucleic acid. The RSV target 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 oligonucleotide 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 oligonucleotide 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 presence 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 RSV target 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 RSV target 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 chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprises a sense strand or sense region having two or more (e.g., 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 or more) 2′-O-alkyl (e.g. 2′-O-methyl) modifications or any combination thereof. In another embodiment, the 2′-O-alkyl modification is at alternating position in the sense strand or sense region of the siNA, such as position 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 etc. or position 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 etc.
In one embodiment, a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprises an antisense strand or antisense region having two or more (e.g., 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 or more) 2′-O-alkyl (e.g. 2′-O-methyl) modifications or any combination thereof. In another embodiment, the 2′-O-alkyl modification is at alternating position in the antisense strand or antisense region of the siNA, such as position 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 etc. or position 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 etc.
In one embodiment, a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprises a sense strand or sense region and an antisense strand or antisense region, each having two or more (e.g., 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 or more) 2′-O-alkyl (e.g. 2′-O-methyl), 2′-deoxy-2′-fluoro, 2′-deoxy, or abasic chemical modifications or any combination thereof. In another embodiment, the 2′-O-alkyl modification is at alternating position in the sense strand or sense region of the siNA, such as position 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 etc. or position 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 etc. In another embodiment, the 2′-O-alkyl modification is at alternating position in the antisense strand or antisense region of the siNA, such as position 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 etc. or position 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 etc.
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:




    
    
        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 pyrimidine 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:




    
    
        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 pyrimidine 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:




    
    
        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 pyrimidine 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:




    
    
        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 pyrimidine 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:




    
    
        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 pyrimidine 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:




    
    
        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 pyrimidine 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:




    
    
        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 30; 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 pyrimidine 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 X1 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, SIII, 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 RSV target 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 RSV target 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 RSV target 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 RSV target 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 RSV target 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 composition comprising a siNA molecule or double stranded nucleic acid molecule formulated as any of formulation LNP-051; LNP-053; LNP-054; LNP-069; LNP-073; LNP-077; LNP-080; LNP-082; LNP-083; LNP-060; LNP-061; LNP-086; LNP-097; LNP-098; LNP-099; LNP-100; LNP-101; LNP-102; LNP-103; or LNP-104 (see Table VI).
In one embodiment, the invention features a composition comprising a first double stranded nucleic and a second double stranded nucleic acid molecule each having a first strand and a second strand that are complementary to each other, wherein the second strand of the first double stranded nucleic acid molecule comprises sequence complementary to a first RSV target sequence and the second strand of the second double stranded nucleic acid molecule comprises sequence complementary to a second RSV target sequence. In one embodiment, the composition further comprises a cationic lipid, a neutral lipid, and a polyethyleneglycol-conjugate. In one embodiment, the composition further comprises a cationic lipid, a neutral lipid, a polyethyleneglycol-conjugate, and a cholesterol. In one embodiment, the composition further comprises a polyethyleneglycol-conjugate, a cholesterol, and a surfactant. In one embodiment, the cationic lipid is selected from the group consisting of CLinDMA, pCLinDMA, eCLinDMA, DMOBA, and DMLBA. In one embodiment, the neutral lipid is selected from the group consisting of DSPC, DOBA, and cholesterol. In one embodiment, the polyethyleneglycol-conjugate is selected from the group consisting of a PEG-dimyristoyl glycerol and PEG-cholesterol. In one embodiment, the PEG is 2 KPEG. In one embodiment, the surfactant is selected from the group consisting of palmityl alcohol, stearyl alcohol, oleyl alcohol and linoleyl alcohol. In one embodiment, the cationic lipid is CLinDMA, the neutral lipid is DSPC, the polyethylene glycol conjugate is 2 KPEG-DMG, the cholesterol is cholesterol, and the surfactant is linoleyl alcohol. In one embodiment, the CLinDMA, the DSPC, the 2 KPEG-DMG, the cholesterol, and the linoleyl alcohol are present in molar ratio of 43:38:10:2:7 respectively.
In one embodiment, the invention features a method for modulating the expression of a RSV target 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 RSV target gene; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the RSV target gene in the cell.
In one embodiment, the invention features a method for modulating the expression of a RSV target 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 RSV target gene and wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequence of the RSV target RNA; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the RSV target gene in the cell.
In another embodiment, the invention features a method for modulating the expression of more than one RSV target 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 RSV target genes; and (b) introducing the siNA molecules into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the RSV target genes in the cell.
In another embodiment, the invention features a method for modulating the expression of two or more RSV target 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 RSV target genes and wherein the sense strand sequences of the siNAs comprise sequences identical or substantially similar to the sequences of the RSV target RNAs; and (b) introducing the siNA molecules into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the RSV target genes in the cell.
In another embodiment, the invention features a method for modulating the expression of more than one RSV target 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 RSV target gene and wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequences of the RSV target RNAs; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the RSV target genes in the cell.
In another embodiment, the invention features a method for modulating the expression of a RSV target 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 RSV target gene, wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequences of the RSV target RNA; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the RSV target 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 RSV target cells from a patient are extracted. These extracted cells are contacted with siNAs RSV targeting 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 RSV target 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 RSV target 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 RSV target 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 RSV target gene in that organism.
In one embodiment, the invention features a method of modulating the expression of a RSV target 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 RSV target gene and wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequence of the RSV target 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 RSV target 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 RSV target gene in that organism.
In another embodiment, the invention features a method of modulating the expression of more than one RSV target 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 RSV target 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 RSV target 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 RSV target genes in that organism.
In one embodiment, the invention features a method of modulating the expression of a RSV target 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 RSV target gene; and (b) introducing the siNA molecule into the subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the RSV target gene in the subject or organism. The level of RSV target 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 RSV target 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 RSV target genes; and (b) introducing the siNA molecules into the subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the RSV target genes in the subject or organism. The level of RSV target 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 RSV target gene within a cell (e.g., a lung or lung epithelial 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 RSV target gene; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the RSV target gene in the cell.
In another embodiment, the invention features a method for modulating the expression of more than one RSV target gene within a cell (e.g., a lung or lung epithelial 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 RSV target 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 RSV target genes in the cell.
In one embodiment, the invention features a method of modulating the expression of a RSV target gene in a tissue explant ((e.g., lung 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 RSV target 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 RSV target 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 RSV target gene in that subject or organism.
In another embodiment, the invention features a method of modulating the expression of more than one RSV target gene in a tissue explant (e.g., lung 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 RSV target 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 RSV target 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 RSV target genes in that subject or organism.
In one embodiment, the invention features a method of modulating the expression of a RSV target 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 RSV target gene; and (b) introducing the siNA molecule into the subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the RSV target gene in the subject or organism.
In another embodiment, the invention features a method of modulating the expression of more than one RSV target 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 RSV target gene; and (b) introducing the siNA molecules into the subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the RSV target genes in the subject or organism.
In one embodiment, the invention features a method of modulating the expression of a RSV target 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 RSV target 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 RSV 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 RSV infection 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 RSV target gene in the subject or organism whereby the treatment or prevention of RSV infection 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 liver 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 RSV infection 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 RSV infection in a subject or organism.
In one embodiment, the invention features a method for treating or preventing respiratory distress 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 RSV target gene in the subject or organism whereby the treatment or prevention of respiratory failure 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 lung cells and tissues involved in respiratory failure. 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 failure 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 failure in a subject or organism.
In one embodiment, the invention features a method for treating or preventing bronchiolitis 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 RSV target gene in the subject or organism whereby the treatment or prevention of bronchiolitis 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 liver cells and tissues involved in bronchiolitis. 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 bronchiolitis 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 bronchiolitis in a subject or organism.
In one embodiment, the invention features a method for treating or preventing pneumonia 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 RSV target gene in the subject or organism whereby the treatment or prevention of pneumonia 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 pneumonia. 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 pneumonia 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 pneumonia in a subject or organism.
In one embodiment, the invention features a method for treating or preventing RSV infection 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 an inhibitor of RSV gene expression in the subject or organism.
In one embodiment, the invention features a method for treating or preventing respiratory failure 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 an inhibitor of RSV gene expression in the subject or organism.
In one embodiment, the invention features a method for treating or preventing bronchiolitis 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 an inhibitor of RSV gene expression in the subject or organism.
In one embodiment, the invention features a method for treating or preventing pneumonia 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 an inhibitor of RSV gene expression in the subject or organism.
In one embodiment, the invention features a method for treating or preventing Respiratory syncytial virus (RSV) infection in a subject, comprising administering to the subject PEG Interferon in combination with a siNA molecule of the invention; wherein the PEG Interferon and the siNA molecule are administered under conditions suitable for reducing or inhibiting the level of Respiratory syncytial virus (RSV) in the subject compared to a subject not treated with the PEG Interferon and the siNA molecule. 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, the invention features a method for treating or preventing Respiratory syncytial virus (RSV) infection in a subject, comprising administering to the subject ribavirin in combination with a siNA molecule of the invention; wherein the ribavirin and the siNA are administered under conditions suitable for reducing or inhibiting the level of Respiratory syncytial virus (RSV) in the subject compared to a subject not treated with the ribavirin and the siNA molecule. 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 Respiratory syncytial virus (RSV) infection in a subject, comprising administering to the subject PEG Interferon and ribavirin in combination with a siNA molecule of the invention; wherein the PEG Interferon and ribavirin and the siNA molecule are administered under conditions suitable for reducing or inhibiting the level of Respiratory syncytial virus (RSV) in the subject compared to a subject not treated with the PEG Interferon and ribavirin and the siNA molecule. 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 Respiratory syncytial virus (RSV) infection in a subject, comprising administering to the subject PEG Interferon in combination with a chemically synthesized double stranded nucleic acid molecule; wherein (a) the double stranded nucleic acid molecule comprises a sense strand and an antisense strand; (b) each strand of the double stranded nucleic acid molecule is 15 to 28 nucleotides in length; (c) at least 15 nucleotides of the sense strand are complementary to the antisense strand (d) the antisense strand of the double stranded nucleic acid molecule has complementarity to a Respiratory syncytial virus (RSV) RSV target RNA; and wherein the PEG Interferon and the double stranded nucleic acid molecule are administered under conditions suitable for reducing or inhibiting the level of Respiratory syncytial virus (RSV) in the subject compared to a subject not treated with the PEG Interferon and the double stranded nucleic acid molecule. 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 Respiratory syncytial virus (RSV) infection in a subject, comprising administering to the subject ribavirin in combination with a chemically synthesized double stranded nucleic acid molecule; wherein (a) the double stranded nucleic acid molecule comprises a sense strand and an antisense strand; (b) each strand of the double stranded nucleic acid molecule is 15 to 28 nucleotides in length; (c) at least 15 nucleotides of the sense strand are complementary to the antisense strand (d) the antisense strand of the double stranded nucleic acid molecule has complementarity to a Respiratory syncytial virus (RSV) RSV target RNA; and wherein the ribavirin and the double stranded nucleic acid molecule are administered under conditions suitable for reducing or inhibiting the level of Respiratory syncytial virus (RSV) in the subject compared to a subject not treated with the ribavirin and the double stranded nucleic acid molecule. 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 Respiratory syncytial virus (RSV) infection in a subject, comprising administering to the subject PEG Interferon and ribavirin in combination with a chemically synthesized double stranded nucleic acid molecule; wherein (a) the double stranded nucleic acid molecule comprises a sense strand and an antisense strand; (b) each strand of the double stranded nucleic acid molecule is 15 to 28 nucleotides in length; (c) at least 15 nucleotides of the sense strand are complementary to the antisense strand (d) the antisense strand of the double stranded nucleic acid molecule has complementarity to a Respiratory syncytial virus (RSV) RSV target RNA; and wherein the PEG Interferon and ribavirin and the double stranded nucleic acid molecule are administered under conditions suitable for reducing or inhibiting the level of Respiratory syncytial virus (RSV) in the subject compared to a subject not treated with the PEG Interferon and ribavirin and the double stranded nucleic acid molecule. 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, in addition to the methods described herein or in combination with the methods described herein, a subject is further treated with palivizumab, RespiGam, A-60444, or other antiviral compounds and fusion inhibitors that may be used to treat RSV infection, alone, or in combination with other therapeutic modalities.
In one embodiment, the invention features a method for treating or preventing Respiratory syncytial virus (RSV) infection in a subject, comprising administering to the subject PEG Interferon in combination with a chemically synthesized double stranded nucleic acid molecule; wherein (a) the double stranded nucleic acid molecule comprises a sense strand and an antisense strand; (b) each strand of the double stranded nucleic acid molecule is 15 to 28 nucleotides in length; (c) at least 15 nucleotides of the sense strand are complementary to the antisense strand (d) the antisense strand of the double stranded nucleic acid molecule has complementarity to a Respiratory syncytial virus (RSV) RSV target RNA; (e) at least 20% of the internal nucleotides of each strand of the double stranded nucleic acid molecule are modified nucleosides having a chemical modification; and (f) at least two of the chemical modifications are different from each other, and wherein the PEG Interferon and the double stranded nucleic acid molecule are administered under conditions suitable for reducing or inhibiting the level of Respiratory syncytial virus (RSV) in the subject compared to a subject not treated with the PEG Interferon and the double stranded nucleic acid molecule. 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 Respiratory syncytial virus (RSV) infection in a subject, comprising administering to the subject ribavirin in combination with a chemically synthesized double stranded nucleic acid molecule; wherein (a) the double stranded nucleic acid molecule comprises a sense strand and an antisense strand; (b) each strand of the double stranded nucleic acid molecule is 15 to 28 nucleotides in length; (c) at least 15 nucleotides of the sense strand are complementary to the antisense strand (d) the antisense strand of the double stranded nucleic acid molecule has complementarity to a Respiratory syncytial virus (RSV) RSV target RNA; (e) at least 20% of the internal nucleotides of each strand of the double stranded nucleic acid molecule are modified nucleosides having a chemical modification; and (f) at least two of the chemical modifications are different from each other, and wherein the ribavirin and the double stranded nucleic acid molecule are administered under conditions suitable for reducing or inhibiting the level of Respiratory syncytial virus (RSV) in the subject compared to a subject not treated with the ribavirin and the double stranded nucleic acid molecule. 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 Respiratory syncytial virus (RSV) infection in a subject, comprising administering to the subject PEG Interferon and ribavirin in combination with a chemically synthesized double stranded nucleic acid molecule; wherein (a) the double stranded nucleic acid molecule comprises a sense strand and an antisense strand; (b) each strand of the double stranded nucleic acid molecule is 15 to 28 nucleotides in length; (c) at least 15 nucleotides of the sense strand are complementary to the antisense strand (d) the antisense strand of the double stranded nucleic acid molecule has complementarity to a Respiratory syncytial virus (RSV) RSV target RNA; (e) at least 20% of the internal nucleotides of each strand of the double stranded nucleic acid molecule are modified nucleosides having a chemical modification; and (f) at least two of the chemical modifications are different from each other, and wherein the PEG Interferon and ribavirin and the double stranded nucleic acid molecule are administered under conditions suitable for reducing or inhibiting the level of Respiratory syncytial virus (RSV) in the subject compared to a subject not treated with the PEG Interferon and ribavirin and the double stranded nucleic acid molecule. 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 Respiratory syncytial virus (RSV) infection in a subject, comprising administering to the subject PEG Interferon in combination with a chemically synthesized double stranded nucleic acid molecule; wherein (a) the double stranded nucleic acid molecule comprises a sense strand and an antisense strand; (b) each strand of the double stranded nucleic acid molecule is 15 to 28 nucleotides in length; (c) at least 15 nucleotides of the sense strand are complementary to the antisense strand (d) the antisense strand of the double stranded nucleic acid molecule has complementarity to a Respiratory syncytial virus (RSV) RSV target RNA; (e) at least 20% of the internal nucleotides of each strand of the double stranded nucleic acid molecule are modified nucleosides having a sugar modification; and (f) at least two of the sugar modifications are different from each other, and wherein the PEG Interferon and the double stranded nucleic acid molecule are administered under conditions suitable for reducing or inhibiting the level of Respiratory syncytial virus (RSV) in the subject compared to a subject not treated with the PEG Interferon and the double stranded nucleic acid molecule. 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 Respiratory syncytial virus (RSV) infection in a subject, comprising administering to the subject ribavirin in combination with a chemically synthesized double stranded nucleic acid molecule; wherein (a) the double stranded nucleic acid molecule comprises a sense strand and an antisense strand; (b) each strand of the double stranded nucleic acid molecule is 15 to 28 nucleotides in length; (c) at least 15 nucleotides of the sense strand are complementary to the antisense strand (d) the antisense strand of the double stranded nucleic acid molecule has complementarity to a Respiratory syncytial virus (RSV) RSV target RNA; (e) at least 20% of the internal nucleotides of each strand of the double stranded nucleic acid molecule are modified nucleosides having a sugar modification; and (f) at least two of the sugar modifications are different from each other, and wherein the ribavirin and the double stranded nucleic acid molecule are administered under conditions suitable for reducing or inhibiting the level of Respiratory syncytial virus (RSV) in the subject compared to a subject not treated with the ribavirin and the double stranded nucleic acid molecule. 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 Respiratory syncytial virus (RSV) infection in a subject, comprising administering to the subject PEG Interferon and ribavirin in combination with a chemically synthesized double stranded nucleic acid molecule; wherein (a) the double stranded nucleic acid molecule comprises a sense strand and an antisense strand; (b) each strand of the double stranded nucleic acid molecule is 15 to 28 nucleotides in length; (c) at least 15 nucleotides of the sense strand are complementary to the antisense strand (d) the antisense strand of the double stranded nucleic acid molecule has complementarity to a Respiratory syncytial virus (RSV) RSV target RNA; (e) at least 20% of the internal nucleotides of each strand of the double stranded nucleic acid molecule are modified nucleosides having a sugar modification; and (f) at least two of the sugar modifications are different from each other, and wherein the PEG Interferon and ribavirin and the double stranded nucleic acid molecule are administered under conditions suitable for reducing or inhibiting the level of Respiratory syncytial virus (RSV) in the subject compared to a subject not treated with the PEG Interferon and ribavirin and the double stranded nucleic acid molecule. 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 any of the above method for treating or preventing respiratory syncytial virus (RSV) infection in a subject, the treatment is combined with administration of a corticosteroid composition as is generally recognized in the art, including Triamcinolone acetonide, methylprednisolone, and dexamethasone.
In any of the above method for treating or preventing respiratory syncytial virus (RSV) infection in a subject, the treatment is combined with administration of a beta-2 agonist composition as is generally recognized in the art, including for example, albuterol or albuterol sulfate.
In one embodiment, the invention features a composition comprising PEG Interferon and one or more double stranded nucleic acid molecules or siNA molecules of the invention in a pharmaceutically acceptable carrier or diluent. In another embodiment, the invention features a composition comprising PEG Interferon, ribavirin, Vertex VX-950, Actilon (CPG 10101), and/or Isatoribine (TLR-7 agonist) and one or more double stranded nucleic acid molecules or siNA molecules of the invention in a pharmaceutically acceptable carrier or diluent.
In one embodiment, a method of treatment of the invention features administration of a double stranded nucleic acid molecule of the invention in combination with one or more other therapeutic modalities, including Interferon (e.g., Interferon-alpha, or PEG interferon such as PEG-Intron, Rebetol, Rebetron, or Pegasys), ribavirin, Vertex VX-950, Actilon (CPG 10101), or Isatoribine (TLR-7 agonist). In another embodiment, such combination therapies can be utilized in any of the embodiments herein.
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 described herein or as are known in the art. Systemic administration can include, for example, pulmonary (inhalation, nebulization etc.) 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, inhalation, nebulization, 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.
In another embodiment, the invention features a method of modulating the expression of more than one RSV target gene in a subject or organism comprising contacting the subject or organism with one or more siNA molecules of the invention under conditions suitable to modulate (e.g., inhibit) the expression of the RSV target genes in the subject or organism.
The siNA molecules of the invention can be designed to down regulate or inhibit target gene expression through RNAi targeting of a variety of nucleic acid molecules. In one embodiment, the siNA molecules of the invention are used to target various DNA corresponding to a target 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 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 RSV family genes (e.g., all known RSV strains, groups of related RSV strains, or groups of divergent RSV strains). As such, siNA molecules targeting multiple RSV 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 proliferative diseases, disorders and conditions.
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.
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, target genes encoding RNA sequence(s) referred to herein by Genbank Accession number, for example, Genbank Accession Nos. shown herein (e.g. in Table I) and in U.S. Ser. No. 10/923,536 and PCT/US03/05028, 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 treating or preventing a disease, trait, or condition, such as hearing loss, deafness, tinnitus, and/or motion and balance disorders in a subject, comprising administering to the subject a composition of the invention under conditions suitable for the treatment or prevention of the disease, trait, or condition in the subject, alone or in conjunction with one or more other therapeutic compounds.
In another embodiment, the invention features a method for validating a target gene 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 target gene; (b) introducing the siNA molecule into a cell, tissue, subject, or organism under conditions suitable for modulating expression of the 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 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 target gene; (b) introducing the siNA molecule into a biological system under conditions suitable for modulating expression of the 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 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 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 target polynucleotide (e.g., RNA or DNA 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 one embodiment, the invention features siNA constructs that mediate RNAi against a 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 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 target RNA sequence within a cell.
In one embodiment, the invention features siNA constructs that mediate RNAi against a 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 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 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 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 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 target DNA sequence.
In one embodiment, the invention features siNA constructs that mediate RNAi against a 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 target polynucleotide in a cell, wherein the chemical modifications do not significantly effect the interaction of siNA with a 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 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 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 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 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 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 target RNA.
In yet another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against a 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 target DNA.
In one embodiment, the invention features siNA constructs that mediate RNAi against a 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 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 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 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 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 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 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-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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 Tables II and III 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. 16-28 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 RSV RNA (see for example target sequences in Tables II and III). In one embodiment, the multifunctional siNA of the invention can comprise sequence targeting RSV RNA and one or more cellular targets involved in the RSV lifecycle, such as cellular receptors, cell surface molecules, cellular enzymes, cellular transcription factors, and/or cytokines, second messengers, and cellular accessory molecules including, but not limited to, La antigen (see for example Costa-Mattioli et al., 2004, Mol Cell Biol., 24, 6861-70, e.g., Genbank Accession No. NM—003142) (e.g., interferon regulatory factors (IRFs; e.g., Genbank Accession No. AF082503.1); cellular PKR protein kinase (e.g., Genbank Accession No. XM—002661.7); human eukaryotic initiation factors 2B (elF2Bgamma; e.g., Genbank Accession No. AF256223, and/or elF2gamma; e.g., Genbank Accession No. NM—006874.1); human DEAD Box protein (DDX3; e.g., Genbank Accession No. XM—018021.2); and cellular proteins that bind to the poly(U) tract of the RSV 3′-UTR, such as polypyrimidine tract-binding protein (e.g., Genbank Accession Nos. NM—031991.1 and XM—042972.3).
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-2-carbonyl, and GU imino amino-2-carbonyl base pairs.
By “RSV” as used herein is meant, any respiratory syncytial virus or RSV protein, peptide, or polypeptide having RSV activity, such as encoded by RSV Genbank Accession Nos. shown in Table I. The term RSV also refers to nucleic acid sequences encoding any RSV protein, peptide, or polypeptide having RSV activity (e.g., any of protein such as nucleopretein (N), large (L) and phosphoproteins (P), matrix (M), fusion (F), glycoprotein (G), NS1 and 2 non-structural proteins, including small hydrophobic (SH) and M2 proteins). The term “RSV” is also meant to include other RSV encoding sequence, such as other RSV isoforms, mutant RSV genes, splice variants of RSV genes, and RSV gene polymorphisms. In one embodiment, the term RSV as used herein refers to cellular or host proteins or polynucleotides encoding such proteins or that are otherwise involved in RSV infection and/or replication.
By “target” as used herein is meant, any target protein, peptide, or polypeptide, such as encoded by Genbank Accession Nos. herein and in U.S. Ser. No. 10/923,536 and PCT/US03/05028, 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 herein or in U.S. Ser. No. 10/923,536 and PCT/US03/05028. 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.
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 “microRNA” 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; Ying et al., 2004, Gene, 342, 25-28; and Sethupathy et al., 2006, RNA, 12:192-197). 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, the invention features nucleic acids that inhibit, down-regulate, or disrupt miRNAs that are involved in RSV infection and/or the RSV life-cycle. For example, a double stranded nucleic acid molecule of the invention (e.g., siNA) can be used to inhibit the function of a miRNA. In certain embodiments herein, the micro RNA is the target RNA in any of the embodiments herein. Double stranded nucleic acid molecules of the invention (e.g., siNA) having a antisense strand or antisense region that is complementary to a target miRNA sequence and a sense strand complementary to the antisense strand can be used to inhibit the activity of miRNAs involved in the RSV life-cycle or in RSV infection to prevent RSV activity or treat RSV infection in a cell or organism. Similarly, an single stranded nucleic acid molecule having complementary to a target miRNA sequence can be used to inhibit the activity of miRNAs involved in the RSV life-cycle or in RSV infection to prevent RSV activity or treat RSV infection in a cell or organism (see for example Zamore et al., US 2005/0227256 and Tuschl et al., US 2005/0182005 both incorporated by reference herein in their entirety; Zamore et al., 2005, Science, 309: 1519-24; Czech, 2006, NEJM, 354:1194-5; Krutzfeldt et al, 2005, Nature, 438:685-9).
In one embodiment, siNA molecules of the invention that down regulate or reduce target gene expression are used for treating, preventing or reducing RSV infection, respiratory distress, bronchiolitis and/or pneumonia in a subject or organism as described herein or otherwise known in the art.
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 Tables II and III 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 IV 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. In one embodiment, the subject is an infant (e.g., subjects that are less than 1 month old, or 1, 2, 3, 4, 5, 6, 7, 8, 9 10, 11, or 12 months old). In one embodiment, the subject is a toddler (e.g., 1, 2, 3, 4, 5 or 6 years old). In one embodiment, the subject is a senior (e.g., anyone over the age of about 65 years of age).
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. In one embodiment, the nucleic acid molecules of the invention (e.g, dsRNA, siNA etc.) are partially modified (e.g., about 5%, 10,%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% modified) with chemical modifications. In another embodiment, the nucleic acid molecules of the invention (e.g, dsRNA, siNA etc.) are completely modified (e.g., about 100% modified) with chemical modifications.
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 for preventing or treating diseases, disorders, conditions, and traits described herein or otherwise known in the art, in a subject or organism.
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 prevent or treat RSV infection, respiratory distress, bronchiolitis and/or pneumonia 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 prevent or treat diseases, disorders, conditions, and traits described herein 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 herein or in U.S. Ser. No. 10/923,536 and PCT/US03/05028, 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 target RNA molecules and down-regulate gene encoding target RNA molecules (for example 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 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 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 RSV siNA sequence. Such chemical modifications can be applied to any RSV sequence and/or cellular target sequence. Furthermore, the sequences shown in FIG. 5 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). In addition, the sequences shown in FIG. 5 can optionally include terminal ribonucleotides at up to about 4 positions at the 5′-end of the antisense strand (e.g., about 1, 2, 3, or 4 terminal ribonucleotides at the 5′-end of the antisense strand).
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′-modifications, 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 bifunctional 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 bifunctional 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 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 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 30mer 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.



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 RSV 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 RSV 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 RSV 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 RSV target nucleic acid molecule (e.g., RSV 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 RSV target nucleic acid (e.g., RNA). 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 RSV 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 RSV target nucleic acid molecule or a portion thereof (e.g., RSV 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, 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 RSV target nucleic acid sequence or a portion thereof and is of length sufficient to interact (e.g., base pair) with the RSV target nucleic acid sequence or a portion thereof (e.g., RSV 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 RSV 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 RSV target RNA or a portion thereof (e.g., RSV 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 RSV 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 RSV target RNA or a portion thereof (e.g., RSV 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 RSV target nucleic acid sequence or a portion thereof (e.g., RSV RNA target) and is of length sufficient to interact with the RSV target nucleic acid sequence of a portion thereof (e.g., RSV 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 RSV target RNA or a portion thereof (e.g., RSV 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 RSV 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 RSV 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 RSV target RNA or a portion thereof (e.g., RSV 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 RSV 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 RSV target nucleic acid sequence (e.g., RSV target RNA) or a portion thereof and is of length sufficient to interact (e.g., base pair) with the RSV 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 RSV target nucleic acid sequence or a portion thereof (e.g., RSV RNA target) and is of length sufficient to interact (e.g., base pair) with the RSV target nucleic acid sequence (e.g., RSV 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 RSV 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 RSV target nucleic acid (e.g., RSV 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 RSV 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 of the RSV or cellular/host target nucleic acid sequence or can target sequences of more than one distinct target nucleic acid molecules (e.g., RSV RNA or cellular/host 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 virus or viral proteins (e.g. nucleopretein (N), large (L) and phosphoproteins (P), matrix (M), fusion (F), glycoprotein (G), NS1 and 2 non-structural proteins, including small hydrophobic (SH) and M2 protein targets) and corresponding cellular proteins required for viral infection and/or replication, or differing strains or subtypes of a particular virus (e.g., RSV subtype A and subtype B and different strains thereof). 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 or RSV 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., RSV RNA), thereby allowing down regulation or inhibition of different strain variants or a virus, or splice variants encoded by a single host gene, or allowing for targeting of both coding and non-coding regions of the host 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, 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 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 (see for example Schwarz et al., 2003, Cell, 115, 199-208). In this instance the sense sequence is believed to direct the RISC 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 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 FIGS. 16-28.
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., RSV or host 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., RSV or host 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 first 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 viral genome (e.g., RSV) 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 host proteins involved in the RSV life-cycle) can be used to down regulate, inhibit, or shut down a particular biologic pathway by targeting, for example, a viral RNA (e.g., RSV RNA) and one or more host RNAs that are involved in viral infection or the viral life-cycle (e.g., Rho-A, ICAM-1, or interferon regulatory factors).
In another non-limiting example, a multifunctional siNA molecule comprising a region in one strand having a nucleotide sequence complementarity to a first target nucleic acid sequence derived from a target nucleic acid molecule encoding a virus or a viral protein (e.g., RSV) and the second strand comprising a region having a nucleotide sequence complementarity to a second target nucleic acid sequence present in target nucleic acid molecule encoding a cellular protein (e.g., a host cell receptor for the virus) can be used to down regulate, inhibit, or shut down the viral replication and infection by targeting the virus and cellular proteins necessary for viral infection or replication.
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 (e.g., conserved sequence) present in a target nucleic acid molecule such as a viral genome (e.g., RSV RNA) and the second strand comprising a region having a nucleotide sequence complementarity to a second target nucleic acid sequence (e.g., conserved sequence) present in target nucleic acid molecule derived from a gene encoding a viral protein (e.g., RSV proteins) to down regulate, inhibit, or shut down the viral replication and infection by targeting the viral genome and viral encoded proteins necessary for viral infection or replication.
In one embodiment the invention takes advantage of conserved nucleotide sequences present in different strains, isotypes or forms of a virus and genes encoded by these different strains, isotypes and forms of the virus (e.g., RSV). By designing multifunctional siNAs in a manner where one strand includes a sequence that is complementary to target nucleic acid sequence conserved among various strains, isotypes or forms of a virus and the other strand includes sequence that is complementary to target nucleic acid sequence conserved in a protein encoded by the virus, it is possible to selectively and effectively inhibit viral replication or infection using a single multifunctional siNA.
In one embodiment, a multifunctional short interfering nucleic acid (multifunctional siNA) of the invention comprises a first region and a second region, wherein the first region comprises nucleotide sequence complementary to a RSV viral RNA of a first viral strain and the second region comprises nucleotide sequence complementary to a RSV viral RNA of a second viral strain. In one embodiment, the first and second regions can comprise nucleotide sequence complementary to shared or conserved RNA sequences of differing viral strains or classes or viral strains.
In one embodiment, a multifunctional short interfering nucleic acid (multifunctional siNA) of the invention comprises a first region and a second region, wherein the first region comprises a nucleotide sequence complementary to a RSV viral RNA encoding one or more RSV viruses (e.g., one or more strains of RSV) and the second region comprises a nucleotide sequence complementary to a viral RNA encoding one or more interferon agonist proteins. In one embodiment, the first region can comprise a nucleotide sequence complementary to shared or conserved RNA sequences of differing RSV viral strains or classes of RSV viral strains. Non-limiting example of interferon agonist proteins include any protein that is capable of inhibition or suppressing RNA silencing (e.g., RNA binding proteins such as E3L or NS1 or equivalents thereof.
In one embodiment, a multifunctional short interfering nucleic acid (multifunctional siNA) of the invention comprises a first region and a second region, wherein the first region comprises nucleotide sequence complementary to a RSV viral RNA and the second region comprises nucleotide sequence complementary to a cellular RNA that is involved in RSV viral infection and/or replication. Non-limiting examples of cellular RNAs involved in viral infection and/or replication include cellular receptors (see for example Ghildyal et al., 2005, J Gen Virol., 86: 1879-94), cell surface molecules, cellular enzymes, cellular transcription factors, and/or cytokines, second messengers, and cellular accessory molecules including, but not limited to, La antigen, FAS, interferon agonist protein, interferon regulatory factors (IRFs); cellular PKR protein kinase (PKR); human eukaryotic initiation factors 2B (elF2B gamma and/or elF2gamma); human DEAD Box protein (DDX3); and cellular proteins that bind to the poly(U) tract of the RSV 3′-UTR, such as polypyrimidine tract-binding protein.
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 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; XZ comprises a nucleic acid sequence that is complementary to a first RSV target nucleic acid sequence; YZ is an oligonucleotide comprising nucleic acid sequence that is complementary to a second RSV 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 complementary; 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 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 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 RSV 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 RSV 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 RSV target nucleic acid sequence and the second RSV target nucleic acid sequence are present in the same target nucleic acid molecule (e.g., RSV RNA or host RNA). In another embodiment, the first RSV target nucleic acid sequence and the second RSV target nucleic acid sequence are present in different target nucleic acid molecules (e.g., RSV RNA and host 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 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 RSV target RNAs or a portion thereof. In one embodiment, the first RSV target nucleic acid sequence and the second RSV target nucleic acid sequence are present in the same target nucleic acid molecule (e.g., RSV RNA or host RNA). In another embodiment, the first RSV target nucleic acid sequence and the second RSV target nucleic acid sequence are present in different target nucleic acid molecules (e.g., RSV RNA and host 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 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 RSV 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 RSV target sequence via RNA interference. In one embodiment, the first RSV target nucleic acid sequence and the second RSV target nucleic acid sequence are present in the same target nucleic acid molecule (e.g., RSV RNA or host RNA). In another embodiment, the first RSV target nucleic acid sequence and the second RSV target nucleic acid sequence are present in different target nucleic acid molecules (e.g., RSV RNA and host 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 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 RSV 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 RSV target sequence via RNA interference. In one embodiment, the first RSV target nucleic acid sequence and the second RSV target nucleic acid sequence are present in the same target nucleic acid molecule (e.g., RSV RNA or host RNA). In another embodiment, the first RSV target nucleic acid sequence and the second RSV target nucleic acid sequence are present in different target nucleic acid molecules (e.g., RSV RNA and host 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 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 RSV 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 RSV target nucleic acid sequence are all present in the same target nucleic acid molecule (e.g., RSV RNA or host RNA). In another embodiment, the first, second, third and fourth RSV target nucleic acid sequence are independently present in different target nucleic acid molecules (e.g., RSV RNA and host 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 (see FIG. 22). 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 (see FIG. 22 (A)). 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 (see FIG. 22 (B)). 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 (see FIG. 22 (C-D)). 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 (see FIG. 22 (G-H)). 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 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 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 (see FIG. 22 (E)). 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 (see FIG. 22 (F)). 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 RSV RNA or a portion thereof or a polynucleotide coding or non-coding sequence of cellular or host target that is involved in RSV infection or replication, or disease processes associated with RSV infection such as such as cellular receptors, cell surface molecules, cellular enzymes, cellular transcription factors, and/or cytokines, second messengers, and cellular accessory molecules including, but not limited to, ICAM-1, RhoA (see for example Budge et al., 2004, Journal of Antimicrobial Chemotherapy, 54(2):299-302, e.g., Genbank Accession No. NM—044472); FAS (e.g., Genbank Accession No. NM—000043) or FAS ligand (e.g., Genbank Accession No. NM—000639); interferon regulatory factors (IRFs; e.g., Genbank Accession No. AF082503.1); cellular PKR protein kinase (e.g., Genbank Accession No. XM—002661.7); human eukaryotic initiation factors 2B (elF2Bgamma; e.g., Genbank Accession No. AF256223, and/or elF2gamma; e.g., Genbank Accession No. NM—006874.1); human DEAD Box protein (DDX3; e.g., Genbank Accession No. XM—018021.2); and cellular proteins that bind to the poly(U) tract of the RSV 3′-UTR, such as polypyrimidine tract-binding protein (e.g., Genbank Accession Nos. NM—031991.1 and XM—042972.3). In one embodiment, the first RSV target nucleic acid sequence is a RSV RNA or a portion thereof and the second RSV target nucleic acid sequence is a RSV RNA of a portion thereof. In one embodiment, the first RSV target nucleic acid sequence is a RSV RNA or a portion thereof and the second RSV target nucleic acid sequence is a host RNA or a portion thereof. In one embodiment, the first RSV target nucleic acid sequence is a host RNA or a portion thereof and the second RSV target nucleic acid sequence is a host RNA or a portion thereof. In one embodiment, the first RSV target nucleic acid sequence is a host RNA or a portion thereof and the second RSV target nucleic acid sequence is a RSV 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. In one embodiment, the nucleic acid molecules of the invention are synthesized, deprotected, and analyzed according to methods described in U.S. Pat. Nos. 6,995,259, 6,686,463, 6,673,918, 6,649,751, 6,989,442, and U.S. Ser. No. 10/190,359, all incorporated by reference herein in their entirety.
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. In one embodiment, the nucleic acid molecules of the invention are synthesized, deprotected, and analyzed according to methods described in U.S. Pat. Nos. 6,995,259, 6,686,463, 6,673,918, 6,649,751, 6,989,442, and U.S. Ser. No. 10/190,359, all incorporated by reference herein in their entirety.
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, TIBS17, 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.
In one embodiment, a nucleic acid molecule of the invention is chemically modified as described in US 20050020521, incorporated by reference herein in its entirety.
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 RSV infection, respiratory distress, bronchiolitis and pneumonia and/or any other trait, disease or condition that is related to or will respond to the levels of RSV in a cell or tissue, alone or in combination with other therapies. In one embodiment, the siNA molecules of the invention and formulations or compositions thereof are administered to the lung as is described herein and as is generally known in the art.
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, U.S. Provisional patent application No. 60/737,024, filed Nov. 15, 2005, and U.S. Ser. No. 11/353,630, filed Feb. 14, 2006 (Vargeese et al.), all of which are incorporated by reference herein in their entirety. Such siNA formuations are generally referred to as “lipid nucleic acid particles” (LNP). In one embodiment, a siNA molecule of the invention is formulated with one or more LNP compositions described herein in Table IV (see U.S. Ser. No. 11/353,630 supra).
In one embodiment, the siNA molecules of the invention and formulations or compositions thereof are administered to lung tissues and cells as is described in US 2006/0062758; US 2006/0014289; and US 2004/0077540.
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, the nucleic acid molecules of the invention and formulations thereof (e.g., LNP formulations of double stranded 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 liquified 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 siNA and LNP compositions and formulations provided herein for use in pulmonary delivery further comprise one or more surfactants. Suitable surfactants or surfactant components for enhancing the uptake of the compositions of the invention include synthetic and natural as well as full and truncated forms of surfactant protein A, surfactant protein B, surfactant protein C, surfactant protein D and surfactant Protein E, di-saturated phosphatidylcholine (other than dipalmitoyl), dipalmitoylphosphatidylcholine, phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine; phosphatidic acid, ubiquinones, lysophosphatidylethanolamine, lysophosphatidylcholine, palmitoyl-lysophosphatidylcholine, dehydroepiandrosterone, dolichols, sulfatidic acid, glycerol-3-phosphate, dihydroxyacetone phosphate, glycerol, glycero-3-phosphocholine, dihydroxyacetone, palmitate, cytidine diphosphate (CDP) diacylglycerol, CDP choline, choline, choline phosphate; as well as natural and artificial lamelar bodies which are the natural carrier vehicles for the components of surfactant, omega-3 fatty acids, polyenic acid, polyenoic acid, lecithin, palmitinic acid, non-ionic block copolymers of ethylene or propylene oxides, polyoxypropylene, monomeric and polymeric, polyoxyethylene, monomeric and polymeric, poly (vinyl amine) with dextran and/or alkanoyl side chains, Brij 35, Triton X-100 and synthetic surfactants ALEC, Exosurf, Survan and Atovaquone, among others. These surfactants may be used either as single or part of a multiple component surfactant in a formulation, or as covalently bound additions to the 5′ and/or 3′ ends of the nucleic acid component of a pharmaceutical composition herein.
The composition of the present invention may be administered into the respiratory system as a formulation including particles of respirable size, e.g. particles of a size sufficiently small to pass through the nose, mouth and larynx upon inhalation and through the bronchi and alveoli of the lungs. In general, respirable particles range from about 0.5 to 10 microns in size. Particles of non-respirable size which are included in the aerosol tend to deposit in the throat and be swallowed, and the quantity of non-respirable particles in the aerosol is thus minimized. For nasal administration, a particle size in the range of 10-500 um is preferred to ensure retention in the nasal cavity.
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 15mer 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, siNA compounds and compositions of the invention are administered either systemically or locally 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 therapeis 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, a siNA molecule of the invention is administered iontophoretically, for example to a particular organ or compartment (e.g., lung, liver, 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 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 oligonucleotide, 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 certain embodiment, the nucleic acid molecules of the invention are delivered to hematopoetic cells as is 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, and U.S. Ser. No. 11/353,630, filed Feb. 14, 2006 (Vargeese et al.), all of which are incorporated by reference herein in their entirety.
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, 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 (e.g., lung). 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.
In one embodiment, a liposomal formulation of the invention comprises a double stranded nucleic acid molecule of the invention (e.g, siNA) formulated or complexed with compounds and compositions described in U.S. Pat. Nos. 6,858,224; 6,534,484; 6,287,591; 6,835,395; 6,586,410; 6,858,225; 6,815,432; 6,586,001; 6,120,798; 6,977,223; 6,998,115; 5,981,501; 5,976,567; 5,705,385; US 2006/0019912; US 2006/0019258; US 2006/0008909; US 2005/0255153; US 2005/0079212; US 2005/0008689; US 2003/0077829, US 2005/0064595, US 2005/0175682, US 2005/0118253; US 2004/0071654; US 2005/0244504; US 2005/0265961 and US 2003/0077829, all of which are incorporated by reference herein in their entirety.
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; propulic 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 (po 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. U S A, 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. U.S. A, 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.
RSV Biology and Biochemistry
Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and pneumonia among infants and children under 1 year of age. Illness begins most frequently with fever, runny nose, cough, and sometimes wheezing. During their first RSV infection, between 25% and 40% of infants and young children have signs or symptoms of bronchiolitis or pneumonia, and 0.5% to 2% require hospitalization. Most children recover from illness in 8 to 15 days. The majority of children hospitalized for RSV infection are under 6 months of age. RSV also causes repeated infections throughout life, usually associated with moderate-to-severe cold-like symptoms; however, severe lower respiratory tract disease may occur at any age, especially among the elderly or among those with compromised cardiac, pulmonary, or immune systems.
Human respiratory syncytial virus (hRSV or RSV) is the type species of the genus Pneumovirus. Along with other members of the family Paramyxoviridae, hRSV is an enveloped virus with a negative sense, single-stranded RNA genome. These viruses are 150-200 nm in diameter with a helical nucleocapsid. Other members of the family include human parainfluenzavirus 1 (HPIV-1; genus Respirovirus), mumps virus (MuV), human parainfluenzavirus 2 and 4 (HPIV-2 and HPIV-4; genus Rubulavirus), measles virus (MeV; genus Morbillivirus), Hendravirus and Nipahvirus (genus Henipavirus) and human metapneumovirus (hMPV; genus Metapneumovirus).
The hRSV virion enters its target cell by fusion of the hRSV envelope with the cell membrane and release of the viral genome into the cell's cytoplasm where translation will occur. The nucleopretein (N), large (L) and phosphoproteins (P) together with the RNA genome form the nucleoprotein core. These together with the matrix (M), fusion (F), and glycoprotein (G) are classified as structural proteins. The nonstructural proteins include NS1 and 2, small hydrophobic (SH) and M2 (formerly 22-kDa). The respiratory syncytial virus genome is transcribed from the 3′ end into monocistronic (each species only encodes a single protein) mRNA molecules. New viruses are released form the infected cell buy budding. In the presence of newly synthesized hRSV fusion (F) protein, neighbouring infected cells may form a clump whose membranes have fused to form a “giant cell” called a syncytium. New virions can then spread more effectively from cell-to-cell.
RSV is spread from respiratory secretions through close contact with infected persons or contact with contaminated surfaces or objects. Infection can occur when infectious material contacts mucous membranes of the eyes, mouth, or nose, and possibly through the inhalation of droplets generated by a sneeze or cough. In temperate climates, RSV infections usually occur during annual community outbreaks, often lasting 4 to 6 months, during the late fall, winter, or early spring months. The timing and severity of outbreaks in a community vary from year to year. RSV spreads efficiently among children during the annual outbreaks, and most children will have serologic evidence of RSV infection by 2 years of age.
Diagnosis of RSV infection can be made by virus isolation, detection of viral antigens, detection of viral RNA, demonstration of a rise in serum antibodies, or a combination of these approaches. Most clinical laboratories use antigen detection assays to diagnose infection.
For children with mild disease, no specific treatment is necessary other than the treatment of symptoms (e.g., acetaminophen to reduce fever). Children with severe disease may require oxygen therapy and sometimes mechanical ventilation. Ribavirin aerosol may be used in the treatment of some patients with severe disease. Some investigators have used a combination of immune globulin intravenous (IGIV) with high titers of neutralizing RSV antibody (RSV-IGIV) and ribavirin to treat patients with compromised immune systems.
The use of nucleic molecules of the invention targeting RSV genes and cellular/host gene targets associated with the RSV life cycle therefore provides a class of novel therapeutic agents that can be used in the treatment and diagnosis of RSV infection, respiratory distress, stridor, bronchiolitis and pneumonia, or any other disease or condition that responds to modulation (e.g., inhibition) of RSV genes in a subject or organism.
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 Bromotripyrrolidinophosphoniumhexafluororophosphate (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′ UU 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 target RNA sequence.
In one embodiment, siNA molecules of the invention are selected using the following methodology. The following guidelines were compiled to predict hyper-active siNAs that contain chemical modifications described herein. These rules emerged from a comparative analysis of hyper-active (>75% knockdown of target mRNA levels) and inactive (<75% knockdown of target mRNA levels) siNAs against several different targets. A total of 242 siNA sequences were analyzed. Thirty-five siNAs out of 242 siRNAs were grouped into hyper-active and the remaining siNAs were grouped into inactive groups. The hyper-active siNAs clearly showed a preference for certain bases at particular nucleotide positions within the siNA sequence. For example, A or U nucleobase was overwhelmingly present at position 19 of the sense strand in hyper-active siNAs and opposite was true for inactive siNAs. There was also a pattern of a A/U rich (3 out of 5 bases as A or U) region between positions 15-19 and G/C rich region between positions 1-5 (3 out of 5 bases as G or C) of the sense strand in hyperactive siNAs. As shown in Table VII, 12 such patterns were identified that were characteristics of hyper-active siNAs. It is to be noted that not every pattern was present in each hyper-active siNA. Thus, to design an algorithm for predicting hyper-active siNAs, a different score was assigned for each pattern. Depending on how frequently such patterns occur in hyper-active siRNAs versus inactive siRNAs, the design parameters were assigned a score with the highest being 10. If a certain nucleobase is not preferred at a position, then a negative score was assigned. For example, at positions 9 and 13 of the sense strand, a G nucleotide was not preferred in hyper-active siNAs and therefore they were given score of −3(minus 3). The differential score for each pattern is given in Table VII. The pattern # 4 was given a maximum score of −100. This is mainly to weed out any sequence that contains string of 4Gs or 4Cs as they can be highly incompatible for synthesis and can allow sequences to self-aggregate, thus rendering the siNA inactive. Using this algorithm, the highest score possible for any siNA is 66. As there are numerous siNA sequences possible against any given target of reasonable size ( ̃1000 nucleotides), this algorithm is useful to generate hyper-active siNAs.
In one embodiment, rules 1-11 are used to generate active siNA molecules of the invention. In another embodiment, rules 1-12 are used to generate active siNA molecules of the invention.
Example 4
RSV siNA Design
siNA target sites were chosen by analyzing sequences of the RSV target and optionally prioritizing the target sites on the basis of the rules presented in Example 3 above, and alternately on the basis of folding (structure of any given sequence analyzed to determine siNA accessibility to the target), or 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 selected using the algorithm above 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.
RSV RNA sequences (Table I) were analysed for homology with an 80% cutoff to generate a consensus RSV target RNA molecule from which double stranded siNA molecules were designed (Table II). To generate synthetic siNA constructs (Table III), the algorithm described in Example 3 was utilized to pick active double stranded constructs and chemically modified versions thereof. In each of Tables II and III, the target sequence is shown, along with the upper (sense strand) and lower (antisense strand) of the siNA duplex. The following sequences, referred to by GenBank accession number, were used to generate the siNA sequences shown in Tables II and III.















