# Patent

## Title
Compositions and methods for inhibiting expression of Eg5 and VEGF genes

## Abstract

This invention relates to compositions containing double-stranded ribonucleic acid (dsRNA) in a SNALP formulation, and methods of using the compositions to inhibit the expression of the Eg5 and Vascular Endothelial Growth Factor (VEGF), and methods of using the compositions to treat pathological processes mediated by Eg5 and VEGF expression, such as cancer.

## Background
RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 12/552,207, filed Sep. 1, 2009, which is a continuation of PCT Application No. PCT/US2009/036233, filed Mar. 5, 2009 which claims the benefit of U.S. Provisional Application No. 61/034,019, filed Mar. 5, 2008, and U.S. Provisional Application No. 61/083,367, filed Jul. 24, 2008, and U.S. Provisional Application No. 61/086,381, filed Aug. 5, 2008, and U.S. Provisional Application No. 61/112,079, filed Nov. 6, 2008, and U.S. Provisional Application No. 61/150,664, filed Feb. 6, 2009 which are hereby incorporated in their entirety by reference.


FIELD OF THE INVENTION
This invention relates to compositions containing double-stranded ribonucleic acid (dsRNA), and their use in mediating RNA interference to inhibit the expression of a combination of genes, e.g., the Eg5 and Vascular Endothelial Growth Factor (VEGF) genes formulated in SNALP, and the use of the compositions to treat pathological processes mediated by Eg5 and VEGF expression, such as cancer.
REFERENCE TO SEQUENCE LISTING
This application includes a Sequence Listing submitted electronically as a text file named 20876_sequence_listing.txt, created on Apr. 9, 2012, with a size of 640,482 bytes. The sequence listing is incorporated by reference.
BACKGROUND OF THE INVENTION
The maintenance of cell populations within an organism is governed by the cellular processes of cell division and programmed cell death. Within normal cells, the cellular events associated with the initiation and completion of each process is highly regulated. In proliferative disease such as cancer, one or both of these processes may be perturbed. For example, a cancer cell may have lost its regulation (checkpoint control) of the cell division cycle through either the overexpression of a positive regulator or the loss of a negative regulator, perhaps by mutation.
Alternatively, a cancer cell may have lost the ability to undergo programmed cell death through the overexpression of a negative regulator. Hence, there is a need to develop new chemotherapeutic drugs that will restore the processes of checkpoint control and programmed cell death to cancerous cells.
One approach to the treatment of human cancers is to target a protein that is essential for cell cycle progression. In order for the cell cycle to proceed from one phase to the next, certain prerequisite events must be completed. There are checkpoints within the cell cycle that enforce the proper order of events and phases. One such checkpoint is the spindle checkpoint that occurs during the metaphase stage of mitosis. Small molecules that target proteins with essential functions in mitosis may initiate the spindle checkpoint to arrest cells in mitosis. Of the small molecules that arrest cells in mitosis, those which display anti-tumor activity in the clinic also induce apoptosis, the morphological changes associated with programmed cell death. An effective chemotherapeutic for the treatment of cancer may thus be one which induces checkpoint control and programmed cell death. Unfortunately, there are few compounds available for controlling these processes within the cell. Most compounds known to cause mitotic arrest and apoptosis act as tubulin binding agents. These compounds alter the dynamic instability of microtubules and indirectly alter the function/structure of the mitotic spindle thereby causing mitotic arrest. Because most of these compounds specifically target the tubulin protein which is a component of all microtubules, they may also affect one or more of the numerous normal cellular processes in which microtubules have a role. Hence, there is also a need for agents that more specifically target proteins associated with proliferating cells.
Eg5 is one of several kinesin-like motor proteins that are localized to the mitotic spindle and known to be required for formation and/or function of the bipolar mitotic spindle. Recently, there was a report of a small molecule that disturbs bipolarity of the mitotic spindle (Mayer, T. U. et. al. 1999. Science 286(5441) 971-4, herein incorporated by reference). More specifically, the small molecule induced the formation of an aberrant mitotic spindle wherein a monoastral array of microtubules emanated from a central pair of centrosomes, with chromosomes attached to the distal ends of the microtubules. The small molecule was dubbed “monastrol” after the monoastral array. This monoastral array phenotype had been previously observed in mitotic cells that were immunodepleted of the Eg5 motor protein. This distinctive monoastral array phenotype facilitated identification of monastrol as a potential inhibitor of Eg5. Indeed, monastrol was further shown to inhibit the Eg5 motor-driven motility of microtubules in an in vitro assay. The Eg5 inhibitor monastrol had no apparent effect upon the related kinesin motor or upon the motor(s) responsible for golgi apparatus movement within the cell. Cells that display the monoastral array phenotype either through immunodepletion of Eg5 or monastrol inhibition of Eg5 arrest in M-phase of the cell cycle. However, the mitotic arrest induced by either immunodepletion or inhibition of Eg5 is transient (Kapoor, T. M., 2000. J Cell Biol 150(5) 975-80). Both the monoastral array phenotype and the cell cycle arrest in mitosis induced by monastrol are reversible. Cells recover to form a normal bipolar mitotic spindle, to complete mitosis and to proceed through the cell cycle and normal cell proliferation. These data suggest that an inhibitor of Eg5 which induced a transient mitotic arrest may not be effective for the treatment of cancer cell proliferation. Nonetheless, the discovery that monastrol causes mitotic arrest is intriguing and hence there is a need to further study and identify compounds which can be used to modulate the Eg5 motor protein in a manner that would be effective in the treatment of human cancers. There is also a need to explore the use of these compounds in combination with other antineoplastic agents.
VEGF (also known as vascular permeability factor, VPF) is a multifunctional cytokine that stimulates angiogenesis, epithelial cell proliferation, and endothelial cell survival. VEGF can be produced by a wide variety of tissues, and its overexpression or aberrant expression can result in a variety disorders, including cancers and retinal disorders such as age-related macular degeneration and other angiogenic disorders.
Recently, double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et al.) discloses the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of genes in C. elegans. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanism has now become the focus for the development of a new class of pharmaceutical agents for treating disorders that are caused by the aberrant or unwanted regulation of a gene.
SUMMARY OF THE INVENTION
Disclosed are compositions having two double-stranded ribonucleic acids (dsRNA) for inhibiting the expression of a human kinesin family member 11 (Eg5/KSP) and a human VEGF gene in a cell. The dsRNAs are formulated in a stable nucleic acid lipid particle (SNALP). Also disclosed are method for using the composition to decrease expression of Eg5/KSP and/or VEGF in a cell, and method of treatment of a disease, e.g., liver cancer, using the compositions of the invention.
Accordingly, disclosed herein is a composition having a first double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a human kinesin family member 11 (Eg5/KSP) gene in a cell and a second dsRNA for inhibiting expression of a human VEGF in a cell, wherein both said first and said second dsRNA are formulated in a stable nucleic acid lipid particle (SNALP); said first dsRNA consists of a first sense strand and a first antisense strand, and said first sense strand has a first sequence and said first antisense strand has a second sequence complementary to at least 15 contiguous nucleotides of SEQ ID NO: 1311 (5′-UCGAGAAUCUAAACUAACU-3′), wherein said first sequence is complementary to said second sequence and wherein said first dsRNA is between 15 and 30 base pairs in length; and said second dsRNA consists of a second sense strand and a second antisense strand, said second sense strand having a third sequence and said second antisense strand having a fourth sequence complementary to at least 15 contiguous nucleotides of SEQ ID NO: 1538 (5′-GCACAUAGGAGAGAUGAGCUU-3′), wherein said third sequence is complementary to said fourth sequence and wherein each strand is between 15 and 30 base pairs in length.