gi|3089371|gb|AF035006.1|



gi|60549163|gb|AY911262.1|



Ref Seq (NC_001803.1)



gi|1912287|gb|U39662.1|HRU39662



gi|1695254|gb|U63644.1|HRU63644



gi|2582022|gb|AF013254.1|AF013254



gi|2627296|gb|U50362.1|HRU50362



gi|38230482|gb|AY353550.1|



gi|1912298|gb|U39661.1|RSU39661



gi|9629198|ref|NC_001781.1|



gi|2627309|gb|U50363.1|HRU50363



gi|2582034|gb|AF013255.1|AF013255










Chemically modified siNA constructs were 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. 5,889,136; 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-diisopropylphosphoroamidite 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 target 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 μM 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 target RNA target for siNA mediated RNAi cleavage, wherein a plurality of siNA constructs are screened for RNAi mediated cleavage of the target 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 RSV Target RNA
siNA molecules targeted to RSV 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 RSV RNA are given in Table II and III.
Two formats are used to test the efficacy of siNAs targeting RSV. First, the reagents are tested in cell culture using, for example, RSV infected Jurkat, HeLa, A549 or 293T cells, to determine the extent of RNA and protein inhibition. siNA reagents (e.g.; see Tables II and III) are selected against the RSV target as described herein. RNA inhibition is measured after delivery of these reagents by a suitable transfection agent to, for example, RSV infected Jurkat, HeLa, A549 or 293T 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., Jurkat, HeLa, A549 or 293T 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., LNP formulations herein, or another suitable lipid such as Lipofectamine, final concentration 2 μg/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, 10 U RNase Inhibitor (Promega), 1.25 U AMPLITAQ GOLD® (DNA polymerase) (PE-Applied Biosystems) and 10 U 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
Models Useful to Evaluate the Down-Regulation of RSV Gene Expression
Evaluating the efficacy of anti-iRSV agents in vitro and in animal models is an important prerequisite to human clinical trials. Bitco et al., 2005, Nature Medicine, 11, 50-55, describes the use of certain nasally administered vector expressed siRNA constructs targeting RSV. Zhang et al., 2005, Nature Medicine, 11, 56-62, describes the use of certain nasally administered vector expressed siRNA constructs targeting the N1 gene of RSV. As such, these models can be used in evaluating the efficacy of siNA molecules of the invention in inhibiting RSV expression. As such, these models and others can be adapted to evaluate the safety and efficacy of siNA molecules of the invention in a pre-clinical setting. For example, the models used by Bitco et al. and Zhang et al. supra, can be adapted for use with synthetic siNA LNP formulations that are administered to the lungs of mice via intranasal inhalation or nebulization as is generally known in the art.
Example 9
RNAi Mediated Inhibition of Target Gene Expression
In Vitro siNA Mediated Inhibition of Target RNA
siNA constructs (Table III) are tested for efficacy in reducing RSV RNA expression in, for example, A549 cells that are infected with RSV. Cells are plated approximately 24 hours before transfection in 96-well plates at 5,000-7,500 cells/well, 100 μl/well, such that at the time of transfection cells are 70-90% confluent. For transfection, annealed siNAs are mixed with the transfection reagent (Lipofectamine 2000, Invitrogen) in a volume of 50 μl/well and incubated for 20 minutes at room temperature. The siNA transfection mixtures are added to cells to give a final siNA concentration of 25 nM in a volume of 150 μl. Each siNA transfection mixture is added to 3 wells for triplicate siNA treatments. Cells are incubated at 37° for 24 hours in the continued presence of the siNA transfection mixture. At 24 hours, RNA is prepared from each well of treated cells. The supernatants with the transfection mixtures are first removed and discarded, then the cells are lysed and RNA prepared from each well. Target RSV gene expression following treatment is evaluated by RT-PCR for the target gene and for a control gene (36B4, an RNA polymerase subunit) for normalization. The triplicate data is averaged and the standard deviations determined for each treatment. Normalized data are graphed and the percent reduction of target mRNA by active siNAs in comparison to their respective inverted control siNAs is determined.
Example 10
Indications
The present body of knowledge in RSV research indicates the need for methods to assay RSV activity and for compounds that can regulate RSV 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 RSV levels. In addition, the nucleic acid molecules can be used to treat disease state related to RSV levels. Particular disease states that can be associated with RSV expression modulation include, but are not limited to, RSV infection, respiratory distress, stridor, bronchiolitis and pneumonia.
Example 11
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 multifunctional 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 constructs 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 differing 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 30mer 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 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.
Example 12
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 full-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. 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





RSV Accession Numbers















1: AF035006


Human respiratory syncytial virus, recombinant mutant rA2cp, complete


genome


gi|3089371|gb|AF035006.1|[3089371]


2: U50362


Human respiratory syncytial virus, mutant cp-RSV, complete genome


gi|2627296|gb|U50362.1|HRU50362[2627296]


3: U50363


Human respiratory syncytial virus, mutant cpts-248, complete genome


gi|2627309|gb|U50363.1|HRU50363[2627309]


4: U63644


Human respiratory syncytial virus, mutant cpts-248/404, complete genome


gi|1695254|gb|U63644.1|HRU63644[1695254]


5: M74568


Human respiratory syncytial virus nonstructural protein 1,


nonstructural protein 2, nucleocapsid protein, phosphoprotein, matrix


protein, small hydrophobic protein, glycoprotein, fusion glycoprotein,


22K/M2 protein and L protein mRNA, complete cds


gi|333959|gb|M74568.1|RSHSEQ[333959]


6: AY911262


Human respiratory syncytial virus strain ATCC VR-26, complete genome


gi|60549163|gb|AY911262.1|[60549163]


7: U39662


Human respiratory syncytial virus, complete genome


gi|1912287|gb|U39662.1|HRU39662[1912287]


8: U39661


Respiratory syncytial virus, complete genome


gi|1912298|gb|U39661.1|RSU39661[1912298]


9: M11486


Human respiratory syncytial virus nonstructural protein (1C),


nonstructural protein (1B), major nucleocapsid (N), phosphoprotein (P),


protein (M), 1A (1A), G (G), protein (F) and envelope-associated


protein (22K) gene, complete cds


gi|333925|gb|M11486.1|RSH1CE[333925]


10: M75730


Human respiratory syncytial virus polymerase L RNA, complete cds


gi|333955|gb|M75730.1|RSHPOLLA[333955]


11: U27298


Human respiratory syncytial virus RNA-dependent RNA polymerase (L)


gene, complete cds


gi|1143829|gb|U27298.1|HRU27298[1143829]


12: AF254574


Human respiratory syncytial virus M2 gene, partial sequence; and


polymerase L gene, complete cds


gi|7960297|gb|AF254574.1|AF254574[7960297]


13: U35343


Human respiratory syncytial virus RNA polymerase large subunit (L)


gene, complete cds


gi|1016280|gb|U35343.1|HRU35343[1016280]


14: D00151


Human respiratory syncytial virus genes for fusion protein and 22K


protein, complete cds


gi|222548|dbj|D00151.1|RSH22K[222548]


15: X02221


Human respiratory syncytial virus (A2) mRNA for fusion glycoprotein Fo


gi|61210|emb|X02221.1|PNRSF0[61210]


16: M22643


Human respiratory syncytial virus fusion (F) protein mRNA, complete cds


gi|333938|gb|M22643.1|RSHF1[333938]


17: AY330616


Human respiratory syncytial virus strain Long fusion protein precursor,


gene, complete cds


gi|37674753|gb|AY330616.1|[37674753]


18: AY330613


Human respiratory syncytial virus strain Long clone T1480G fusion


protein precursor, gene, complete cds


gi|37674747|gb|AY330613.1|[37674747]


19: AY330614


Human respiratory syncytial virus strain Long clone T433A fusion


protein precursor, gene, complete cds


gi|37674749|gb|AY330614.1|[37674749]


20: AY330615


Human respiratory syncytial virus strain Long clone T444C fusion


protein precursor, gene, complete cds


gi|37674751|gb|AY330615.1|[37674751]


21: AY330611


Human respiratory syncytial virus strain Long clone A1188G fusion


protein precursor, gene, complete cds


gi|37674743|gb|AY330611.1|[37674743]


22: AY330612


Human respiratory syncytial virus strain Long clone A1194G fusion


protein precursor, gene, complete cds


gi|37674745|gb|AY330612.1|[37674745]


23: Z26524


Human respiratory syncytial virus F gene for fusion protein


gi|403378|emb|Z26524.1|HRSVFG[403378]


24: L25351


Human respiratory syncytial virus fusion protein (F) mRNA, complete cds


gi|409060|gb|L25351.1|RSHFUSP[409060]


25: AY198176


Human respiratory syncytial virus strain E49 fusion protein gene,


complete cds


gi|29290040|gb|AY198176.1|[29290040]


26: U31559


Human respiratory syncytial virus, strain RSB89-5857, fusion protein


(F) mRNA, complete cds


gi|961608|gb|U31559.1|HRU31559[961608]


27: U31560


Human respiratory syncytial virus, strain RSB89-1734, fusion protein


(F) mRNA, complete cds


gi|961610|gb|U31560.1|HRU31560[961610]


28: U31558


Human respiratory syncytial virus, strain RSB89-6190, fusion protein


(F) mRNA, complete cds


gi|961606|gb|U31558.1|HRU31558[961606]


29: U31562


Human respiratory syncytial virus, strain RSB89-6614, fusion protein


(F) mRNA, complete cds


gi|961614|gb|U31562.1|HRU31562[961614]


30: AY526556


Human respiratory syncytial virus isolate SAA1SA98D707 fusion protein


gene, partial cds


gi|46405973|gb|AY526556.1|[46405973]


31: AY526553


Human respiratory syncytial virus isolate GA5SA97D1131 fusion protein


gene, partial cds


gi|46405967|gb|AY526553.1|[46405967]


32: AF067125


Human respiratory syncytial virus fusion protein (F) mRNA, partial cds


gi|3172521|gb|AF067125.1|AF067125[3172521]


33: X00001


Human respiratory syncytial (RS) virus nucleocapsid gene


gi|61215|emb|X00001.1|PNRSO1[61215]


34: X03149


Respiratory syncytial virus mRNA for envelope glycoprotein G


gi|60997|emb|X03149.1|PARSENVG[60997]


35: M22644


Human respiratory syncytial virus phosphoprotein (P) mRNA,


complete cds


gi|333949|gb|M22644.1|RSHP1[333949]


36: AF065405


Human respiratory syncytial virus strain WV2780 attachment glycoprotein


G mRNA, complete cds


gi|4106742|gb|AF065405.1|AF065405[4106742]


37: M17212


Human respiratory syncytial virus (subgroup A) attachment protein (G)


mRNA, complete cds


gi|333940|gb|M17212.1|RSHGLYG[333940]


38: M29342


Human respiratory syncytial virus phosphoprotein mRNA, complete cds


gi|333951|gb|M29342.1|RSHPHOS[333951]


39: AF065407


Human respiratory syncytial virus strain WV6973 attachment glycoprotein


G mRNA, partial cds


gi|4106746|gb|AF065407.1|AF065407[4106746]


40: AY151197


Human respiratory syncytial virus isolate sa97D1131 nucleoprotein (N)


mRNA, partial cds


gi|32140842|gb|AY151197.1|[32140842]


41: AY151194


Human respiratory syncytial virus isolate SA98V173 nucleoprotein (N)


mRNA, partial cds


gi|32140836|gb|AY151194.1|[32140836]


42: AY151196


Human respiratory syncytial virus isolate sa97D540 nucleoprotein (N)


mRNA, partial cds


gi|32140840|gb|AY151196.1|[32140840]


43: AY151198


Human respiratory syncytial virus isolate Ab3061CT01 nucleoprotein (N)


mRNA, partial cds


gi|32140844|gb|AY151198.1|[32140844]


44: AY151199


Human respiratory syncytial virus isolate Ab4029B101 nucleoprotein (N)


mRNA, partial cds


gi|32140846|gb|AY151199.1|[32140846]


45: AY151195


Human respiratory syncytial virus isolate SA98d707 nucleoprotein (N)


mRNA, partial cds


gi|32140838|gb|AY151195.1|[32140838]


46: AM040047


Human respiratory syncytial virus partial fusion protein gene for


fusion protein, genomic RNA


gi|68161825|emb|AM040047.1|[68161825]


47: U35030


Respiratory syncytial virus nonstructural protein 1 mRNA, partial cds


gi|1016239|gb|U35030.1|RSU35030[1016239]


48: U35029


Respiratory syncytial virus nonstructural protein 2 mRNA, complete cds


gi|1016237|gb|U35029.1|RSU35029[1016237]


49: M17245


Human respiratory syncytial virus (RS) polymerase L gene, 5′ end, and


22K protein gene, 3′ end


gi|333953|gb|M17245.1|RSHPOLL[333953]


50: S70183


glycoprotein G = secreted form {alternative initiation} [respiratory


syncytial virus, mRNA Partial, 240 nt]


gi|547067|bbm|339653|bbs|148059|gb|S70183.1|[547067]


51: AJ492167


respiratory syncytial virus partial N gene for nucleoprotein, genomic


RNA, isolate CT343/00


gi|32329733|emb|AJ492167.1|RSY492167[32329733]


52: AJ492163


respiratory syncytial virus partial N gene for nucleoprotein, genomic


RNA, isolate CT243/00


gi|32329725|emb|AJ492163.1|RSY492163[32329725]


53: AJ492159


respiratory syncytial virus partial N gene for nucleoprotein, genomic


RNA, isolate CT127/00


gi|32329717|emb|AJ492159.1|RSY492159[32329717]


54: AF280466


Respiratory syncytial virus isolate A SH protein (SH) gene, complete


cds


gi|11055422|gb|AF280466.1|AF280466[11055422]


55: AJ492166


respiratory syncytial virus partial N gene for nucleoprotein, genomic


RNA, isolate CT328/00


gi|32329731|emb|AJ492166.1|RSY492166[32329731]


56: AJ492155


respiratory syncytial virus partial N gene for nucleoprotein, genomic


RNA, isolate CT107A/00


gi|32329709|emb|AJ492155.1|RSY492155[32329709]


57: AJ492165


respiratory syncytial virus partial N gene for nucleoprotein, genomic


RNA, isolate CT287/00


gi|32329729|emb|AJ492165.1|RSY492165[32329729]


58: AJ492158


respiratory syncytial virus partial N gene for nucleoprotein, genomic


RNA, isolate CT122/00


gi|32329715|emb|AJ492158.1|RSY492158[32329715]


59: AF280490


Respiratory syncytial virus isolate 181P2 SH protein (SH) gene,


complete cds


gi|11055470|gb|AF280490.1|AF280490[11055470]


60: AF280469


Respiratory syncytial virus isolate 022 SH protein (SH) gene, complete


cds


gi|11055428|gb|AF280469.1|AF280469[11055428]


61: AF280482


Respiratory syncytial virus isolate 136 SH protein (SH) gene, complete


cds


gi|11055454|gb|AF280482.1|AF280482[11055454]


62: AF280487


Respiratory syncytial virus isolate 159P2 SH protein (SH) gene,


complete cds


gi|11055464|gb|AF280487.1|AF280487[11055464]


63: AF280483


Respiratory syncytial virus isolate 139P2 SH protein (SH) gene,


complete cds


gi|11055456|gb|AF280483.1|AF280483[11055456]


64: AF280492


Respiratory syncytial virus isolate 194 SH protein (SH) gene, complete


cds


gi|11055474|gb|AF280492.1|AF280492[11055474]


65: AF280471


Respiratory syncytial virus isolate 037A SH protein (SH) gene, complete


cds


gi|11055432|gb|AF280471.1|AF280471[11055432]


66: AF280488


Respiratory syncytial virus isolate 160 SH protein (SH) gene, complete


cds


gi|11055466|gb|AF280488.1|AF280488[11055466]


67: AF280476


Respiratory syncytial virus isolate 081P2 SH protein (SH) gene,


complete cds


gi|11055442|gb|AF280476.1|AF280476[11055442]


68: AJ492164


respiratory syncytial virus partial N gene for nucleoprotein, genomic


RNA, isolate CT25/00


gi|32329727|emb|AJ492164.1|RSY492164[32329727]


69: AF280475


Respiratory syncytial virus isolate 073 SH protein (SH) gene, complete


cds


gi|11055440|gb|AF280475.1|AF280475[11055440]


70: AF280467


Respiratory syncytial virus isolate 013A SH protein (SH) gene, complete


cds


gi|11055424|gb|AF280467.1|AF280467[11055424]


71: AF280484


Respiratory syncytial virus isolate 143BCE SH protein (SH) gene,


complete cds


gi|11055458|gb|AF280484.1|AF280484[11055458]


72: AF280468


Respiratory syncytial virus isolate 021C SH protein (SH) gene, complete


cds


gi|11055426|gb|AF280468.1|AF280468[11055426]


73: AF280485


Respiratory syncytial virus isolate 151P2 SH protein (SH) gene,


complete cds


gi|110554460|gb|AF280485.1|AF280485[11055460]


74: AF280481


Respiratory syncytial virus isolate 132S SH protein (SH) gene, complete


cds


gi|11055452|gb|AF280481.1|AF280481[11055452]


75: AF280477


Respiratory syncytial virus isolate 084P1 SH protein (SH) gene,


complete cds


gi|11055444|gb|AF280477.1|AF280477[11055444]


76: AF280473


Respiratory syncytial virus isolate 050 SH protein (SH) gene, complete


cds


gi|11055436|gb|AF280473.1|AF280473[11055436]


77: AF280486


Respiratory syncytial virus isolate 154 SH protein (SH) gene, complete


cds


gi|11055462|gb|AF280486.1|AF280486[11055462]


78: AF280478


Respiratory syncytial virus isolate 091 SH protein (SH) gene, complete


cds


gi|11055446|gb|AF280478.1|AF280478[11055446]


79: AF280491


Respiratory syncytial virus isolate 191 SH protein (SH) gene, complete


cds


gi|11055472|gb|AF280491.1|AF280491[11055472]


80: AF280470


Respiratory syncytial virus isolate 026A SH protein (SH) gene, complete


cds


gi|1055430|gb|AF280470.1|AF280470[11055430]


81: AF280480


Respiratory syncytial virus isolate 117 SH protein (SH) gene, complete


cds


gi|11055450|gb|AF280480.1|AF280480[11055450]


82: AF280472


Respiratory syncytial virus isolate 044 SH protein (SH) gene, complete


cds


gi|11055434|gb|AF280472.1|AF280472[11055434]


83: AF280489


Respiratory syncytial virus isolate 173P3 SH protein (SH) gene,


complete cds


gi|11055468|gb|AF280489.1|AF280489[11055468]


84: AJ492162


respiratory syncytial virus partial N gene for nucleoprotein, genomic


RNA, isolate CT20/00


gi|32329723|emb|AJ492162.1|RSY492162[32329723]

















TABLE II







RSV siNA AND TARGET SEQUENCES



















Seq


Seq


Seq



Pos
Seq
ID
UPos
Upper seq
ID
LPos
Lower seq
ID





   38
AAAAAAUGGGGCAAAUAAG
   1
   38
AAAAAAUGGGGCAAAUAAG
   1
   56
CUUAUUUGCCCCAUUUUUU
1268



 


   39
AAAAAUGGGGCAAAUAAGA
   2
   39
AAAAAUGGGGCAAAUAAGA
   2
   57
UCUUAUUUGCCCCAUUUUU
1269


 


   40
AAAAUGGGGCAAAUAAGAA
   3
   40
AAAAUGGGGCAAAUAAGAA
   3
   58
UUCUUAUUUGCCCCAUUUU
1270


 


   41
AAAUGGGGCAAAUAAGAAU
   4
   41
AAAUGGGGCAAAUAAGAAU
   4
   59
AUUCUUAUUUGCCCCAUUU
1271


 


   42
AAUGGGGCAAAUAAGAAUU
   5
   42
AAUGGGGCAAAUAAGAAUU
   5
   60
AAUUCUUAUUUGCCCCAUU
1272


 


   43
AUGGGGCAAAUAAGAAUUU
   6
   43
AUGGGGCAAAUAAGAAUUU
   6
   61
AAAUUCUUAUUUGCCCCAU
1273


 


   44
UGGGGCAAAUAAGAAUUUG
   7
   44
UGGGGCAAAUAAGAAUUUG
   7
   62
CAAAUUCUUAUUUGCCCCA
1274


 


   45
GGGGCAAAUAAGAAUUUGA
   8
   45
GGGGCAAAUAAGAAUUUGA
   8
   63
UCAAAUUCUUAUUUGCCCC
1275


 


   46
GGGCAAAUAAGAAUUUGAU
   9
   46
GGGCAAAUAAGAAUUUGAU
   9
   64
AUCAAAUUCUUAUUUGCCC
1276


 


   47
GGCAAAUAAGAAUUUGAUA
  10
   47
GGCAAAUAAGAAUUUGAUA
  10
   65
UAUCAAAUUCUUAUUUGCC
1277


 


   48
GCAAAUAAGAAUUUGAUAA
  11
   48
GCAAAUAAGAAUUUGAUAA
  11
   66
UUAUCAAAUUCUUAUUUGC
1278


 


   49
CAAAUAAGAAUUUGAUAAG
  12
   49
CAAAUAAGAAUUUGAUAAG
  12
   67
CUUAUCAAAUUCUUAUUUG
1279


 


   50
AAAUAAGAAUUUGAUAAGU
  13
   50
AAAUAAGAAUUUGAUAAGU
  13
   68
ACUUAUCAAAUUCUUAUUU
1280


 


  159
GAAGUAGCAUUGUUAAAAA
  14
  159
GAAGUAGCAUUGUUAAAAA
  14
  177
UUUUUAACAAUGCUACUUC
1281


 


  160
AAGUAGCAUUGUUAAAAAU
  15
  160
AAGUAGCAUUGUUAAAAAU
  15
  178
AUUUUUAACAAUGCUACUU
1282


 


  161
AGUAGCAUUGUUAAAAAUA
  16
  161
AGUAGCAUUGUUAAAAAUA
  16
  179
UAUUUUUAACAAUGCUACU
1283


 


  162
GUAGCAUUGUUAAAAAUAA
  17
  162
GUAGCAUUGUUAAAAAUAA
  17
  180
UUAUUUUUAACAAUGCUAC
1284


 


  163
UAGCAUUGUUAAAAAUAAC
  18
  163
UAGCAUUGUUAAAAAUAAC
  18
  181
GUUAUUUUUAACAAUGCUA
1285


 


  164
AGCAUUGUUAAAAAUAACA
  19
  164
AGCAUUGUUAAAAAUAACA
  19
  182
UGUUAUUUUUAACAAUGCU
1286


 


  165
GCAUUGUUAAAAAUAACAU
  20
  165
GCAUUGUUAAAAAUAACAU
  20
  183
AUGUUAUUUUUAACAAUGC
1287


 


  166
CAUUGUUAAAAAUAACAUG
  21
  166
CAUUGUUAAAAAUAACAUG
  21
  184
CAUGUUAUUUUUAACAAUG
1288


 


  775
AGAAAACUUGAUGAAAGAC
  22
  775
AGAAAACUUGAUGAAAGAC
  22
  793
GUCUUUCAUCAAGUUUUCU
1289


 


  776
GAAAACUUGAUGAAAGACA
  23
  776
GAAAACUUGAUGAAAGACA
  23
  794
UGUCUUUCAUCAAGUUUUC
1290


 


  943
AUUGGCAUUAAGCCUACAA
  24
  943
AUUGGCAUUAAGCCUACAA
  24
  961
UUGUAGGCUUAAUGCCAAU
1291


 


  944
UUGGCAUUAAGCCUACAAA
  25
  944
UUGGCAUUAAGCCUACAAA
  25
  962
UUUGUAGGCUUAAUGCCAA
1292


 


 1140
AUGGCUCUUAGCAAAGUCA
  26
 1140
AUGGCUCUUAGCAAAGUCA
  26
 1158
UGACUUUGCUAAGAGCCAU
1293


 


 1141
UGGCUCUUAGCAAAGUCAA
  27
 1141
UGGCUCUUAGCAAAGUCAA
  27
 1159
UUGACUUUGCUAAGAGCCA
1294


 


 1142
GGCUCUUAGCAAAGUCAAG
  28
 1142
GGCUCUUAGCAAAGUCAAG
  28
 1160
CUUGACUUUGCUAAGAGCC
1295


 


 1143
GCUCUUAGCAAAGUCAAGU
  29
 1143
GCUCUUAGCAAAGUCAAGU
  29
 1161
ACUUGACUUUGCUAAGAGC
1296


 


 1144
CUCUUAGCAAAGUCAAGUU
  30
 1144
CUCUUAGCAAAGUCAAGUU
  30
 1162
AACUUGACUUUGCUAAGAG
1297


 


 1329
UUAAUAGGUAUGUUAUAUG
  31
 1329
UUAAUAGGUAUGUUAUAUG
  31
 1347
CAUAUAACAUACCUAUUAA
1298


 


 1330
UAAUAGGUAUGUUAUAUGC
  32
 1330
UAAUAGGUAUGUUAUAUGC
  32
 1348
GCAUAUAACAUACCUAUUA
1299


 


 1518
AUUGAGAUAGAAUCUAGAA
  33
 1518
AUUGAGAUAGAAUCUAGAA
  33
 1536
UUCUAGAUUCUAUCUCAAU
1300


 


 1519
UUGAGAUAGAAUCUAGAAA
  34
 1519
UUGAGAUAGAAUCUAGAAA
  34
 1537
UUUCUAGAUUCUAUCUCAA
1301


 


 1539
UCCUACAAAAAAAUGCUAA
  35
 1539
UCCUACAAAAAAAUGCUAA
  35
 1557
UUAGCAUUUUUUUGUAGGA
1302


 


 1540
CCUACAAAAAAAUGCUAAA
  36
 1540
CCUACAAAAAAAUGCUAAA
  36
 1558
UUUAGCAUUUUUUUGUAGG
1303


 


 1541
CUACAAAAAAAUGCUAAAA
  37
 1541
CUACAAAAAAAUGCUAAAA
  37
 1559
UUUUAGCAUUUUUUUGUAG
1304


 


 1542
UACAAAAAAAUGCUAAAAG
  38
 1542
UACAAAAAAAUGCUAAAAG
  38
 1560
CUUUUAGCAUUUUUUUGUA
1305


 


 1543
ACAAAAAAAUGCUAAAAGA
  39
 1543
ACAAAAAAAUGCUAAAAGA
  39
 1561
UCUUUUAGCAUUUUUUUGU
1306


 


 2037
UUCUACCAUAUAUUGAACA
  40
 2037
UUCUACCAUAUAUUGAACA
  40
 2055
UGUUCAAUAUAUGGUAGAA
1307


 


 2038
UCUACCAUAUAUUGAACAA
  41
 2038
UCUACCAUAUAUUGAACAA
  41
 2056
UUGUUCAAUAUAUGGUAGA
1308


 


 2402
AAAUUCCUAGAAUCAAUAA
  42
 2402
AAAUUCCUAGAAUCAAUAA
  42
 2420
UUAUUGAUUCUAGGAAUUU
1309


 


 2403
AAUUCCUAGAAUCAAUAAA
  43
 2403
AAUUCCUAGAAUCAAUAAA
  43
 2421
UUUAUUGAUUCUAGGAAUU
1310


 


 2404
AUUCCUAGAAUCAAUAAAG
  44
 2404
AUUCCUAGAAUCAAUAAAG
  44
 2422
CUUUAUUGAUUCUAGGAAU
1311


 


 2405
UUCCUAGAAUCAAUAAAGG
  45
 2405
UUCCUAGAAUCAAUAAAGG
  45
 2423
CCUUUAUUGAUUCUAGGAA
1312


 


 2406
UCCUAGAAUCAAUAAAGGG
  46
 2406
UCCUAGAAUCAAUAAAGGG
  46
 2424
CCCUUUAUUGAUUCUAGGA
1313


 


 2407
CCUAGAAUCAAUAAAGGGC
  47
 2407
CCUAGAAUCAAUAAAGGGC
  47
 2425
GCCCUUUAUUGAUUCUAGG
1314


 


 2408
CUAGAAUCAAUAAAGGGCA
  48
 2408
CUAGAAUCAAUAAAGGGCA
  48
 2426
UGCCCUUUAUUGAUUCUAG
1315


 


 2409
UAGAAUCAAUAAAGGGCAA
  49
 2409
UAGAAUCAAUAAAGGGCAA
  49
 2427
UUGCCCUUUAUUGAUUCUA
1316


 


 2477
AACUCAAUAGAUAUAGAAG
  50
 2477
AACUCAAUAGAUAUAGAAG
  50
 2495
CUUCUAUAUCUAUUGAGUU
1317


 


 2478
ACUCAAUAGAUAUAGAAGU
  51
 2478
ACUCAAUAGAUAUAGAAGU
  51
 2496
ACUUCUAUAUCUAUUGAGU
1318


 


 2479
CUCAAUAGAUAUAGAAGUA
  52
 2479
CUCAAUAGAUAUAGAAGUA
  52
 2497
UACUUCUAUAUCUAUUGAG
1319


 


 2480
UCAAUAGAUAUAGAAGUAA
  53
 2480
UCAAUAGAUAUAGAAGUAA
  53
 2498
UUACUUCUAUAUCUAUUGA
1320


 


 2481
CAAUAGAUAUAGAAGUAAC
  54
 2481
CAAUAGAUAUAGAAGUAAC
  54
 2499
GUUACUUCUAUAUCUAUUG
1321


 


 2669
ACAUUUGAUAACAAUGAAG
  55
 2669
ACAUUUGAUAACAAUGAAG
  55
 2687
CUUCAUUGUUAUCAAAUGU
1322


 


 2670
CAUUUGAUAACAAUGAAGA
  56
 2670
CAUUUGAUAACAAUGAAGA
  56
 2688
UCUUCAUUGUUAUCAAAUG
1323


 


 2671
AUUUGAUAACAAUGAAGAA
  57
 2671
AUUUGAUAACAAUGAAGAA
  57
 2689
UUCUUCAUUGUUAUCAAAU
1324


 


 2672
UUUGAUAACAAUGAAGAAG
  58
 2672
UUUGAUAACAAUGAAGAAG
  58
 2690
CUUCUUCAUUGUUAUCAAA
1325


 


 2673
UUGAUAACAAUGAAGAAGA
  59
 2673
UUGAUAACAAUGAAGAAGA
  59
 2691
UCUUCUUCAUUGUUAUCAA
1326


 


 2674
UGAUAACAAUGAAGAAGAA
  60
 2674
UGAUAACAAUGAAGAAGAA
  60
 2692
UUCUUCUUCAUUGUUAUCA
1327


 


 2675
GAUAACAAUGAAGAAGAAU
  61
 2675
GAUAACAAUGAAGAAGAAU
  61
 2693
AUUCUUCUUCAUUGUUAUC
1328


 


 2676
AUAACAAUGAAGAAGAAUC
  62
 2676
AUAACAAUGAAGAAGAAUC
  62
 2694
GAUUCUUCUUCAUUGUUAU
1329


 


 2759
AUUGAUGAAAAAUUAAGUG
  63
 2759
AUUGAUGAAAAAUUAAGUG
  63
 2777
CACUUAAUUUUUCAUCAAU
1330


 


 2760
UUGAUGAAAAAUUAAGUGA
  64
 2760
UUGAUGAAAAAUUAAGUGA
  64
 2778
UCACUUAAUUUUUCAUCAA
1331


 


 2761
UGAUGAAAAAUUAAGUGAA
  65
 2761
UGAUGAAAAAUUAAGUGAA
  65
 2779
UUCACUUAAUUUUUCAUCA
1332


 


 2762
GAUGAAAAAUUAAGUGAAA
  66
 2762
GAUGAAAAAUUAAGUGAAA
  66
 2780
UUUCACUUAAUUUUUCAUC
1333


 


 2763
AUGAAAAAUUAAGUGAAAU
  67
 2763
AUGAAAAAUUAAGUGAAAU
  67
 2781
AUUUCACUUAAUUUUUCAU
1334


 


 2764
UGAAAAAUUAAGUGAAAUA
  68
 2764
UGAAAAAUUAAGUGAAAUA
  68
 2782
UAUUUCACUUAAUUUUUCA
1335


 


 2897
GAAGCAUUAAUGACCAAUG
  69
 2897
GAAGCAUUAAUGACCAAUG
  69
 2915
CAUUGGUCAUUAAUGCUUC
1336


 


 2898
AAGCAUUAAUGACCAAUGA
  70
 2898
AAGCAUUAAUGACCAAUGA
  70
 2916
UCAUUGGUCAUUAAUGCUU
1337


 


 3223
UGGGGCAAAUAUGGAAACA
  71
 3223
UGGGGCAAAUAUGGAAACA
  71
 3241
UGUUUCCAUAUUUGCCCCA
1338


 


 3224
GGGGCAAAUAUGGAAACAU
  72
 3224
GGGGCAAAUAUGGAAACAU
  72
 3242
AUGUUUCCAUAUUUGCCCC
1339


 


 3225
GGGCAAAUAUGGAAACAUA
  73
 3225
GGGCAAAUAUGGAAACAUA
  73
 3243
UAUGUUUCCAUAUUUGCCC
1340


 


 3226
GGCAAAUAUGGAAACAUAC
  74
 3226
GGCAAAUAUGGAAACAUAC
  74
 3244
GUAUGUUUCCAUAUUUGCC
1341


 


 3227
GCAAAUAUGGAAACAUACG
  75
 3227
GCAAAUAUGGAAACAUACG
  75
 3245
CGUAUGUUUCCAUAUUUGC
1342


 


 3228
CAAAUAUGGAAACAUACGU
  76
 3228
CAAAUAUGGAAACAUACGU
  76
 3246
ACGUAUGUUUCCAUAUUUG
1343


 


 3229
AAAUAUGGAAACAUACGUG
  77
 3229
AAAUAUGGAAACAUACGUG
  77
 3247
CACGUAUGUUUCCAUAUUU
1344


 


 3230
AAUAUGGAAACAUACGUGA
  78
 3230
AAUAUGGAAACAUACGUGA
  78
 3248
UCACGUAUGUUUCCAUAUU
1345


 


 3231
AUAUGGAAACAUACGUGAA
  79
 3231
AUAUGGAAACAUACGUGAA
  79
 3249
UUCACGUAUGUUUCCAUAU
1346


 


 3232
UAUGGAAACAUACGUGAAC
  80
 3232
UAUGGAAACAUACGUGAAC
  80
 3250
GUUCACGUAUGUUUCCAUA
1347


 


 3233
AUGGAAACAUACGUGAACA
  81
 3233
AUGGAAACAUACGUGAACA
  81
 3251
UGUUCACGUAUGUUUCCAU
1348


 


 3234
UGGAAACAUACGUGAACAA
  82
 3234
UGGAAACAUACGUGAACAA
  82
 3252
UUGUUCACGUAUGUUUCCA
1349


 


 3254
CUUCACGAAGGCUCCACAU
  83
 3254
CUUCACGAAGGCUCCACAU
  83
 3272
AUGUGGAGCCUUCGUGAAG
1350


 


 3255
UUCACGAAGGCUCCACAUA
  84
 3255
UUCACGAAGGCUCCACAUA
  84
 3273
UAUGUGGAGCCUUCGUGAA
1351


 


 3256
UCACGAAGGCUCCACAUAC
  85
 3256
UCACGAAGGCUCCACAUAC
  85
 3274
GUAUGUGGAGCCUUCGUGA
1352


 


 3257
CACGAAGGCUCCACAUACA
  86
 3257
CACGAAGGCUCCACAUACA
  86
 3275
UGUAUGUGGAGCCUUCGUG
1353


 


 3258
ACGAAGGCUCCACAUACAC
  87
 3258
ACGAAGGCUCCACAUACAC
  87
 3276
GUGUAUGUGGAGCCUUCGU
1354


 


 3259
CGAAGGCUCCACAUACACA
  88
 3259
CGAAGGCUCCACAUACACA
  88
 3277
UGUGUAUGUGGAGCCUUCG
1355


 


 3260
GAAGGCUCCACAUACACAG
  89
 3260
GAAGGCUCCACAUACACAG
  89
 3278
CUGUGUAUGUGGAGCCUUC
1356


 


 3261
AAGGCUCCACAUACACAGC
  90
 3261
AAGGCUCCACAUACACAGC
  90
 3279
GCUGUGUAUGUGGAGCCUU
1357


 


 6193
UUAACCAGCAAAGUGUUAG
  91
 6193
UUAACCAGCAAAGUGUUAG
  91
 6211
CUAACACUUUGCUGGUUAA
1358


 