In some embodiments, the first antisense strand has a second sequence complementary to SEQ ID NO: 1311 (5′-UCGAGAAUCUAAACUAACU-3′) and the second antisense strand has a fourth sequence complementary to SEQ ID NO: 1538 (5′-GCACAUAGGAGAGAUGAGCUU-3′). In other embodiments, the first dsRNA consists of a sense strand consisting of SEQ ID NO: 1534 (5′-UCGAGAAUCUAAACUAACUTT-3′) and an antisense strand consisting of SEQ ID NO: 1535 (5′-AGUUAGUUUAGAUUCUCGATT-3′) and the second dsRNA consists of a sense strand consisting of SEQ ID NO:1536 (5′-GCACAUAGGAGAGAUGAGCUU-3′), and an antisense strand consisting of SEQ ID NO: 1537 (5′-AAGCUCAUCUCUCCUAUGUGCUG-3′). In further embodiments, each strand is modified as follows to include a 2′-O-methyl ribonucleotide as indicated by a lower case letter “c” or “u” and a phosphorothioate as indicated by a lower case letter “s”: the first dsRNA consists of a sense strand consisting of SEQ ID NO: 1240 (5′-ucGAGAAucuAAAcuAAcuTsT-3′), and an antisense strand consisting of SEQ ID NO: 1241 (5′-AGUuAGUUuAGAUUCUCGATsT); the second dsRNA consists of a sense strand consisting of SEQ ID NO: 1242 (5′-GcAcAuAGGAGAGAuGAGCUsU-3′) and an antisense strand consisting of SEQ ID NO: 1243 (5′-AAGCUcAUCUCUCCuAuGuGCusG-3′).
In some embodiments, the first dsRNA contains two overhangs and the second dsRNA contains an overhang at the 3′ of the antisense and a blunt end at the 5′ end of the antisense strand.
The first and second dsRNA can have at least one modified nucleotide. For example, each dsRNA can have at least one modified nucleotide chosen from the group of: a 2′-O-methyl modified nucleotide, a nucleotide having a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group. The modified nucleotide can be chosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base having nucleotide. In some embodiments, the first and second dsRNA each comprise at least one 2′-O-methyl modified ribonucleotide and at least one nucleotide having a 5′-phosphorothioate group.
Each strand of each dsRNA can be, e.g., 19-23 bases in length, or, alternatively 21-23 bases in length. In one embodiment, each strand of the first dsRNA is 21 bases in length and the sense strand of the second dsRNA is 21 bases in length and the antisense strand of the second dsRNA is 23 bases in length.
In some embodiments, the first and second dsRNA are present in an equimolar ratio.
As described herein, the dsRNAs are formulated as SNALPS. In some embodiments, the SNALP formulation includes DLinDMA, cholesterol, DPPC, and PEG2000-C-DMA. For example, the SNALP can have the components in the proportions listed in Table 17.
The composition of the invention can be used to reduce expression of Eg5 and/or VAGF. In some embodiments, the composition of the invention, upon contact with a cell expressing Eg5, inhibits expression of Eg5 by at least 40, 50, 60, 70, 80, or by at least 90%. In other embodiments, the composition of the invention, upon contact with a cell expressing VEGF, inhibits expression of VEGF by at least 40, 50, 60, 70, 80, or by at least 90%. Administration of the composition to a cell can expression of both Eg5 and VEGF in said cell. The composition of claims 1-17, wherein the composition is administered in a nM concentration.
Administration of the composition of the invention to a cell can result in, e.g., an increase in mono-aster formation in the cell. Administration of the composition to a mammal can result in at least one effect selected from the group consisting of prevention of tumor growth, reduction in tumor growth, or prolonged survival in said mammal. The effect can be measured using at least one assay selected from the group consisting of determination of body weight, determination of organ weight, visual inspection, mRNA analysis, serum AFP analysis and survival monitoring. Included are compositions with these effect when administered in a nM concentration.
In a further embodiment the composition of the invention includes Sorafenib.
Also included in the invention are methods of suing the compositions of the invention. In one embodiment is are methods for inhibiting the expression of Eg5/KSP and VEGF in a cell by administering any of the compositions of the invention to the cell. Other embodiments are methods for preventing tumor growth, reducing tumor growth, or prolonging survival in a mammal in need of treatment for cancer by administering the composition to said mammal. In some embodiments the mammal has liver cancer, e.g., the mammal is a human with liver cancer. The method can include a further step of administering Sorafenib.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a graph showing liver weights as percentage of body weight following administration of SNALP-siRNAs in a Hep3B mouse model.