 6194
UAACCAGCAAAGUGUUAGA
  92
 6194
UAACCAGCAAAGUGUUAGA
  92
 6212
UCUAACACUUUGCUGGUUA
1359


 


 6427
AUAACAAAUGAUCAGAAAA
  93
 6427
AUAACAAAUGAUCAGAAAA
  93
 6445
UUUUCUGAUCAUUUGUUAU
1360


 


 6428
UAACAAAUGAUCAGAAAAA
  94
 6428
UAACAAAUGAUCAGAAAAA
  94
 6446
UUUUUCUGAUCAUUUGUUA
1361


 


 6718
AAUCGAGUAUUUUGUGACA
  95
 6718
AAUCGAGUAUUUUGUGACA
  95
 6736
UGUCACAAAAUACUCGAUU
1362


 


 6719
AUCGAGUAUUUUGUGACAC
  96
 6719
AUCGAGUAUUUUGUGACAC
  96
 6737
GUGUCACAAAAUACUCGAU
1363


 


 7093
UUUGAUGCAUCAAUAUCUC
  97
 7093
UUUGAUGCAUCAAUAUCUC
  97
 7111
GAGAUAUUGAUGCAUCAAA
1364


 


 7094
UUGAUGCAUCAAUAUCUCA
  98
 7094
UUGAUGCAUCAAUAUCUCA
  98
 7112
UGAGAUAUUGAUGCAUCAA
1365


 


 7095
UGAUGCAUCAAUAUCUCAA
  99
 7095
UGAUGCAUCAAUAUCUCAA
  99
 7113
UUGAGAUAUUGAUGCAUCA
1366


 


 7096
GAUGCAUCAAUAUCUCAAG
 100
 7096
GAUGCAUCAAUAUCUCAAG
 100
 7114
CUUGAGAUAUUGAUGCAUC
1367


 


 7097
AUGCAUCAAUAUCUCAAGU
 101
 7097
AUGCAUCAAUAUCUCAAGU
 101
 7115
ACUUGAGAUAUUGAUGCAU
1368


 


 7098
UGCAUCAAUAUCUCAAGUC
 102
 7098
UGCAUCAAUAUCUCAAGUC
 102
 7116
GACUUGAGAUAUUGAUGCA
1369


 


 7099
GCAUCAAUAUCUCAAGUCA
 103
 7099
GCAUCAAUAUCUCAAGUCA
 103
 7117
UGACUUGAGAUAUUGAUGC
1370


 


 7100
CAUCAAUAUCUCAAGUCAA
 104
 7100
CAUCAAUAUCUCAAGUCAA
 104
 7118
UUGACUUGAGAUAUUGAUG
1371


 


 7802
UAGUUGGAGUGCUAGAGAG
 105
 7802
UAGUUGGAGUGCUAGAGAG
 105
 7820
CUCUCUAGCACUCCAACUA
1372


 


 7803
AGUUGGAGUGCUAGAGAGU
 106
 7803
AGUUGGAGUGCUAGAGAGU
 106
 7821
ACUCUCUAGCACUCCAACU
1373


 


 7804
GUUGGAGUGCUAGAGAGUU
 107
 7804
GUUGGAGUGCUAGAGAGUU
 107
 7822
AACUCUCUAGCACUCCAAC
1374


 


 7805
UUGGAGUGCUAGAGAGUUA
 108
 7805
UUGGAGUGCUAGAGAGUUA
 108
 7823
UAACUCUCUAGCACUCCAA
1375


 


 7849
AAACAAUCAGCAUGUGUUG
 109
 7849
AAACAAUCAGCAUGUGUUG
 109
 7867
CAACACAUGCUGAUUGUUU
1376


 


 7850
AACAAUCAGCAUGUGUUGC
 110
 7850
AACAAUCAGCAUGUGUUGC
 110
 7868
GCAACACAUGCUGAUUGUU
1377


 


 7945
AAGAUAAGAGUGUACAAUA
 111
 7945
AAGAUAAGAGUGUACAAUA
 111
 7963
UAUUGUACACUCUUAUCUU
1378


 


 7946
AGAUAAGAGUGUACAAUAC
 112
 7946
AGAUAAGAGUGUACAAUAC
 112
 7964
GUAUUGUACACUCUUAUCU
1379


 


 7947
GAUAAGAGUGUACAAUACU
 113
 7947
GAUAAGAGUGUACAAUACU
 113
 7965
AGUAUUGUACACUCUUAUC
1380


 


 7948
AUAAGAGUGUACAAUACUG
 114
 7948
AUAAGAGUGUACAAUACUG
 114
 7966
CAGUAUUGUACACUCUUAU
1381


 


 7949
UAAGAGUGUACAAUACUGU
 115
 7949
UAAGAGUGUACAAUACUGU
 115
 7967
ACAGUAUUGUACACUCUUA
1382


 


 8382
UAUAUAUAUUAGUGUCAUA
 116
 8382
UAUAUAUAUUAGUGUCAUA
 116
 8400
UAUGACACUAAUAUAUAUA
1383


 


 8383
AUAUAUAUUAGUGUCAUAA
 117
 8383
AUAUAUAUUAGUGUCAUAA
 117
 8401
UUAUGACACUAAUAUAUAU
1384


 


 8459
UGGGACAAAAUGGAUCCCA
 118
 8459
UGGGACAAAAUGGAUCCCA
 118
 8477
UGGGAUCCAUUUUGUCCCA
1385


 


 8460
GGGACAAAAUGGAUCCCAU
 119
 8460
GGGACAAAAUGGAUCCCAU
 119
 8478
AUGGGAUCCAUUUUGUCCC
1386


 


 8461
GGACAAAAUGGAUCCCAUU
 120
 8461
GGACAAAAUGGAUCCCAUU
 120
 8479
AAUGGGAUCCAUUUUGUCC
1387


 


 8462
GACAAAAUGGAUCCCAUUA
 121
 8462
GACAAAAUGGAUCCCAUUA
 121
 8480
UAAUGGGAUCCAUUUUGUC
1388


 


 8463
ACAAAAUGGAUCCCAUUAU
 122
 8463
ACAAAAUGGAUCCCAUUAU
 122
 8481
AUAAUGGGAUCCAUUUUGU
1389


 


 8464
CAAAAUGGAUCCCAUUAUU
 123
 8464
CAAAAUGGAUCCCAUUAUU
 123
 8482
AAUAAUGGGAUCCAUUUUG
1390


 


 8465
AAAAUGGAUCCCAUUAUUA
 124
 8465
AAAAUGGAUCCCAUUAUUA
 124
 8483
UAAUAAUGGGAUCCAUUUU
1391


 


 8466
AAAUGGAUCCCAUUAUUAA
 125
 8466
AAAUGGAUCCCAUUAUUAA
 125
 8484
UUAAUAAUGGGAUCCAUUU
1392


 


 8467
AAUGGAUCCCAUUAUUAAU
 126
 8467
AAUGGAUCCCAUUAUUAAU
 126
 8485
AUUAAUAAUGGGAUCCAUU
1393


 


 8468
AUGGAUCCCAUUAUUAAUG
 127
 8468
AUGGAUCCCAUUAUUAAUG
 127
 8486
CAUUAAUAAUGGGAUCCAU
1394


 


 8469
UGGAUCCCAUUAUUAAUGG
 128
 8469
UGGAUCCCAUUAUUAAUGG
 128
 8487
CCAUUAAUAAUGGGAUCCA
1395


 


 8470
GGAUCCCAUUAUUAAUGGA
 129
 8470
GGAUCCCAUUAUUAAUGGA
 129
 8488
UCCAUUAAUAAUGGGAUCC
1396


 


 8471
GAUCCCAUUAUUAAUGGAA
 130
 8471
GAUCCCAUUAUUAAUGGAA
 130
 8489
UUCCAUUAAUAAUGGGAUC
1397


 


 8472
AUCCCAUUAUUAAUGGAAA
 131
 8472
AUCCCAUUAUUAAUGGAAA
 131
 8490
UUUCCAUUAAUAAUGGGAU
1398


 


 8606
AACUUAAUUAGUAGACAAA
 132
 8606
AACUUAAUUAGUAGACAAA
 132
 8624
UUUGUCUACUAAUUAAGUU
1399


 


 8729
CAGUCAUUACUUAUGACAU
 133
 8729
CAGUCAUUACUUAUGACAU
 133
 8747
AUGUCAUAAGUAAUGACUG
1400


 


 8730
AGUCAUUACUUAUGACAUA
 134
 8730
AGUCAUUACUUAUGACAUA
 134
 8748
UAUGUCAUAAGUAAUGACU
1401


 


 9020
AUCAAAACAACACUCUUGA
 135
 9020
AUCAAAACAACACUCUUGA
 135
 9038
UCAAGAGUGUUGUUUUGAU
1402


 


 9021
UCAAAACAACACUCUUGAA
 136
 9021
UCAAAACAACACUCUUGAA
 136
 9039
UUCAAGAGUGUUGUUUUGA
1403


 


 9062
CAUCCUCCAUCAUGGUUAA
 137
 9062
CAUCCUCCAUCAUGGUUAA
 137
 9080
UUAACCAUGAUGGAGGAUG
1404


 


 9063
AUCCUCCAUCAUGGUUAAU
 138
 9063
AUCCUCCAUCAUGGUUAAU
 138
 9081
AUUAACCAUGAUGGAGGAU
1405


 


 9064
UCCUCCAUCAUGGUUAAUA
 139
 9064
UCCUCCAUCAUGGUUAAUA
 139
 9082
UAUUAACCAUGAUGGAGGA
1406


 


 9065
CCUCCAUCAUGGUUAAUAC
 140
 9065
CCUCCAUCAUGGUUAAUAC
 140
 9083
GUAUUAACCAUGAUGGAGG
1407


 


 9066
CUCCAUCAUGGUUAAUACA
 141
 9066
CUCCAUCAUGGUUAAUACA
 141
 9084
UGUAUUAACCAUGAUGGAG
1408


 


 9347
ACAUUAAAUAAAAGCUUAG
 142
 9347
ACAUUAAAUAAAAGCUUAG
 142
 9365
CUAAGCUUUUAUUUAAUGU
1409


 


 9348
CAUUAAAUAAAAGCUUAGG
 143
 9348
CAUUAAAUAAAAGCUUAGG
 143
 9366
CCUAAGCUUUUAUUUAAUG
1410


 


 9470
GUAGAGGGAUUUAUUAUGU
 144
 9470
GUAGAGGGAUUUAUUAUGU
 144
 9488
ACAUAAUAAAUCCCUCUAC
1411


 


 9471
UAGAGGGAUUUAUUAUGUC
 145
 9471
UAGAGGGAUUUAUUAUGUC
 145
 9489
GACAUAAUAAAUCCCUCUA
1412


 


 9472
AGAGGGAUUUAUUAUGUCU
 146
 9472
AGAGGGAUUUAUUAUGUCU
 146
 9490
AGACAUAAUAAAUCCCUCU
1413


 


 9503
AUAACAGAAGAAGAUCAAU
 147
 9503
AUAACAGAAGAAGAUCAAU
 147
 9521
AUUGAUCUUCUUCUGUUAU
1414


 


 9504
UAACAGAAGAAGAUCAAUU
 148
 9504
UAACAGAAGAAGAUCAAUU
 148
 9522
AAUUGAUCUUCUUCUGUUA
1415


 


10047
UGCCUAAAAAAGUGGAUCU
 149
10047
UGCCUAAAAAAGUGGAUCU
 149
10065
AGAUCCACUUUUUUAGGCA
1416


 


10048
GCCUAAAAAAGUGGAUCUU
 150
10048
GCCUAAAAAAGUGGAUCUU
 150
10066
AAGAUCCACUUUUUUAGGC
1417


 


10049
CCUAAAAAAGUGGAUCUUG
 151
10049
CCUAAAAAAGUGGAUCUUG
 151
10067
CAAGAUCCACUUUUUUAGG
1418


 


10050
CUAAAAAAGUGGAUCUUGA
 152
10050
CUAAAAAAGUGGAUCUUGA
 152
10068
UCAAGAUCCACUUUUUUAG
1419


 


10051
UAAAAAAGUGGAUCUUGAA
 153
10051
UAAAAAAGUGGAUCUUGAA
 153
10069
UUCAAGAUCCACUUUUUUA
1420


 


10052
AAAAAAGUGGAUCUUGAAA
 154
10052
AAAAAAGUGGAUCUUGAAA
 154
10070
UUUCAAGAUCCACUUUUUU
1421


 


10053
AAAAAGUGGAUCUUGAAAU
 155
10053
AAAAAGUGGAUCUUGAAAU
 155
10071
AUUUCAAGAUCCACUUUUU
1422


 


10054
AAAAGUGGAUCUUGAAAUG
 156
10054
AAAAGUGGAUCUUGAAAUG
 156
10072
CAUUUCAAGAUCCACUUUU
1423


 


10055
AAAGUGGAUCUUGAAAUGA
 157
10055
AAAGUGGAUCUUGAAAUGA
 157
10073
UCAUUUCAAGAUCCACUUU
1424


 


10056
AAGUGGAUCUUGAAAUGAU
 158
10056
AAGUGGAUCUUGAAAUGAU
 158
10074
AUCAUUUCAAGAUCCACUU
1425


 


10334
CUCAGUGUAGGUAGAAUGU
 159
10334
CUCAGUGUAGGUAGAAUGU
 159
10352
ACAUUCUACCUACACUGAG
1426


 


10335
UCAGUGUAGGUAGAAUGUU
 160
10335
UCAGUGUAGGUAGAAUGUU
 160
10353
AACAUUCUACCUACACUGA
1427


 


10336
CAGUGUAGGUAGAAUGUUU
 161
10336
CAGUGUAGGUAGAAUGUUU
 161
10354
AAACAUUCUACCUACACUG
1428


 


10337
AGUGUAGGUAGAAUGUUUG
 162
10337
AGUGUAGGUAGAAUGUUUG
 162
10355
CAAACAUUCUACCUACACU
1429


 


10338
GUGUAGGUAGAAUGUUUGC
 163
10338
GUGUAGGUAGAAUGUUUGC
 163
10356
GCAAACAUUCUACCUACAC
1430


 


10445
ACAAGAUAUGGUGAUCUAG
 164
10445
ACAAGAUAUGGUGAUCUAG
 164
10463
CUAGAUCACCAUAUCUUGU
1431


 


10446
CAAGAUAUGGUGAUCUAGA
 165
10446
CAAGAUAUGGUGAUCUAGA
 165
10464
UCUAGAUCACCAUAUCUUG
1432


 


10571
AGCAAAUUCAAUCAAGCAU
 166
10571
AGCAAAUUCAAUCAAGCAU
 166
10589
AUGCUUGAUUGAAUUUGCU
1433


 


10572
GCAAAUUCAAUCAAGCAUU
 167
10572
GCAAAUUCAAUCAAGCAUU
 167
10590
AAUGCUUGAUUGAAUUUGC
1434


 


10573
CAAAUUCAAUCAAGCAUUU
 168
10573
CAAAUUCAAUCAAGCAUUU
 168
10591
AAAUGCUUGAUUGAAUUUG
1435


 


10757
GAUGAACAAAGUGGAUUAU
 169
10757
GAUGAACAAAGUGGAUUAU
 169
10775
AUAAUCCACUUUGUUCAUC
1436


 


10758
AUGAACAAAGUGGAUUAUA
 170
10758
AUGAACAAAGUGGAUUAUA
 170
10776
UAUAAUCCACUUUGUUCAU
1437


 


11232
UAUUAUGCAGUUUAAUAUU
 171
11232
UAUUAUGCAGUUUAAUAUU
 171
11250
AAUAUUAAACUGCAUAAUA
1438


 


11233
AUUAUGCAGUUUAAUAUUU
 172
11233
AUUAUGCAGUUUAAUAUUU
 172
11251
AAAUAUUAAACUGCAUAAU
1439


 


11234
UUAUGCAGUUUAAUAUUUA
 173
11234
UUAUGCAGUUUAAUAUUUA
 173
11252
UAAAUAUUAAACUGCAUAA
1440


 


11235
UAUGCAGUUUAAUAUUUAG
 174
11235
UAUGCAGUUUAAUAUUUAG
 174
11253
CUAAAUAUUAAACUGCAUA
1441


 


11795
AUUAUGCAAAAUAUAGAAC
 175
11795
AUUAUGCAAAAUAUAGAAC
 175
11813
GUUCUAUAUUUUGCAUAAU
1442


 


11796
UUAUGCAAAAUAUAGAACC
 176
11796
UUAUGCAAAAUAUAGAACC
 176
11814
GGUUCUAUAUUUUGCAUAA
1443


 


11832
UAAGAGUUGUUUAUGAAAG
 177
11832
UAAGAGUUGUUUAUGAAAG
 177
11850
CUUUCAUAAACAACUCUUA
1444


 


11833
AAGAGUUGUUUAUGAAAGU
 178
11833
AAGAGUUGUUUAUGAAAGU
 178
11851
ACUUUCAUAAACAACUCUU
1445


 


12272
GAGAAAAAAACAAUGCCAG
 179
12272
GAGAAAAAAACAAUGCCAG
 179
12290
CUGGCAUUGUUUUUUUCUC
1446


 


12273
AGAAAAAAACAAUGCCAGU
 180
12273
AGAAAAAAACAAUGCCAGU
 180
12291
ACUGGCAUUGUUUUUUUCU
1447


 


12383
GAUGAAUUCAUGGAAGAAC
 181
12383
GAUGAAUUCAUGGAAGAAC
 181
12401
GUUCUUCCAUGAAUUCAUC
1448


 


12384
AUGAAUUCAUGGAAGAACU
 182
12384
AUGAAUUCAUGGAAGAACU
 182
12402
AGUUCUUCCAUGAAUUCAU
1449


 


12503
CCAUGUGAAUUCCCUGCAU
 183
12503
CCAUGUGAAUUCCCUGCAU
 183
12521
AUGCAGGGAAUUCACAUGG
1450


 


12504
CAUGUGAAUUCCCUGCAUC
 184
12504
CAUGUGAAUUCCCUGCAUC
 184
12522
GAUGCAGGGAAUUCACAUG
1451


 


12505
AUGUGAAUUCCCUGCAUCA
 185
12505
AUGUGAAUUCCCUGCAUCA
 185
12523
UGAUGCAGGGAAUUCACAU
1452


 


12506
UGUGAAUUCCCUGCAUCAA
 186
12506
UGUGAAUUCCCUGCAUCAA
 186
12524
UUGAUGCAGGGAAUUCACA
1453


 


12507
GUGAAUUCCCUGCAUCAAU
 187
12507
GUGAAUUCCCUGCAUCAAU
 187
12525
AUUGAUGCAGGGAAUUCAC
1454


 


12508
UGAAUUCCCUGCAUCAAUA
 188
12508
UGAAUUCCCUGCAUCAAUA
 188
12526
UAUUGAUGCAGGGAAUUCA
1455


 


12509
GAAUUCCCUGCAUCAAUAC
 189
12509
GAAUUCCCUGCAUCAAUAC
 189
12527
GUAUUGAUGCAGGGAAUUC
1456


 


12510
AAUUCCCUGCAUCAAUACC
 190
12510
AAUUCCCUGCAUCAAUACC
 190
12528
GGUAUUGAUGCAGGGAAUU
1457


 


12511
AUUCCCUGCAUCAAUACCA
 191
12511
AUUCCCUGCAUCAAUACCA
 191
12529
UGGUAUUGAUGCAGGGAAU
1458


 


12512
UUCCCUGCAUCAAUACCAG
 192
12512
UUCCCUGCAUCAAUACCAG
 192
12530
CUGGUAUUGAUGCAGGGAA
1459


 


12513
UCCCUGCAUCAAUACCAGC
 193
12513
UCCCUGCAUCAAUACCAGC
 193
12531
GCUGGUAUUGAUGCAGGGA
1460


 


12514
CCCUGCAUCAAUACCAGCU
 194
12514
CCCUGCAUCAAUACCAGCU
 194
12532
AGCUGGUAUUGAUGCAGGG
1461


 


12515
CCUGCAUCAAUACCAGCUU
 195
12515
CCUGCAUCAAUACCAGCUU
 195
12533
AAGCUGGUAUUGAUGCAGG
1462


 


12516
CUGCAUCAAUACCAGCUUA
 196
12516
CUGCAUCAAUACCAGCUUA
 196
12534
UAAGCUGGUAUUGAUGCAG
1463


 


12517
UGCAUCAAUACCAGCUUAU
 197
12517
UGCAUCAAUACCAGCUUAU
 197
12535
AUAAGCUGGUAUUGAUGCA
1464


 


12518
GCAUCAAUACCAGCUUAUA
 198
12518
GCAUCAAUACCAGCUUAUA
 198
12536
UAUAAGCUGGUAUUGAUGC
1465


 


12519
CAUCAAUACCAGCUUAUAG
 199
12519
CAUCAAUACCAGCUUAUAG
 199
12537
CUAUAAGCUGGUAUUGAUG
1466


 


12520
AUCAAUACCAGCUUAUAGA
 200
12520
AUCAAUACCAGCUUAUAGA
 200
12538
UCUAUAAGCUGGUAUUGAU
1467


 


12521
UCAAUACCAGCUUAUAGAA
 201
12521
UCAAUACCAGCUUAUAGAA
 201
12539
UUCUAUAAGCUGGUAUUGA
1468


 


12522
CAAUACCAGCUUAUAGAAC
 202
12522
CAAUACCAGCUUAUAGAAC
 202
12540
GUUCUAUAAGCUGGUAUUG
1469


 


12579
UAUUAACAGAAAAGUAUGG
 203
12579
UAUUAACAGAAAAGUAUGG
 203
12597
CCAUACUUUUCUGUUAAUA
1470


 


12779
CAAGUGAUACAAAAACAGC
 204
12779
CAAGUGAUACAAAAACAGC
 204
12797
GCUGUUUUUGUAUCACUUG
1471


 


12780
AAGUGAUACAAAAACAGCA
 205
12780
AAGUGAUACAAAAACAGCA
 205
12798
UGCUGUUUUUGUAUCACUU
1472


 


12938
AUUUUAAGUACUAAUUUAG
 206
12938
AUUUUAAGUACUAAUUUAG
 206
12956
CUAAAUUAGUACUUAAAAU
1473


 


12939
UUUUAAGUACUAAUUUAGC
 207
12939
UUUUAAGUACUAAUUUAGC
 207
12957
GCUAAAUUAGUACUUAAAA
1474


 


12940
UUUAAGUACUAAUUUAGCU
 208
12940
UUUAAGUACUAAUUUAGCU
 208
12958
AGCUAAAUUAGUACUUAAA
1475


 


12941
UUAAGUACUAAUUUAGCUG
 209
12941
UUAAGUACUAAUUUAGCUG
 209
12959
CAGCUAAAUUAGUACUUAA
1476


 


12942
UAAGUACUAAUUUAGCUGG
 210
12942
UAAGUACUAAUUUAGCUGG
 210
12960
CCAGCUAAAUUAGUACUUA
1477


 


12943
AAGUACUAAUUUAGCUGGA
 211
12943
AAGUACUAAUUUAGCUGGA
 211
12961
UCCAGCUAAAUUAGUACUU
1478


 


12944
AGUACUAAUUUAGCUGGAC
 212
12944
AGUACUAAUUUAGCUGGAC
 212
12962
GUCCAGCUAAAUUAGUACU
1479


 


12945
GUACUAAUUUAGCUGGACA
 213
12945
GUACUAAUUUAGCUGGACA
 213
12963
UGUCCAGCUAAAUUAGUAC
1480


 


12946
UACUAAUUUAGCUGGACAU
 214
12946
UACUAAUUUAGCUGGACAU
 214
12964
AUGUCCAGCUAAAUUAGUA
1481


 


12947
ACUAAUUUAGCUGGACAUU
 215
12947
ACUAAUUUAGCUGGACAUU
 215
12965
AAUGUCCAGCUAAAUUAGU
1482


 


12948
CUAAUUUAGCUGGACAUUG
 216
12948
CUAAUUUAGCUGGACAUUG
 216
12966
CAAUGUCCAGCUAAAUUAG
1483


 


12949
UAAUUUAGCUGGACAUUGG
 217
12949
UAAUUUAGCUGGACAUUGG
 217
12967
CCAAUGUCCAGCUAAAUUA
1484


 


12950
AAUUUAGCUGGACAUUGGA
 218
12950
AAUUUAGCUGGACAUUGGA
 218
12968
UCCAAUGUCCAGCUAAAUU
1485


 


12951
AUUUAGCUGGACAUUGGAU
 219
12951
AUUUAGCUGGACAUUGGAU
 219
12969
AUCCAAUGUCCAGCUAAAU
1486


 


12952
UUUAGCUGGACAUUGGAUU
 220
12952
UUUAGCUGGACAUUGGAUU
 220
12970
AAUCCAAUGUCCAGCUAAA
1487


 


12953
UUAGCUGGACAUUGGAUUC
 221
12953
UUAGCUGGACAUUGGAUUC
 221
12971
GAAUCCAAUGUCCAGCUAA
1488


 


12954
UAGCUGGACAUUGGAUUCU
 222
12954
UAGCUGGACAUUGGAUUCU
 222
12972
AGAAUCCAAUGUCCAGCUA
1489


 


13001
GGUAUUUUUGAAAAAGAUU
 223
13001
GGUAUUUUUGAAAAAGAUU
 223
13019
AAUCUUUUUCAAAAAUACC
1490


 


13002
GUAUUUUUGAAAAAGAUUG
 224
13002
GUAUUUUUGAAAAAGAUUG
 224
13020
CAAUCUUUUUCAAAAAUAC
1491


 


13003
UAUUUUUGAAAAAGAUUGG
 225
13003
UAUUUUUGAAAAAGAUUGG
 225
13021
CCAAUCUUUUUCAAAAAUA
1492


 


13004
AUUUUUGAAAAAGAUUGGG
 226
13004
AUUUUUGAAAAAGAUUGGG
 226
13022
CCCAAUCUUUUUCAAAAAU
1493


 


13005
UUUUUGAAAAAGAUUGGGG
 227
13005
UUUUUGAAAAAGAUUGGGG
 227
13023
CCCCAAUCUUUUUCAAAAA
1494


 


13006
UUUUGAAAAAGAUUGGGGA
 228
13006
UUUUGAAAAAGAUUGGGGA
 228
13024
UCCCCAAUCUUUUUCAAAA
1495


 


13007
UUUGAAAAAGAUUGGGGAG
 229
13007
UUUGAAAAAGAUUGGGGAG
 229
13025
CUCCCCAAUCUUUUUCAAA
1496


 


13008
UUGAAAAAGAUUGGGGAGA
 230
13008
UUGAAAAAGAUUGGGGAGA
 230
13026
UCUCCCCAAUCUUUUUCAA
1497


 


13009
UGAAAAAGAUUGGGGAGAG
 231
13009
UGAAAAAGAUUGGGGAGAG
 231
13027
CUCUCCCCAAUCUUUUUCA
1498


 


13010
GAAAAAGAUUGGGGAGAGG
 232
13010
GAAAAAGAUUGGGGAGAGG
 232
13028
CCUCUCCCCAAUCUUUUUC
1499


 


13011
AAAAAGAUUGGGGAGAGGG
 233
13011
AAAAAGAUUGGGGAGAGGG
 233
13029
CCCUCUCCCCAAUCUUUUU
1500


 


13334
UGCCCUUGGGUUGUUAACA
 234
13334
UGCCCUUGGGUUGUUAACA
 234
13352
UGUUAACAACCCAAGGGCA
1501


 


13335
GCCCUUGGGUUGUUAACAU
 235
13335
GCCCUUGGGUUGUUAACAU
 235
13353
AUGUUAACAACCCAAGGGC
1502


 


13336
CCCUUGGGUUGUUAACAUA
 236
13336
CCCUUGGGUUGUUAACAUA
 236
13354
UAUGUUAACAACCCAAGGG
1503


 


13337
CCUUGGGUUGUUAACAUAG
 237
13337
CCUUGGGUUGUUAACAUAG
 237
13355
CUAUGUUAACAACCCAAGG
1504


 


13338
CUUGGGUUGUUAACAUAGA
 238
13338
CUUGGGUUGUUAACAUAGA
 238
13356
UCUAUGUUAACAACCCAAG
1505


 


13339
UUGGGUUGUUAACAUAGAU
 239
13339
UUGGGUUGUUAACAUAGAU
 239
13357
AUCUAUGUUAACAACCCAA
1506


 


13340
UGGGUUGUUAACAUAGAUU
 240
13340
UGGGUUGUUAACAUAGAUU
 240
13358
AAUCUAUGUUAACAACCCA
1507


 


13341
GGGUUGUUAACAUAGAUUA
 241
13341
GGGUUGUUAACAUAGAUUA
 241
13359
UAAUCUAUGUUAACAACCC
1508


 


13342
GGUUGUUAACAUAGAUUAU
 242
13342
GGUUGUUAACAUAGAUUAU
 242
13360
AUAAUCUAUGUUAACAACC
1509


 


13343
GUUGUUAACAUAGAUUAUC
 243
13343
GUUGUUAACAUAGAUUAUC
 243
13361
GAUAAUCUAUGUUAACAAC
1510


 


13344
UUGUUAACAUAGAUUAUCA
 244
13344
UUGUUAACAUAGAUUAUCA
 244
13362
UGAUAAUCUAUGUUAACAA
1511


 


13452
ACAAAUUCAAUGAUGAAUU
 245
13452
ACAAAUUCAAUGAUGAAUU
 245
13470
AAUUCAUCAUUGAAUUUGU
1512


 


13453
CAAAUUCAAUGAUGAAUUU
 246
13453
CAAAUUCAAUGAUGAAUUU
 246
13471
AAAUUCAUCAUUGAAUUUG
1513


 


13454
AAAUUCAAUGAUGAAUUUU
 247
13454
AAAUUCAAUGAUGAAUUUU
 247
13472
AAAAUUCAUCAUUGAAUUU
1514


 


13455
AAUUCAAUGAUGAAUUUUA
 248
13455
AAUUCAAUGAUGAAUUUUA
 248
13473
UAAAAUUCAUCAUUGAAUU
1515


 


13898
UGCAUGCUUCCUUGGCAUC
 249
13898
UGCAUGCUUCCUUGGCAUC
 249
13916
GAUGCCAAGGAAGCAUGCA
1516


 


13899
GCAUGCUUCCUUGGCAUCA
 250
13899
GCAUGCUUCCUUGGCAUCA
 250
13917
UGAUGCCAAGGAAGCAUGC
1517


 


13900
CAUGCUUCCUUGGCAUCAU
 251
13900
CAUGCUUCCUUGGCAUCAU
 251
13918
AUGAUGCCAAGGAAGCAUG
1518


 


13964
AGUAUAGAGUAUAUUUUAA
 252
13964
AGUAUAGAGUAUAUUUUAA
 252
13982
UUAAAAUAUACUCUAUACU
1519


 


13965
GUAUAGAGUAUAUUUUAAA
 253
13965
GUAUAGAGUAUAUUUUAAA
 253
13983
UUUAAAAUAUACUCUAUAC
1520


 


13966
UAUAGAGUAUAUUUUAAAA
 254
13966
UAUAGAGUAUAUUUUAAAA
 254
13984
UUUUAAAAUAUACUCUAUA
1521


 


13967
AUAGAGUAUAUUUUAAAAG
 255
13967
AUAGAGUAUAUUUUAAAAG
 255
13985
CUUUUAAAAUAUACUCUAU
1522


 


13968
UAGAGUAUAUUUUAAAAGA
 256
13968
UAGAGUAUAUUUUAAAAGA
 256
13986
UCUUUUAAAAUAUACUCUA
1523


 


14008
UUGUAUAGCAUUCAUAGGU
 257
14008
UUGUAUAGCAUUCAUAGGU
 257
14026
ACCUAUGAAUGCUAUACAA
1524


 


14009
UGUAUAGCAUUCAUAGGUG
 258
14009
UGUAUAGCAUUCAUAGGUG
 258
14027
CACCUAUGAAUGCUAUACA
1525


 


14010
GUAUAGCAUUCAUAGGUGA
 259
14010
GUAUAGCAUUCAUAGGUGA
 259
14028
UCACCUAUGAAUGCUAUAC
1526


 


14011
UAUAGCAUUCAUAGGUGAA
 260
14011
UAUAGCAUUCAUAGGUGAA
 260
14029
UUCACCUAUGAAUGCUAUA
1527


 


14012
AUAGCAUUCAUAGGUGAAG
 261
14012
AUAGCAUUCAUAGGUGAAG
 261
14030
CUUCACCUAUGAAUGCUAU
1528


 


14013
UAGCAUUCAUAGGUGAAGG
 262
14013
UAGCAUUCAUAGGUGAAGG
 262
14031
CCUUCACCUAUGAAUGCUA
1529


 


14014
AGCAUUCAUAGGUGAAGGA
 263
14014
AGCAUUCAUAGGUGAAGGA
 263
14032
UCCUUCACCUAUGAAUGCU
1530


 


14015
GCAUUCAUAGGUGAAGGAG
 264
14015
GCAUUCAUAGGUGAAGGAG
 264
14033
CUCCUUCACCUAUGAAUGC
1531


 


14016
CAUUCAUAGGUGAAGGAGC
 265
14016
CAUUCAUAGGUGAAGGAGC
 265
14034
GCUCCUUCACCUAUGAAUG
1532


 


14186
ACCAUUCCUGCUACAGAUG
 266
14186
ACCAUUCCUGCUACAGAUG
 266
14204
CAUCUGUAGCAGGAAUGGU
1533


 


14187
CCAUUCCUGCUACAGAUGC
 267
14187
CCAUUCCUGCUACAGAUGC
 267
14205
GCAUCUGUAGCAGGAAUGG
1534


 


14188
CAUUCCUGCUACAGAUGCA
 268
14188
CAUUCCUGCUACAGAUGCA
 268
14206
UGCAUCUGUAGCAGGAAUG
1535


 


14189
AUUCCUGCUACAGAUGCAA
 269
14189
AUUCCUGCUACAGAUGCAA
 269
14207
UUGCAUCUGUAGCAGGAAU
1536


 


14190
UUCCUGCUACAGAUGCAAC
 270
14190
UUCCUGCUACAGAUGCAAC
 270
14208
GUUGCAUCUGUAGCAGGAA
1537


 


14213
AACAUUCAUUGGUCUUAUU
 271
14213
AACAUUCAUUGGUCUUAUU
 271
14231
AAUAAGACCAAUGAAUGUU
1538


 


14214
ACAUUCAUUGGUCUUAUUU
 272
14214
ACAUUCAUUGGUCUUAUUU
 272
14232
AAAUAAGACCAAUGAAUGU
1539


 


14215
CAUUCAUUGGUCUUAUUUA
 273
14215
CAUUCAUUGGUCUUAUUUA
 273
14233
UAAAUAAGACCAAUGAAUG
1540


 


14216
AUUCAUUGGUCUUAUUUAC
 274
14216
AUUCAUUGGUCUUAUUUAC
 274
14234
GUAAAUAAGACCAAUGAAU
1541


 


14217
UUCAUUGGUCUUAUUUACA
 275
14217
UUCAUUGGUCUUAUUUACA
 275
14235
UGUAAAUAAGACCAAUGAA
1542


 


14218
UCAUUGGUCUUAUUUACAU
 276
14218
UCAUUGGUCUUAUUUACAU
 276
14236
AUGUAAAUAAGACCAAUGA
1543


 


14219
CAUUGGUCUUAUUUACAUA
 277
14219
CAUUGGUCUUAUUUACAUA
 277
14237
UAUGUAAAUAAGACCAAUG
1544


 


14220
AUUGGUCUUAUUUACAUAU
 278
14220
AUUGGUCUUAUUUACAUAU
 278
14238
AUAUGUAAAUAAGACCAAU
1545


 


14221
UUGGUCUUAUUUACAUAUA
 279
14221
UUGGUCUUAUUUACAUAUA
 279
14239
UAUAUGUAAAUAAGACCAA
1546


 


14222
UGGUCUUAUUUACAUAUAA
 280
14222
UGGUCUUAUUUACAUAUAA
 280
14240
UUAUAUGUAAAUAAGACCA
1547


 


14223
GGUCUUAUUUACAUAUAAA
 281
14223
GGUCUUAUUUACAUAUAAA
 281
14241
UUUAUAUGUAAAUAAGACC
1548


 


14383
AAAAUAUCAUGCUCAAGAU
 282
14383
AAAAUAUCAUGCUCAAGAU
 282
14401
AUCUUGAGCAUGAUAUUUU
1549


 


14384
AAAUAUCAUGCUCAAGAUG
 283
14384
AAAUAUCAUGCUCAAGAUG
 283
14402
CAUCUUGAGCAUGAUAUUU
1550


 


14385
AAUAUCAUGCUCAAGAUGA
 284
14385
AAUAUCAUGCUCAAGAUGA
 284
14403
UCAUCUUGAGCAUGAUAUU
1551


 


14386
AUAUCAUGCUCAAGAUGAU
 285
14386
AUAUCAUGCUCAAGAUGAU
 285
14404
AUCAUCUUGAGCAUGAUAU
1552


 


14387
UAUCAUGCUCAAGAUGAUA
 286
14387
UAUCAUGCUCAAGAUGAUA
 286
14405
UAUCAUCUUGAGCAUGAUA
1553


 


14388
AUCAUGCUCAAGAUGAUAU
 287
14388
AUCAUGCUCAAGAUGAUAU
 287
14406
AUAUCAUCUUGAGCAUGAU
1554


 


14480
GUUUACUUAGUCCUUACAA
 288
14480
GUUUACUUAGUCCUUACAA
 288
14498
UUGUAAGGACUAAGUAAAC
1555


 


14481
UUUACUUAGUCCUUACAAU
 289
14481
UUUACUUAGUCCUUACAAU
 289
14499
AUUGUAAGGACUAAGUAAA
1556


 


14482
UUACUUAGUCCUUACAAUA
 290
14482
UUACUUAGUCCUUACAAUA
 290
14500
UAUUGUAAGGACUAAGUAA
1557


 


14483
UACUUAGUCCUUACAAUAG
 291
14483
UACUUAGUCCUUACAAUAG
 291
14501
CUAUUGUAAGGACUAAGUA
1558


 


14484
ACUUAGUCCUUACAAUAGG
 292
14484
ACUUAGUCCUUACAAUAGG
 292
14502
CCUAUUGUAAGGACUAAGU
1559


 


14639
CUUUGUUACCCUAUAACAA
 293
14639
CUUUGUUACCCUAUAACAA
 293
14657
UUGUUAUAGGGUAACAAAG
1560


 


14640
UUUGUUACCCUAUAACAAA
 294
14640
UUUGUUACCCUAUAACAAA
 294
14658
UUUGUUAUAGGGUAACAAA
1561


 


14641
UUGUUACCCUAUAACAAAA
 295
14641
UUGUUACCCUAUAACAAAA
 295
14659
UUUUGUUAUAGGGUAACAA
1562


 


14642
UGUUACCCUAUAACAAAAA
 296
14642
UGUUACCCUAUAACAAAAA
 296
14660
UUUUUGUUAUAGGGUAACA
1563


 


14643
GUUACCCUAUAACAAAAAA
 297
14643
GUUACCCUAUAACAAAAAA
 297
14661
UUUUUUGUUAUAGGGUAAC
1564


 


14644
UUACCCUAUAACAAAAAAA
 298
14644
UUACCCUAUAACAAAAAAA
 298
14662
UUUUUUUGUUAUAGGGUAA
1565


 


14645
UACCCUAUAACAAAAAAAG
 299
14645
UACCCUAUAACAAAAAAAG
 299
14663
CUUUUUUUGUUAUAGGGUA
1566


 


14646
ACCCUAUAACAAAAAAAGG
 300
14646
ACCCUAUAACAAAAAAAGG
 300
14664
CCUUUUUUUGUUAUAGGGU
1567


 


14647
CCCUAUAACAAAAAAAGGA
 301
14647
CCCUAUAACAAAAAAAGGA
 301
14665
UCCUUUUUUUGUUAUAGGG
1568


 


14648
CCUAUAACAAAAAAAGGAA
 302
14648
CCUAUAACAAAAAAAGGAA
 302
14666
UUCCUUUUUUUGUUAUAGG
1569


 


14649
CUAUAACAAAAAAAGGAAU
 303
14649
CUAUAACAAAAAAAGGAAU
 303
14667
AUUCCUUUUUUUGUUAUAG
1570


 


14650
UAUAACAAAAAAAGGAAUU
 304
14650
UAUAACAAAAAAAGGAAUU
 304
14668
AAUUCCUUUUUUUGUUAUA
1571


 


14651
AUAACAAAAAAAGGAAUUA
 305
14651
AUAACAAAAAAAGGAAUUA
 305
14669
UAAUUCCUUUUUUUGUUAU
1572


 


14652
UAACAAAAAAAGGAAUUAA
 306
14652
UAACAAAAAAAGGAAUUAA
 306
14670
UUAAUUCCUUUUUUUGUUA
1573


 


15165
AUUAGUUUUUGACACUUUU
 307
15165
AUUAGUUUUUGACACUUUU
 307
15183
AAAAGUGUCAAAAACUAAU
1574


 


15166
UUAGUUUUUGACACUUUUU
 308
15166
UUAGUUUUUGACACUUUUU
 308
15184
AAAAAGUGUCAAAAACUAA
1575


 


15167
UAGUUUUUGACACUUUUUU
 309
15167
UAGUUUUUGACACUUUUUU
 309
15185
AAAAAAGUGUCAAAAACUA
1576


 


15168
AGUUUUUGACACUUUUUUU
 310
15168
AGUUUUUGACACUUUUUUU
 310
15186
AAAAAAAGUGUCAAAAACU
1577


 


15169
GUUUUUGACACUUUUUUUC
 311
15169
GUUUUUGACACUUUUUUUC
 311
15187
GAAAAAAAGUGUCAAAAAC
1578


 


15170
UUUUUGACACUUUUUUUCU
 312
15170
UUUUUGACACUUUUUUUCU
 312
15188
AGAAAAAAAGUGUCAAAAA
1579


 


15171
UUUUGACACUUUUUUUCUC
 313
15171
UUUUGACACUUUUUUUCUC
 313
15189
GAGAAAAAAAGUGUCAAAA
1580


 


 1520
UGAGAUAGAAUCUAGAAAA
 314
 1520
UGAGAUAGAAUCUAGAAAA
 314
 1538
UUUUCUAGAUUCUAUCUCA
1581


 


 1521
GAGAUAGAAUCUAGAAAAU
 315
 1521
GAGAUAGAAUCUAGAAAAU
 315
 1539
AUUUUCUAGAUUCUAUCUC
1582


 


 1522
AGAUAGAAUCUAGAAAAUC
 316
 1522
AGAUAGAAUCUAGAAAAUC
 316
 1540
GAUUUUCUAGAUUCUAUCU
1583


 


 1523
GAUAGAAUCUAGAAAAUCC
 317
 1523
GAUAGAAUCUAGAAAAUCC
 317
 1541
GGAUUUUCUAGAUUCUAUC
1584


 


 1524
AUAGAAUCUAGAAAAUCCU
 318
 1524
AUAGAAUCUAGAAAAUCCU
 318
 1542
AGGAUUUUCUAGAUUCUAU
1585


 


 1525
UAGAAUCUAGAAAAUCCUA
 319
 1525
UAGAAUCUAGAAAAUCCUA
 319
 1543
UAGGAUUUUCUAGAUUCUA
1586


 


 1526
AGAAUCUAGAAAAUCCUAC
 320
 1526
AGAAUCUAGAAAAUCCUAC
 320
 1544
GUAGGAUUUUCUAGAUUCU
1587


 


 1527
GAAUCUAGAAAAUCCUACA
 321
 1527
GAAUCUAGAAAAUCCUACA
 321
 1545
UGUAGGAUUUUCUAGAUUC
1588


 


 1528
AAUCUAGAAAAUCCUACAA
 322
 1528
AAUCUAGAAAAUCCUACAA
 322
 1546
UUGUAGGAUUUUCUAGAUU
1589


 


 1529
AUCUAGAAAAUCCUACAAA
 323
 1529
AUCUAGAAAAUCCUACAAA
 323
 1547
UUUGUAGGAUUUUCUAGAU
1590


 


 1530
UCUAGAAAAUCCUACAAAA
 324
 1530
UCUAGAAAAUCCUACAAAA
 324
 1548
UUUUGUAGGAUUUUCUAGA
1591


 


 1531
CUAGAAAAUCCUACAAAAA
 325
 1531
CUAGAAAAUCCUACAAAAA
 325
 1549
UUUUUGUAGGAUUUUCUAG
1592


 


 1532
UAGAAAAUCCUACAAAAAA
 326
 1532
UAGAAAAUCCUACAAAAAA
 326
 1550
UUUUUUGUAGGAUUUUCUA
1593


 


 1533
AGAAAAUCCUACAAAAAAA
 327
 1533
AGAAAAUCCUACAAAAAAA
 327
 1551
UUUUUUUGUAGGAUUUUCU
1594


 


 1534
GAAAAUCCUACAAAAAAAU
 328
 1534
GAAAAUCCUACAAAAAAAU
 328
 1552
AUUUUUUUGUAGGAUUUUC
1595


 


 1535
AAAAUCCUACAAAAAAAUG
 329
 1535
AAAAUCCUACAAAAAAAUG
 329
 1553
CAUUUUUUUGUAGGAUUUU
1596


 


 1536
AAAUCCUACAAAAAAAUGC
 330
 1536
AAAUCCUACAAAAAAAUGC
 330
 1554
GCAUUUUUUUGUAGGAUUU
1597


 


 1537
AAUCCUACAAAAAAAUGCU
 331
 1537
AAUCCUACAAAAAAAUGCU
 331
 1555
AGCAUUUUUUUGUAGGAUU
1598


 


 1538
AUCCUACAAAAAAAUGCUA
 332
 1538
AUCCUACAAAAAAAUGCUA
 332
 1556
UAGCAUUUUUUUGUAGGAU
1599


 


 1872
GGAUUGUUUAUGAAUGCCU
 333
 1872
GGAUUGUUUAUGAAUGCCU
 333
 1890
AGGCAUUCAUAAACAAUCC
1600


 


 1873
GAUUGUUUAUGAAUGCCUA
 334
 1873
GAUUGUUUAUGAAUGCCUA
 334
 1891
UAGGCAUUCAUAAACAAUC
1601


 


 1874
AUUGUUUAUGAAUGCCUAU
 335
 1874
AUUGUUUAUGAAUGCCUAU
 335
 1892
AUAGGCAUUCAUAAACAAU
1602


 


 1875
UUGUUUAUGAAUGCCUAUG
 336
 1875
UUGUUUAUGAAUGCCUAUG
 336
 1893
CAUAGGCAUUCAUAAACAA
1603


 


 1876
UGUUUAUGAAUGCCUAUGG
 337
 1876
UGUUUAUGAAUGCCUAUGG
 337
 1894
CCAUAGGCAUUCAUAAACA
1604


 


 1877
GUUUAUGAAUGCCUAUGGU
 338
 1877
GUUUAUGAAUGCCUAUGGU
 338
 1895
ACCAUAGGCAUUCAUAAAC
1605


 


 2482
AAUAGAUAUAGAAGUAACC
 339
 2482
AAUAGAUAUAGAAGUAACC
 339
 2500
GGUUACUUCUAUAUCUAUU
1606


 


 2483
AUAGAUAUAGAAGUAACCA
 340
 2483
AUAGAUAUAGAAGUAACCA
 340
 2501
UGGUUACUUCUAUAUCUAU
1607


 


 2484
UAGAUAUAGAAGUAACCAA
 341
 2484
UAGAUAUAGAAGUAACCAA
 341
 2502
UUGGUUACUUCUAUAUCUA
1608


 


 2485
AGAUAUAGAAGUAACCAAA
 342
 2485
AGAUAUAGAAGUAACCAAA
 342
 2503
UUUGGUUACUUCUAUAUCU
1609


 


 2486
GAUAUAGAAGUAACCAAAG
 343
 2486
GAUAUAGAAGUAACCAAAG
 343
 2504
CUUUGGUUACUUCUAUAUC
1610


 


 2487
AUAUAGAAGUAACCAAAGA
 344
 2487
AUAUAGAAGUAACCAAAGA
 344
 2505
UCUUUGGUUACUUCUAUAU
1611


 


 2663
AUAGAAACAUUUGAUAACA
 345
 2663
AUAGAAACAUUUGAUAACA
 345
 2681
UGUUAUCAAAUGUUUCUAU
1612


 


 2664
UAGAAACAUUUGAUAACAA
 346
 2664
UAGAAACAUUUGAUAACAA
 346
 2682
UUGUUAUCAAAUGUUUCUA
1613


 


 2665
AGAAACAUUUGAUAACAAU
 347
 2665
AGAAACAUUUGAUAACAAU
 347
 2683
AUUGUUAUCAAAUGUUUCU
1614


 


 2666
GAAACAUUUGAUAACAAUG
 348
 2666
GAAACAUUUGAUAACAAUG
 348
 2684
CAUUGUUAUCAAAUGUUUC
1615


 


 2667
AAACAUUUGAUAACAAUGA
 349
 2667
AAACAUUUGAUAACAAUGA
 349
 2685
UCAUUGUUAUCAAAUGUUU
1616


 


 2668
AACAUUUGAUAACAAUGAA
 350
 2668
AACAUUUGAUAACAAUGAA
 350
 2686
UUCAUUGUUAUCAAAUGUU
1617


 


 4795
UGGCAAUGAUAAUCUCAAC
 351
 4795
UGGCAAUGAUAAUCUCAAC
 351
 4813
GUUGAGAUUAUCAUUGCCA
1618


 


 5866
AUAAAACAAGAAUUAGAUA
 352
 5866
AUAAAACAAGAAUUAGAUA
 352
 5884
UAUCUAAUUCUUGUUUUAU
1619


 


 5867
UAAAACAAGAAUUAGAUAA
 353
 5867
UAAAACAAGAAUUAGAUAA
 353
 5885
UUAUCUAAUUCUUGUUUUA
1620


 


 6412
AUCAAUGAUAUGCCUAUAA
 354
 6412
AUCAAUGAUAUGCCUAUAA
 354
 6430
UUAUAGGCAUAUCAUUGAU
1621


 


 6413
UCAAUGAUAUGCCUAUAAC
 355
 6413
UCAAUGAUAUGCCUAUAAC
 355
 6431
GUUAUAGGCAUAUCAUUGA
1622


 


 6414
CAAUGAUAUGCCUAUAACA
 356
 6414
CAAUGAUAUGCCUAUAACA
 356
 6432
UGUUAUAGGCAUAUCAUUG
1623


 


 6415
AAUGAUAUGCCUAUAACAA
 357
 6415
AAUGAUAUGCCUAUAACAA
 357
 6433
UUGUUAUAGGCAUAUCAUU
1624


 


 6416
AUGAUAUGCCUAUAACAAA
 358
 6416
AUGAUAUGCCUAUAACAAA
 358
 6434
UUUGUUAUAGGCAUAUCAU
1625


 


 6417
UGAUAUGCCUAUAACAAAU
 359
 6417
UGAUAUGCCUAUAACAAAU
 359
 6435
AUUUGUUAUAGGCAUAUCA
1626


 


 6418
GAUAUGCCUAUAACAAAUG
 360
 6418
GAUAUGCCUAUAACAAAUG
 360
 6436
CAUUUGUUAUAGGCAUAUC
1627


 


 6419
AUAUGCCUAUAACAAAUGA
 361
 6419
AUAUGCCUAUAACAAAUGA
 361
 6437
UCAUUUGUUAUAGGCAUAU
1628


 


 6420
UAUGCCUAUAACAAAUGAU
 362
 6420
UAUGCCUAUAACAAAUGAU
 362
 6438
AUCAUUUGUUAUAGGCAUA
1629


 


 6421
AUGCCUAUAACAAAUGAUC
 363
 6421
AUGCCUAUAACAAAUGAUC
 363
 6439
GAUCAUUUGUUAUAGGCAU
1630


 


 6422
UGCCUAUAACAAAUGAUCA
 364
 6422
UGCCUAUAACAAAUGAUCA
 364
 6440
UGAUCAUUUGUUAUAGGCA
1631


 


 6423
GCCUAUAACAAAUGAUCAG
 365
 6423
GCCUAUAACAAAUGAUCAG
 365
 6441
CUGAUCAUUUGUUAUAGGC
1632


 


 6424
CCUAUAACAAAUGAUCAGA
 366
 6424
CCUAUAACAAAUGAUCAGA
 366
 6442
UCUGAUCAUUUGUUAUAGG
1633


 


 6425
CUAUAACAAAUGAUCAGAA
 367
 6425
CUAUAACAAAUGAUCAGAA
 367
 6443
UUCUGAUCAUUUGUUAUAG
1634


 


 6426
UAUAACAAAUGAUCAGAAA
 368
 6426
UAUAACAAAUGAUCAGAAA
 368
 6444
UUUCUGAUCAUUUGUUAUA
1635


 


 8378
ACAAUAUAUAUAUUAGUGU
 369
 8378
ACAAUAUAUAUAUUAGUGU
 369
 8396
ACACUAAUAUAUAUAUUGU
1636


 


 8379
CAAUAUAUAUAUUAGUGUC
 370
 8379
CAAUAUAUAUAUUAGUGUC
 370
 8397
GACACUAAUAUAUAUAUUG
1637


 


 8380
AAUAUAUAUAUUAGUGUCA
 371
 8380
AAUAUAUAUAUUAGUGUCA
 371
 8398
UGACACUAAUAUAUAUAUU
1638


 


 8381
AUAUAUAUAUUAGUGUCAU
 372
 8381
AUAUAUAUAUUAGUGUCAU
 372
 8399
AUGACACUAAUAUAUAUAU
1639


 


 8513
GAUAGUUAUUUAAAAGGUG
 373
 8513
GAUAGUUAUUUAAAAGGUG
 373
 8531
CACCUUUUAAAUAACUAUC
1640


 


 8514
AUAGUUAUUUAAAAGGUGU
 374
 8514
AUAGUUAUUUAAAAGGUGU
 374
 8532
ACACCUUUUAAAUAACUAU
1641


 


 8515
UAGUUAUUUAAAAGGUGUU
 375
 8515
UAGUUAUUUAAAAGGUGUU
 375
 8533
AACACCUUUUAAAUAACUA
1642


 


 8516
AGUUAUUUAAAAGGUGUUA
 376
 8516
AGUUAUUUAAAAGGUGUUA
 376
 8534
UAACACCUUUUAAAUAACU
1643


 


 8517
GUUAUUUAAAAGGUGUUAU
 377
 8517
GUUAUUUAAAAGGUGUUAU
 377
 8535
AUAACACCUUUUAAAUAAC
1644


 


 8582
CCUUAUCUCAAAAAUGAUU
 378
 8582
CCUUAUCUCAAAAAUGAUU
 378
 8600
AAUCAUUUUUGAGAUAAGG
1645


 


 8583
CUUAUCUCAAAAAUGAUUA
 379
 8583
CUUAUCUCAAAAAUGAUUA
 379
 8601
UAAUCAUUUUUGAGAUAAG
1646


 


 8603
ACCAACUUAAUUAGUAGAC
 380
 8603
ACCAACUUAAUUAGUAGAC
 380
 8621
GUCUACUAAUUAAGUUGGU
1647


 


 8604
CCAACUUAAUUAGUAGACA
 381
 8604
CCAACUUAAUUAGUAGACA
 381
 8622
UGUCUACUAAUUAAGUUGG
1648


 


 8605
CAACUUAAUUAGUAGACAA
 382
 8605
CAACUUAAUUAGUAGACAA
 382
 8623
UUGUCUACUAAUUAAGUUG
1649


 


 9047
AUGUGUUCAAUGCAACAUC
 383
 9047
AUGUGUUCAAUGCAACAUC
 383
 9065
GAUGUUGCAUUGAACACAU
1650


 


 9048
UGUGUUCAAUGCAACAUCC
 384
 9048
UGUGUUCAAUGCAACAUCC
 384
 9066
GGAUGUUGCAUUGAACACA
1651


 


 9049
GUGUUCAAUGCAACAUCCU
 385
 9049
GUGUUCAAUGCAACAUCCU
 385
 9067
AGGAUGUUGCAUUGAACAC
1652


 


 9050
UGUUCAAUGCAACAUCCUC
 386
 9050
UGUUCAAUGCAACAUCCUC
 386
 9068
GAGGAUGUUGCAUUGAACA
1653


 


 9051
GUUCAAUGCAACAUCCUCC
 387
 9051
GUUCAAUGCAACAUCCUCC
 387
 9069
GGAGGAUGUUGCAUUGAAC
1654


 


 9052
UUCAAUGCAACAUCCUCCA
 388
 9052
UUCAAUGCAACAUCCUCCA
 388
 9070
UGGAGGAUGUUGCAUUGAA
1655


 


 9053
UCAAUGCAACAUCCUCCAU
 389
 9053
UCAAUGCAACAUCCUCCAU
 389
 9071
AUGGAGGAUGUUGCAUUGA
1656


 


 9054
CAAUGCAACAUCCUCCAUC
 390
 9054
CAAUGCAACAUCCUCCAUC
 390
 9072
GAUGGAGGAUGUUGCAUUG
1657


 


 9055
AAUGCAACAUCCUCCAUCA
 391
 9055
AAUGCAACAUCCUCCAUCA
 391
 9073
UGAUGGAGGAUGUUGCAUU
1658


 


 9056
AUGCAACAUCCUCCAUCAU
 392
 9056
AUGCAACAUCCUCCAUCAU
 392
 9074
AUGAUGGAGGAUGUUGCAU
1659


 


 9057
UGCAACAUCCUCCAUCAUG
 393
 9057
UGCAACAUCCUCCAUCAUG
 393
 9075
CAUGAUGGAGGAUGUUGCA
1660


 


 9058
GCAACAUCCUCCAUCAUGG
 394
 9058
GCAACAUCCUCCAUCAUGG
 394
 9076
CCAUGAUGGAGGAUGUUGC
1661


 


 9059
CAACAUCCUCCAUCAUGGU
 395
 9059
CAACAUCCUCCAUCAUGGU
 395
 9077
ACCAUGAUGGAGGAUGUUG
1662


 


 9060
AACAUCCUCCAUCAUGGUU
 396
 9060
AACAUCCUCCAUCAUGGUU
 396
 9078
AACCAUGAUGGAGGAUGUU
1663


 


 9061
ACAUCCUCCAUCAUGGUUA
 397
 9061
ACAUCCUCCAUCAUGGUUA
 397
 9079
UAACCAUGAUGGAGGAUGU
1664


 


10847
GAUCUAAUAUCUCUCAAAG
 398
10847
GAUCUAAUAUCUCUCAAAG
 398
10865
CUUUGAGAGAUAUUAGAUC
1665


 


10848
AUCUAAUAUCUCUCAAAGG
 399
10848
AUCUAAUAUCUCUCAAAGG
 399
10866
CCUUUGAGAGAUAUUAGAU
1666


 


10849
UCUAAUAUCUCUCAAAGGG
 400
10849
UCUAAUAUCUCUCAAAGGG
 400
10867
CCCUUUGAGAGAUAUUAGA
1667


 


10850
CUAAUAUCUCUCAAAGGGA
 401
10850
CUAAUAUCUCUCAAAGGGA
 401
10868
UCCCUUUGAGAGAUAUUAG
1668


 


10851
UAAUAUCUCUCAAAGGGAA
 402
10851
UAAUAUCUCUCAAAGGGAA
 402
10869
UUCCCUUUGAGAGAUAUUA
1669


 


10852
AAUAUCUCUCAAAGGGAAA
 403
10852
AAUAUCUCUCAAAGGGAAA
 403
10870
UUUCCCUUUGAGAGAUAUU
1670


 


10853
AUAUCUCUCAAAGGGAAAU
 404
10853
AUAUCUCUCAAAGGGAAAU
 404
10871
AUUUCCCUUUGAGAGAUAU
1671


 


10854
UAUCUCUCAAAGGGAAAUU
 405
10854
UAUCUCUCAAAGGGAAAUU
 405
10872
AAUUUCCCUUUGAGAGAUA
1672


 


10855
AUCUCUCAAAGGGAAAUUC
 406
10855
AUCUCUCAAAGGGAAAUUC
 406
10873
GAAUUUCCCUUUGAGAGAU
1673


 


10856
UCUCUCAAAGGGAAAUUCU
 407
10856
UCUCUCAAAGGGAAAUUCU
 407
10874
AGAAUUUCCCUUUGAGAGA
1674


 


10857
CUCUCAAAGGGAAAUUCUC
 408
10857
CUCUCAAAGGGAAAUUCUC
 408
10875
GAGAAUUUCCCUUUGAGAG
1675


 


12488
ACAGUCAGUAGUAGACCAU
 409
12488
ACAGUCAGUAGUAGACCAU
 409
12506
AUGGUCUACUACUGACUGU
1676


 


12489
CAGUCAGUAGUAGACCAUG
 410
12489
CAGUCAGUAGUAGACCAUG
 410
12507
CAUGGUCUACUACUGACUG
1677


 


12490
AGUCAGUAGUAGACCAUGU
 411
12490
AGUCAGUAGUAGACCAUGU
 411
12508
ACAUGGUCUACUACUGACU
1678


 


12491
GUCAGUAGUAGACCAUGUG
 412
12491
GUCAGUAGUAGACCAUGUG
 412
12509
CACAUGGUCUACUACUGAC
1679


 


12492
UCAGUAGUAGACCAUGUGA
 413
12492
UCAGUAGUAGACCAUGUGA
 413
12510
UCACAUGGUCUACUACUGA
1680


 


12493
CAGUAGUAGACCAUGUGAA
 414
12493
CAGUAGUAGACCAUGUGAA
 414
12511
UUCACAUGGUCUACUACUG
1681


 


12494
AGUAGUAGACCAUGUGAAU
 415
12494
AGUAGUAGACCAUGUGAAU
 415
12512
AUUCACAUGGUCUACUACU
1682


 


12495
GUAGUAGACCAUGUGAAUU
 416
12495
GUAGUAGACCAUGUGAAUU
 416
12513
AAUUCACAUGGUCUACUAC
1683


 


12496
UAGUAGACCAUGUGAAUUC
 417
12496
UAGUAGACCAUGUGAAUUC
 417
12514
GAAUUCACAUGGUCUACUA
1684


 


12497
AGUAGACCAUGUGAAUUCC
 418
12497
AGUAGACCAUGUGAAUUCC
 418
12515
GGAAUUCACAUGGUCUACU
1685


 


12498
GUAGACCAUGUGAAUUCCC
 419
12498
GUAGACCAUGUGAAUUCCC
 419
12516
GGGAAUUCACAUGGUCUAC
1686


 


12499
UAGACCAUGUGAAUUCCCU
 420
12499
UAGACCAUGUGAAUUCCCU
 420
12517
AGGGAAUUCACAUGGUCUA
1687


 


12500
AGACCAUGUGAAUUCCCUG
 421
12500
AGACCAUGUGAAUUCCCUG
 421
12518
CAGGGAAUUCACAUGGUCU
1688


 


12501
GACCAUGUGAAUUCCCUGC
 422
12501
GACCAUGUGAAUUCCCUGC
 422
12519
GCAGGGAAUUCACAUGGUC
1689


 


12502
ACCAUGUGAAUUCCCUGCA
 423
12502
ACCAUGUGAAUUCCCUGCA
 423
12520
UGCAGGGAAUUCACAUGGU
1690


 


12781
AGUGAUACAAAAACAGCAU
 424
12781
AGUGAUACAAAAACAGCAU
 424
12799
AUGCUGUUUUUGUAUCACU
1691


 


12782
GUGAUACAAAAACAGCAUA
 425
12782
GUGAUACAAAAACAGCAUA
 425
12800
UAUGCUGUUUUUGUAUCAC
1692


 


12783
UGAUACAAAAACAGCAUAU
 426
12783
UGAUACAAAAACAGCAUAU
 426
12801
AUAUGCUGUUUUUGUAUCA
1693


 


12784
GAUACAAAAACAGCAUAUG
 427
12784
GAUACAAAAACAGCAUAUG
 427
12802
CAUAUGCUGUUUUUGUAUC
1694


 


12785
AUACAAAAACAGCAUAUGU
 428
12785
AUACAAAAACAGCAUAUGU
 428
12803
ACAUAUGCUGUUUUUGUAU
1695


 


12786
UACAAAAACAGCAUAUGUU
 429
12786
UACAAAAACAGCAUAUGUU
 429
12804
AACAUAUGCUGUUUUUGUA
1696


 


14105
GAUUGCAAUGAUCAUAGUU
 430
14105
GAUUGCAAUGAUCAUAGUU
 430
14123
AACUAUGAUCAUUGCAAUC
1697


 


14106
AUUGCAAUGAUCAUAGUUU
 431
14106
AUUGCAAUGAUCAUAGUUU
 431
14124
AAACUAUGAUCAUUGCAAU
1698


 


14107
UUGCAAUGAUCAUAGUUUA
 432
14107
UUGCAAUGAUCAUAGUUUA
 432
14125
UAAACUAUGAUCAUUGCAA
1699


 


14108
UGCAAUGAUCAUAGUUUAC
 433
14108
UGCAAUGAUCAUAGUUUAC
 433
14126
GUAAACUAUGAUCAUUGCA
1700


 


14109
GCAAUGAUCAUAGUUUACC
 434
14109
GCAAUGAUCAUAGUUUACC
 434
14127
GGUAAACUAUGAUCAUUGC
1701


 


14110
CAAUGAUCAUAGUUUACCU
 435
14110
CAAUGAUCAUAGUUUACCU
 435
14128
AGGUAAACUAUGAUCAUUG
1702


 


14111
AAUGAUCAUAGUUUACCUA
 436
14111
AAUGAUCAUAGUUUACCUA
 436
14129
UAGGUAAACUAUGAUCAUU
1703


 


14112
AUGAUCAUAGUUUACCUAU
 437
14112
AUGAUCAUAGUUUACCUAU
 437
14130
AUAGGUAAACUAUGAUCAU
1704


 


14113
UGAUCAUAGUUUACCUAUU
 438
14113
UGAUCAUAGUUUACCUAUU
 438
14131
AAUAGGUAAACUAUGAUCA
1705


 


14114
GAUCAUAGUUUACCUAUUG
 439
14114
GAUCAUAGUUUACCUAUUG
 439
14132
CAAUAGGUAAACUAUGAUC
1706


 


14115
AUCAUAGUUUACCUAUUGA
 440
14115
AUCAUAGUUUACCUAUUGA
 440
14133
UCAAUAGGUAAACUAUGAU
1707


 


 1191
CUGUCAUCCAGCAAAUACA
 441
 1191
CUGUCAUCCAGCAAAUACA
 441
 1209
UGUAUUUGCUGGAUGACAG
1708


 


 1192
UGUCAUCCAGCAAAUACAC
 442
 1192
UGUCAUCCAGCAAAUACAC
 442
 1210
GUGUAUUUGCUGGAUGACA
1709


 


 4792
UUCUGGCAAUGAUAAUCUC
 443
 4792
UUCUGGCAAUGAUAAUCUC
 443
 4810
GAGAUUAUCAUUGCCAGAA
1710


 


 4793
UCUGGCAAUGAUAAUCUCA
 444
 4793
UCUGGCAAUGAUAAUCUCA
 444
 4811
UGAGAUUAUCAUUGCCAGA
1711


 


 4794
CUGGCAAUGAUAAUCUCAA
 445
 4794
CUGGCAAUGAUAAUCUCAA
 445
 4812
UUGAGAUUAUCAUUGCCAG
1712


 


10952
CAUGCUCAAGCAGAUUAUU
 446
10952
CAUGCUCAAGCAGAUUAUU
 446
10970
AAUAAUCUGCUUGAGCAUG
1713


 


10953
AUGCUCAAGCAGAUUAUUU
 447
10953
AUGCUCAAGCAGAUUAUUU
 447
10971
AAAUAAUCUGCUUGAGCAU
1714


 


10954
UGCUCAAGCAGAUUAUUUG
 448
10954
UGCUCAAGCAGAUUAUUUG
 448
10972
CAAAUAAUCUGCUUGAGCA
1715


 


11293
AAAUCAUGCAUUAUGUAAC
 449
11293
AAAUCAUGCAUUAUGUAAC
 449
11311
GUUACAUAAUGCAUGAUUU
1716


 


11294
AAUCAUGCAUUAUGUAACA
 450
11294
AAUCAUGCAUUAUGUAACA
 450
11312
UGUUACAUAAUGCAUGAUU
1717


 


11295
AUCAUGCAUUAUGUAACAA
 451
11295
AUCAUGCAUUAUGUAACAA
 451
11313
UUGUUACAUAAUGCAUGAU
1718


 


11296
UCAUGCAUUAUGUAACAAU
 452
11296
UCAUGCAUUAUGUAACAAU
 452
11314
AUUGUUACAUAAUGCAUGA
1719


 


11297
CAUGCAUUAUGUAACAAUA
 453
11297
CAUGCAUUAUGUAACAAUA
 453
11315
UAUUGUUACAUAAUGCAUG
1720


 


11298
AUGCAUUAUGUAACAAUAA
 454
11298
AUGCAUUAUGUAACAAUAA
 454
11316
UUAUUGUUACAUAAUGCAU
1721


 


 3045
ACAAUGAUCUAUCACUUGA
 455
 3045
ACAAUGAUCUAUCACUUGA
 455
 3063
UCAAGUGAUAGAUCAUUGU
1722


 


11001
AUAAAGAGUAUGCAGGCAU
 456
11001
AUAAAGAGUAUGCAGGCAU
 456
11019
AUGCCUGCAUACUCUUUAU
1723


 


11793
AUAUUAUGCAAAAUAUAGA
 457
11793
AUAUUAUGCAAAAUAUAGA
 457
11811
UCUAUAUUUUGCAUAAUAU
1724


 


13449
AACACAAAUUCAAUGAUGA
 458
13449
AACACAAAUUCAAUGAUGA
 458
13467
UCAUCAUUGAAUUUGUGUU
1725


 


   57
AAUUUGAUAAGUACCACUU
 459
   57
AAUUUGAUAAGUACCACUU
 459
   75
AAGUGGUACUUAUCAAAUU
1726


 


   75
UAAAUUUAACUCCCUUGGU
 460
   75
UAAAUUUAACUCCCUUGGU
 460
   93
ACCAAGGGAGUUAAAUUUA
1727


 


   93
UUAGAGAUGGGCAGCAAUU
 461
   93
UUAGAGAUGGGCAGCAAUU
 461
  111
AAUUGCUGCCCAUCUCUAA
1728


 


  129
GUUAGAUUACAAAAUUUGU
 462
  129
GUUAGAUUACAAAAUUUGU
 462
  147
ACAAAUUUUGUAAUCUAAC
1729


 


  147
UUUGACAAUGAUGAAGUAG
 463
  147
UUUGACAAUGAUGAAGUAG
 463
  165
CUACUUCAUCAUUGUCAAA
1730


 


  219
UUGGCUAAGGCAGUGAUAC
 464
  219
UUGGCUAAGGCAGUGAUAC
 464
  237
GUAUCACUGCCUUAGCCAA
1731


 


  237
CAUACAAUCAAAUUGAAUG
 465
  237
CAUACAAUCAAAUUGAAUG
 465
  255
CAUUCAAUUUGAUUGUAUG
1732


 


  273
GUUAUUACAAGUAGUGAUA
 466
  273
GUUAUUACAAGUAGUGAUA
 466
  291
UAUCACUACUUGUAAUAAC
1733


 


  363
AUAUGGGAAAUGAUGGAAU
 467
  363
AUAUGGGAAAUGAUGGAAU
 467
  381
AUUCCAUCAUUUCCCAUAU
1734


 


  417
GACAAUUGUGAAAUUAAAU
 468
  417
GACAAUUGUGAAAUUAAAU
 468
  435
AUUUAAUUUCACAAUUGUC
1735


 


  435
UUCUCCAAAAAACUAAGUG
 469
  435
UUCUCCAAAAAACUAAGUG
 469
  453
CACUUAGUUUUUUGGAGAA
1736


 


  453
GAUUCAACAAUGACCAAUU
 470
  453
GAUUCAACAAUGACCAAUU
 470
  471
AAUUGGUCAUUGUUGAAUC
1737


 


  471
UAUAUGAAUCAAUUAUCUG
 471
  471
UAUAUGAAUCAAUUAUCUG
 471
  489
CAGAUAAUUGAUUCAUAUA
1738


 


  489
GAAUUACUUGGAUUUGAUC
 472
  489
GAAUUACUUGGAUUUGAUC
 472
  507
GAUCAAAUCCAAGUAAUUC
1739


 


  507
CUUAAUCCAUAAAUUAUAA
 473
  507
CUUAAUCCAUAAAUUAUAA
 473
  525
UUAUAAUUUAUGGAUUAAG
1740


 


  561
AUUAGUUAAUAUAAAACUU
 474
  561
AUUAGUUAAUAUAAAACUU
 474
  579
AAGUUUUAUAUUAACUAAU
1741


 


  777
AAAACUUGAUGAAAGACAG
 475
  777
AAAACUUGAUGAAAGACAG
 475
  795
CUGUCUUUCAUCAAGUUUU
1742


 


  795
GGCCACAUUUACAUUCCUG
 476
  795
GGCCACAUUUACAUUCCUG
 476
  813
CAGGAAUGUAAAUGUGGCC
1743


 


  813
GGUCAACUAUGAAAUGAAA
 477
  813
GGUCAACUAUGAAAUGAAA
 477
  831
UUUCAUUUCAUAGUUGACC
1744


 


  867
AUAUACUGAAUACAACACA
 478
  867
AUAUACUGAAUACAACACA
 478
  885
UGUGUUGUAUUCAGUAUAU
1745


 


  921
UCAUGAUGGGUUCUUAGAA
 479
  921
UCAUGAUGGGUUCUUAGAA
 479
  939
UUCUAAGAACCCAUCAUGA
1746


 


  939
AUGCAUUGGCAUUAAGCCU
 480
  939
AUGCAUUGGCAUUAAGCCU
 480
  957
AGGCUUAAUGCCAAUGCAU
1747


 


  975
AAUAUACAAGUAUGAUCUC
 481
  975
AAUAUACAAGUAUGAUCUC
 481
  993
GAGAUCAUACUUGUAUAUU
1748


 


 1155
GUCAAGUUGAAUGAUACAC
 482
 1155
GUCAAGUUGAAUGAUACAC
 482
 1173
GUGUAUCAUUCAACUUGAC
1749


 


 1173
CUCAACAAAGAUCAACUUC
 483
 1173
CUCAACAAAGAUCAACUUC
 483
 1191
GAAGUUGAUCUUUGUUGAG
1750


 


 1209
ACCAUCCAACGGAGCACAG
 484
 1209
ACCAUCCAACGGAGCACAG
 484
 1227
CUGUGCUCCGUUGGAUGGU
1751


 


 1227
GGAGAUAGUAUUGAUACUC
 485
 1227
GGAGAUAGUAUUGAUACUC
 485
 1245
GAGUAUCAAUACUAUCUCC
1752


 


 1245
CCUAAUUAUGAUGUGCAGA
 486
 1245
CCUAAUUAUGAUGUGCAGA
 486
 1263
UCUGCACAUCAUAAUUAGG
1753


 


 1263
AAACACAUCAAUAAGUUAU
 487
 1263
AAACACAUCAAUAAGUUAU
 487
 1281
AUAACUUAUUGAUGUGUUU
1754


 


 1281
UGUGGCAUGUUAUUAAUCA
 488
 1281
UGUGGCAUGUUAUUAAUCA
 488
 1299
UGAUUAAUAACAUGCCACA
1755


 


 1299
ACAGAAGAUGCUAAUCAUA
 489
 1299
ACAGAAGAUGCUAAUCAUA
 489
 1317
UAUGAUUAGCAUCUUCUGU
1756


 


 1317
AAAUUCACUGGGUUAAUAG
 490
 1317
AAAUUCACUGGGUUAAUAG
 490
 1335
CUAUUAACCCAGUGAAUUU
1757


 


 1335
GGUAUGUUAUAUGCUAUGU
 491
 1335
GGUAUGUUAUAUGCUAUGU
 491
 1353
ACAUAGCAUAUAACAUACC
1758


 


 1371
GACACCAUAAAAAUACUCA
 492
 1371
GACACCAUAAAAAUACUCA
 492
 1389
UGAGUAUUUUUAUGGUGUC
1759


 


 1389
AGAGAUGCGGGAUAUCAUG
 493
 1389
AGAGAUGCGGGAUAUCAUG
 493
 1407
CAUGAUAUCCCGCAUCUCU
1760


 


 1425
GAUGUAACAACACAUCGUC
 494
 1425
GAUGUAACAACACAUCGUC
 494
 1443
GACGAUGUGUUGUUACAUC
1761


 


 1461
GAAAUGAAAUUUGAAGUGU
 495
 1461
GAAAUGAAAUUUGAAGUGU
 495
 1479
ACACUUCAAAUUUCAUUUC
1762


 


 1497
ACAACUGAAAUUCAAAUCA
 496
 1497
ACAACUGAAAUUCAAAUCA
 496
 1515
UGAUUUGAAUUUCAGUUGU
1763


 


 1515
AACAUUGAGAUAGAAUCUA
 497
 1515
AACAUUGAGAUAGAAUCUA
 497
 1533
UAGAUUCUAUCUCAAUGUU
1764


 


 1551
AUGCUAAAAGAAAUGGGAG
 498
 1551
AUGCUAAAAGAAAUGGGAG
 498
 1569
CUCCCAUUUCUUUUAGCAU
1765


 


 1569
GAGGUAGCUCCAGAAUACA
 499
 1569
GAGGUAGCUCCAGAAUACA
 499
 1587
UGUAUUCUGGAGCUACCUC
1766


 


 1605
UGUGGGAUGAUAAUAUUAU
 500
 1605
UGUGGGAUGAUAAUAUUAU
 500
 1623
AUAAUAUUAUCAUCCCACA
1767


 


 1695
GCUAAUAAUGUCCUAAAAA
 501
 1695
GCUAAUAAUGUCCUAAAAA
 501
 1713
UUUUUAGGACAUUAUUAGC
1768


 


 1731
AAAGGCUUACUACCCAAGG
 502
 1731
AAAGGCUUACUACCCAAGG
 502
 1749
CCUUGGGUAGUAAGCCUUU
1769


 


 1803
GUUUUUGUUCAUUUUGGUA
 503
 1803
GUUUUUGUUCAUUUUGGUA
 503
 1821
UACCAAAAUGAACAAAAAC
1770


 


 1839
AGAGGUGGCAGUAGAGUUG
 504
 1839
AGAGGUGGCAGUAGAGUUG
 504
 1857
CAACUCUACUGCCACCUCU
1771


 


 1965
AGUGUGCAAGCAGAAAUGG
 505
 1965
AGUGUGCAAGCAGAAAUGG
 505
 1983
CCAUUUCUGCUUGCACACU
1772


 


 2001
UAUGAAUAUGCCCAAAAAU
 506
 2001
UAUGAAUAUGCCCAAAAAU
 506
 2019
AUUUUUGGGCAUAUUCAUA
1773


 


 2055
AACCCAAAAGCAUCAUUAU
 507
 2055
AACCCAAAAGCAUCAUUAU
 507
 2073
AUAAUGAUGCUUUUGGGUU
1774


 


 2109
GUAUUAGGCAAUGCUGCUG
 508
 2109
GUAUUAGGCAAUGCUGCUG
 508
 2127
CAGCAGCAUUGCCUAAUAC
1775


 


 2127
GGCCUAGGCAUAAUGGGAG
 509
 2127
GGCCUAGGCAUAAUGGGAG
 509
 2145
CUCCCAUUAUGCCUAGGCC
1776


 


 2163
AGGAAUCAAGAUCUAUAUG
 510
 2163
AGGAAUCAAGAUCUAUAUG
 510
 2181
CAUAUAGAUCUUGAUUCCU
1777


 


 2199
GCUGAACAACUCAAAGAAA
 511
 2199
GCUGAACAACUCAAAGAAA
 511
 2217
UUUCUUUGAGUUGUUCAGC
1778


 


 2217
AAUGGUGUGAUUAACUACA
 512
 2217
AAUGGUGUGAUUAACUACA
 512
 2235
UGUAGUUAAUCACACCAUU
1779


 


 2253
GCAGAAGAACUAGAGGCUA
 513
 2253
GCAGAAGAACUAGAGGCUA
 513
 2271
UAGCCUCUAGUUCUUCUGC
1780


 


 2271
AUCAAACAUCAGCUUAAUC
 514
 2271
AUCAAACAUCAGCUUAAUC
 514
 2289
GAUUAAGCUGAUGUUUGAU
1781


 


 2289
CCAAAAGAUAAUGAUGUAG
 515
 2289
CCAAAAGAUAAUGAUGUAG
 515
 2307
CUACAUCAUUAUCUUUUGG
1782


 


 2307
GAGCUUUGAGUUAAUAAAA
 516
 2307
GAGCUUUGAGUUAAUAAAA
 516
 2325
UUUUAUUAACUCAAAGCUC
1783


 


 2361
CUCCUGAAUUCCAUGGAGA
 517
 2361
CUCCUGAAUUCCAUGGAGA
 517
 2379
UCUCCAUGGAAUUCAGGAG
1784


 


 2415
CAAUAAAGGGCAAAUUCAC
 518
 2415
CAAUAAAGGGCAAAUUCAC
 518
 2433
GUGAAUUUGCCCUUUAUUG
1785


 


 2451
AGAAAAAAGAUAGUAUCAU
 519
 2451
AGAAAAAAGAUAGUAUCAU
 519
 2469
AUGAUACUAUCUUUUUUCU
1786


 


 2469
UAUCUGUCAACUCAAUAGA
 520
 2469
UAUCUGUCAACUCAAUAGA
 520
 2487
UCUAUUGAGUUGACAGAUA
1787


 


 2505
AAAGCCCUAUAACAUCAAA
 521
 2505
AAAGCCCUAUAACAUCAAA
 521
 2523
UUUGAUGUUAUAGGGCUUU
1788


 


 2541
CAACAAAUGAGACAGAUGA
 522
 2541
CAACAAAUGAGACAGAUGA
 522
 2559
UCAUCUGUCUCAUUUGUUG
1789


 


 2577
CCAAUUAUCAAAGAAAACC
 523
 2577
CCAAUUAUCAAAGAAAACC
 523
 2595
GGUUUUCUUUGAUAAUUGG
1790


 


 2595
CUCUAGUAAGUUUCAAAGA
 524
 2595
CUCUAGUAAGUUUCAAAGA
 524
 2613
UCUUUGAAACUUACUAGAG
1791


 


 2649
UAUACAAAGAAACCAUAGA
 525
 2649
UAUACAAAGAAACCAUAGA
 525
 2667
UCUAUGGUUUCUUUGUAUA
1792


 


 2757
GGAUUGAUGAAAAAUUAAG
 526
 2757
GGAUUGAUGAAAAAUUAAG
 526
 2775
CUUAAUUUUUCAUCAAUCC
1793


 


 2775
GUGAAAUACUAGGAAUGCU
 527
 2775
GUGAAAUACUAGGAAUGCU
 527
 2793
AGCAUUCCUAGUAUUUCAC
1794


 


 2847
GAGAUGCCAUGGUUGGUUU
 528
 2847
GAGAUGCCAUGGUUGGUUU
 528
 2865
AAACCAACCAUGGCAUCUC
1795


 


 2883
AAAAAAUCAGAACUGAAGC
 529
 2883
AAAAAAUCAGAACUGAAGC
 529
 2901
GCUUCAGUUCUGAUUUUUU
1796


 


 2901
CAUUAAUGACCAAUGACAG
 530
 2901
CAUUAAUGACCAAUGACAG
 530
 2919
CUGUCAUUGGUCAUUAAUG
1797


 


 2937
GACUCAGGAAUGAGGAAAG
 531
 2937
GACUCAGGAAUGAGGAAAG
 531
 2955
CUUUCCUCAUUCCUGAGUC
1798


 


 2955
GUGAAAAGAUGGCAAAAGA
 532
 2955
GUGAAAAGAUGGCAAAAGA
 532
 2973
UCUUUUGCCAUCUUUUCAC
1799


 


 2973
ACACAUCAGAUGAAGUGUC
 533
 2973
ACACAUCAGAUGAAGUGUC
 533
 2991
GACACUUCAUCUGAUGUGU
1800


 


 2991
CUCUCAAUCCAACAUCAGA
 534
 2991
CUCUCAAUCCAACAUCAGA
 534
 3009
UCUGAUGUUGGAUUGAGAG
1801


 


 3027
UGGAAGGGAAUGAUAGUGA
 535
 3027
UGGAAGGGAAUGAUAGUGA
 535
 3045
UCACUAUCAUUCCCUUCCA
1802


 


 3279
CUGCUGUUCAAUACAAUGU
 536
 3279
CUGCUGUUCAAUACAAUGU
 536
 3297
ACAUUGUAUUGAACAGCAG
1803


 


 3315
ACCCUGCAUCACUUACAAU
 537
 3315
ACCCUGCAUCACUUACAAU
 537
 3333
AUUGUAAGUGAUGCAGGGU
1804


 


 3333
UAUGGGUGCCCAUGUUCCA
 538
 3333
UAUGGGUGCCCAUGUUCCA
 538
 3351
UGGAACAUGGGCACCCAUA
1805


 


 3369
AUUUACUUAUAAAAGAACU
 539
 3369
AUUUACUUAUAAAAGAACU
 539
 3387
AGUUCUUUUAUAAGUAAAU
1806


 


 3387
UAGCUAAUGUCAACAUACU
 540
 3387
UAGCUAAUGUCAACAUACU
 540
 3405
AGUAUGUUGACAUUAGCUA
1807


 


 3405
UAGUGAAACAAAUAUCCAC
 541
 3405
UAGUGAAACAAAUAUCCAC
 541
 3423
GUGGAUAUUUGUUUCACUA
1808


 


 3441
UAAGAGUCAUGAUAAACUC
 542
 3441
UAAGAGUCAUGAUAAACUC
 542
 3459
GAGUUUAUCAUGACUCUUA
1809


 


 3477
CACAAAUGCCCAGCAAAUU
 543
 3477
CACAAAUGCCCAGCAAAUU
 543
 3495
AAUUUGCUGGGCAUUUGUG
1810


 


 3513
UGUCCUUGGAUGAAAGAAG
 544
 3513
UGUCCUUGGAUGAAAGAAG
 544
 3531
CUUCUUUCAUCCAAGGACA
1811


 


 3549
UAACCACACCCUGUGAAAU
 545
 3549
UAACCACACCCUGUGAAAU
 545
 3567
AUUUCACAGGGUGUGGUUA
1812


 


 3567
UCAAGGCAUGUAGUCUAAC
 546
 3567
UCAAGGCAUGUAGUCUAAC
 546
 3585
GUUAGACUACAUGCCUUGA
1813


 


 3585
CAUGCCUAAAAUCAAAAAA
 547
 3585
CAUGCCUAAAAUCAAAAAA
 547
 3603
UUUUUUGAUUUUAGGCAUG
1814


 


 3693
CAUCAAAAAAAGUCAUAAU
 548
 3693
CAUCAAAAAAAGUCAUAAU
 548
 3711
AUUAUGACUUUUUUUGAUG
1815


 


 3711
UACCAACAUACCUAAGAUC
 549
 3711
UACCAACAUACCUAAGAUC
 549
 3729
GAUCUUAGGUAUGUUGGUA
1816


 


 3729
CCAUCAGUGUCAGAAAUAA
 550
 3729
CCAUCAGUGUCAGAAAUAA
 550
 3747
UUAUUUCUGACACUGAUGG
1817


 


 3747
AAGAUCUGAACACACUUGA
 551
 3747
AAGAUCUGAACACACUUGA
 551
 3765
UCAAGUGUGUUCAGAUCUU
1818


 


 3765
AAAAUAUAACAACCACUGA
 552
 3765
AAAAUAUAACAACCACUGA
 552
 3783
UCAGUGGUUGUUAUAUUUU
1819


 


 3801
CAAAUGCAAAAAUCAUCCC
 553
 3801
CAAAUGCAAAAAUCAUCCC
 553
 3819
GGGAUGAUUUUUGCAUUUG
1820


 


 3837
UAGUCAUCACAGUGACUGA
 554
 3837
UAGUCAUCACAGUGACUGA
 554
 3855
UCAGUCACUGUGAUGACUA
1821


 


 3855
ACAACAAAGGAGCAUUCAA
 555
 3855
ACAACAAAGGAGCAUUCAA
 555
 3873
UUGAAUGCUCCUUUGUUGU
1822


 


 3909
UUGGAGCUUACCUAGAAAA
 556
 3909
UUGGAGCUUACCUAGAAAA
 556
 3927
UUUUCUAGGUAAGCUCCAA
1823


 


 3927
AAGAAAGUAUAUAUUAUGU
 557
 3927
AAGAAAGUAUAUAUUAUGU
 557
 3945
ACAUAAUAUAUACUUUCUU
1824


 


 3963
ACACAGCUACACGAUUUGC
 558
 3963
ACACAGCUACACGAUUUGC
 558
 3981
GCAAAUCGUGUAGCUGUGU
1825


 


 3981
CAAUCAAACCCAUGGAAGA
 559
 3981
CAAUCAAACCCAUGGAAGA
 559
 3999
UCUUCCAUGGGUUUGAUUG
1826


 


 4035
UACAAACUUUCUACCUACA
 560
 4035
UACAAACUUUCUACCUACA
 560
 4053
UGUAGGUAGAAAGUUUGUA
1827


 


 4125
CAGAUCAUCCCAAGUCAUU
 561
 4125
CAGAUCAUCCCAAGUCAUU
 561
 4143
AAUGACUUGGGAUGAUCUG
1828


 


 4215
CCAACUAAUCACAAUAUCU
 562
 4215
CCAACUAAUCACAAUAUCU
 562
 4233
AGAUAUUGUGAUUAGUUGG
1829


 


 4269
AACCAAUGGAAAAUACAUC
 563
 4269
AACCAAUGGAAAAUACAUC
 563
 4287
GAUGUAUUUUCCAUUGGUU
1830


 


 4287
CCAUAACAAUAGAAUUCUC
 564
 4287
CCAUAACAAUAGAAUUCUC
 564
 4305
GAGAAUUCUAUUGUUAUGG
1831


 


 4305
CAAGCAAAUUCUGGCCUUA
 565
 4305
CAAGCAAAUUCUGGCCUUA
 565
 4323
UAAGGCCAGAAUUUGCUUG
1832


 


 4323
ACUUUACACUAAUACACAU
 566
 4323
ACUUUACACUAAUACACAU
 566
 4341
AUGUGUAUUAGUGUAAAGU
1833


 


 4359
CUUUGCUAAUCAUAAUCUC
 567
 4359
CUUUGCUAAUCAUAAUCUC
 567
 4377
GAGAUUAUGAUUAGCAAAG
1834


 


 4413
AUAACGUAUUCCAUAACAA
 568
 4413
AUAACGUAUUCCAUAACAA
 568
 4431
UUGUUAUGGAAUACGUUAU
1835


 


 4647
GCAAAUGCAAACAUGUCCA
 569
 4647
GCAAAUGCAAACAUGUCCA
 569
 4665
UGGACAUGUUUGCAUUUGC
1836


 


 4665
AAAAACAAGGACCAACGCA
 570
 4665
AAAAACAAGGACCAACGCA
 570
 4683
UGCGUUGGUCCUUGUUUUU
1837


 


 4773
GCACAAAUCACAUUAUCCA
 571
 4773
GCACAAAUCACAUUAUCCA
 571
 4791
UGGAUAAUGUGAUUUGUGC
1838


 


 4791
AUUCUGGCAAUGAUAAUCU
 572
 4791
AUUCUGGCAAUGAUAAUCU
 572
 4809
AGAUUAUCAUUGCCAGAAU
1839


 


 4845
GCCUCGGCAAACCACAAAG
 573
 4845
GCCUCGGCAAACCACAAAG
 573
 4863
CUUUGUGGUUUGCCGAGGC
1840


 


 4881
AUCAUACAAGAUGCAACAA
 574
 4881
AUCAUACAAGAUGCAACAA
 574
 4899
UUGUUGCAUCUUGUAUGAU
1841


 


 4899
AGCCAGAUCAAGAACACAA
 575
 4899
AGCCAGAUCAAGAACACAA
 575
 4917
UUGUGUUCUUGAUCUGGCU
1842


 


 5133
CCCAAUAAUGAUUUUCACU
 576
 5133
CCCAAUAAUGAUUUUCACU
 576
 5151
AGUGAAAAUCAUUAUUGGG
1843


 


 5187
AGCAACAAUCCAACCUGCU
 577
 5187
AGCAACAAUCCAACCUGCU
 577
 5205
AGCAGGUUGGAUUGUUGCU
1844


 


 5367
AACACCACCAAAACAAACA
 578
 5367
AACACCACCAAAACAAACA
 578
 5385
UGUUUGUUUUGGUGGUGUU
1845


 


 5619
GGGGCAAAUAACAAUGGAG
 579
 5619
GGGGCAAAUAACAAUGGAG
 579
 5637
CUCCAUUGUUAUUUGCCCC
1846


 


 5709
AAACAUCACUGAAGAAUUU
 580
 5709
AAACAUCACUGAAGAAUUU
 580
 5727
AAAUUCUUCAGUGAUGUUU
1847


 


 5799
UAUAACUAUAGAAUUAAGU
 581
 5799
UAUAACUAUAGAAUUAAGU
 581
 5817
ACUUAAUUCUAUAGUUAUA
1848


 


 5907
AUUGCAGUUGCUCAUGCAA
 582
 5907
AUUGCAGUUGCUCAUGCAA
 582
 5925
UUGCAUGAGCAACUGCAAU
1849


 


 5943
CAAUCGAGCCAGAAGAGAA
 583
 5943
CAAUCGAGCCAGAAGAGAA
 583
 5961
UUCUCUUCUGGCUCGAUUG
1850


 


 5961
ACUACCAAGGUUUAUGAAU
 584
 5961
ACUACCAAGGUUUAUGAAU
 584
 5979
AUUCAUAAACCUUGGUAGU
1851


 


 6015
AUUAAGCAAGAAAAGGAAA
 585
 6015
AUUAAGCAAGAAAAGGAAA
 585
 6033
UUUCCUUUUCUUGCUUAAU
1852


 


 6051
UUUGUUAGGUGUUGGAUCU
 586
 6051
UUUGUUAGGUGUUGGAUCU
 586
 6069
AGAUCCAACACCUAACAAA
1853


 


 6087
UGCUGUAUCUAAGGUCCUG
 587
 6087
UGCUGUAUCUAAGGUCCUG
 587
 6105
CAGGACCUUAGAUACAGCA
1854


 


 6141
UCUACUAUCCACAAACAAG
 588
 6141
UCUACUAUCCACAAACAAG
 588
 6159
CUUGUUUGUGGAUAGUAGA
1855


 


 6195
AACCAGCAAAGUGUUAGAC
 589
 6195
AACCAGCAAAGUGUUAGAC
 589
 6213
GUCUAACACUUUGCUGGUU
1856


 


 6213
CCUCAAAAACUAUAUAGAU
 590
 6213
CCUCAAAAACUAUAUAGAU
 590
 6231
AUCUAUAUAGUUUUUGAGG
1857


 


 6303
ACAAAAGAACAACAGACUA
 591
 6303
ACAAAAGAACAACAGACUA
 591
 6321
UAGUCUGUUGUUCUUUUGU
1858


 


 6321
ACUAGAGAUUACCAGGGAA
 592
 6321
ACUAGAGAUUACCAGGGAA
 592
 6339
UUCCCUGGUAAUCUCUAGU
1859


 


 6411
AAUCAAUGAUAUGCCUAUA
 593
 6411
AAUCAAUGAUAUGCCUAUA
 593
 6429
UAUAGGCAUAUCAUUGAUU
1860


 


 6429
AACAAAUGAUCAGAAAAAG
 594
 6429
AACAAAUGAUCAGAAAAAG
 594
 6447
CUUUUUCUGAUCAUUUGUU
1861


 


 6447
GUUAAUGUCCAACAAUGUU
 595
 6447
GUUAAUGUCCAACAAUGUU
 595
 6465
AACAUUGUUGGACAUUAAC
1862


 


 6465
UCAAAUAGUUAGACAGCAA
 596
 6465
UCAAAUAGUUAGACAGCAA
 596
 6483
UUGCUGUCUAACUAUUUGA
1863


 


 6483
AAGUUACUCUAUCAUGUCC
 597
 6483
AAGUUACUCUAUCAUGUCC
 597
 6501
GGACAUGAUAGAGUAACUU
1864


 


 6519
CUUAGCAUAUGUAGUACAA
 598
 6519
CUUAGCAUAUGUAGUACAA
 598
 6537
UUGUACUACAUAUGCUAAG
1865


 


 6591
UCUAUGUACAACCAACACA
 599
 6591
UCUAUGUACAACCAACACA
 599
 6609
UGUGUUGGUUGUACAUAGA
1866


 


 6645
CAGAGGAUGGUACUGUGAC
 600
 6645
CAGAGGAUGGUACUGUGAC
 600
 6663
GUCACAGUACCAUCCUCUG
1867


 


 6663
CAAUGCAGGAUCAGUAUCU
 601
 6663
CAAUGCAGGAUCAGUAUCU
 601
 6681
AGAUACUGAUCCUGCAUUG
1868


 


 6735
CACAAUGAACAGUUUAACA
 602
 6735
CACAAUGAACAGUUUAACA
 602
 6753
UGUUAAACUGUUCAUUGUG
1869


 


 6753
AUUACCAAGUGAAGUAAAU
 603
 6753
AUUACCAAGUGAAGUAAAU
 603
 6771
AUUUACUUCACUUGGUAAU
1870


 


 6825
AAAAACAGAUGUAAGCAGC
 604
 6825
AAAAACAGAUGUAAGCAGC
 604
 6843
GCUGCUUACAUCUGUUUUU
1871


 


 6843
CUCCGUUAUCACAUCUCUA
 605
 6843
CUCCGUUAUCACAUCUCUA
 605
 6861
UAGAGAUGUGAUAACGGAG
1872


 


 6861
AGGAGCCAUUGUGUCAUGC
 606
 6861
AGGAGCCAUUGUGUCAUGC
 606
 6879
GCAUGACACAAUGGCUCCU
1873


 


 6879
CUAUGGCAAAACUAAAUGU
 607
 6879
CUAUGGCAAAACUAAAUGU
 607
 6897
ACAUUUAGUUUUGCCAUAG
1874


 


 6897
UACAGCAUCCAAUAAAAAU
 608
 6897
UACAGCAUCCAAUAAAAAU
 608
 6915
AUUUUUAUUGGAUGCUGUA
1875


 


 6915
UCGUGGAAUCAUAAAGACA
 609
 6915
UCGUGGAAUCAUAAAGACA
 609
 6933
UGUCUUUAUGAUUCCACGA
1876


 


 7041
AGGUGAACCAAUAAUAAAU
 610
 7041
AGGUGAACCAAUAAUAAAU
 610
 7059
AUUUAUUAUUGGUUCACCU
1877


 


 7149
UCGUAAAUCCGAUGAAUUA
 611
 7149
UCGUAAAUCCGAUGAAUUA
 611
 7167
UAAUUCAUCGGAUUUACGA
1878


 


 7203
UAUCAUGAUAACUACUAUA
 612
 7203
UAUCAUGAUAACUACUAUA
 612
 7221
UAUAGUAGUUAUCAUGAUA
1879


 


 7221
AAUUAUAGUGAUUAUAGUA
 613
 7221
AAUUAUAGUGAUUAUAGUA
 613
 7239
UACUAUAAUCACUAUAAUU
1880


 


 7239
AAUAUUGUUAUCAUUAAUU
 614
 7239
AAUAUUGUUAUCAUUAAUU
 614
 7257
AAUUAAUGAUAACAAUAUU
1881


 


 7293
CACACCAGUCACACUAAGC
 615
 7293
CACACCAGUCACACUAAGC
 615
 7311
GCUUAGUGUGACUGGUGUG
1882


 


 7329
UAUAAAUAAUAUUGCAUUU
 616
 7329
UAUAAAUAAUAUUGCAUUU
 616
 7347
AAAUGCAAUAUUAUUUAUA
1883


 


 7365
AGCACCUAAUCAUGUUCUU
 617
 7365
AGCACCUAAUCAUGUUCUU
 617
 7383
AAGAACAUGAUUAGGUGCU
1884


 


 7401
GCUCAUAGACAACCCAUCU
 618
 7401
GCUCAUAGACAACCCAUCU
 618
 7419
AGAUGGGUUGUCUAUGAGC
1885


 


 7437
AAAUCUGAACUUCAUCGAA
 619
 7437
AAAUCUGAACUUCAUCGAA
 619
 7455
UUCGAUGAAGUUCAGAUUU
1886


 


 7491
AAGUAGAUUCCUAGUUUAU
 620
 7491
AAGUAGAUUCCUAGUUUAU
 620
 7509
AUAAACUAGGAAUCUACUU
1887


 


 7581
ACGAAGGAAUCCUUGCAAA
 621
 7581
ACGAAGGAAUCCUUGCAAA
 621
 7599
UUUGCAAGGAUUCCUUCGU
1888


 


 7599
AUUUGAAAUUCGAGGUCAU
 622
 7599
AUUUGAAAUUCGAGGUCAU
 622
 7617
AUGACCUCGAAUUUCAAAU
1889


 


 7653
UUAUUUUGAAUGGCCACCC
 623
 7653
UUAUUUUGAAUGGCCACCC
 623
 7671
GGGUGGCCAUUCAAAAUAA
1890


 


 7689
ACAAAACUUUAUGUUAAAC
 624
 7689
ACAAAACUUUAUGUUAAAC
 624
 7707
GUUUAACAUAAAGUUUUGU
1891


 


 7707
CAGAAUACUUAAGUCUAUG
 625
 7707
CAGAAUACUUAAGUCUAUG
 625
 7725
CAUAGACUUAAGUAUUCUG
1892


 


 7761
AGCUGCAGAGUUGGACAGA
 626
 7761
AGCUGCAGAGUUGGACAGA
 626
 7779
UCUGUCCAACUCUGCAGCU
1893


 


 7851
ACAAUCAGCAUGUGUUGCC
 627
 7851
ACAAUCAGCAUGUGUUGCC
 627
 7869
GGCAACACAUGCUGAUUGU
1894


 


 7869
CAUGAGCAAACUCCUCACU
 628
 7869
CAUGAGCAAACUCCUCACU
 628
 7887
AGUGAGGAGUUUGCUCAUG
1895


 


 7941
ACCCAAGAUAAGAGUGUAC
 629
 7941
ACCCAAGAUAAGAGUGUAC
 629
 7959
GUACACUCUUAUCUUGGGU
1896


 


 7959
CAAUACUGUCAUAUCAUAU
 630
 7959
CAAUACUGUCAUAUCAUAU
 630
 7977
AUAUGAUAUGACAGUAUUG
1897


 


 7977
UAUUGAAAGCAACAGGAAA
 631
 7977
UAUUGAAAGCAACAGGAAA
 631
 7995
UUUCCUGUUGCUUUCAAUA
1898


 


 7995
AAACAAUAAACAAACUAUC
 632
 7995
AAACAAUAAACAAACUAUC
 632
 8013
GAUAGUUUGUUUAUUGUUU
1899


 


 8013
CCAUCUGUUAAAAAGAUUG
 633
 8013
CCAUCUGUUAAAAAGAUUG
 633
 8031
CAAUCUUUUUAACAGAUGG
1900


 


 8031
GCCAGCAGACGUAUUGAAG
 634
 8031
GCCAGCAGACGUAUUGAAG
 634
 8049
CUUCAAUACGUCUGCUGGC
1901


 


 8139
CAAAAAUAAUGAUACUACC
 635
 8139
CAAAAAUAAUGAUACUACC
 635
 8157
GGUAGUAUCAUUAUUUUUG
1902


 


 8157
CUGACAAAUAUCCUUGUAG
 636
 8157
CUGACAAAUAUCCUUGUAG
 636
 8175
CUACAAGGAUAUUUGUCAG
1903


 


 8229
AGAACACACUAUAUUUCAA
 637
 8229
AGAACACACUAUAUUUCAA
 637
 8247
UUGAAAUAUAGUGUGUUCU
1904


 


 8391
UAGUGUCAUAACACUCAAU
 638
 8391
UAGUGUCAUAACACUCAAU
 638
 8409
AUUGAGUGUUAUGACACUA
1905


 


 8481
UUAAUGGAAAUUCUGCUAA
 639
 8481
UUAAUGGAAAUUCUGCUAA
 639
 8499
UUAGCAGAAUUUCCAUUAA
1906


 


 8553
AUGCUUUAGGAAGUUACAU
 640
 8553
AUGCUUUAGGAAGUUACAU
 640
 8571
AUGUAACUUCCUAAAGCAU
1907


 


 8571
UAUUCAAUGGUCCUUAUCU
 641
 8571
UAUUCAAUGGUCCUUAUCU
 641
 8589
AGAUAAGGACCAUUGAAUA
1908


 


 8589
UCAAAAAUGAUUAUACCAA
 642
 8589
UCAAAAAUGAUUAUACCAA
 642
 8607
UUGGUAUAAUCAUUUUUGA
1909


 


 8607
ACUUAAUUAGUAGACAAAA
 643
 8607
ACUUAAUUAGUAGACAAAA
 643
 8625
UUUUGUCUACUAAUUAAGU
1910


 


 8625
AUCCAUUAAUAGAACACAU
 644
 8625
AUCCAUUAAUAGAACACAU
 644
 8643
AUGUGUUCUAUUAAUGGAU
1911


 


 8661
AUAUAACACAGUCCUUAAU
 645
 8661
AUAUAACACAGUCCUUAAU
 645
 8679
AUUAAGGACUGUGUUAUAU
1912


 


 8733
CAUUACUUAUGACAUACAA
 646
 8733
CAUUACUUAUGACAUACAA
 646
 8751
UUGUAUGUCAUAAGUAAUG
1913


 


 8787
AUUUACUUAAAAAGAUAAU
 647
 8787
AUUUACUUAAAAAGAUAAU
 647
 8805
AUUAUCUUUUUAAGUAAAU
1914


 


 8805
UAAGAAGAGCUAUAGAAAU
 648
 8805
UAAGAAGAGCUAUAGAAAU
 648
 8823
AUUUCUAUAGCUCUUCUUA
1915


 


 8841
AUGCUAUAUUGAAUAAACU
 649
 8841
AUGCUAUAUUGAAUAAACU
 649
 8859
AGUUUAUUCAAUAUAGCAU
1916


 


 8877
ACAAGAUUAAAUCCAACAA
 650
 8877
ACAAGAUUAAAUCCAACAA
 650
 8895
UUGUUGGAUUUAAUCUUGU
1917


 


 8931
UAAUCAAAGAUGAUAUACU
 651
 8931
UAAUCAAAGAUGAUAUACU
 651
 8949
AGUAUAUCAUCUUUGAUUA
1918


 


 8967
AUCAAUCUCAUCUUAAAGC
 652
 8967
AUCAAUCUCAUCUUAAAGC
 652
 8985
GCUUUAAGAUGAGAUUGAU
1919


 


 9075
GGUUAAUACAUUGGUUUAA
 653
 9075
GGUUAAUACAUUGGUUUAA
 653
 9093
UUAAACCAAUGUAUUAACC
1920


 


 9111
ACAACAUAUUAACACAGUA
 654
 9111
ACAACAUAUUAACACAGUA
 654
 9129
UACUGUGUUAAUAUGUUGU
1921


 


 9219
GUAUAGUUUAUCAUAAGGA
 655
 9219
GUAUAGUUUAUCAUAAGGA
 655
 9237
UCCUUAUGAUAAACUAUAC
1922


 


 9237
AACUCAAAAGAAUUACUGU
 656
 9237
AACUCAAAAGAAUUACUGU
 656
 9255
ACAGUAAUUCUUUUGAGUU
1923


 


 9255
UGACAACCUAUAAUCAAUU
 657
 9255
UGACAACCUAUAAUCAAUU
 657
 9273
AAUUGAUUAUAGGUUGUCA
1924


 


 9291
UUAGCCUUAGUAGAUUAAA
 658
 9291
UUAGCCUUAGUAGAUUAAA
 658
 9309
UUUAAUCUACUAAGGCUAA
1925


 


 9309
AUGUUUGUUUAAUUACAUG
 659
 9309
AUGUUUGUUUAAUUACAUG
 659
 9327
CAUGUAAUUAAACAAACAU
1926


 


 9345
ACACAUUAAAUAAAAGCUU
 660
 9345
ACACAUUAAAUAAAAGCUU
 660
 9363
AAGCUUUUAUUUAAUGUGU
1927


 


 9381
UCAAUAAUGUUAUCUUGAC
 661
 9381
UCAAUAAUGUUAUCUUGAC
 661
 9399
GUCAAGAUAACAUUAUUGA
1928


 


 9453
UCUACAUAAUAAAAGAGGU
 662
 9453
UCUACAUAAUAAAAGAGGU
 662
 9471
ACCUCUUUUAUUAUGUAGA
1929


 


 9489
CUCUAAUUUUAAAUAUAAC
 663
 9489
CUCUAAUUUUAAAUAUAAC
 663
 9507
GUUAUAUUUAAAAUUAGAG
1930


 


 9507
CAGAAGAAGAUCAAUUCAG
 664
 9507
CAGAAGAAGAUCAAUUCAG
 664
 9525
CUGAAUUGAUCUUCUUCUG
1931


 


 9543
GUAUGCUCAACAACAUCAC
 665
 9543
GUAUGCUCAACAACAUCAC
 665
 9561
GUGAUGUUGUUGAGCAUAC
1932


 


 9561
CAGAUGCUGCUAAUAAAGC
 666
 9561
CAGAUGCUGCUAAUAAAGC
 666
 9579
GCUUUAUUAGCAGCAUCUG
1933


 


 9597
CAAGAGUAUGUCAUACAUU
 667
 9597
CAAGAGUAUGUCAUACAUU
 667
 9615
AAUGUAUGACAUACUCUUG
1934


 


 9633
CCGAUAAUAUAAUAAAUGG
 668
 9633
CCGAUAAUAUAAUAAAUGG
 668
 9651
CCAUUUAUUAUAUUAUCGG
1935


 


 9651
GCAGAUGGAUAAUUCUAUU
 669
 9651
GCAGAUGGAUAAUUCUAUU
 669
 9669
AAUAGAAUUAUCCAUCUGC
1936


 


 9669
UAAGUAAGUUCCUUAAAUU
 670
 9669
UAAGUAAGUUCCUUAAAUU
 670
 9687
AAUUUAAGGAACUUACUUA
1937


 


 9687
UAAUUAAGCUUGCAGGUGA
 671
 9687
UAAUUAAGCUUGCAGGUGA
 671
 9705
UCACCUGCAAGCUUAAUUA
1938


 


 9705
ACAAUAACCUUAACAAUCU
 672
 9705
ACAAUAACCUUAACAAUCU
 672
 9723
AGAUUGUUAAGGUUAUUGU
1939


 


 9741
UGUUCAGAAUAUUUGGACA
 673
 9741
UGUUCAGAAUAUUUGGACA
 673
 9759
UGUCCAAAUAUUCUGAACA
1940


 


 9759
ACCCAAUGGUAGAUGAAAG
 674
 9759
ACCCAAUGGUAGAUGAAAG
 674
 9777
CUUUCAUCUACCAUUGGGU
1941


 


 9777
GACAAGCCAUGGAUGCUGU
 675
 9777
GACAAGCCAUGGAUGCUGU
 675
 9795
ACAGCAUCCAUGGCUUGUC
1942


 


 9813
AGACCAAAUUUUACUUGUU
 676
 9813
AGACCAAAUUUUACUUGUU
 676
 9831
AACAAGUAAAAUUUGGUCU
1943


 


 9849
UAAGAGGUGCCUUUAUAUA
 677
 9849
UAAGAGGUGCCUUUAUAUA
 677
 9867
UAUAUAAAGGCACCUCUUA
1944


 


 9885
UUGUAAAUAAUUACAACAG
 678
 9885
UUGUAAAUAAUUACAACAG
 678
 9903
CUGUUGUAAUUAUUUACAA
1945


 


 9921
AUGCUAUUGUUUUACCCUU
 679
 9921
AUGCUAUUGUUUUACCCUU
 679
 9939
AAGGGUAAAACAAUAGCAU
1946


 


 9939
UAAGAUGGUUAACUUACUA
 680
 9939
UAAGAUGGUUAACUUACUA
 680
 9957
UAGUAAGUUAACCAUCUUA
1947


 


 9957
AUAAACUAAACACUUAUCC
 681
 9957
AUAAACUAAACACUUAUCC
 681
 9975
GGAUAAGUGUUUAGUUUAU
1948


 


 9993
CAGAAAGAGAUUUGAUUGU
 682
 9993
CAGAAAGAGAUUUGAUUGU
 682
10011
ACAAUCAAAUCUCUUUCUG
1949


 


10029
UCUAUCGUGAGUUUCGGUU
 683
10029
UCUAUCGUGAGUUUCGGUU
 683
10047
AACCGAAACUCACGAUAGA
1950


 


10101
CUAAAAAUUUGAUAUGGAC
 684
10101
CUAAAAAUUUGAUAUGGAC
 684
10119
GUCCAUAUCAAAUUUUUAG
1951


 


10119
CUAGUUUCCCUAGAAAUUA
 685
10119
CUAGUUUCCCUAGAAAUUA
 685
10137
UAAUUUCUAGGGAAACUAG
1952


 


10173
AAAAAUUAAAAUUUUCCGA
 686
10173
AAAAAUUAAAAUUUUCCGA
 686
10191
UCGGAAAAUUUUAAUUUUU
1953


 


10191
AGAGUGAUAAAUCAAGAAG
 687
10191
AGAGUGAUAAAUCAAGAAG
 687
10209
CUUCUUGAUUUAUCACUCU
1954


 


10227
UAAGAGAUAACAAAUUCAA
 688
10227
UAAGAGAUAACAAAUUCAA
 688
10245
UUGAAUUUGUUAUCUCUUA
1955


 


10245
AUGAAUGUGAUUUAUACAA
 689
10245
AUGAAUGUGAUUUAUACAA
 689
10263
UUGUAUAAAUCACAUUCAU
1956


 


10263
ACUGUGUAGUUAAUCAAAG
 690
10263
ACUGUGUAGUUAAUCAAAG
 690
10281
CUUUGAUUAACUACACAGU
1957


 


10317
CAGGCAAAGAAAGAGAACU
 691
10317
CAGGCAAAGAAAGAGAACU
 691
10335
AGUUCUCUUUCUUUGCCUG
1958


 


10407
UAGCUGAAAACAUUUUACA
 692
10407
UAGCUGAAAACAUUUUACA
 692
10425
UGUAAAAUGUUUUCAGCUA
1959


 


10425
AAUUCUUUCCUGAAAGUCU
 693
10425
AAUUCUUUCCUGAAAGUCU
 693
10443
AGACUUUCAGGAAAGAAUU
1960


 


10443
UUACAAGAUAUGGUGAUCU
 694
10443
UUACAAGAUAUGGUGAUCU
 694
10461
AGAUCACCAUAUCUUGUAA
1961


 


10497
UAAGUAACAAAUCAAAUCG
 695
10497
UAAGUAACAAAUCAAAUCG
 695
10515
CGAUUUGAUUUGUUACUUA
1962


 


10533
ACAAUUACAUUAGUAAGUG
 696
10533
ACAAUUACAUUAGUAAGUG
 696
10551
CACUUACUAAUGUAAUUGU
1963


 


10551
GCUCUAUCAUCACAGAUCU
 697
10551
GCUCUAUCAUCACAGAUCU
 697
10569
AGAUCUGUGAUGAUAGAGC
1964


 


10569
UCAGCAAAUUCAAUCAAGC
 698
10569
UCAGCAAAUUCAAUCAAGC
 698
10587
GCUUGAUUGAAUUUGCUGA
1965


 


10605
CAUGUAUUUGUAGUGAUGU
 699
10605
CAUGUAUUUGUAGUGAUGU
 699
10623
ACAUCACUACAAAUACAUG
1966


 


10677
UUCCUCAUGUCACAAUAAU
 700
10677
UUCCUCAUGUCACAAUAAU
 700
10695
AUUAUUGUGACAUGAGGAA
1967


 


10695
UAUGCACAUAUAGGCAUGC
 701
10695
UAUGCACAUAUAGGCAUGC
 701
10713
GCAUGCCUAUAUGUGCAUA
1968


 


10731
AUCAUAUUGUAGAUCUUAA
 702
10731
AUCAUAUUGUAGAUCUUAA
 702
10749
UUAAGAUCUACAAUAUGAU
1969


 


10749
ACAAUGUAGAUGAACAAAG
 703
10749
ACAAUGUAGAUGAACAAAG
 703
10767
CUUUGUUCAUCUACAUUGU
1970


 


10767
GUGGAUUAUAUAGAUAUCA
 704
10767
GUGGAUUAUAUAGAUAUCA
 704
10785
UGAUAUCUAUAUAAUCCAC
1971


 


10803
GGUGGUGUCAAAAACUAUG
 705
10803
GGUGGUGUCAAAAACUAUG
 705
10821
CAUAGUUUUUGACACCACC
1972


 


10821
GGACCAUAGAAGCUAUAUC
 706
10821
GGACCAUAGAAGCUAUAUC
 706
10839
GAUAUAGCUUCUAUGGUCC
1973


 


10839
CACUAUUGGAUCUAAUAUC
 707
10839
CACUAUUGGAUCUAAUAUC
 707
10857
GAUAUUAGAUCCAAUAGUG
1974


 


10875
CAAUUACUGCUUUAAUUAA
 708
10875
CAAUUACUGCUUUAAUUAA
 708
10893
UUAAUUAAAGCAGUAAUUG
1975


 


10893
AUGGUGACAAUCAAUCAAU
 709
10893
AUGGUGACAAUCAAUCAAU
 709
10911
AUUGAUUGAUUGUCACCAU
1976


 


10929
UCAGACUCAUGGAAGGUCA
 710
10929
UCAGACUCAUGGAAGGUCA
 710
10947
UGACCUUCCAUGAGUCUGA
1977


 


10965
AUUAUUUGCUAGCAUUAAA
 711
10965
AUUAUUUGCUAGCAUUAAA
 711
10983
UUUAAUGCUAGCAAAUAAU
1978


 


11037
GAACUGAGACUUAUAUAUC
 712
11037
GAACUGAGACUUAUAUAUC
 712
11055
GAUAUAUAAGUCUCAGUUC
1979


 


11073
UGAGUAAAACAAUUCAACA
 713
11073
UGAGUAAAACAAUUCAACA
 713
11091
UGUUGAAUUGUUUUACUCA
1980


 


11091
AUAACGGUGUAUAUUACCC
 714
11091
AUAACGGUGUAUAUUACCC
 714
11109
GGGUAAUAUACACCGUUAU
1981


 


11127
UCCUAAGAGUGGGACCGUG
 715
11127
UCCUAAGAGUGGGACCGUG
 715
11145
CACGGUCCCACUCUUAGGA
1982


 


11145
GGAUAAACACUAUACUUGA
 716
11145
GGAUAAACACUAUACUUGA
 716
11163
UCAAGUAUAGUGUUUAUCC
1983


 


11163
AUGAUUUCAAAGUGAGUCU
 717
11163
AUGAUUUCAAAGUGAGUCU
 717
11181
AGACUCACUUUGAAAUCAU
1984


 


11181
UAGAAUCUAUAGGUAGUUU
 718
11181
UAGAAUCUAUAGGUAGUUU
 718
11199
AAACUACCUAUAGAUUCUA
1985


 


11199
UGACACAAGAAUUAGAAUA
 719
11199
UGACACAAGAAUUAGAAUA
 719
11217
UAUUCUAAUUCUUGUGUCA
1986


 


11253
GAAAUGUAUGGUUAUAUAA
 720
11253
GAAAUGUAUGGUUAUAUAA
 720
11271
UUAUAUAACCAUACAUUUC
1987


 


11325
UGGACAUAUUAAAGGUUCU
 721
11325
UGGACAUAUUAAAGGUUCU
 721
11343
AGAACCUUUAAUAUGUCCA
1988


 


11343
UGAAACACUUAAAAACCUU
 722
11343
UGAAACACUUAAAAACCUU
 722
11361
AAGGUUUUUAAGUGUUUCA
1989


 


11361
UUUUUAAUCUUGAUAAUAU
 723
11361
UUUUUAAUCUUGAUAAUAU
 723
11379
AUAUUAUCAAGAUUAAAAA
1990


 


11379
UUGAUACAGCAUUAACAUU
 724
11379
UUGAUACAGCAUUAACAUU
 724
11397
AAUGUUAAUGCUGUAUCAA
1991


 


11415
UGUUAUUUGGUGGUGGUGA
 725
11415
UGUUAUUUGGUGGUGGUGA
 725
11433
UCACCACCACCAAAUAACA
1992


 


11433
AUCCCAACUUGUUAUAUCG
 726
11433
AUCCCAACUUGUUAUAUCG
 726
11451
CGAUAUAACAAGUUGGGAU
1993


 


11451
GAAGUUUCUAUAGAAGAAC
 727
11451
GAAGUUUCUAUAGAAGAAC
 727
11469
GUUCUUCUAUAGAAACUUC
1994


 


11487
AGGCUAUAGUUCACUCUGU
 728
11487
AGGCUAUAGUUCACUCUGU
 728
11505
ACAGAGUGAACUAUAGCCU
1995


 


11505
UGUUCAUACUUAGUUAUUA
 729
11505
UGUUCAUACUUAGUUAUUA
 729
11523
UAAUAACUAAGUAUGAACA
1996


 


11559
UGUCAGAUGAUAGAUUGAA
 730
11559
UGUCAGAUGAUAGAUUGAA
 730
11577
UUCAAUCUAUCAUCUGACA
1997


 


11577
AUAAGUUCUUAACAUGCAU
 731
11577
AUAAGUUCUUAACAUGCAU
 731
11595
AUGCAUGUUAAGAACUUAU
1998


 


11613
ACCCUAAUGCUGAAUUCGU
 732
11613
ACCCUAAUGCUGAAUUCGU
 732
11631
ACGAAUUCAGCAUUAGGGU
1999


 


11631
UAACAUUGAUGAGAGAUCC
 733
11631
UAACAUUGAUGAGAGAUCC
 733
11649
GGAUCUCUCAUCAAUGUUA
2000


 


11649
CUCAAGCUUUAGGGUCUGA
 734
11649
CUCAAGCUUUAGGGUCUGA
 734
11667
UCAGACCCUAAAGCUUGAG
2001


 


11667
AGAGACAAGCUAAAAUUAC
 735
11667
AGAGACAAGCUAAAAUUAC
 735
11685
GUAAUUUUAGCUUGUCUCU
2002


 


11739
AAAUAUUCUCCAAAAGUGC
 736
11739
AAAUAUUCUCCAAAAGUGC
 736
11757
GCACUUUUGGAGAAUAUUU
2003


 


11775
CAGAGAUAGAUCUAAAUGA
 737
11775
CAGAGAUAGAUCUAAAUGA
 737
11793
UCAUUUAGAUCUAUCUCUG
2004


 


11829
GGCUAAGAGUUGUUUAUGA
 738
11829
GGCUAAGAGUUGUUUAUGA
 738
11847
UCAUAAACAACUCUUAGCC
2005


 


11847
AAAGUUUACCCUUUUAUAA
 739
11847
AAAGUUUACCCUUUUAUAA
 739
11865
UUAUAAAAGGGUAAACUUU
2006


 


11865
AAGCAGAGAAAAUAGUAAA
 740
11865
AAGCAGAGAAAAUAGUAAA
 740
11883
UUUACUAUUUUCUCUGCUU
2007


 


11901
AAUCUAUAACUAACAUACU
 741
11901
AAUCUAUAACUAACAUACU
 741
11919
AGUAUGUUAGUUAUAGAUU
2008


 


11937
UAGACUUAACAGAUAUUGA
 742
11937
UAGACUUAACAGAUAUUGA
 742
11955
UCAAUAUCUGUUAAGUCUA
2009


 


11955
AUAGAGCCACUGAGAUGAU
 743
11955
AUAGAGCCACUGAGAUGAU
 743
11973
AUCAUCUCAGUGGCUCUAU
2010


 


11973
UGAGGAAAAACAUAACUUU
 744
11973
UGAGGAAAAACAUAACUUU
 744
11991
AAAGUUAUGUUUUUCCUCA
2011


 


12045
GUAUGGAAAACCUAAGUAU
 745
12045
GUAUGGAAAACCUAAGUAU
 745
12063
AUACUUAGGUUUUCCAUAC
2012


 


12063
UUACUGAAUUAAGCAAAUA
 746
12063
UUACUGAAUUAAGCAAAUA
 746
12081
UAUUUGCUUAAUUCAGUAA
2013


 


12081
AUGUUAGGGAAAGAUCUUG
 747
12081
AUGUUAGGGAAAGAUCUUG
 747
12099
CAAGAUCUUUCCCUAACAU
2014


 


12099
GGUCUUUAUCCAAUAUAGU
 748
12099
GGUCUUUAUCCAAUAUAGU
 748
12117
ACUAUAUUGGAUAAAGACC
2015


 


12135
GUAUCAUGUAUACAAUGGA
 749
12135
GUAUCAUGUAUACAAUGGA
 749
12153
UCCAUUGUAUACAUGAUAC
2016


 


12207
AUGUUAACAGUUUAACACG
 750
12207
AUGUUAACAGUUUAACACG
 750
12225
CGUGUUAAACUGUUAACAU
2017


 


12243
CUAAACCAUGGGUUGGUUC
 751
12243
CUAAACCAUGGGUUGGUUC
 751
12261
GAACCAACCCAUGGUUUAG
2018


 


12261
CAUCUACACAAGAGAAAAA
 752
12261
CAUCUACACAAGAGAAAAA
 752
12279
UUUUUCUCUUGUGUAGAUG
2019


 


12279
AAACAAUGCCAGUUUAUAA
 753
12279
AAACAAUGCCAGUUUAUAA
 753
12297
UUAUAAACUGGCAUUGUUU
2020


 


12297
AUAGACAAGUUUUAACCAA
 754
12297
AUAGACAAGUUUUAACCAA
 754
12315
UUGGUUAAAACUUGUCUAU
2021


 


12333
UAGAUCUAUUAGCAAAAUU
 755
12333
UAGAUCUAUUAGCAAAAUU
 755
12351
AAUUUUGCUAAUAGAUCUA
2022


 


12351
UGGAUUGGGUGUAUGCAUC
 756
12351
UGGAUUGGGUGUAUGCAUC
 756
12369
GAUGCAUACACCCAAUCCA
2023


 


12369
CUAUAGAUAACAAGGAUGA
 757
12369
CUAUAGAUAACAAGGAUGA
 757
12387
UCAUCCUUGUUAUCUAUAG
2024


 


12477
UGCAUCGCCUUACAGUCAG
 758
12477
UGCAUCGCCUUACAGUCAG
 758
12495
CUGACUGUAAGGCGAUGCA
2025


 


12567
CUAUUAAUCGCAUAUUAAC
 759
12567
CUAUUAAUCGCAUAUUAAC
 759
12585
GUUAAUAUGCGAUUAAUAG
2026


 


12585
CAGAAAAGUAUGGUGAUGA
 760
12585
CAGAAAAGUAUGGUGAUGA
 760
12603
UCAUCACCAUACUUUUCUG
2027


 


12621
UCCAAAACUGUAUAAGCUU
 761
12621
UCCAAAACUGUAUAAGCUU
 761
12639
AAGCUUAUACAGUUUUGGA
2028


 


12657
CAGUAGUAGAACAAUUUAC
 762
12657
CAGUAGUAGAACAAUUUAC
 762
12675
GUAAAUUGUUCUACUACUG
2029


 


12675
CUAAUGUAUGUCCUAACAG
 763
12675
CUAAUGUAUGUCCUAACAG
 763
12693
CUGUUAGGACAUACAUUAG
2030


 


12711
AGCUUAAUGAGAUACAUUU
 764
12711
AGCUUAAUGAGAUACAUUU
 764
12729
AAAUGUAUCUCAUUAAGCU
2031


 


12747
UCACAGGUGAUGUUGAUAU
 765
12747
UCACAGGUGAUGUUGAUAU
 765
12765
AUAUCAACAUCACCUGUGA
2032


 


12765
UUCACAAGUUAAAACAAGU
 766
12765
UUCACAAGUUAAAACAAGU
 766
12783
ACUUGUUUUAACUUGUGAA
2033


 


12801
UGUUUUUACCAGACAAAAU
 767
12801
UGUUUUUACCAGACAAAAU
 767
12819
AUUUUGUCUGGUAAAAACA
2034


 


12819
UAAGUUUGACUCAAUAUGU
 768
12819
UAAGUUUGACUCAAUAUGU
 768
12837
ACAUAUUGAGUCAAACUUA
2035


 


12837
UGGAAUUAUUCUUAAGUAA
 769
12837
UGGAAUUAUUCUUAAGUAA
 769
12855
UUACUUAAGAAUAAUUCCA
2036


 


12873
GAUCUCAUGUUAAUUCUAA
 770
12873
GAUCUCAUGUUAAUUCUAA
 770
12891
UUAGAAUUAACAUGAGAUC
2037


 


12927
AUAAUACUUACAUUUUAAG
 771
12927
AUAAUACUUACAUUUUAAG
 771
12945
CUUAAAAUGUAAGUAUUAU
2038


 


12981
AACUUAUGAAAGAUUCUAA
 772
12981
AACUUAUGAAAGAUUCUAA
 772
12999
UUAGAAUCUUUCAUAAGUU
2039


 


13017
AUUGGGGAGAGGGAUAUAU
 773
13017
AUUGGGGAGAGGGAUAUAU
 773
13035
AUAUAUCCCUCUCCCCAAU
2040


 


13053
UUAAUUUGAAAGUUUUCUU
 774
13053
UUAAUUUGAAAGUUUUCUU
 774
13071
AAGAAAACUUUCAAAUUAA
2041


 


13071
UCAAUGCUUAUAAGACCUA
 775
13071
UCAAUGCUUAUAAGACCUA
 775
13089
UAGGUCUUAUAAGCAUUGA
2042


 


13089
AUCUCUUGUGUUUUCAUAA
 776
13089
AUCUCUUGUGUUUUCAUAA
 776
13107
UUAUGAAAACACAAGAGAU
2043


 


13161
GUGUAUUGGAAUUAAUAGA
 777
13161
GUGUAUUGGAAUUAAUAGA
 777
13179
UCUAUUAAUUCCAAUACAC
2044


 


13179
ACAGUAGUUAUUGGAAGUC
 778
13179
ACAGUAGUUAUUGGAAGUC
 778
13197
GACUUCCAAUAACUACUGU
2045


 


13197
CUAUGUCUAAGGUAUUUUU
 779
13197
CUAUGUCUAAGGUAUUUUU
 779
13215
AAAAAUACCUUAGACAUAG
2046


 


13215
UAGAACAAAAAGUUAUCAA
 780
13215
UAGAACAAAAAGUUAUCAA
 780
13233
UUGAUAACUUUUUGUUCUA
2047


 


13251
AUGCAAGUUUACAUAGAGU
 781
13251
AUGCAAGUUUACAUAGAGU
 781
13269
ACUCUAUGUAAACUUGCAU
2048


 


13269
UAAAAGGAUGUCAUAGCUU
 782
13269
UAAAAGGAUGUCAUAGCUU
 782
13287
AAGCUAUGACAUCCUUUUA
2049


 


13359
AUCAUCCAACACAUAUGAA
 783
13359
AUCAUCCAACACAUAUGAA
 783
13377
UUCAUAUGUGUUGGAUGAU
2050


 


13377
AAGCAAUAUUAACUUAUAU
 784
13377
AAGCAAUAUUAACUUAUAU
 784
13395
AUAUAAGUUAAUAUUGCUU
2051


 


13395
UAGAUCUUGUUAGAAUGGG
 785
13395
UAGAUCUUGUUAGAAUGGG
 785
13413
CCCAUUCUAACAAGAUCUA
2052


 


13467
AAUUUUAUACUUCUAAUCU
 786
13467
AAUUUUAUACUUCUAAUCU
 786
13485
AGAUUAGAAGUAUAAAAUU
2053


 


13503
ACUUCUCAGAUAAUACUCA
 787
13503
ACUUCUCAGAUAAUACUCA
 787
13521
UGAGUAUUAUCUGAGAAGU
2054


 


13557
AAUUAGAAAAUAAUUACAA
 788
13557
AAUUAGAAAAUAAUUACAA
 788
13575
UUGUAAUUAUUUUCUAAUU
2055


 


13575
ACAAAUUAUAUCAUCCUAC
 789
13575
ACAAAUUAUAUCAUCCUAC
 789
13593
GUAGGAUGAUAUAAUUUGU
2056


 


13665
GUAUAGGUAAAAAUGUUGA
 790
13665
GUAUAGGUAAAAAUGUUGA
 790
13683
UCAACAUUUUUACCUAUAC
2057


 


13683
ACUCAAUAAUGUUACCAUU
 791
13683
ACUCAAUAAUGUUACCAUU
 791
13701
AAUGGUAACAUUAUUGAGU
2058


 


13701
UGUUAUCUAAUAAGAAGCU
 792
13701
UGUUAUCUAAUAAGAAGCU
 792
13719
AGCUUCUUAUUAGAUAACA
2059


 


13737
UGAUUAGAACCAAUUACAG
 793
13737
UGAUUAGAACCAAUUACAG
 793
13755
CUGUAAUUGGUUCUAAUCA
2060


 


13917
AUAUUAAUAGAUUCAAUUU
 794
13917
AUAUUAAUAGAUUCAAUUU
 794
13935
AAAUUGAAUCUAUUAAUAU
2061


 


13935
UUGUAUUUAGUUCUACAGG
 795
13935
UUGUAUUUAGUUCUACAGG
 795
13953
CCUGUAGAACUAAAUACAA
2062


 


13953
GUUGUAAAAUUAGUAUAGA
 796
13953
GUUGUAAAAUUAGUAUAGA
 796
13971
UCUAUACUAAUUUUACAAC
2063


 


14007
AUUGUAUAGCAUUCAUAGG
 797
14007
AUUGUAUAGCAUUCAUAGG
 797
14025
CCUAUGAAUGCUAUACAAU
2064


 


14025
GUGAAGGAGCAGGGAAUUU
 798
14025
GUGAAGGAGCAGGGAAUUU
 798
14043
AAAUUCCCUGCUCCUUCAC
2065


 


14079
UAAGAUAUAUUUACAGAAG
 799
14079
UAAGAUAUAUUUACAGAAG
 799
14097
CUUCUGUAAAUAUAUCUUA
2066


 


14151
AUGGACAUAUCAACAUUGA
 800
14151
AUGGACAUAUCAACAUUGA
 800
14169
UCAAUGUUGAUAUGUCCAU
2067


 


14169
AUUAUGGUGAAAAUUUGAC
 801
14169
AUUAUGGUGAAAAUUUGAC
 801
14187
GUCAAAUUUUCACCAUAAU
2068


 


14205
CAACCAACAACAUUCAUUG
 802
14205
CAACCAACAACAUUCAUUG
 802
14223
CAAUGAAUGUUGUUGGUUG
2069


 


14241
AGUUUGCUGAACCUAUCAG
 803
14241
AGUUUGCUGAACCUAUCAG
 803
14259
CUGAUAGGUUCAGCAAACU
2070


 


14295
UCAACUGGAGUAAAAUUAU
 804
14295
UCAACUGGAGUAAAAUUAU
 804
14313
AUAAUUUUACUCCAGUUGA
2071


 


14349
ACUGUUCCUCAGUUAAUAA
 805
14349
ACUGUUCCUCAGUUAAUAA
 805
14367
UUAUUAACUGAGGAACAGU
2072


 


14421
ACAAUAUAACUAUAUUAAA
 806
14421
ACAAUAUAACUAUAUUAAA
 806
14439
UUUAAUAUAGUUAUAUUGU
2073


 


14529
AUGUAGUACAAAAUGCUAA
 807
14529
AUGUAGUACAAAAUGCUAA
 807
14547
UUAGCAUUUUGUACUACAU
2074


 


14547
AAUUGAUACUAUCAAGAAC
 808
14547
AAUUGAUACUAUCAAGAAC
 808
14565
GUUCUUGAUAGUAUCAAUU
2075


 


14565
CCAAAAAUUUCAUCAUGCC
 809
14565
CCAAAAAUUUCAUCAUGCC
 809
14583
GGCAUGAUGAAAUUUUUGG
2076


 


14601
AGUCUAUUGAUGCAAAUAU
 810
14601
AGUCUAUUGAUGCAAAUAU
 810
14619
AUAUUUGCAUCAAUAGACU
2077


 


14619
UUAAAAGUUUGAUACCCUU
 811
14619
UUAAAAGUUUGAUACCCUU
 811
14637
AAGGGUAUCAAACUUUUAA
2078


 


14637
UUCUUUGUUACCCUAUAAC
 812
14637
UUCUUUGUUACCCUAUAAC
 812
14655
GUUAUAGGGUAACAAAGAA
2079


 


14655
CAAAAAAAGGAAUUAAUAC
 813
14655
CAAAAAAAGGAAUUAAUAC
 813
14673
GUAUUAAUUCCUUUUUUUG
2080


 


14673
CUGCAUUGUCAAAACUAAA
 814
14673
CUGCAUUGUCAAAACUAAA
 814
14691
UUUAGUUUUGACAAUGCAG
2081


 


14691
AGAGUGUUGUUAGUGGAGA
 815
14691
AGAGUGUUGUUAGUGGAGA
 815
14709
UCUCCACUAACAACACUCU
2082


 


14709
AUAUACUAUCAUAUUCUAU
 816
14709
AUAUACUAUCAUAUUCUAU
 816
14727
AUAGAAUAUGAUAGUAUAU
2083


 


14745
UUUUCAGCAAUAAACUUAU
 817
14745
UUUUCAGCAAUAAACUUAU
 817
14763
AUAAGUUUAUUGCUGAAAA
2084


 


14763
UAAAUCAUAAGCAUAUGAA
 818
14763
UAAAUCAUAAGCAUAUGAA
 818
14781
UUCAUAUGCUUAUGAUUUA
2085


 


14799
AUCAUGUUUUAAAUUUCAG
 819
14799
AUCAUGUUUUAAAUUUCAG
 819
14817
CUGAAAUUUAAAACAUGAU
2086


 


14817
GAUCAACAGAACUAAACUA
 820
14817
GAUCAACAGAACUAAACUA
 820
14835
UAGUUUAGUUCUGUUGAUC
2087


 


14853
UAGAAUCUACAUAUCCUUA
 821
14853
UAGAAUCUACAUAUCCUUA
 821
14871
UAAGGAUAUGUAGAUUCUA
2088


 


14907
AACUUAAAAAACUGAUUAA
 822
14907
AACUUAAAAAACUGAUUAA
 822
14925
UUAAUCAGUUUUUUAAGUU
2089


 


14943
UAUACAACUUUCAUAAUGA
 823
14943
UAUACAACUUUCAUAAUGA
 823
14961
UCAUUAUGAAAGUUGUAUA
2090


 


15159
CGAGAUAUUAGUUUUUGAC
 824
15159
CGAGAUAUUAGUUUUUGAC
 824
15177
GUCAAAAACUAAUAUCUCG
2091


 


    3
GCGAAAAAAUGCGUACAAC
 825
    3
GCGAAAAAAUGCGUACAAC
 825
   21
GUUGUACGCAUUUUUUCGC
2092


 


  111
UCAUUGAGUAUGAUAAAAG
 826
  111
UCAUUGAGUAUGAUAAAAG
 826
  129
CUUUUAUCAUACUCAAUGA
2093


 


  291
AUUUGCCCUAAUAAUAAUA
 827
  291
AUUUGCCCUAAUAAUAAUA
 827
  309
UAUUAUUAUUAGGGCAAAU
2094


 


  543
AUCAAUGUCACUAACACCA
 828
  543
AUCAAUGUCACUAACACCA
 828
  561
UGGUGUUAGUGACAUUGAU
2095


 


  597
GGGCAAAUAAAUCAAUUCA
 829
  597
GGGCAAAUAAAUCAAUUCA
 829
  615
UGAAUUGAUUUAUUUGCCC
2096


 


  741
AUUUAUAUACUUGAUAAAU
 830
  741
AUUUAUAUACUUGAUAAAU
 830
  759
AUUUAUCAAGUAUAUAAAU
2097


 


 1083
UUAUAGUAAUUUAAAAUUA
 831
 1083
UUAUAGUAAUUUAAAAUUA
 831
 1101
UAAUUUUAAAUUACUAUAA
2098


 


 1587
AGGCAUGACUCUCCUGAUU
 832
 1587
AGGCAUGACUCUCCUGAUU
 832
 1605
AAUCAGGAGAGUCAUGCCU
2099


 


 1641
AUAACCAAAUUAGCAGCAG
 833
 1641
AUAACCAAAUUAGCAGCAG
 833
 1659
CUGCUGCUAAUUUGGUUAU
2100


 


 1821
AUAGCACAAUCUUCUACCA
 834
 1821
AUAGCACAAUCUUCUACCA
 834
 1839
UGGUAGAAGAUUGUGCUAU
2101


 


 1893
GGUGCAGGGCAAGUGAUGU
 835
 1893
GGUGCAGGGCAAGUGAUGU
 835
 1911
ACAUCACUUGCCCUGCACC
2102


 


 2091
CCUCACUUCUCCAGUGUAG
 836
 2091
CCUCACUUCUCCAGUGUAG
 836
 2109
CUACACUGGAGAAGUGAGG
2103


 


 2397
CUACCAAAUUCCUAGAAUC
 837
 2397
CUACCAAAUUCCUAGAAUC
 837
 2415
GAUUCUAGGAAUUUGGUAG
2104


 


 2721
AUCAGACAAACGAUAAUAU
 838
 2721
AUCAGACAAACGAUAAUAU
 838
 2739
AUAUUAUCGUUUGUCUGAU
2105


 


 2739
UAACAGCAAGAUUAGAUAG
 839
 2739
UAACAGCAAGAUUAGAUAG
 839
 2757
CUAUCUAAUCUUGCUGUUA
2106


 


 2829
CUGCUCGGGAUGGUAUAAG
 840
 2829
CUGCUCGGGAUGGUAUAAG
 840
 2847
CUUAUACCAUCCCGAGCAG
2107


 


 3297
UCUUAGAAAAAGACGAUGA
 841
 3297
UCUUAGAAAAAGACGAUGA
 841
 3315
UCAUCGUCUUUUUCUAAGA
2108


 


 3459
CAAGAAGUGCAGUGCUAGC
 842
 3459
CAAGAAGUGCAGUGCUAGC
 842
 3477
GCUAGCACUGCACUUCUUG
2109


 


 3873
AAUACAUAAAGCCACAAAG
 843
 3873
AAUACAUAAAGCCACAAAG
 843
 3891
CUUUGUGGCUUUAUGUAUU
2110


 


 3891
GUCAAUUUAUAGUAGAUCU
 844
 3891
GUCAAUUUAUAGUAGAUCU
 844
 3909
AGAUCUACUAUAAAUUGAC
2111


 


 4161
CUCAAAUAAGUUAAUAAAA
 845
 4161
CUCAAAUAAGUUAAUAAAA
 845
 4179
UUUUAUUAACUUAUUUGAG
2112


 


 4197
AUAAUCAUUGGAGGAAAUC
 846
 4197
AUAAUCAUUGGAGGAAAUC
 846
 4215
GAUUUCCUCCAAUGAUUAU
2113


 


 4377
CCAUCAUGAUUGCAAUACU
 847
 4377
CCAUCAUGAUUGCAAUACU
 847
 4395
AGUAUUGCAAUCAUGAUGG
2114


 


 4809
UCAACUUCACUUAUAAUUG
 848
 4809
UCAACUUCACUUAUAAUUG
 848
 4827
CAAUUAUAAGUGAAGUUGA
2115


 


 4827
GCAGCCAUCAUAUUCAUAG
 849
 4827
GCAGCCAUCAUAUUCAUAG
 849
 4845
CUAUGAAUAUGAUGGCUGC
2116


 


 4917
ACCCCAACAUACCUCACCC
 850
 4917
ACCCCAACAUACCUCACCC
 850
 4935
GGGUGAGGUAUGUUGGGGU
2117


 


 5025
AAGUCAACCCUGCAAUCCA
 851
 5025
AAGUCAACCCUGCAAUCCA
 851
 5043
UGGAUUGCAGGGUUGACUU
2118


 


 5043
ACAACAGUCAAGACCAAAA
 852
 5043
ACAACAGUCAAGACCAAAA
 852
 5061
UUUUGGUCUUGACUGUUGU
2119


 


 5097
ACCACAAAACAACGCCAAA
 853
 5097
ACCACAAAACAACGCCAAA
 853
 5115
UUUGGCGUUGUUUUGUGGU
2120


 


 5151
UUUGAAGUGUUCAACUUUG
 854
 5151
UUUGAAGUGUUCAACUUUG
 854
 5169
CAAAGUUGAACACUUCAAA
2121


 


 5259
AAGCCCACAAAAAAACCAA
 855
 5259
AAGCCCACAAAAAAACCAA
 855
 5277
UUGGUUUUUUUGUGGGCUU
2122


 


 5817
UAAUAUCAAGGAAAAUAAG
 856
 5817
UAAUAUCAAGGAAAAUAAG
 856
 5835
CUUAUUUUCCUUGAUAUUA
2123


 


 5853
UAAGGUAAAAUUGAUAAAA
 857
 5853
UAAGGUAAAAUUGAUAAAA
 857
 5871
UUUUAUCAAUUUUACCUUA
2124


 


 5871
ACAAGAAUUAGAUAAAUAU
 858
 5871
ACAAGAAUUAGAUAAAUAU
 858
 5889
AUAUUUAUCUAAUUCUUGU
2125


 


 6105
GCACCUAGAAGGGGAAGUG
 859
 6105
GCACCUAGAAGGGGAAGUG
 859
 6123
CACUUCCCCUUCUAGGUGC
2126


 


 6159
GGCUGUAGUCAGCUUAUCA
 860
 6159
GGCUGUAGUCAGCUUAUCA
 860
 6177
UGAUAAGCUGACUACAGCC
2127


 


 6177
AAAUGGAGUUAGUGUCUUA
 861
 6177
AAAUGGAGUUAGUGUCUUA
 861
 6195
UAAGACACUAACUCCAUUU
2128


 


 6285
AACUGUGAUAGAGUUCCAA
 862
 6285
AACUGUGAUAGAGUUCCAA
 862
 6303
UUGGAACUCUAUCACAGUU
2129


 


 6339
AUUUAGUGUUAAUGCAGGU
 863
 6339
AUUUAGUGUUAAUGCAGGU
 863
 6357
ACCUGCAUUAACACUAAAU
2130


 


 6357
UGUAACUACACCUGUAAGC
 864
 6357
UGUAACUACACCUGUAAGC
 864
 6375
GCUUACAGGUGUAGUUACA
2131


 


 6789
AUUCAACCCCAAAUAUGAU
 865
 6789
AUUCAACCCCAAAUAUGAU
 865
 6807
AUCAUAUUUGGGGUUGAAU
2132


 


 6933
AUUUUCUAACGGGUGCGAU
 866
 6933
AUUUUCUAACGGGUGCGAU
 866
 6951
AUCGCACCCGUUAGAAAAU
2133


 


 7131
CCAGAGCCUAGCAUUUAUU
 867
 7131
CCAGAGCCUAGCAUUUAUU
 867
 7149
AAUAAAUGCUAGGCUCUGG
2134


 


 7167
AUUACAUAAUGUAAAUGCU
 868
 7167
AUUACAUAAUGUAAAUGCU
 868
 7185
AGCAUUUACAUUAUGUAAU
2135


 


 7347
UAGUAACUAAAUAAAAAUA
 869
 7347
UAGUAACUAAAUAAAAAUA
 869
 7365
UAUUUUUAUUUAGUUACUA
2136


 


 7455
AACUCUCAUCUAUAAACCA
 870
 7455
AACUCUCAUCUAUAAACCA
 870
 7473
UGGUUUAUAGAUGAGAGUU
2137


 


 7563
AACUGGGGCAAAUAUGUCA
 871
 7563
AACUGGGGCAAAUAUGUCA
 871
 7581
UGACAUAUUUGCCCCAGUU
2138


 


 7635
GUGUCAUUUUAGUCAUAAU
 872
 7635
GUGUCAUUUUAGUCAUAAU
 872
 7653
AUUAUGACUAAAAUGACAC
2139


 


 7671
CCAUGCACUGCUUGUAAGA
 873
 7671
CCAUGCACUGCUUGUAAGA
 873
 7689
UCUUACAAGCAGUGCAUGG
2140


 


 8103
AAAAGAAUCAACUGUUAGU
 874
 8103
AAAAGAAUCAACUGUUAGU
 874
 8121
ACUAACAGUUGAUUCUUUU
2141


 


 8211
UUACUAUGUAUAAUCAAAA
 875
 8211
UUACUAUGUAUAAUCAAAA
 875
 8229
UUUUGAUUAUACAUAGUAA
2142


 


 8301
UCCAUUGGACCUCUCAAGA
 876
 8301
UCCAUUGGACCUCUCAAGA
 876
 8319
UCUUGAGAGGUCCAAUGGA
2143


 


 8337
AAAAUUUUCUACAACAUCU
 877
 8337
AAAAUUUUCUACAACAUCU
 877
 8355
AGAUGUUGUAGAAAAUUUU
2144


 


 8715
AACCUACUUAUUUUCAGUC
 878
 8715
AACCUACUUAUUUUCAGUC
 878
 8733
GACUGAAAAUAAGUAGGUU
2145


 


 8895
AUGGACAAGAUGAAGACAA
 879
 8895
AUGGACAAGAUGAAGACAA
 879
 8913
UUGUCUUCAUCUUGUCCAU
2146


 


 9363
UAGGCUUAAGAUGCGGAUU
 880
 9363
UAGGCUUAAGAUGCGGAUU
 880
 9381
AAUCCGCAUCUUAAGCCUA
2147


 


 9579
CUCAGAAAAAUCUGCUAUC
 881
 9579
CUCAGAAAAAUCUGCUAUC
 881
 9597
GAUAGCAGAUUUUUCUGAG
2148


 


10155
AAAAUUAUAUAGAACAUGA
 882
10155
AAAAUUAUAUAGAACAUGA
 882
10173
UCAUGUUCUAUAUAAUUUU
2149


 


10479
UAGAAUUGAAAGCAGGAAU
 883
10479
UAGAAUUGAAAGCAGGAAU
 883
10497
AUUCCUGCUUUCAAUUCUA
2150


 


11019
UAGGCCACAAAUUAAAAGG
 884
11019
UAGGCCACAAAUUAAAAGG
 884
11037
CCUUUUAAUUUGUGGCCUA
2151


 


11109
CAGCUAGUAUAAAGAAAGU
 885
11109
CAGCUAGUAUAAAGAAAGU
 885
11127
ACUUUCUUUAUACUAGCUG
2152


 


11289
UAAAAAAUCAUGCAUUAUG
 886
11289
UAAAAAAUCAUGCAUUAUG
 886
11307
CAUAAUGCAUGAUUUUUUA
2153


 


11541
AAGAUAAACUUCAAGAUCU
 887
11541
AAGAUAAACUUCAAGAUCU
 887
11559
AGAUCUUGAAGUUUAUCUU
2154


 


11685
CUAGCGAAAUCAAUAGACU
 888
11685
CUAGCGAAAUCAAUAGACU
 888
11703
AGUCUAUUGAUUUCGCUAG
2155


 


12225
GUGGUGAGAGAGGACCCAC
 889
12225
GUGGUGAGAGAGGACCCAC
 889
12243
GUGGGUCCUCUCUCACCAC
2156


 


12315
AAAAACAGAGAGAUCAAAU
 890
12315
AAAAACAGAGAGAUCAAAU
 890
12333
AUUUGAUCUCUCUGUUUUU
2157


 


12855
AUAAAACACUCAAAUCUGG
 891
12855
AUAAAACACUCAAAUCUGG
 891
12873
CCAGAUUUGAGUGUUUUAU
2158


 


12963
AUUGGAUUCUGAUUAUACA
 892
12963
AUUGGAUUCUGAUUAUACA
 892
12981
UGUAUAAUCAGAAUCCAAU
2159


 


13035
UAACUGAUCAUAUGUUCAU
 893
13035
UAACUGAUCAUAUGUUCAU
 893
13053
AUGAACAUAUGAUCAGUUA
2160


 


13125
AGCUGGAGUGUGAUAUGAA
 894
13125
AGCUGGAGUGUGAUAUGAA
 894
13143
UUCAUAUCACACUCCAGCU
2161


 


13305
AACGUCUUAAUGUAGCAGA
 895
13305
AACGUCUUAAUGUAGCAGA
 895
13323
UCUGCUACAUUAAGACGUU
2162


 


13323
AAUUCACAGUUUGCCCUUG
 896
13323
AAUUCACAGUUUGCCCUUG
 896
13341
CAAGGGCAAACUGUGAAUU
2163


 


13539
UAAGGAUUGCUAAUUCUGA
 897
13539
UAAGGAUUGCUAAUUCUGA
 897
13557
UCAGAAUUAGCAAUCCUUA
2164


 


13647
AGACACUGAAUGACUAUUG
 898
13647
AGACACUGAAUGACUAUUG
 898
13665
CAAUAGUCAUUCAGUGUCU
2165


 


13755
GCAAACAAGAUUUGUAUAA
 899
13755
GCAAACAAGAUUUGUAUAA
 899
13773
UUAUACAAAUCUUGUUUGC
2166


 


13809
AUCAUUCAGGUAAUACAGC
 900
13809
AUCAUUCAGGUAAUACAGC
 900
13827
GCUGUAUUACCUGAAUGAU
2167


 


14133
AGUUUUUAAGGCUGUACAA
 901
14133
AGUUUUUAAGGCUGUACAA
 901
14151
UUGUACAGCCUUAAAAACU
2168


 


14259
GUCUUUUUGUCUGUGAUGC
 902
14259
GUCUUUUUGUCUGUGAUGC
 902
14277
GCAUCACAGACAAAAAGAC
2169


 


14439
AAACUUAUGUAUGCUUAGG
 903
14439
AAACUUAUGUAUGCUUAGG
 903
14457
CCUAAGCAUACAUAAGUUU
2170


 


14475
CUGAAGUUUACUUAGUCCU
 904
14475
CUGAAGUUUACUUAGUCCU
 904
14493
AGGACUAAGUAAACUUCAG
2171


 


14781
ACAUCUUAAAAUGGUUCAA
 905
14781
ACAUCUUAAAAUGGUUCAA
 905
14799
UUGAACCAUUUUAAGAUGU
2172


 


 4053
AUUCUUCACUUCACCAUCA
 906
 4053
AUUCUUCACUUCACCAUCA
 906
 4071
UGAUGGUGAAGUGAAGAAU
2173


 


10947
AAACUCAUGCUCAAGCAGA
 907
10947
AAACUCAUGCUCAAGCAGA
 907
10965
UCUGCUUGAGCAUGAGUUU
2174


 


14043
UAUUAUUGCGUACAGUAGU
 908
14043
UAUUAUUGCGUACAGUAGU
 908
14061
ACUACUGUACGCAAUAAUA
2175


 


 2703
CAUAUGAAGAAAUAAAUGA
 909
 2703
CAUAUGAAGAAAUAAAUGA
 909
 2721
UCAUUUAUUUCUUCAUAUG
2176


 


 7185
UGGUAAAUCCACCAUAAAU
 910
 7185
UGGUAAAUCCACCAUAAAU
 910
 7203
AUUUAUGGUGGAUUUACCA
2177


 


   21
CAAACUUGCGUAAACCAAA
 911
   21
CAAACUUGCGUAAACCAAA
 911
   39
UUUGGUUUACGCAAGUUUG
2178


 


  183
UGCUAUACUGACAAAUUAA
 912
  183
UGCUAUACUGACAAAUUAA
 912
  201
UUAAUUUGUCAGUAUAGCA
2179


 


  651
CACACCACAAAGACUGAUG
 913
  651
CACACCACAAAGACUGAUG
 913
  669
CAUCAGUCUUUGUGGUGUG
2180


 


  723
AGACAUCAUAACACACAGA
 914
  723
AGACAUCAUAACACACAGA
 914
  741
UCUGUGUGUUAUGAUGUCU
2181


 


  993
CAAUCCAUGAAUUUCAACA
 915
  993
CAAUCCAUGAAUUUCAACA
 915
 1011
UGUUGAAAUUCAUGGAUUG
2182


 


 1659
GGGGAUAGAUCUGGUCUUA
 916
 1659
GGGGAUAGAUCUGGUCUUA
 916
 1677
UAAGACCAGAUCUAUCCCC
2183


 


 1749
GAUAUAGCCAACAGCUUCU
 917
 1749
GAUAUAGCCAACAGCUUCU
 917
 1767
AGAAGCUGUUGGCUAUAUC
2184


 


 1929
GCAAAAUCAGUUAAAAAUA
 918
 1929
GCAAAAUCAGUUAAAAAUA
 918
 1947
UAUUUUUAACUGAUUUUGC
2185


 


 2235
AGUGUAUUAGACUUGACAG
 919
 2235
AGUGUAUUAGACUUGACAG
 919
 2253
CUGUCAAGUCUAAUACACU
2186


 


 2433
CAUCACCUAAAGAUCCCAA
 920
 2433
CAUCACCUAAAGAUCCCAA
 920
 2451
UUGGGAUCUUUAGGUGAUG
2187


 


 2631
AUAAUCCCUUUUCAAAACU
 921
 2631
AUAAUCCCUUUUCAAAACU
 921
 2649
AGUUUUGAAAAGGGAUUAU
2188


 


 2685
AAGAAGAAUCUAGCUAUUC
 922
 2685
AAGAAGAAUCUAGCUAUUC
 922
 2703
GAAUAGCUAGAUUCUUCUU
2189


 


 2793
UUCACACAUUAGUAGUAGC
 923
 2793
UUCACACAUUAGUAGUAGC
 923
 2811
GCUACUACUAAUGUGUGAA
2190


 


 3009
AGAAAUUGAACAACCUGUU
 924
 3009
AGAAAUUGAACAACCUGUU
 924
 3027
AACAGGUUGUUCAAUUUCU
2191


 


 3099
CACAACACCAACAGAAGAC
 925
 3099
CACAACACCAACAGAAGAC
 925
 3117
GUCUUCUGUUGGUGUUGUG
2192


 


 3603
AUAUGUUAACUACAGUUAA
 926
 3603
AUAUGUUAACUACAGUUAA
 926
 3621
UUAACUGUAGUUAACAUAU
2193


 


 4629
ACUAACAAUAACGUUGGGG
 927
 4629
ACUAACAAUAACGUUGGGG
 927
 4647
CCCCAACGUUAUUGUUAGU
2194


 


 4701
AAGACCUGGGACACUCUCA
 928
 4701
AAGACCUGGGACACUCUCA
 928
 4719
UGAGAGUGUCCCAGGUCUU
2195


 


 4755
UUAAAUCUUAAAUCUAUAG
 929
 4755
UUAAAUCUUAAAUCUAUAG
 929
 4773
CUAUAGAUUUAAGAUUUAA
2196


 


 4863
GUCACACUAACAACUGCAA
 930
 4863
GUCACACUAACAACUGCAA
 930
 4881
UUGCAGUUGUUAGUGUGAC
2197


 


 4953
AUCAGCUUCUCCAAUCUGU
 931
 4953
AUCAGCUUCUCCAAUCUGU
 931
 4971
ACAGAUUGGAGAAGCUGAU
2198


 


 4989
ACCACCACCAUACUAGCUU
 932
 4989
ACCACCACCAUACUAGCUU
 932
 5007
AAGCUAGUAUGGUGGUGGU
2199


 


 5115
AACAAACCACCAAACAAAC
 933
 5115
AACAAACCACCAAACAAAC
 933
 5133
GUUUGUUUGGUGGUUUGUU
2200


 


 5241
GGAAAGAAAACCACCACCA
 934
 5241
GGAAAGAAAACCACCACCA
 934
 5259
UGGUGGUGGUUUUCUUUCC
2201


 


 5295
AAAGAUCUCAAACCUCAAA
 935
 5295
AAAGAUCUCAAACCUCAAA
 935
 5313
UUUGAGGUUUGAGAUCUUU
2202


 


 5763
UCUUAGUGCUCUAAGAACU
 936
 5763
UCUUAGUGCUCUAAGAACU
 936
 5781
AGUUCUUAGAGCACUAAGA
2203


 


 5781
UGGUUGGUAUACUAGUGUU
 937
 5781
UGGUUGGUAUACUAGUGUU
 937
 5799
AACACUAGUAUACCAACCA
2204


 


 5979
UUAUACACUCAACAAUACC
 938
 5979
UUAUACACUCAACAAUACC
 938
 5997
GGUAUUGUUGAGUGUAUAA
2205


 


 6069
UGCAAUCGCCAGUGGCAUU
 939
 6069
UGCAAUCGCCAGUGGCAUU
 939
 6087
AAUGCCACUGGCGAUUGCA
2206


 


 6699
AACAUGUAAAGUUCAAUCG
 940
 6699
AACAUGUAAAGUUCAAUCG
 940
 6717
CGAUUGAACUUUACAUGUU
2207


 


 6717
GAAUCGAGUAUUUUGUGAC
 941
 6717
GAAUCGAGUAUUUUGUGAC
 941
 6735
GUCACAAAAUACUCGAUUC
2208


 


 7005
UGUAAAUAAGCAAGAAGGC
 942
 7005
UGUAAAUAAGCAAGAAGGC
 942
 7023
GCCUUCUUGCUUAUUUACA
2209


 


 7023
CAAAAGUCUCUAUGUAAAA
 943
 7023
CAAAAGUCUCUAUGUAAAA
 943
 7041
UUUUACAUAGAGACUUUUG
2210


 


 7113
AGUCAAUGAGAAGAUUAAC
 944
 7113
AGUCAAUGAGAAGAUUAAC
 944
 7131
GUUAAUCUUCUCAUUGACU
2211


 


 7311
CAAGGAUCAACUGAGUGGU
 945
 7311
CAAGGAUCAACUGAGUGGU
 945
 7329
ACCACUCAGUUGAUCCUUG
2212


 


 7419
UAUCAUUGGAUUUUCUUAA
 946
 7419
UAUCAUUGGAUUUUCUUAA
 946
 7437
UUAAGAAAAUCCAAUGAUA
2213


 


 7527
GAAUACCAGAUUAACUUAC
 947
 7527
GAAUACCAGAUUAACUUAC
 947
 7545
GUAAGUUAAUCUGGUAUUC
2214


 


 9039
AGAAAUUAAUGUGUUCAAU
 948
 9039
AGAAAUUAAUGUGUUCAAU
 948
 9057
AUUGAACACAUUAAUUUCU
2215


 


 9093
AUUUAUACACAAAAUUAAA
 949
 9093
AUUUAUACACAAAAUUAAA
 949
 9111
UUUAAUUUUGUGUAUAAAU
2216


 


 9201
UUUUGAAUCAAUAUGGUUG
 950
 9201
UUUUGAAUCAAUAUGGUUG
 950
 9219
CAACCAUAUUGAUUCAAAA
2217


 


 9615
UAUUAGAUAAGACAGUAUC
 951
 9615
UAUUAGAUAAGACAGUAUC
 951
 9633
GAUACUGUCUUAUCUAAUA
2218


 


 9831
UAAGCAGUUUGAGUAUGUU
 952
 9831
UAAGCAGUUUGAGUAUGUU
 952
 9849
AACAUACUCAAACUGCUUA
2219


 


10137
AUAUGCCGUCACACAUACA
 953
10137
AUAUGCCGUCACACAUACA
 953
10155
UGUAUGUGUGACGGCAUAU
2220


 


10713
CACCCCCCUAUAUAAGAGA
 954
10713
CACCCCCCUAUAUAAGAGA
 954
10731
UCUCUUAUAUAGGGGGGUG
2221


 


11307
GUAACAAUAAAUUAUAUUU
 955
11307
GUAACAAUAAAUUAUAUUU
 955
11325
AAAUAUAAUUUAUUGUUAC
2222


 


11469
CUCCUGAUUUCCUCACAGA
 956
11469
CUCCUGAUUUCCUCACAGA
 956
11487
UCUGUGAGGAAAUCAGGAG
2223


 


11811
AACCUACAUAUCCUCACGG
 957
11811
AACCUACAUAUCCUCACGG
 957
11829
CCGUGAGGAUAUGUAGGUU
2224


 


11919
UGGAAAAGACUUCUGCCAU
 958
11919
UGGAAAAGACUUCUGCCAU
 958
11937
AUGGCAGAAGUCUUUUCCA
2225


 


12153
ACAUCAAAUAUACAACAAG
 959
12153
ACAUCAAAUAUACAACAAG
 959
12171
CUUGUUGUAUAUUUGAUGU
2226


 


12171
GCACUAUAGCUAGUGGCAU
 960
12171
GCACUAUAGCUAGUGGCAU
 960
12189
AUGCCACUAGCUAUAGUGC
2227


 


12441
AAAAAUUAUUUCCACAAUA
 961
12441
AAAAAUUAUUUCCACAAUA
 961
12459
UAUUGUGGAAAUAAUUUUU
2228


 


12639
UUGGCCUUAGCUUAAUGUC
 962
12639
UUGGCCUUAGCUUAAUGUC
 962
12657
GACAUUAAGCUAAGGCCAA
2229


 


13485
UCUUUUACAUUAAUUAUAA
 963
13485
UCUUUUACAUUAAUUAUAA
 963
13503
UUAUAAUUAAUGUAAAAGA
2230


 


13521
AUCUAUUAACUAAACAUAU
 964
13521
AUCUAUUAACUAAACAUAU
 964
13539
AUAUGUUUAGUUAAUAGAU
2231


 


13881
AUAGCACAUCACUUUAUUG
 965
13881
AUAGCACAUCACUUUAUUG
 965
13899
CAAUAAAGUGAUGUGCUAU
2232


 


13971
AGUAUAUUUUAAAAGACCU
 966
13971
AGUAUAUUUUAAAAGACCU
 966
13989
AGGUCUUUUAAAAUAUACU
2233


 


14331
AUGUAAGAAAAUGCAAGUA
 967
14331
AUGUAAGAAAAUGCAAGUA
 967
14349
UACUUGCAUUUUCUUACAU
2234


 


14367
AAUGUACGUUAAUAGUAAA
 968
14367
AAUGUACGUUAAUAGUAAA
 968
14385
UUUACUAUUAACGUACAUU
2235


 


14493
UUACAAUAGGUCCUGCAAA
 969
14493
UUACAAUAGGUCCUGCAAA
 969
14511
UUUGCAGGACCUAUUGUAA
2236


 


14889
ACAGCUUGACAACUAAUGA
 970
14889
ACAGCUUGACAACUAAUGA
 970
14907
UCAUUAGUUGUCAAGCUGU
2237


 


15087
UUAUCAUUUUGAUCUAGGA
 971
15087
UUAUCAUUUUGAUCUAGGA
 971
15105
UCCUAGAUCAAAAUGAUAA
2238


 


  525
AUAAAUAUCAACUAGCAAA
 972
  525
AUAAAUAUCAACUAGCAAA
 972
  543
UUUGCUAGUUGAUAUUUAU
2239


 


  579
UGACAGAAGAUAAAAAUGG
 973
  579
UGACAGAAGAUAAAAAUGG
 973
  597
CCAUUUUUAUCUUCUGUCA
2240


 


  615
AGCCGACCCAACCAUGGAC
 974
  615
AGCCGACCCAACCAUGGAC
 974
  633
GUCCAUGGUUGGGUCGGCU
2241


 


  633
CACAACACACAAUGACACC
 975
  633
CACAACACACAAUGACACC
 975
  651
GGUGUCAUUGUGUGUUGUG
2242


 


  669
GAUCACAGACAUGAGACCA
 976
  669
GAUCACAGACAUGAGACCA
 976
  687
UGGUCUCAUGUCUGUGAUC
2243


 


  687
AUUGUCACUUGAGACUAUA
 977
  687
AUUGUCACUUGAGACUAUA
 977
  705
UAUAGUCUCAAGUGACAAU
2244


 


  705
AAUAAUAUCACUAACCAGA
 978
  705
AAUAAUAUCACUAACCAGA
 978
  723
UCUGGUUAGUGAUAUUAUU
2245


 


  759
UCAUGAAUGUAUAGUGAGA
 979
  759
UCAUGAAUGUAUAGUGAGA
 979
  777
UCUCACUAUACAUUCAUGA
2246


 


  831
ACUAUUGCACAAAGUGGGA
 980
  831
ACUAUUGCACAAAGUGGGA
 980
  849
UCCCACUUUGUGCAAUAGU
2247


 


  849
AAGCACUAAAUACAAAAAA
 981
  849
AAGCACUAAAUACAAAAAA
 981
  867
UUUUUUGUAUUUAGUGCUU
2248


 


  885
AAAAUAUGGCACUUUUCCU
 982
  885
AAAAUAUGGCACUUUUCCU
 982
  903
AGGAAAAGUGCCAUAUUUU
2249


 


  903
UAUGCCAAUAUUUAUCAAU
 983
  903
UAUGCCAAUAUUUAUCAAU
 983
  921
AUUGAUAAAUAUUGGCAUA
2250


 


  957
UACAAAGCACACUCCCAUA
 984
  957
UACAAAGCACACUCCCAUA
 984
  975
UAUGGGAGUGUGCUUUGUA
2251


 


 1011
ACAAGAGUCACACAAUCUG
 985
 1011
ACAAGAGUCACACAAUCUG
 985
 1029
CAGAUUGUGUGACUCUUGU
2252


 


 1029
GAAAUAACAACUUCAUGCA
 986
 1029
GAAAUAACAACUUCAUGCA
 986
 1047
UGCAUGAAGUUGUUAUUUC
2253


 


 1047
AUAACCACACUCCAUAGUU
 987
 1047
AUAACCACACUCCAUAGUU
 987
 1065
AACUAUGGAGUGUGGUUAU
2254


 


 1065
UCAAAUGGAGCCUGAAAAU
 988
 1065
UCAAAUGGAGCCUGAAAAU
 988
 1083
AUUUUCAGGCUCCAUUUGA
2255


 


 1101
AAGGAGAGACAUAAGAUGA
 989
 1101
AAGGAGAGACAUAAGAUGA
 989
 1119
UCAUCUUAUGUCUCUCCUU
2256


 


 1119
AAAGAUGGGGCAAAUACAA
 990
 1119
AAAGAUGGGGCAAAUACAA
 990
 1137
UUGUAUUUGCCCCAUCUUU
2257


 


 1137
AAAAUGGCUCUUAGCAAAG
 991
 1137
AAAAUGGCUCUUAGCAAAG
 991
 1155
CUUUGCUAAGAGCCAUUUU
2258


 


 1353
UCUAGAUUAGGAAGAGAAG
 992
 1353
UCUAGAUUAGGAAGAGAAG
 992
 1371
CUUCUCUUCCUAAUCUAGA
2259


 


 1407
GUAAAAGCAAAUGGAGUGG
 993
 1407
GUAAAAGCAAAUGGAGUGG
 993
 1425
CCACUCCAUUUGCUUUUAC
2260


 


 1479
UUAACAUUGUCAAGCUUAA
 994
 1479
UUAACAUUGUCAAGCUUAA
 994
 1497
UUAAGCUUGACAAUGUUAA
2261


 


 1623
UGUAUAGCGGCAUUAGUAA
 995
 1623
UGUAUAGCGGCAUUAGUAA
 995
 1641
UUACUAAUGCCGCUAUACA
2262


 


 1677
ACAGCUGUGAUUAGGAGGG
 996
 1677
ACAGCUGUGAUUAGGAGGG
 996
 1695
CCCUCCUAAUCACAGCUGU
2263


 


 1713
AAUGAAAUGAAACGUUAUA
 997
 1713
AAUGAAAUGAAACGUUAUA
 997
 1731
UAUAACGUUUCAUUUCAUU
2264


 


 1767
UAUGAAGUGUUUGAAAAAU
 998
 1767
UAUGAAGUGUUUGAAAAAU
 998
 1785
AUUUUUCAAACACUUCAUA
2265


 


 1785
UAUCCUCACUUUAUAGAUG
 999
 1785
UAUCCUCACUUUAUAGAUG
 999
 1803
CAUCUAUAAAGUGAGGAUA
2266


 


 1857
GAAGGGAUUUUUGCUGGAU
1000
 1857
GAAGGGAUUUUUGCUGGAU
1000
 1875
AUCCAGCAAAAAUCCCUUC
2267


 


 1911
UUACGGUGGGGGGUCUUAG
1001
 1911
UUACGGUGGGGGGUCUUAG
1001
 1929
CUAAGACCCCCCACCGUAA
2268


 


 1947
AUUAUGCUAGGACACGCUA
1002
 1947
AUUAUGCUAGGACACGCUA
1002
 1965
UAGCGUGUCCUAGCAUAAU
2269


 


 1983
GAACAAGUUGUGGAGGUUU
1003
 1983
GAACAAGUUGUGGAGGUUU
1003
 2001
AAACCUCCACAACUUGUUC
2270


 


 2019
UUGGGUGGAGAAGCAGGGU
1004
 2019
UUGGGUGGAGAAGCAGGGU
1004
 2037
ACCCUGCUUCUCCACCCAA
2271


 


 2073
UUGUCUUUGACUCAAUUUC
1005
 2073
UUGUCUUUGACUCAAUUUC
1005
 2091
GAAAUUGAGUCAAAGACAA
2272


 


 2145
GAAUACAGAGGUACACCAA
1006
 2145
GAAUACAGAGGUACACCAA
1006
 2163
UUGGUGUACCUCUGUAUUC
2273


 


 2181
GAUGCUGCAAAAGCAUAUG
1007
 2181
GAUGCUGCAAAAGCAUAUG
1007
 2199
CAUAUGCUUUUGCAGCAUC
2274


 


 2379
AAGAUGCAAACAACAGAGC
1008
 2379
AAGAUGCAAACAACAGAGC
1008
 2397
GCUCUGUUGUUUGCAUCUU
2275


 


 2523
AUUCAACCAUUAUAAACCC
1009
 2523
AUUCAACCAUUAUAAACCC
1009
 2541
GGGUUUAUAAUGGUUGAAU
2276


 


 2559
AUACUGUAGGGAACAAGCC
1010
 2559
AUACUGUAGGGAACAAGCC
1010
 2577
GGCUUGUUCCCUACAGUAU
2277


 


 2613
AAGACCCUACGCCAAGUGA
1011
 2613
AAGACCCUACGCCAAGUGA
1011
 2631
UCACUUGGCGUAGGGUCUU
2278


 


 2811
CGAGUGCAGGACCUACAUC
1012
 2811
CGAGUGCAGGACCUACAUC
1012
 2829
GAUGUAGGUCCUGCACUCG
2279


 


 2919
GACUAGAAGCUAUGGCAAG
1013
 2919
GACUAGAAGCUAUGGCAAG
1013
 2937
CUUGCCAUAGCUUCUAGUC
2280


 


 3063
AUGAUUUCUGAUCAGUUAC
1014
 3063
AUGAUUUCUGAUCAGUUAC
1014
 3081
GUAACUGAUCAGAAAUCAU
2281


 


 3081
CCAAUCUGUACAUCAACAC
1015
 3081
CCAAUCUGUACAUCAACAC
1015
 3099
GUGUUGAUGUACAGAUUGG
2282


 


 3117
CCAACAAACAAACCAACUC
1016
 3117
CCAACAAACAAACCAACUC
1016
 3135
GAGUUGGUUUGUUUGUUGG
2283


 


 3135
CACCCAUCCAACCAAACAU
1017
 3135
CACCCAUCCAACCAAACAU
1017
 3153
AUGUUUGGUUGGAUGGGUG
2284


 


 3153
UCUAUACGCCAAUCAGCCA
1018
 3153
UCUAUACGCCAAUCAGCCA
1018
 3171
UGGCUGAUUGGCGUAUAGA
2285


 


 3171
AAUCCAAAACUAGCCACCC
1019
 3171
AAUCCAAAACUAGCCACCC
1019
 3189
GGGUGGCUAGUUUUGGAUU
2286


 


 3189
CGGAAAAAAUAGAUACUAU
1020
 3189
CGGAAAAAAUAGAUACUAU
1020
 3207
AUAGUAUCUAUUUUUUCCG
2287


 


 3207
UAGUUACAAAAAAAGAUGG
1021
 3207
UAGUUACAAAAAAAGAUGG
1021
 3225
CCAUCUUUUUUUGUAACUA
2288


 


 3243
ACGUGAACAAACUUCACGA
1022
 3243
ACGUGAACAAACUUCACGA
1022
 3261
UCGUGAAGUUUGUUCACGU
2289


 


 3351
AAUCAUCCAUGCCAGCAGA
1023
 3351
AAUCAUCCAUGCCAGCAGA
1023
 3369
UCUGCUGGCAUGGAUGAUU
2290


 


 3495
UUACCAUAUGUGCCAAUGU
1024
 3495
UUACCAUAUGUGCCAAUGU
1024
 3513
ACAUUGGCACAUAUGGUAA
2291


 


 3531
GCAAGCUGGCAUAUGAUGU
1025
 3531
GCAAGCUGGCAUAUGAUGU
1025
 3549
ACAUCAUAUGCCAGCUUGC
2292


 


 3621
AAGAUCUCACUAUGAAAAC
1026
 3621
AAGAUCUCACUAUGAAAAC
1026
 3639
GUUUUCAUAGUGAGAUCUU
2293


 


 3639
CACUCAACCCAACACAUGA
1027
 3639
CACUCAACCCAACACAUGA
1027
 3657
UCAUGUGUUGGGUUGAGUG
2294


 


 3657
ACAUCAUUGCUUUAUGUGA
1028
 3657
ACAUCAUUGCUUUAUGUGA
1028
 3675
UCACAUAAAGCAAUGAUGU
2295


 


 3675
AAUUUGAAAAUAUAGUAAC
1029
 3675
AAUUUGAAAAUAUAGUAAC
1029
 3693
GUUACUAUAUUUUCAAAUU
2296


 


 3783
AAUUCAAAAAUGCCAUCAC
1030
 3783
AAUUCAAAAAUGCCAUCAC
1030
 3801
GUGAUGGCAUUUUUGAAUU
2297


 


 3819
CUUACUCAGGAUUACUGUU
1031
 3819
CUUACUCAGGAUUACUGUU
1031
 3837
AACAGUAAUCCUGAGUAAG
2298


 


 3945
UUACAACAAAUUGGAAGCA
1032
 3945
UUACAACAAAUUGGAAGCA
1032
 3963
UGCUUCCAAUUUGUUGUAA
2299


 


 3999
AUUAACCUUUUUCUUCUAC
1033
 3999
AUUAACCUUUUUCUUCUAC
1033
 4017
GUAGAAGAAAAAGGUUAAU
2300


 


 4017
CAUCAGUGAGUUGAUUCAU
1034
 4017
CAUCAGUGAGUUGAUUCAU
1034
 4035
AUGAAUCAACUCACUGAUG
2301


 


 4071
AUAAUCACCAACCCUCUGU
1035
 4071
AUAAUCACCAACCCUCUGU
1035
 4089
ACAGAGGGUUGGUGAUUAU
2302


 


 4089
UGGUUCAACUAAUCAAACA
1036
 4089
UGGUUCAACUAAUCAAACA
1036
 4107
UGUUUGAUUAGUUGAACCA
2303


 


 4107
AAAACCCAUCUGGAGCCUC
1037
 4107
AAAACCCAUCUGGAGCCUC
1037
 4125
GAGGCUCCAGAUGGGUUUU
2304


 


 4143
UGUUCAUCAGAUCUAGUAC
1038
 4143
UGUUCAUCAGAUCUAGUAC
1038
 4161
GUACUAGAUCUGAUGAACA
2305


 


 4179
AAUAUCCACAUGGGGCAAA
1039
 4179
AAUAUCCACAUGGGGCAAA
1039
 4197
UUUGCCCCAUGUGGAUAUU
2306


 


 4233
UGUCAACAUAGACAAGUCA
1040
 4233
UGUCAACAUAGACAAGUCA
1040
 4251
UGACUUGUCUAUGUUGACA
2307


 


 4341
UGAUAACAACAAUAAUCUC
1041
 4341
UGAUAACAACAAUAAUCUC
1041
 4359
GAGAUUAUUGUUGUUAUCA
2308


 


 4431
AAACCUUUGAGCUACCAAG
1042
 4431
AAACCUUUGAGCUACCAAG
1042
 4449
CUUGGUAGCUCAAAGGUUU
2309


 


 4449
GAGCUCGAGUCAAUACAUA
1043
 4449
GAGCUCGAGUCAAUACAUA
1043
 4467
UAUGUAUUGACUCGAGCUC
2310


 


 4467
AGCAUUCACCAAUCUGAUG
1044
 4467
AGCAUUCACCAAUCUGAUG
1044
 4485
CAUCAGAUUGGUGAAUGCU
2311


 


 4485
GGCACAAAACAGUAACCUU
1045
 4485
GGCACAAAACAGUAACCUU
1045
 4503
AAGGUUACUGUUUUGUGCC
2312


 


 4503
UGCAUUUGUAAGUGAACAA
1046
 4503
UGCAUUUGUAAGUGAACAA
1046
 4521
UUGUUCACUUACAAAUGCA
2313


 


 4521
ACCCUCACCUCUUUACAAA
1047
 4521
ACCCUCACCUCUUUACAAA
1047
 4539
UUUGUAAAGAGGUGAGGGU
2314


 


 4539
AACCACAUCAACAUCUCAC
1048
 4539
AACCACAUCAACAUCUCAC
1048
 4557
GUGAGAUGUUGAUGUGGUU
2315


 


 4557
CCAUGCAAGCCAUCAUCCA
1049
 4557
CCAUGCAAGCCAUCAUCCA
1049
 4575
UGGAUGAUGGCUUGCAUGG
2316


 


 4575
AUAUUAUAAAGUAGUUAAU
1050
 4575
AUAUUAUAAAGUAGUUAAU
1050
 4593
AUUAACUACUUUAUAAUAU
2317


 


 4593
UUAAAAAUAAUCAUAACAA
1051
 4593
UUAAAAAUAAUCAUAACAA
1051
 4611
UUGUUAUGAUUAUUUUUAA
2318


 


 4611
AUGAACUAAGAUAUUAAGA
1052
 4611
AUGAACUAAGAUAUUAAGA
1052
 4629
UCUUAAUAUCUUAGUUCAU
2319


 


 4719
AAUCAUCUAUUAUUCAUAU
1053
 4719
AAUCAUCUAUUAUUCAUAU
1053
 4737
AUAUGAAUAAUAGAUGAUU
2320


 


 4737
UCAUCGUGCUUAUACAAGU
1054
 4737
UCAUCGUGCUUAUACAAGU
1054
 4755
ACUUGUAUAAGCACGAUGA
2321


 


 4935
CAGAAUCCCCAGCUUGGAA
1055
 4935
CAGAAUCCCCAGCUUGGAA
1055
 4953
UUCCAAGCUGGGGAUUCUG
2322


 


 4971
UCUGAAACUACAUCACAAA
1056
 4971
UCUGAAACUACAUCACAAA
1056
 4989
UUUGUGAUGUAGUUUCAGA
2323


 


 5007
UCAACAACACCAAGUGUCA
1057
 5007
UCAACAACACCAAGUGUCA
1057
 5025
UGACACUUGGUGUUGUUGA
2324


 


 5061
AACACAACAACAACCAAAA
1058
 5061
AACACAACAACAACCAAAA
1058
 5079
UUUUGGUUGUUGUUGUGUU
2325


 


 5079
AUACAACCCAGCAAGCCCA
1059
 5079
AUACAACCCAGCAAGCCCA
1059
 5097
UGGGCUUGCUGGGUUGUAU
2326


 


 5169
GUACCUUGCAGCAUAUGCA
1060
 5169
GUACCUUGCAGCAUAUGCA
1060
 5187
UGCAUAUGCUGCAAGGUAC
2327


 


 5205
UGGGCUAUCUGUAAAAGAA
1061
 5205
UGGGCUAUCUGUAAAAGAA
1061
 5223
UUCUUUUACAGAUAGCCCA
2328


 


 5223
AUACCAAACAAAAAACCUG
1062
 5223
AUACCAAACAAAAAACCUG
1062
 5241
CAGGUUUUUUGUUUGGUAU
2329


 


 5277
ACCAUCAAGACAACCAAAA
1063
 5277
ACCAUCAAGACAACCAAAA
1063
 5295
UUUUGGUUGUCUUGAUGGU
2330


 


 5313
ACCACAAAACCAAAGGAAG
1064
 5313
ACCACAAAACCAAAGGAAG
1064
 5331
CUUCCUUUGGUUUUGUGGU
2331


 


 5331
GUACCUACCACCAAGCCCA
1065
 5331
GUACCUACCACCAAGCCCA
1065
 5349
UGGGCUUGGUGGUAGGUAC
2332


 


 5349
ACAGAAAAGCCAACCAUCA
1066
 5349
ACAGAAAAGCCAACCAUCA
1066
 5367
UGAUGGUUGGCUUUUCUGU
2333


 


 5385
AUCAGAACUACACUGCUCA
1067
 5385
AUCAGAACUACACUGCUCA
1067
 5403
UGAGCAGUGUAGUUCUGAU
2334


 


 5403
ACCAACAAUACCACAGGAA
1068
 5403
ACCAACAAUACCACAGGAA
1068
 5421
UUCCUGUGGUAUUGUUGGU
2335


 


 5421
AAUCCAGAACACACAAGUC
1069
 5421
AAUCCAGAACACACAAGUC
1069
 5439
GACUUGUGUGUUCUGGAUU
2336


 


 5439
CAAAAGGGAACCCUCCACU
1070
 5439
CAAAAGGGAACCCUCCACU
1070
 5457
AGUGGAGGGUUCCCUUUUG
2337


 


 5457
UCAACCUCCUCCGAUGGCA
1071
 5457
UCAACCUCCUCCGAUGGCA
1071
 5475
UGCCAUCGGAGGAGGUUGA
2338


 


 5475
AAUCCAAGCCCUUCACAAG
1072
 5475
AAUCCAAGCCCUUCACAAG
1072
 5493
CUUGUGAAGGGCUUGGAUU
2339


 


 5493
GUCUAUACAACAUCCGAGU
1073
 5493
GUCUAUACAACAUCCGAGU
1073
 5511
ACUCGGAUGUUGUAUAGAC
2340


 


 5511
UACCUAUCACAACCUCCAU
1074
 5511
UACCUAUCACAACCUCCAU
1074
 5529
AUGGAGGUUGUGAUAGGUA
2341


 


 5529
UCUCCAUCCAACACAACAA
1075
 5529
UCUCCAUCCAACACAACAA
1075
 5547
UUGUUGUGUUGGAUGGAGA
2342


 


 5547
AACCAGUAGUCAUUAAAAA
1076
 5547
AACCAGUAGUCAUUAAAAA
1076
 5565
UUUUUAAUGACUACUGGUU
2343


 


 5565
AGCGUAUUAUUGCAAAAAG
1077
 5565
AGCGUAUUAUUGCAAAAAG
1077
 5583
CUUUUUGCAAUAAUACGCU
2344


 


 5583
GCCAUGACCAAAUCAACCA
1078
 5583
GCCAUGACCAAAUCAACCA
1078
 5601
UGGUUGAUUUGGUCAUGGC
2345


 


 5601
AGAAUCAAAAUCAACUCUG
1079
 5601
AGAAUCAAAAUCAACUCUG
1079
 5619
CAGAGUUGAUUUUGAUUCU
2346


 


 5637
GUUGCCAAUCCUCAAAACA
1080
 5637
GUUGCCAAUCCUCAAAACA
1080
 5655
UGUUUUGAGGAUUGGCAAC
2347


 


 5655
AAAUGCAAUUACCGCAAUC
1081
 5655
AAAUGCAAUUACCGCAAUC
1081
 5673
GAUUGCGGUAAUUGCAUUU
2348


 


 5673
CCUUGCUGCAGUCACACUC
1082
 5673
CCUUGCUGCAGUCACACUC
1082
 5691
GAGUGUGACUGCAGCAAGG
2349


 


 5691
CUGUUUUGCUUCCAGUCAA
1083
 5691
CUGUUUUGCUUCCAGUCAA
1083
 5709
UUGACUGGAAGCAAAACAG
2350


 


 5745
UGCAGUCAGCAAAGGCUAU
1084
 5745
UGCAGUCAGCAAAGGCUAU
1084
 5763
AUAGCCUUUGCUGACUGCA
2351


 


 5889
UAAAAGUGCUGUAACAGAA
1085
 5889
UAAAAGUGCUGUAACAGAA
1085
 5907
UUCUGUUACAGCACUUUUA
2352


 


 5925
AAGCACACCGGCAACCAAC
1086
 5925
AAGCACACCGGCAACCAAC
1086
 5943
GUUGGUUGCCGGUGUGCUU
2353


 


 5997
CAAAAAUACCAAUGUAACA
1087
 5997
CAAAAAUACCAAUGUAACA
1087
 6015
UGUUACAUUGGUAUUUUUG
2354


 


 6033
AAGAAGAUUUCUUGGCUUU
1088
 6033
AAGAAGAUUUCUUGGCUUU
1088
 6051
AAAGCCAAGAAAUCUUCUU
2355


 


 6231
UAAACAGUUGUUACCUAUU
1089
 6231
UAAACAGUUGUUACCUAUU
1089
 6249
AAUAGGUAACAACUGUUUA
2356


 


 6249
UGUGAACAAGCAAAGCUGU
1090
 6249
UGUGAACAAGCAAAGCUGU
1090
 6267
ACAGCUUUGCUUGUUCACA
2357


 


 6267
UAGCAUAUCAAACAUUGAA
1091
 6267
UAGCAUAUCAAACAUUGAA
1091
 6285
UUCAAUGUUUGAUAUGCUA
2358


 


 6375
CACUUAUAUGUUAACAAAU
1092
 6375
CACUUAUAUGUUAACAAAU
1092
 6393
AUUUGUUAACAUAUAAGUG
2359


 


 6393
UAGUGAAUUAUUAUCAUUA
1093
 6393
UAGUGAAUUAUUAUCAUUA
1093
 6411
UAAUGAUAAUAAUUCACUA
2360


 


 6501
CAUAAUAAAGGAGGAAGUC
1094
 6501
CAUAAUAAAGGAGGAAGUC
1094
 6519
GACUUCCUCCUUUAUUAUG
2361


 


 6537
AUUACCACUAUAUGGUGUA
1095
 6537
AUUACCACUAUAUGGUGUA
1095
 6555
UACACCAUAUAGUGGUAAU
2362


 


 6555
AAUAGAUACACCUUGUUGG
1096
 6555
AAUAGAUACACCUUGUUGG
1096
 6573
CCAACAAGGUGUAUCUAUU
2363


 


 6609
AAAGGAAGGGUCCAACAUC
1097
 6609
AAAGGAAGGGUCCAACAUC
1097
 6627
GAUGUUGGACCCUUCCUUU
2364


 


 6627
CUGUUUAACAAGAACCGAC
1098
 6627
CUGUUUAACAAGAACCGAC
1098
 6645
GUCGGUUCUUGUUAAACAG
2365


 


 6681
UUUCUUCCCACUAGCUGAA
1099
 6681
UUUCUUCCCACUAGCUGAA
1099
 6699
UUCAGCUAGUGGGAAGAAA
2366


 


 6771
UCUCUGCAACAUUGACAUA
1100
 6771
UCUCUGCAACAUUGACAUA
1100
 6789
UAUGUCAAUGUUGCAGAGA
2367


 


 6807
UUGCAAAAUUAUGACUUCA
1101
 6807
UUGCAAAAUUAUGACUUCA
1101
 6825
UGAAGUCAUAAUUUUGCAA
2368


 


 6951
UUAUGUAUCAAAUAAGGGG
1102
 6951
UUAUGUAUCAAAUAAGGGG
1102
 6969
CCCCUUAUUUGAUACAUAA
2369


 


 6987
AGGUAAUACAUUAUAUUAU
1103
 6987
AGGUAAUACAUUAUAUUAU
1103
 7005
AUAAUAUAAUGUAUUACCU
2370


 


 7059
UUUCUAUGACCCAUUAGUG
1104
 7059
UUUCUAUGACCCAUUAGUG
1104
 7077
CACUAAUGGGUCAUAGAAA
2371


 


 7077
GUUCCCCUCUGAUGAAUUU
1105
 7077
GUUCCCCUCUGAUGAAUUU
1105
 7095
AAAUUCAUCAGAGGGGAAC
2372


 


 7257
UGCCGUUGGACUGCUCCUA
1106
 7257
UGCCGUUGGACUGCUCCUA
1106
 7275
UAGGAGCAGUCCAACGGCA
2373


 


 7275
AUACUGCAAGGCCAGAAGC
1107
 7275
AUACUGCAAGGCCAGAAGC
1107
 7293
GCUUCUGGCCUUGCAGUAU
2374


 


 7383
UACAAUGGUUUCAUAUCUG
1108
 7383
UACAAUGGUUUCAUAUCUG
1108
 7401
CAGAUAUGAAACCAUUGUA
2375


 


 7473
AUCUCACUUACAUUAUUUA
1109
 7473
AUCUCACUUACAUUAUUUA
1109
 7491
UAAAUAAUGUAAGUGAGAU
2376


 


 7509
UAGUUAUAUAAAACAAUUG
1110
 7509
UAGUUAUAUAAAACAAUUG
1110
 7527
CAAUUGUUUUAUAUAACUA
2377


 


 7545
CUAUUUGUAAAAAAUGAGA
1111
 7545
CUAUUUGUAAAAAAUGAGA
1111
 7563
UCUCAUUUUUUACAAAUAG
2378


 


 7617
UUGCUUGAAUGGUAAGAGG
1112
 7617
UUGCUUGAAUGGUAAGAGG
1112
 7635
CCUCUUACCAUUCAAGCAA
2379


 


 7779
AACAGAAGAGUAUGCCCUC
1113
 7779
AACAGAAGAGUAUGCCCUC
1113
 7797
GAGGGCAUACUCUUCUGUU
2380


 


 7797
CGGUGUAGUUGGAGUGCUA
1114
 7797
CGGUGUAGUUGGAGUGCUA
1114
 7815
UAGCACUCCAACUACACCG
2381


 


 7815
AGAGAGUUAUAUAGGAUCU
1115
 7815
AGAGAGUUAUAUAGGAUCU
1115
 7833
AGAUCCUAUAUAACUCUCU
2382


 


 7833
UAUAAAUAAUAUAACUAAA
1116
 7833
UAUAAAUAAUAUAACUAAA
1116
 7851
UUUAGUUAUAUUAUUUAUA
2383


 


 7887
UGAACUCAACAGUGAUGAC
1117
 7887
UGAACUCAACAGUGAUGAC
1117
 7905
GUCAUCACUGUUGAGUUCA
2384


 


 7905
CAUCAAAAAACUGAGGGAC
1118
 7905
CAUCAAAAAACUGAGGGAC
1118
 7923
GUCCCUCAGUUUUUUGAUG
2385


 


 7923
CAAUGAAGAGCCAAAUUCA
1119
 7923
CAAUGAAGAGCCAAAUUCA
1119
 7941
UGAAUUUGGCUCUUCAUUG
2386


 


 8049
GAAAACCAUAAAAACCACA
1120
 8049
GAAAACCAUAAAAACCACA
1120
 8067
UGUGGUUUUUAUGGUUUUC
2387


 


 8067
AUUGGAUAUCCACAAGAGC
1121
 8067
AUUGGAUAUCCACAAGAGC
1121
 8085
GCUCUUGUGGAUAUCCAAU
2388


 


 8085
CAUAACCAUCAAUAACCCA
1122
 8085
CAUAACCAUCAAUAACCCA
1122
 8103
UGGGUUAUUGAUGGUUAUG
2389


 


 8121
UGAUAUAAACGACCAUGCC
1123
 8121
UGAUAUAAACGACCAUGCC
1123
 8139
GGCAUGGUCGUUUAUAUCA
2390


 


 8175
GUAUAAAUUCCAUACUAAU
1124
 8175
GUAUAAAUUCCAUACUAAU
1124
 8193
AUUAGUAUGGAAUUUAUAC
2391


 


 8193
UAACAAGUAGUUGUAGAGU
1125
 8193
UAACAAGUAGUUGUAGAGU
1125
 8211
ACUCUACAACUACUUGUUA
2392


 


 8247
AUCAAAACAACCAAAAUAA
1126
 8247
AUCAAAACAACCAAAAUAA
1126
 8265
UUAUUUUGGUUGUUUUGAU
2393


 


 8265
ACCAUAUAUACUCACCGAA
1127
 8265
ACCAUAUAUACUCACCGAA
1127
 8283
UUCGGUGAGUAUAUAUGGU
2394


 


 8283
AUCAACCAUUCAAUGAAAU
1128
 8283
AUCAACCAUUCAAUGAAAU
1128
 8301
AUUUCAUUGAAUGGUUGAU
2395


 


 8319
ACUUGAUUGAUGCAAUUCA
1129
 8319
ACUUGAUUGAUGCAAUUCA
1129
 8337
UGAAUUGCAUCAAUCAAGU
2396


 


 8355
UAGGUAUUACUGAUGAUAU
1130
 8355
UAGGUAUUACUGAUGAUAU
1130
 8373
AUAUCAUCAGUAAUACCUA
2397


 


 8373
UAUACACAAUAUAUAUAUU
1131
 8373
UAUACACAAUAUAUAUAUU
1131
 8391
AAUAUAUAUAUUGUGUAUA
2398


 


 8409
UCCUAAUGCUUACCACAUC
1132
 8409
UCCUAAUGCUUACCACAUC
1132
 8427
GAUGUGGUAAGCAUUAGGA
2399


 


 8427
CAUCAAACUAUUAACUCAA
1133
 8427
CAUCAAACUAUUAACUCAA
1133
 8445
UUGAGUUAAUAGUUUGAUG
2400


 


 8445
AACAAUUCAAGCCAUGGGA
1134
 8445
AACAAUUCAAGCCAUGGGA
1134
 8463
UCCCAUGGCUUGAAUUGUU
2401


 


 8499
AUGUGUAUCUAACCGAUAG
1135
 8499
AUGUGUAUCUAACCGAUAG
1135
 8517
CUAUCGGUUAGAUACACAU
2402


 


 8535
UUUCUUUCUCAGAAUGUAA
1136
 8535
UUUCUUUCUCAGAAUGUAA
1136
 8553
UUACAUUCUGAGAAAGAAA
2403


 


 8643
UAAAUCUAAAGAAACUAAA
1137
 8643
UAAAUCUAAAGAAACUAAA
1137
 8661
UUUAGUUUCUUUAGAUUUA
2404


 


 8697
GUGAAAUAAAAAUAGAAGA
1138
 8697
GUGAAAUAAAAAUAGAAGA
1138
 8715
UCUUCUAUUUUUAUUUCAC
2405


 


 8751
AGAGUAUGACCUCGUUAGA
1139
 8751
AGAGUAUGACCUCGUUAGA
1139
 8769
UCUAACGAGGUCAUACUCU
2406


 


 8769
AACAGAUUACUACCACUAA
1140
 8769
AACAGAUUACUACCACUAA
1140
 8787
UUAGUGGUAGUAAUCUGUU
2407


 


 8823
UUAGUGAUGUCAAAGUCUA
1141
 8823
UUAGUGAUGUCAAAGUCUA
1141
 8841
UAGACUUUGACAUCACUAA
2408


 


 8859
UGGGGCUUAAAGAAAAAGA
1142
 8859
UGGGGCUUAAAGAAAAAGA
1142
 8877
UCUUUUUCUUUAAGCCCCA
2409


 


 8913
ACUCAGUUAUUACAACCAU
1143
 8913
ACUCAGUUAUUACAACCAU
1143
 8931
AUGGUUGUAAUAACUGAGU
2410


 


 8949
UUUUAGCUGUUAAGGAUAA
1144
 8949
UUUUAGCUGUUAAGGAUAA
1144
 8967
UUAUCCUUAACAGCUAAAA
2411


 


 8985
CAGUCAAAAAUCACUCUAC
1145
 8985
CAGUCAAAAAUCACUCUAC
1145
 9003
GUAGAGUGAUUUUUGACUG
2412


 


 9003
CAAAACAAAAAGAUACAAU
1146
 9003
CAAAACAAAAAGAUACAAU
1146
 9021
AUUGUAUCUUUUUGUUUUG
2413


 


 9129
AUCGAUCAAGUGAGGUAAA
1147
 9129
AUCGAUCAAGUGAGGUAAA
1147
 9147
UUUACCUCACUUGAUCGAU
2414


 


 9147
AAAACCAUGGUUUUAUAUU
1148
 9147
AAAACCAUGGUUUUAUAUU
1148
 9165
AAUAUAAAACCAUGGUUUU
2415


 


 9165
UGAUAGACAAUCAUACUCU
1149
 9165
UGAUAGACAAUCAUACUCU
1149
 9183
AGAGUAUGAUUGUCUAUCA
2416


 


 9183
UCAAUGGAUUCCAAUUUAU
1150
 9183
UCAAUGGAUUCCAAUUUAU
1150
 9201
AUAAAUUGGAAUCCAUUGA
2417


 


 9327
GGAUUAGUAACUGUUUGAA
1151
 9327
GGAUUAGUAACUGUUUGAA
1151
 9345
UUCAAACAGUUACUAAUCC
2418


 


 9399
CACAACUAUUCCUCUAUGG
1152
 9399
CACAACUAUUCCUCUAUGG
1152
 9417
CCAUAGAGGAAUAGUUGUG
2419


 


 9417
GAGAUUGUAUACUAAAACU
1153
 9417
GAGAUUGUAUACUAAAACU
1153
 9435
AGUUUUAGUAUACAAUCUC
2420


 


 9435
UAUUCCACAAUGAGGGGUU
1154
 9435
UAUUCCACAAUGAGGGGUU
1154
 9453
AACCCCUCAUUGUGGAAUA
2421


 


 9525
GAAAACGGUUUUAUAAUAG
1155
 9525
GAAAACGGUUUUAUAAUAG
1155
 9543
CUAUUAUAAAACCGUUUUC
2422


 


 9723
UGAGUGAAUUAUAUUUUUU
1156
 9723
UGAGUGAAUUAUAUUUUUU
1156
 9741
AAAAAAUAUAAUUCACUCA
2423


 


 9795
UUAAAGUUAAUUGCAACGA
1157
 9795
UUAAAGUUAAUUGCAACGA
1157
 9813
UCGUUGCAAUUAACUUUAA
2424


 


 9867
AUAGAAUUAUAAAAGGAUU
1158
 9867
AUAGAAUUAUAAAAGGAUU
1158
 9885
AAUCCUUUUAUAAUUCUAU
2425


 


 9903
GAUGGCCUACUUUAAGGAA
1159
 9903
GAUGGCCUACUUUAAGGAA
1159
 9921
UUCCUUAAAGUAGGCCAUC
2426


 


 9975
CUUCCUUGUUGGAACUUAC
1160
 9975
CUUCCUUGUUGGAACUUAC
1160
 9993
GUAAGUUCCAACAAGGAAG
2427


 


10011
UUUUAUCAGGACUACGUUU
1161
10011
UUUUAUCAGGACUACGUUU
1161
10029
AAACGUAGUCCUGAUAAAA
2428


 


10065
UUGAAAUGAUCAUAAAUGA
1162
10065
UUGAAAUGAUCAUAAAUGA
1162
10083
UCAUUUAUGAUCAUUUCAA
2429


 


10083
AUAAGGCUAUAUCACCUCC
1163
10083
AUAAGGCUAUAUCACCUCC
1163
10101
GGAGGUGAUAUAGCCUUAU
2430


 


10209
GAGUAUUAGAGUACUAUUU
1164
10209
GAGUAUUAGAGUACUAUUU
1164
10227
AAAUAGUACUCUAAUACUC
2431


 


10281
GUUAUCUUAACAACCCUAA
1165
10281
GUUAUCUUAACAACCCUAA
1165
10299
UUAGGGUUGUUAAGAUAAC
2432


 


10299
AUCAUGUGGUAUCUUUGAC
1166
10299
AUCAUGUGGUAUCUUUGAC
1166
10317
GUCAAAGAUACCACAUGAU
2433


 


10353
UUGCAAUGCAACCAGGAAU
1167
10353
UUGCAAUGCAACCAGGAAU
1167
10371
AUUCCUGGUUGCAUUGCAA
2434


 


10371
UGUUCAGACAAGUUCAAAU
1168
10371
UGUUCAGACAAGUUCAAAU
1168
10389
AUUUGAACUUGUCUGAACA
2435


 


10389
UAUUAGCAGAGAAAAUGAU
1169
10389
UAUUAGCAGAGAAAAUGAU
1169
10407
AUCAUUUUCUCUGCUAAUA
2436


 


10461
UAGAACUACAGAAAAUAUU
1170
10461
UAGAACUACAGAAAAUAUU
1170
10479
AAUAUUUUCUGUAGUUCUA
2437


 


10515
GUUACAAUGAUAAUUACAA
1171
10515
GUUACAAUGAUAAUUACAA
1171
10533
UUGUAAUUAUCAUUGUAAC
2438


 


10587
CAUUUCGAUAUGAAACAUC
1172
10587
CAUUUCGAUAUGAAACAUC
1172
10605
GAUGUUUCAUAUCGAAAUG
2439


 


10623
UACUGGAUGAACUGCAUGG
1173
10623
UACUGGAUGAACUGCAUGG
1173
10641
CCAUGCAGUUCAUCCAGUA
2440


 


10785
AUAUGGGUGGUAUCGAAGG
1174
10785
AUAUGGGUGGUAUCGAAGG
1174
10803
CCUUCGAUACCACCCAUAU
2441


 


10911
UAGAUAUAAGUAAACCAGU
1175
10911
UAGAUAUAAGUAAACCAGU
1175
10929
ACUGGUUUACUUAUAUCUA
2442


 


10983
AUAGUCUUAAAUUACUGUA
1176
10983
AUAGUCUUAAAUUACUGUA
1176
11001
UACAGUAAUUUAAGACUAU
2443


 


11055
CAAGAGAUAUGCAAUUUAU
1177
11055
CAAGAGAUAUGCAAUUUAU
1177
11073
AUAAAUUGCAUAUCUCUUG
2444


 


11217
AUAGAGGAGAAAGUCUAUU
1178
11217
AUAGAGGAGAAAGUCUAUU
1178
11235
AAUAGACUUUCUCCUCUAU
2445


 


11271
AUCAAAUUGCUUUACAACU
1179
11271
AUCAAAUUGCUUUACAACU
1179
11289
AGUUGUAAAGCAAUUUGAU
2446


 


11397
UGUAUAUGAAUUUGCCCAU
1180
11397
UGUAUAUGAAUUUGCCCAU
1180
11415
AUGGGCAAAUUCAUAUACA
2447


 


11523
AUACAAACCAUGAUUUAAA
1181
11523
AUACAAACCAUGAUUUAAA
1181
11541
UUUAAAUCAUGGUUUGUAU
2448


 


11595
UAAUCACAUUUGACAAAAA
1182
11595
UAAUCACAUUUGACAAAAA
1182
11613
UUUUUGUCAAAUGUGAUUA
2449


 


11721
UGAGCACAGCUCCAAACAA
1183
11721
UGAGCACAGCUCCAAACAA
1183
11739
UUGUUUGGAGCUGUGCUCA
2450


 


11757
CACAACACUAUACCACUAC
1184
11757
CACAACACUAUACCACUAC
1184
11775
GUAGUGGUAUAGUGUUGUG
2451


 


11883
AUCUUAUAUCCGGUACAAA
1185
11883
AUCUUAUAUCCGGUACAAA
1185
11901
UUUGUACCGGAUAUAAGAU
2452


 


11991
UGCUUAUAAGGAUAUUUCC
1186
11991
UGCUUAUAAGGAUAUUUCC
1186
12009
GGAAAUAUCCUUAUAAGCA
2453


 


12009
CAUUAGAUUGUAACAGAGA
1187
12009
CAUUAGAUUGUAACAGAGA
1187
12027
UCUCUGUUACAAUCUAAUG
2454


 


12027
AUAAAAGGGAAAUAUUGAG
1188
12027
AUAAAAGGGAAAUAUUGAG
1188
12045
CUCAAUAUUUCCCUUUUAU
2455


 


12117
UUGGUGUUACAUCACCUAG
1189
12117
UUGGUGUUACAUCACCUAG
1189
12135
CUAGGUGAUGUAACACCAA
2456


 


12189
UAAUCAUAGAGAAAUAUAA
1190
12189
UAAUCAUAGAGAAAUAUAA
1190
12207
UUAUAUUUCUCUAUGAUUA
2457


 


12387
AAUUCAUGGAAGAACUUAG
1191
12387
AAUUCAUGGAAGAACUUAG
1191
12405
CUAAGUUCUUCCAUGAAUU
2458


 


12423
UAACAUAUGAGAAAGCCAA
1192
12423
UAACAUAUGAGAAAGCCAA
1192
12441
UUGGCUUUCUCAUAUGUUA
2459


 


12459
AUUUAAGUGUUAACUAUUU
1193
12459
AUUUAAGUGUUAACUAUUU
1193
12477
AAAUAGUUAACACUUAAAU
2460


 


12531
CUUAUAGAACUACAAAUUA
1194
12531
CUUAUAGAACUACAAAUUA
1194
12549
UAAUUUGUAGUUCUAUAAG
2461


 


12549
AUCACUUUGAUACUAGCCC
1195
12549
AUCACUUUGAUACUAGCCC
1195
12567
GGGCUAGUAUCAAAGUGAU
2462


 


12603
AAGAUAUUGAUAUAGUAUU
1196
12603
AAGAUAUUGAUAUAGUAUU
1196
12621
AAUACUAUAUCAAUAUCUU
2463


 


12693
GAAUUAUUCUUAUACCUAA
1197
12693
GAAUUAUUCUUAUACCUAA
1197
12711
UUAGGUAUAAGAAUAAUUC
2464


 


12729
UAAUGAAACCUCCCAUAUU
1198
12729
UAAUGAAACCUCCCAUAUU
1198
12747
AAUAUGGGAGGUUUCAUUA
2465


 


12891
AUUUAAUAUUGGCGCAUAA
1199
12891
AUUUAAUAUUGGCGCAUAA
1199
12909
UUAUGCGCCAAUAUUAAAU
2466


 


12909
AGAUAUCUGACUAUUUUCA
1200
12909
AGAUAUCUGACUAUUUUCA
1200
12927
UGAAAAUAGUCAGAUAUCU
2467


 


12999
AGGGUAUUUUUGAAAAAGA
1201
12999
AGGGUAUUUUUGAAAAAGA
1201
13017
UCUUUUUCAAAAAUACCCU
2468


 


13107
AAGGUUACGGCAGAGCAAA
1202
13107
AAGGUUACGGCAGAGCAAA
1202
13125
UUUGCUCUGCCGUAACCUU
2469


 


13143
AUACUUCAGAUCUCCUAUG
1203
13143
AUACUUCAGAUCUCCUAUG
1203
13161
CAUAGGAGAUCUGAAGUAU
2470


 


13233
AAUACAUUCUUAGCCAGGA
1204
13233
AAUACAUUCUUAGCCAGGA
1204
13251
UCCUGGCUAAGAAUGUAUU
2471


 


13287
UCAAACUAUGGUUUCUUAA
1205
13287
UCAAACUAUGGUUUCUUAA
1205
13305
UUAAGAAACCAUAGUUUGA
2472


 


13413
GAUUGAUAAAUAUAGAUAA
1206
13413
GAUUGAUAAAUAUAGAUAA
1206
13431
UUAUCUAUAUUUAUCAAUC
2473


 


13431
AAAUAUACAUUAAAAAUAA
1207
13431
AAAUAUACAUUAAAAAUAA
1207
13449
UUAUUUUUAAUGUAUAUUU
2474


 


13593
CACCAGAAACCCUAGAAAA
1208
13593
CACCAGAAACCCUAGAAAA
1208
13611
UUUUCUAGGGUUUCUGGUG
2475


 


13611
AUAUACUAACCAAUCCGGU
1209
13611
AUAUACUAACCAAUCCGGU
1209
13629
ACCGGAUUGGUUAGUAUAU
2476


 


13629
UUAAAUGUAAUGACAAAAA
1210
13629
UUAAAUGUAAUGACAAAAA
1210
13647
UUUUUGUCAUUACAUUUAA
2477


 


13773
AUUUAUUUCCUACGGUUGU
1211
13773
AUUUAUUUCCUACGGUUGU
1211
13791
ACAACCGUAGGAAAUAAAU
2478


 


13791
UGAUUGAUAAAAUUAUAGA
1212
13791
UGAUUGAUAAAAUUAUAGA
1212
13809
UCUAUAAUUUUAUCAAUCA
2479


 


13827
CCAAAUCUAACCAACUUUA
1213
13827
CCAAAUCUAACCAACUUUA
1213
13845
UAAAGUUGGUUAGAUUUGG
2480


 


13845
ACACUACUACUUCUCAUCA
1214
13845
ACACUACUACUUCUCAUCA
1214
13863
UGAUGAGAAGUAGUAGUGU
2481


 


13863
AAAUACCUUUAGUGCACAA
1215
13863
AAAUACCUUUAGUGCACAA
1215
13881
UUGUGCACUAAAGGUAUUU
2482


 


14277
CUGAAUUGCCUGUAACAGU
1216
14277
CUGAAUUGCCUGUAACAGU
1216
14295
ACUGUUACAGGCAAUUCAG
2483


 


14313
UAAUAGAGUGGAGCAAGCA
1217
14313
UAAUAGAGUGGAGCAAGCA
1217
14331
UGCUUGCUCCACUCUAUUA
2484


 


14403
AUAUCGAUUUCAAAUUAGA
1218
14403
AUAUCGAUUUCAAAUUAGA
1218
14421
UCUAAUUUGAAAUCGAUAU
2485


 


14457
GCAGUAAGUUAAAGGGGUC
1219
14457
GCAGUAAGUUAAAGGGGUC
1219
14475
GACCCCUUUAACUUACUGC
2486


 


14511
AUGUGUUCCCAGUAUUUAA
1220
14511
AUGUGUUCCCAGUAUUUAA
1220
14529
UUAAAUACUGGGAACACAU
2487


 


14583
CUAAGAAGGCUGAUAAAGA
1221
14583
CUAAGAAGGCUGAUAAAGA
1221
14601
UCUUUAUCAGCCUUCUUAG
2488


 


14727
UAGCAGGACGUAAUGAAGU
1222
14727
UAGCAGGACGUAAUGAAGU
1222
14745
ACUUCAUUACGUCCUGCUA
2489


 


14835
AUAAUCAUUUAUAUAUGGU
1223
14835
AUAAUCAUUUAUAUAUGGU
1223
14853
ACCAUAUAUAAAUGAUUAU
2490


 


14871
AUCUAAGUGAAUUGUUAAA
1224
14871
AUCUAAGUGAAUUGUUAAA
1224
14889
UUUAACAAUUCACUUAGAU
2491


 


14925
AAAUCACAGGUAGUUUGUU
1225
14925
AAAUCACAGGUAGUUUGUU
1225
14943
AACAAACUACCUGUGAUUU
2492


 


14961
AAUAAUGAAUAAAAAUCUU
1226
14961
AAUAAUGAAUAAAAAUCUU
1226
14979
AAGAUUUUUAUUCAUUAUU
2493


 


14979
UAUAUUAAAAAUUCCCAUA
1227
14979
UAUAUUAAAAAUUCCCAUA
1227
14997
UAUGGGAAUUUUUAAUAUA
2494


 


14997
AGCUACACACUAACACUGU
1228
14997
AGCUACACACUAACACUGU
1228
15015
ACAGUGUUAGUGUGUAGCU
2495


 


15051
AAUUUUUUAAUAACUUUUA
1229
15051
AAUUUUUUAAUAACUUUUA
1229
15069
UAAAAGUUAUUAAAAAAUU
2496


 


15069
AGUGAACUAAUCCUAAAAU
1230
15069
AGUGAACUAAUCCUAAAAU
1230
15087
AUUUUAGGAUUAGUUCACU
2497


 


15105
AGGAAUAAAUUUAAAUCCA
1231
15105
AGGAAUAAAUUUAAAUCCA
1231
15123
UGGAUUUAAAUUUAUUCCU
2498


 


15123
AAAUCUAAUUGGUUUAUAU
1232
15123
AAAUCUAAUUGGUUUAUAU
1232
15141
AUAUAAACCAAUUAGAUUU
2499


 


15141
UGUAUAUUAACUAAACUAC
1233
15141
UGUAUAUUAACUAAACUAC
1233
15159
GUAGUUUAGUUAAUAUACA
2500


 


  201
AUACAUUUAACUAAUGCAU
1234
  201
AUACAUUUAACUAAUGCAU
1234
  219
AUGCAUUAGUUAAAUGUAU
2501


 


  255
GGCAUUGUAUUUGUGCAUG
1235
  255
GGCAUUGUAUUUGUGCAUG
1235
  273
CAUGCACAAAUACAAUGCC
2502


 


  309
AUUGUAGUGAAAUCCAAUU
1236
  309
AUUGUAGUGAAAUCCAAUU
1236
  327
AAUUGGAUUUCACUACAAU
2503


 


  327
UUCACAACAAUGCCAGUGU
1237
  327
UUCACAACAAUGCCAGUGU
1237
  345
ACACUGGCAUUGUUGUGAA
2504


 


  345
UUACAAAAUGGAGGUUAUA
1238
  345
UUACAAAAUGGAGGUUAUA
1238
  363
UAUAACCUCCAUUUUGUAA
2505


 


  381
UUAACACACUGCUCUCAAC
1239
  381
UUAACACACUGCUCUCAAC
1239
  399
GUUGAGAGCAGUGUGUUAA
2506


 


  399
CCUAAUGGCCUAAUAGAUG
1240
  399
CCUAAUGGCCUAAUAGAUG
1240
  417
CAUCUAUUAGGCCAUUAGG
2507


 


 1443
CAAGAUAUUAAUGGGAAAG
1241
 1443
CAAGAUAUUAAUGGGAAAG
1241
 1461
CUUUCCCAUUAAUAUCUUG
2508


 


 2325
AAAAAUGGGGCAAAUAAAA
1242
 2325
AAAAAUGGGGCAAAUAAAA
1242
 2343
UUUUAUUUGCCCCAUUUUU
2509


 


 2343
ACAUCAUGGAAAAGUUUGC
1243
 2343
ACAUCAUGGAAAAGUUUGC
1243
 2361
GCAAACUUUUCCAUGAUGU
2510


 


 2865
UAAGAGAAGACAUGAUAGA
1244
 2865
UAAGAGAAGACAUGAUAGA
1244
 2883
UCUAUCAUGUCUUCUCUUA
2511


 


 3423
CACCCAAUGGACCUUCAUU
1245
 3423
CACCCAAUGGACCUUCAUU
1245
 3441
AAUGAAGGUCCAUUGGGUG
2512


 


 4251
AACACGCCAGGCAAAAUCA
1246
 4251
AACACGCCAGGCAAAAUCA
1246
 4269
UGAUUUUGCCUGGCGUGUU
2513


 


 4395
UGAACAAACUCUGUGAAUA
1247
 4395
UGAACAAACUCUGUGAAUA
1247
 4413
UAUUCACAGAGUUUGUUCA
2514


 


 4683
ACCACCAAGACACUAGAAA
1248
 4683
ACCACCAAGACACUAGAAA
1248
 4701
UUUCUAGUGUCUUGGUGGU
2515


 


 5727
UUAUCAAACAACAUGCAGU
1249
 5727
UUAUCAAACAACAUGCAGU
1249
 5745
ACUGCAUGUUGUUUGAUAA
2516


 


 5835
GUGUAAUGGAACAGACGCU
1250
 5835
GUGUAAUGGAACAGACGCU
1250
 5853
AGCGUCUGUUCCAUUACAC
2517


 


 6123
GAACAAAAUCAAAAGUGCU
1251
 6123
GAACAAAAUCAAAAGUGCU
1251
 6141
AGCACUUUUGAUUUUGUUC
2518


 


 6573
GAAACUGCACACAUCCCCU
1252
 6573
GAAACUGCACACAUCCCCU
1252
 6591
AGGGGAUGUGUGCAGUUUC
2519


 


 6969
GGUUGACACUGUGUCUGUA
1253
 6969
GGUUGACACUGUGUCUGUA
1253
 6987
UACAGACACAGUGUCAACC
2520


 


 7725
GGAUAAAAGCAUCGAUACU
1254
 7725
GGAUAAAAGCAUCGAUACU
1254
 7743
AGUAUCGAUGCUUUUAUCC
2521


 


 7743
UUUAUCAGAAAUAAGUGGA
1255
 7743
UUUAUCAGAAAUAAGUGGA
1255
 7761
UCCACUUAUUUCUGAUAAA
2522


 


 8679
UGUCUAAGUAUCAUAAAGG
1256
 8679
UGUCUAAGUAUCAUAAAGG
1256
 8697
CCUUUAUGAUACUUAGACA
2523


 


 9273
UCUUGACAUGGAAAAAUAU
1257
 9273
UCUUGACAUGGAAAAAUAU
1257
 9291
AUAUUUUUCCAUGUCAAGA
2524


 


10641
GUGUACAAUCUCUAUUUUU
1258
10641
GUGUACAAUCUCUAUUUUU
1258
10659
AAAAAUAGAGAUUGUACAC
2525


 


10659
UCUGGUUACAUUUAGCUAU
1259
10659
UCUGGUUACAUUUAGCUAU
1259
10677
AUAGCUAAAUGUAACCAGA
2526


 


11703
UGGCAGUUACUGAGGUUUU
1260
11703
UGGCAGUUACUGAGGUUUU
1260
11721
AAAACCUCAGUAACUGCCA
2527


 


12405
GCAUAGGAAUUCUUGGGUU
1261
12405
GCAUAGGAAUUCUUGGGUU
1261
12423
AACCCAAGAAUUCCUAUGC
2528


 


13719
UUAUUAAAUCGCCUACAAU
1262
13719
UUAUUAAAUCGCCUACAAU
1262
13737
AUUGUAGGCGAUUUAAUAA
2529


 


13989
UUAUAAUUAAAGAUCCUAA
1263
13989
UUAUAAUUAAAGAUCCUAA
1263
14007
UUAGGAUCUUUAAUUAUAA
2530


 


14061
UGGAACUUCAUCCCGAUAU
1264
14061
UGGAACUUCAUCCCGAUAU
1264
14079
AUAUCGGGAUGAAGUUCCA
2531


 


14097
GUCUGAAGGAUUGCAAUGA
1265
14097
GUCUGAAGGAUUGCAAUGA
1265
14115
UCAUUGCAAUCCUUCAGAC
2532


 


15015
UAUUCAAUUAUAGUUAUUU
1266
15015
UAUUCAAUUAUAGUUAUUU
1266
15033
AAAUAACUAUAAUUGAAUA
2533


 


15033
UAAAAUUAAAAAUUAUAUA
1267
15033
UAAAAUUAAAAAUUAUAUA
1267
15051
UAUAUAAUUUUUAAUUUUA
2534





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 sequence is also referred to as the sense strand, whereas the lower sequence is also referred to as the antisense strand. The upper and lower sequences in the Table canfurther comprise a chemical modification having Formulae I-VII, such as exemplary siNA constructs shown in FIGS. 4 and 5, or having modifications described in Table IV or any combination thereof.














TABLE III







RSV Synthetic Modified siNA Constructs















Target

Seq



Seq



Pos
Target
ID
Cmpd#
Aliases
Sequence
ID





9524
AACUUGCGUAAACCAAAAAAAUG
2535

RSV: 27U21 SENSE siNA
CUUGCGUAAACCAAAAAAATT
2639



 


1467
UGGGGCAAAUAAGAAUUUGAUAA
2536

RSV: 48U21 SENSE siNA
GGGCAAAUAAGAAUUUGAUTT
2640


 


5090
UUCUCCAAAAAACUAAGUGAUUC
2537

RSV: 439U21 SENSE siNA
CUCCAAAAAACUAAGUGAUTT
2641


 


7837
CUGGUCAACUAUGAAAUGAAACU
2538

RSV: 815U21 SENSE siNA
GGUCAACUAUGAAAUGAAATT
2642


 


1468
GGGAAGCACUAAAUACAAAAAAU
2539

RSV: 850U21 SENSE siNA
GAAGCACUAAAUACAAAAATT
2643


 


6758
CACUCCCAUAAUAUACAAGUAUG
2540

RSV: 970U21 SENSE siNA
CUCCCAUAAUAUACAAGUATT
2644


 


3973
GUCAUCCAGCAAAUACACCAUCC
2541

RSV: 1197U21 SENSE siNA
CAUCCAGCAAAUACACCAUTT
2645


 


3615
AACAGCUUCUAUGAAGUGUUUGA
2542

RSV: 1762U21 SENSE siNA
CAGCUUCUAUGAAGUGUUUTT
2646


 


9524
AACUUGCGUAAACCAAAAAAAUG
2535

RSV: 45L21 ANTISENSE siNA
UUUUUUUGGUUUACGCAAGTT
2647






(27C)


 


1467
UGGGGCAAAUAAGAAUUUGAUAA
2536

RSV: 66L21 ANTISENSE siNA
AUCAAAUUCUUAUUUGCCCTT
2648






(48C)


 


5090
UUCUCCAAAAAACUAAGUGAUUC
2537

RSV: 457L21 ANTISENSE siNA
AUCACUUAGUUUUUUGGAGTT
2649






(439C)


 


7837
CUGGUCAACUAUGAAAUGAAACU
2538

RSV: 833L21 ANTISENSE siNA
UUUCAUUUCAUAGUUGACCTT
2650






(815C)


 


1468
GGGAAGCACUAAAUACAAAAAAU
2539

RSV: 868L21 ANTISENSE siNA
UUUUUGUAUUUAGUGCUUCTT
2651






(850C)


 


6758
CACUCCCAUAAUAUACAAGUAUG
2540

RSV: 988L21 ANTISENSE siNA
UACUUGUAUAUUAUGGGAGTT
2652






(970C)


 


3973
GUCAUCCAGCAAAUACACCAUCC
2541

RSV: 1215L21 ANTISENSE siNA
AUGGUGUAUUUGCUGGAUGTT
2653






(1197C)


 


3615
AACAGCUUCUAUGAAGUGUUUGA
2542

RSV: 1780L21 ANTISENSE siNA
AAACACUUCAUAGAAGCUGTT
2654






(1762C)


 


9524
AACUUGCGUAAACCAAAAAAAUG
2535

RSV: 27U21 SENSE siNA stab04
B cuuGcGuAAAccAAAAAAATT B
2655


 


1467
UGGGGCAAAUAAGAAUUUGAUAA
2536

RSV: 48U21 SENSE siNA stab04
B GGGcAAAuAAGAAuuuGAuTT B
2656


 


5090
UUCUCCAAAAAACUAAGUGAUUC
2537

RSV: 439U21 SENSE siNA stab04
B cuccAAAAAAcuAAGuGAuTT B
2657


 


7837
CUGGUCAACUAUGAAAUGAAACU
2538

RSV: 815U21 SENSE siNA stab04
B GGucAAcuAuGAAAuGAAATT B
2658


 


1468
GGGAAGCACUAAAUACAAAAAAU
2539

RSV: 850U21 SENSE siNA stab04
B GAAGcAcuAAAuAcAAAAATT B
2659


 


6758
CACUCCCAUAAUAUACAAGUAUG
2540

RSV: 970U21 SENSE siNA stab04
B cucccAuAAuAuAcAAGuATT B
2660


 


3973
GUCAUCCAGCAAAUACACCAUCC
2541

RSV: 1197U21 SENSE siNA
B cAuccAGcAAAuAcAccAuTT B
2661






stab04


 


3615
AACAGCUUCUAUGAAGUGUUUGA
2542

RSV: 1762U21 SENSE siNA
B cAGcuucuAuGAAGuGuuuTT B
2662






stab04


 


9524
AACUUGCGUAAACCAAAAAAAUG
2535

RSV: 45L21 ANTISENSE siNA
uuuuuuuGGuuuAcGcAAGTsT
2663






(27C) stab05


 


1467
UGGGGCAAAUAAGAAUUUGAUAA
2536

RSV: 66L21 ANTISENSE siNA
AucAAAuucuuAuuuGcccTsT
2664






(48C) stab05


 


5090
UUCUCCAAAAAACUAAGUGAUUC
2537

RSV: 457L21 ANTISENSE siNA
AucAcuuAGuuuuuuGGAGTsT
2665






(439C) stab05


 


7837
CUGGUCAACUAUGAAAUGAAACU
2538

RSV: 833L21 ANTISENSE siNA
uuucAuuucAuAGuuGAccTsT
2666






(815C) stab05


 


1468
GGGAAGCACUAAAUACAAAAAAU
2539

RSV: 868L21 ANTISENSE siNA
uuuuuGuAuuuAGuGcuucTsT
2667






(850C) stab05


 


6758
CACUCCCAUAAUAUACAAGUAUG
2540

RSV: 988L21 ANTISENSE siNA
uAcuuGuAuAuuAuGGGAGTsT
2668






(970C) stab05


 


3973
GUCAUCCAGCAAAUACACCAUCC
2541

RSV: 1215L21 ANTISENSE siNA
AuGGuGuAuuuGcuGGAuGTsT
2669






(1197C) stab05


 


3615
AACAGCUUCUAUGAAGUGUUUGA
2542

RSV: 1780L21 ANTISENSE siNA
AAAcAcuucAuAGAAGcuGTsT
2670






(1762C) stab05


 


9524
AACUUGCGUAAACCAAAAAAAUG
2535

RSV: 27U21 SENSE siNA stab07
B cuuGcGuAAAccAAAAAAATT B
2671


 


1467
UGGGGCAAAUAAGAAUUUGAUAA
2536

RSV: 48U21 SENSE siNA stab07
B GGGcAAAuAAGAAuuuGAuTT B
2672


 


5090
UUCUCCAAAAAACUAAGUGAUUC
2537

RSV: 439U21 SENSE siNA stab07
B cuccAAAAAAcuAAGuGAuTT B
2673


 


7837
CUGGUCAACUAUGAAAUGAAACU
2538

RSV: 815U21 SENSE siNA stab07
B GGucAAcuAuGAAAuGAAATT B
2674


 


1468
GGGAAGCACUAAAUACAAAAAAU
2539

RSV: 850U21 SENSE siNA stab07
B GAAGcAcuAAAuAcAAAAATT B
2675


 


6758
CACUCCCAUAAUAUACAAGUAUG
2540

RSV: 970U21 SENSE siNA stab07
B cucccAuAAuAuAcAAGuATT B
2676


 


3973
GUCAUCCAGCAAAUACACCAUCC
2541

RSV: 1197U21 SENSE siNA
B cAuccAGcAAAuAcAccAuTT B
2677






stab07


 


3615
AACAGCUUCUAUGAAGUGUUUGA
2542

RSV: 1762U21 SENSE siNA
B cAGcuucuAuGAAGuGuuuTT B
2678






stab07


 


9524
AACUUGCGUAAACCAAAAAAAUG
2535

RSV: 45L21 ANTISENSE siNA
uuuuuuuGGuuuAcGcAAGTsT
2679






(27C) stab11


 


1467
UGGGGCAAAUAAGAAUUUGAUAA
2536

RSV: 66L21 ANTISENSE siNA
AucAAAuucuuAuuuGcccTsT
2680






(48C) stab11


 


5090
UUCUCCAAAAAACUAAGUGAUUC
2537

RSV: 457L21 ANTISENSE siNA
AucAcuuAGuuuuuuGGAGTsT
2681






(439C) stab11


 


7837
CUGGUCAACUAUGAAAUGAAACU
2538

RSV: 833L21 ANTISENSE siNA
uuucAuuucAuAGuuGAccTsT
2682






(815C) stab11


 


1468
GGGAAGCACUAAAUACAAAAAAU
2539

RSV: 868L21 ANTISENSE siNA
uuuuuGuAuuuAGuGcuucTsT
2683






(850C) stab11


 


6758
CACUCCCAUAAUAUACAAGUAUG
2540

RSV: 988L21 ANTISENSE siNA
uAcuuGuAuAuuAuGGGAGTsT
2684






(970C) stab11


 


3973
GUCAUCCAGCAAAUACACCAUCC
2541

RSV: 1215L21 ANTISENSE siNA
AuGGuGuAuuuGcuGGAuGTsT
2685






(1197C) stab11


 


3615
AACAGCUUCUAUGAAGUGUUUGA
2542

RSV: 1780L21 ANTISENSE siNA
AAAcAcuucAuAGAAGcuGTsT
2686






(1762C) stab11


 


9524
AACUUGCGUAAACCAAAAAAAUG
2535

RSV: 27U21 SENSE siNA stab18
B cuuGcGuAAAccAAAAAAATT B
2687


 


1467
UGGGGCAAAUAAGAAUUUGAUAA
2536

RSV: 48U21 SENSE siNA stab18
B GGGcAAAuAAGAAuuuGAuTT B
2688


 


5090
UUCUCCAAAAAACUAAGUGAUUC
2537

RSV: 439U21 SENSE siNA stab18
B cuccAAAAAAcuAAGuGAuTT B
2689


 


7837
CUGGUCAACUAUGAAAUGAAACU
2538

RSV: 815U21 SENSE siNA stab18
B GGucAAcuAuGAAAuGAAATT B
2690


 


1468
GGGAAGCACUAAAUACAAAAAAU
2539

RSV: 850U21 SENSE siNA stab18
B GAAGcAcuAAAuAcAAAAATT B
2691


 


6758
CACUCCCAUAAUAUACAAGUAUG
2540

RSV: 970U21 SENSE siNA stab18
B cucccAuAAuAuAcAAGuATT B
2692


 


3973
GUCAUCCAGCAAAUACACCAUCC
2541

RSV: 1197U21 SENSE siNA
B cAuccAGcAAAuAcAccAuTT B
2693






stab18


 


3615
AACAGCUUCUAUGAAGUGUUUGA
2542

RSV: 1762U21 SENSE siNA
B cAGcuucuAuGAAGuGuuuTT B
2694






stab18


 


9524
AACUUGCGUAAACCAAAAAAAUG
2535

RSV: 45L21 ANTISENSE siNA
uuuuuuuGGuuuAcGcAAGTsT
2695






(27C) stab08


 


1467
UGGGGCAAAUAAGAAUUUGAUAA
2536

RSV: 66L21 ANTISENSE siNA
AucAAAuucuuAuuuGcccTsT
2696






(48C) stab08


 


5090
UUCUCCAAAAAACUAAGUGAUUC
2537

RSV: 457L21 ANTISENSE siNA
AucAcuuAGuuuuuuGGAGTsT
2697






(439C) stab08


 


7837
CUGGUCAACUAUGAAAUGAAACU
2538

RSV: 833L21 ANTISENSE siNA
uuucAuuucAuAGuuGAccTsT
2698






(815C) stab08


 


1468
GGGAAGCACUAAAUACAAAAAAU
2539

RSV: 868L21 ANTISENSE siNA
uuuuuGuAuuuAGuGcuucTsT
2699






(850C) stab08


 


6758
CACUCCCAUAAUAUACAAGUAUG
2540

RSV: 988L21 ANTISENSE siNA
uAcuuGuAuAuuAuGGGAGTsT
2700






(970C) stab08


 


3973
GUCAUCCAGCAAAUACACCAUCC
2541

RSV: 1215L21 ANTISENSE siNA
AuGGuGuAuuuGcuGGAuGTsT
2701






(1197C) stab08


 


3615
AACAGCUUCUAUGAAGUGUUUGA
2542

RSV: 1780L21 ANTISENSE siNA
AAAcAcuucAuAGAAGcuGTsT
2702






(1762C) stab08


 


9524
AACUUGCGUAAACCAAAAAAAUG
2535

RSV: 27U21 SENSE siNA stab09
B CUUGCGUAAACCAAAAAAATT B
2703


 


1467
UGGGGCAAAUAAGAAUUUGAUAA
2536

RSV: 48U21 SENSE siNA stab09
B GGGCAAAUAAGAAUUUGAUTT B
2704


 


5090
UUCUCCAAAAAACUAAGUGAUUC
2537

RSV: 439U21 SENSE siNA stab09
B CUCCAAAAAACUAAGUGAUTT B
2705


 


7837
CUGGUCAACUAUGAAAUGAAACU
2538

RSV: 815U21 SENSE siNA stab09
B GGUCAACUAUGAAAUGAAATT B
2706


 


1468
GGGAAGCACUAAAUACAAAAAAU
2539

RSV: 850U21 SENSE siNA stab09
B GAAGCACUAAAUACAAAAATT B
2707


 


6758
CACUCCCAUAAUAUACAAGUAUG
2540

RSV: 970U21 SENSE siNA stab09
B CUCCCAUAAUAUACAAGUATT B
2708


 


3973
GUCAUCCAGCAAAUACACCAUCC
2541

RSV: 1197U21 SENSE siNA
B CAUCCAGCAAAUACACCAUTT B
2709






stab09


 


3615
AACAGCUUCUAUGAAGUGUUUGA
2542

RSV: 1762U21 SENSE siNA
B CAGCUUCUAUGAAGUGUUUTT B
2710






stab09


 


9524
AACUUGCGUAAACCAAAAAAAUG
2535

RSV: 45L21 ANTISENSE siNA
UUUUUUUGGUUUACGCAAGTsT
2711






(27C) stab10


 


1467
UGGGGCAAAUAAGAAUUUGAUAA
2536

RSV: 66L21 ANTISENSE siNA
AUCAAAUUCUUAUUUGCCCTsT
2712






(48C) stab10


 


5090
UUCUCCAAAAAACUAAGUGAUUC
2537

RSV: 457L21 ANTISENSE siNA
AUCACUUAGUUUUUUGGAGTsT
2713






(439C) stab10


 


7837
CUGGUCAACUAUGAAAUGAAACU
2538

RSV: 833L21 ANTISENSE siNA
UUUCAUUUCAUAGUUGACCTsT
2714






(815C) stab10


 


1468
GGGAAGCACUAAAUACAAAAAAU
2539

RSV: 868L21 ANTISENSE siNA
UUUUUGUAUUUAGUGCUUCTsT
2715






(850C) stab10


 


6758
CACUCCCAUAAUAUACAAGUAUG
2540

RSV: 988L21 ANTISENSE siNA
UACUUGUAUAUUAUGGGAGTsT
2716






(970C) stab10


 


3973
GUCAUCCAGCAAAUACACCAUCC
2541

RSV: 1215L21 ANTISENSE siNA
AUGGUGUAUUUGCUGGAUGTsT
2717






(1197C) stab10


 


3615
AACAGCUUCUAUGAAGUGUUUGA
2542

RSV: 1780L21 ANTISENSE siNA
AAACACUUCAUAGAAGCUGTsT
2718






(1762C) stab10


 


9524
AACUUGCGUAAACCAAAAAAAUG
2535

RSV: 45L21 ANTISENSE siNA
uuuuuuuGGuuuAcGcAAGTT B
2719






(27C) stab19


 


1467
UGGGGCAAAUAAGAAUUUGAUAA
2536

RSV: 66L21 ANTISENSE siNA
AucAAAuucuuAuuuGcccTT B
2720






(48C) stab19


 


5090
UUCUCCAAAAAACUAAGUGAUUC
2537

RSV: 457L21 ANTISENSE siNA
AucAcuuAGuuuuuuGGAGTT B
2721






(439C) stab19


 


7837
CUGGUCAACUAUGAAAUGAAACU
2538

RSV: 833L21 ANTISENSE siNA
uuucAuuucAuAGuuGAccTT B
2722






(815C) stab19


 


1468
GGGAAGCACUAAAUACAAAAAAU
2539

RSV: 868L21 ANTISENSE siNA
uuuuuGuAuuuAGuGcuucTT B
2723






(850C) stab19


 


6758
CACUCCCAUAAUAUACAAGUAUG
2540

RSV: 988L21 ANTISENSE siNA
uAcuuGuAuAuuAuGGGAGTT B
2724






(970C) stab19


 


3973
GUCAUCCAGCAAAUACACCAUCC
2541

RSV: 1215L21 ANTISENSE siNA
AuGGuGuAuuuGcuGGAuGTT B
2725






(1197C) stab19


 


3615
AACAGCUUCUAUGAAGUGUUUGA
2542

RSV: 1780L21 ANTISENSE siNA
AAAcAcuucAuAGAAGcuGTT B
2726






(1762C) stab19


 


9524
AACUUGCGUAAACCAAAAAAAUG
2535

RSV: 45L21 ANTISENSE siNA
UUUUUUUGGUUUACGCAAGTT B
2727






(27C) stab22


 


1467
UGGGGCAAAUAAGAAUUUGAUAA
2536

RSV: 66L21 ANTISENSE siNA
AUCAAAUUCUUAUUUGCCCTT B
2728






(48C) stab22


 


5090
UUCUCCAAAAAACUAAGUGAUUC
2537

RSV: 457L21 ANTISENSE siNA
AUCACUUAGUUUUUUGGAGTT B
2729






(439C) stab22


 


7837
CUGGUCAACUAUGAAAUGAAACU
2538

RSV: 833L21 ANTISENSE siNA
UUUCAUUUCAUAGUUGACCTT B
2730






(815C) stab22


 


1468
GGGAAGCACUAAAUACAAAAAAU
2539

RSV: 868L21 ANTISENSE siNA
UUUUUGUAUUUAGUGCUUCTT B
2731






(850C) stab22


 


6758
CACUCCCAUAAUAUACAAGUAUG
2540

RSV: 988L21 ANTISENSE siNA
UACUUGUAUAUUAUGGGAGTT B
2732






(970C) stab22


 


3973
GUCAUCCAGCAAAUACACCAUCC
2541

RSV: 1215L21 ANTISENSE siNA
AUGGUGUAUUUGCUGGAUGTT B
2733






(1197C) stab22


 


3615
AACAGCUUCUAUGAAGUGUUUGA
2542

RSV: 1780L21 ANTISENSE siNA
AAACACUUCAUAGAAGCUGTT B
2734






(1762C) stab22


 


9524
AACUUGCGUAAACCAAAAAAAUG
2535

RSV: 45L21 ANTISENSE siNA
UUUuuuuGGuuuAcGcAAGTsT
2735






(27C) stab25


 


1467
UGGGGCAAAUAAGAAUUUGAUAA
2536

RSV: 66L21 ANTISENSE siNA
AUCAAAuucuuAuuuGcccTsT
2736






(48C) stab25


 


5090
UUCUCCAAAAAACUAAGUGAUUC
2537

RSV: 457L21 ANTISENSE siNA
AUCAcuuAGuuuuuuGGAGTsT
2737






(439C) stab25


 


7837
CUGGUCAACUAUGAAAUGAAACU
2538

RSV: 833L21 ANTISENSE siNA
UUUcAuuucAuAGuuGAccTsT
2738






(815C) stab25


 


1468
GGGAAGCACUAAAUACAAAAAAU
2539

RSV: 868L21 ANTISENSE siNA
UUUuuGuAuuuAGuGcuucTsT
2739






(850C) stab25


 


6758
CACUCCCAUAAUAUACAAGUAUG
2540

RSV: 988L21 ANTISENSE siNA
UACuuGuAuAuuAuGGGAGTsT
2740






(970C) stab25


 


3973
GUCAUCCAGCAAAUACACCAUCC
2541

RSV: 1215L21 ANTISENSE siNA
AUGGuGuAuuuGcuGGAuGTsT
2741






(1197C) stab25


 


3615
AACAGCUUCUAUGAAGUGUUUGA
2542

RSV: 1780L21 ANTISENSE siNA
AAAcAcuucAuAGAAGcuGTsT
2742






(1762C) stab25





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













TABLE IV







Non-limiting examples of Stabilization Chemistries for chemically modified


siNA constructs












Chemistry
pyrimidine
Purine
cap
p = S
Strand





“Stab 00”
Ribo
Ribo
TT at 3′-ends

S/AS


“Stab 1”
Ribo
Ribo
—
5 at 5′-end
S/AS






1 at 3′-end


“Stab 2”
Ribo
Ribo
—
All linkages
Usually AS


“Stab 3”
2′-fluoro
Ribo
—
4 at 5′-end
Usually S






4 at 3′-end


“Stab 4”
2′-fluoro
Ribo
5′ and 3′-
—
Usually S





ends


“Stab 5”
2′-fluoro
Ribo
—
1 at 3′-end
Usually AS


“Stab 6”
2′-O-Methyl
Ribo
5′ and 3′-
—
Usually S





ends


“Stab 7”
2′-fluoro
2′-deoxy
5′ and 3′-
—
Usually S





ends


“Stab 8”
2′-fluoro
2′-O-
—
1 at 3′-end
S/AS




Methyl


“Stab 9”
Ribo
Ribo
5′ and 3′-
—
Usually S





ends


“Stab 10”
Ribo
Ribo
—
1 at 3′-end
Usually AS


“Stab 11”
2′-fluoro
2′-deoxy
—
1 at 3′-end
Usually AS


“Stab 12”
2′-fluoro
LNA
5′ and 3′-

Usually S





ends


“Stab 13”
2′-fluoro
LNA

1 at 3′-end
Usually AS


“Stab 14”
2′-fluoro
2′-deoxy

2 at 5′-end
Usually AS






1 at 3′-end


“Stab 15”
2′-deoxy
2′-deoxy

2 at 5′-end
Usually AS






1 at 3′-end


“Stab 16”
Ribo
2′-O-
5′ and 3′-

Usually S




Methyl
ends


“Stab 17”
2′-O-Methyl
2′-O-
5′ and 3′-

Usually S




Methyl
ends


“Stab 18”
2′-fluoro
2′-O-
5′ and 3′-

Usually S




Methyl
ends


“Stab 19”
2′-fluoro
2′-O-
3′-end

S/AS




Methyl


“Stab 20”
2′-fluoro
2′-deoxy
3′-end

Usually AS


“Stab 21”
2′-fluoro
Ribo
3′-end

Usually AS


“Stab 22”
Ribo
Ribo
3′-end

Usually AS


“Stab 23”
2′-fluoro*
2′-deoxy*
5′ and 3′-

Usually S





ends


“Stab 24”
2′-fluoro*
2′-O-
—
1 at 3′-end
S/AS




Methyl*


“Stab 25”
2′-fluoro*
2′-O-
—
1 at 3′-end
S/AS




Methyl*


“Stab 26”
2′-fluoro*
2′-O-
—

S/AS




Methyl*


“Stab 27”
2′-fluoro*
2′-O-
3′-end

S/AS




Methyl*


“Stab 28”
2′-fluoro*
2′-O-
3′-end

S/AS




Methyl*


“Stab 29”
2′-fluoro*
2′-O-

1 at 3′-end
S/AS




Methyl*


“Stab 30”
2′-fluoro*
2′-O-


S/AS




Methyl*


“Stab 31”
2′-fluoro*
2′-O-
3′-end

S/AS




Methyl*


“Stab 32”
2′-fluoro
2′-O-


S/AS




Methyl


“Stab 33”
2′-fluoro
2′-deoxy*
5′ and 3′-
—
Usually S





ends


“Stab 34”
2′-fluoro
2′-O-
5′ and 3′-

Usually S




Methyl*
ends


“Stab 35”
2′-fluoro
2′-O-


Usually AS




Methyl**


“Stab 36”
2′-fluoro
2′-O-


Usually AS




Methyl**


“Stab 3F”
2′-OCF3
Ribo
—
4 at 5′-end
Usually S






4 at 3′-end


“Stab 4F”
2′-OCF3
Ribo
5′ and 3′-
—
Usually S





ends


“Stab 5F”
2′-OCF3
Ribo
—
1 at 3′-end
Usually AS


“Stab 7F”
2′-OCF3
2′-deoxy
5′ and 3′-
—
Usually S





ends


“Stab 8F”
2′-OCF3
2′-O-
—
1 at 3′-end
S/AS




Methyl


“Stab 11F”
2′-OCF3
2′-deoxy
—
1 at 3′-end
Usually AS


“Stab 12F”
2′-OCF3
LNA
5′ and 3′-

Usually S





ends


“Stab 13F”
2′-OCF3
LNA

1 at 3′-end
Usually AS


“Stab 14F”
2′-OCF3
2′-deoxy

2 at 5′-end
Usually AS






1 at 3′-end


“Stab 15F”
2′-OCF3
2′-deoxy

2 at 5′-end
Usually AS






1 at 3′-end


“Stab 18F”
2′-OCF3
2′-O-
5′ and 3′-

Usually S




Methyl
ends


“Stab 19F”
2′-OCF3
2′-O-
3′-end

S/AS




Methyl


“Stab 20F”
2′-OCF3
2′-deoxy
3′-end

Usually AS


“Stab 21F”
2′-OCF3
Ribo
3′-end

Usually AS


“Stab 23F”
2′-OCF3*
2′-deoxy*
5′ and 3′-

Usually S





ends


“Stab 24F”
2′-OCF3*
2′-O-
—
1 at 3′-end
S/AS




Methyl*


“Stab 25F”
2′-OCF3*
2′-O-
—
1 at 3′-end
S/AS




Methyl*


“Stab 26F”
2′-OCF3*
2′-O-
—

S/AS




Methyl*


“Stab 27F”
2′-OCF3*
2′-O-
3′-end

S/AS




Methyl*


“Stab 28F”
2′-OCF3*
2′-O-
3′-end

S/AS




Methyl*


“Stab 29F”
2′-OCF3*
2′-O-

1 at 3′-end
S/AS




Methyl*


“Stab 30F”
2′-OCF3*
2′-O-


S/AS




Methyl*


“Stab 31F”
2′-OCF3*
2′-O-
3′-end

S/AS




Methyl*


“Stab 32F”
2′-OCF3
2′-O-


S/AS




Methyl


“Stab 33F”
2′-OCF3
2′-deoxy*
5′ and 3′-
—
Usually S





ends


“Stab 34F”
2′-OCF3
2′-O-
5′ and 3′-

Usually S




Methyl*
ends


“Stab 35”
2′-OCF3
2′-O-


Usually AS




Methyl**


“Stab 36”
2′-OCF3
2′-O-


Usually AS




Methyl**





CAP = any terminal cap, see for example FIG. 10.


All Stab 00-36 chemistries can comprise 3′-terminal thymidine (TT) residues


All Stab 00-36 chemistries typically comprise about 21 nucleotides, but can vary as described herein.


All Stab 00-36 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


**Stab 35 has 2′-O-methyl U at 3′-overhangs


**Stab 36 has 2′-O-methyl overhangs that are complementary to the target sequence (naturually occurring overhangs)


















TABLE V







Reagent
Equivalents
Amount
Wait Time* DNA
Wait Time* 2′-O-methyl
Wait Time* RNA










A. 2.5 μmol Synthesis Cycle ABI 394 Instrument
















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
















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/2′-O-
Wait
Wait Time*
Wait Time*


Reagent
2′-O-methyl/Ribo
methyl/Ribo
Time* DNA
2′-O-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













TABLE VI







Lipid Nanoparticle (LNP) Formulations









Formulation




#
Composition
Molar Ratio





L051
CLinDMA/DSPC/Chol/PEG-n-DMG
48/40/10/2


L053
DMOBA/DSPC/Chol/PEG-n-DMG
30/20/48/2


L054
DMOBA/DSPC/Chol/PEG-n-DMG
50/20/28/2


L069
CLinDMA/DSPC/Cholesterol/PEG-
48/40/10/2



Cholesterol


L073
pCLinDMA or CLin DMA/DMOBA/
25/25/20/28/2



DSPC/Chol/PEG-n-DMG


L077
eCLinDMA/DSPC/Cholesterol/
48/40/10/2



2KPEG-Chol


L080
eCLinDMA/DSPC/Cholesterol/
48/40/10/2



2KPEG-DMG


L082
pCLinDMA/DSPC/Cholesterol/
48/40/10/2



2KPEG-DMG


L083
pCLinDMA/DSPC/Cholesterol/
48/40/10/2



2KPEG-Chol


L086
CLinDMA/DSPC/Cholesterol/2KPEG-
43/38/10/2/7



DMG/Linoleyl alcohol


L061
DMLBA/Cholesterol/2KPEG-DMG
52/45/3


L060
DMOBA/Cholesterol/2KPEG-DMG N/P
52/45/3



ratio of 5


L097
DMLBA/DSPC/Cholesterol/2KPEG-
50/20/28



DMG


L098
DMOBA/Cholesterol/2KPEG-DMG,
52/45/3



N/P ratio of 3


L099
DMOBA/Cholesterol/2KPEG-DMG,
52/45/3



N/P ratio of 4


L100
DMOBA/DOBA/3% PEG-DMG, N/P
52/45/3



ratio of 3


L101
DMOBA/Cholesterol/2KPEG-
52/45/3



Cholesterol


L102
DMOBA/Cholesterol/2KPEG-
52/45/3



Cholesterol, N/P ratio of 5


L103
DMLBA/Cholesterol/2KPEG-
52/45/3



Cholesterol


L104
CLinDMA/DSPC/Cholesterol/2KPEG-
43/38/10/2/7



cholesterol/Linoleyl alcohol


L105
DMOBA/Cholesterol/2KPEG-Chol, N/P
52/45/3



ratio of 2


L106
DMOBA/Cholesterol/2KPEG-Chol, N/P
67/30/3



ratio of 3


L107
DMOBA/Cholesterol/2KPEG-Chol, N/P
52/45/3



ratio of 1.5


L108
DMOBA/Cholesterol/2KPEG-Chol, N/P
67/30/3



ratio of 2


L109
DMOBA/DSPC/Cholesterol/2KPEG-
50/20/28/2



Chol, N/P ratio of 2


L110
DMOBA/Cholesterol/2KPEG-DMG,
52/45/3



N/P ratio of 1.5


L111
DMOBA/Cholesterol/2KPEG-DMG,
67/30/3



N/P ratio of 1.5


L112
DMLBA/Cholesterol/2KPEG-DMG, N/P
52/45/3



ratio of 1.5


L113
DMLBA/Cholesterol/2KPEG-DMG, N/P
67/30/3



ratio of 1.5


L114
DMOBA/Cholesterol/2KPEG-DMG,
52/45/3



N/P ratio of 2


L115
DMOBA/Cholesterol/2KPEG-DMG,
67/30/3



N/P ratio of 2


L116
DMLBA/Cholesterol/2KPEG-DMG,
52/45/3



N/Pratio of 2


L117
DMLBA/Cholesterol/2KPEG-DMG, N/P
52/45/3



ratio of 2





N/P ratio = Nitrogen:Phosphorous ratio between cationic lipid and nucleic acid




















TABLE VII







Sirna algorithm describing patterns with their relative score for


predicting hyperactive siNAs. All the positions given are for the


sense strand of 19-mer siNA.











Description of pattern
Pattern #
Score















G or C at position 1
1
5



A or U at position 19
2
10



A/U rich between position 15-19
3
10



String of 4 Gs or 4 Cs (not preferred)
4
−100



G/C rich between position 1-5
5
10



A or U at position 18
6
5



A or U at position 10
7
10



G at position 13 (not preferred)
8
−3



A at position 13
9
3



G at position 9 (not preferred)
10
−3



A at position 9
11
3



A or U at position 14
12
10

## Claims

1. A chemically modified short interfering nucleic acid (siNA) molecule, wherein:
(a) the siNA molecule comprises a sense strand and a separate antisense strand, each strand having one or more pyrimidine nucleotides and one or more purine nucleotides;
(b) each strand is independently 18 to 27 nucleotides in length, and together comprise a duplex having between 17 and 23 base pairs;
(c) the antisense strand is complementary to a human Respiratory Syncytial Virus (RSV) RNA sequence comprising SEQ ID NO:2764;
(d) a plurality of the pyrimidine nucleotides present in the sense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and a plurality of the purine nucleotides present in the sense strand are 2′-deoxy purine nucleotides; and,
(e) a plurality of the pyrimidine nucleotides in the antisense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and a plurality of the purine nucleotides present in the antisense strand are 2′-O-methyl purine nucleotides.

2. The siNA molecule of claim 1, wherein the sense strand includes a terminal cap moiety at both 5′- and 3′-ends.

3. The siNA molecule of claim 1, wherein the sense strand, the antisense strand, or both the sense strand and the antisense strand comprise a 3′-overhang.

4. The siNA molecule of claim 1, wherein the antisense strand has a phosphorothioate internucleotide linkage at the 3′-end.

5. A composition comprising the siNA molecule of claim 1 and a pharmaceutically acceptable carrier or diluent.