FIGS. 2A-2D are graphs showing the effects of SNALP-siRNAs on body weight in a Hep3B mouse model.
FIG. 3 is a graph showing the effects of SNALP-siRNAs on body weight in a Hep3B mouse model.
FIG. 4 is a graph showing the body weight in untreated control animals.
FIG. 5 is a graph showing the effects of control luciferase-SNALP siRNAs on body weight in a Hep3B mouse model.
FIG. 6 is a graph showing the effects of VSP-SNALP siRNAs on body weight in a Hep3B mouse model.
FIG. 7A is a graph showing the effects of SNALP-siRNAs on human GAPDH levels normalized to mouse GAPDH levels in a Hep3B mouse model.
FIG. 7B is a graph showing the effects of SNALP-siRNAs on serum AFP levels as measured by serum ELISA in a Hep3B mouse model.
FIG. 8 is a graph showing the effects of SNALP-siRNAs on human GAPDH levels normalized to mouse GAPDH levels in a Hep3B mouse model.
FIG. 9 is a graph showing the effects of SNALP-siRNAs on human KSP levels normalized to human GAPDH levels in a Hep3B mouse model.
FIG. 10 is a graph showing the effects of SNALP-siRNAs on human VEGF levels normalized to human GAPDH levels in a Hep3B mouse model.
FIG. 11A is a graph showing the effects of SNALP-siRNAs on mouse VEGF levels normalized to human GAPDH levels in a Hep3B mouse model.
FIG. 11B is a set of graphs showing the effects of SNALP-siRNAs on human GAPDH levels and serum AFP levels in a Hep3B mouse model.
FIGS. 12A-12C are graphs showing the effects of SNALP-siRNAs on tumor KSP, VEGF and GAPDH levels in a Hep3B mouse model.
FIG. 13A and FIG. 13B are graphs showing the effects of SNALP-siRNAs on survival in mice with hepatic tumors. Treatment was started at 18 days (FIG. 13A) and 26 days (FIG. 13B) after tumor cell seeding.
FIG. 14 is a graph showing the effects of SNALP-siRNAs on serum alpha fetoprotein (AFP) levels.
FIGS. 15A and 15B are images of H&E stained sections in tumor bearing animals (three weeks after Hep3B cell implantation) were administered 2 mg/kg SNALP-VSP (A) or 2 mg/kg SNALP-Luc (B). Twenty four hours later, tumor bearing liver lobes were processed for histological analysis. Arrows indicate mono asters.
FIG. 16 is a flow diagram illustrating the manufacturing process of ALN-VSPDS01.
FIG. 17 is a cryo-transmission electron microscope (cryo-TEM) image of ALN-VSP02.
FIG. 18 is a flow diagram illustrating the manufacturing process of ALN-VSP02.
FIG. 19 is a graph illustrating the effects on survival of administration SNALP formulated siRNA and Sorafenib.



DETAILED DESCRIPTION OF THE INVENTION
The invention provides compositions and methods for inhibiting the expression of the Eg5 gene and VEGF gene in a cell or mammal using the dsRNAs. The dsRNAs are preferably packaged in a stable nucleic acid particle (SNALP). The invention also provides compositions and methods for treating pathological conditions and diseases, such as liver cancer, in a mammal caused by the expression of the Eg5 gene and VEGF genes. The dsRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi).
The following detailed description discloses how to make and use the compositions containing dsRNAs to inhibit the expression of the Eg5 gene and VEGF genes, respectively, as well as compositions and methods for treating diseases and disorders caused by the expression of these genes, such as cancer. The pharmaceutical compositions featured in the invention include a dsRNA having an antisense strand comprising a region of complementarity which is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an RNA transcript of the Eg5 gene, together with a pharmaceutically acceptable carrier. The compositions featured in the invention also include a dsRNA having an antisense strand having a region of complementarity which is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an RNA transcript of the VEGF gene.
Accordingly, certain aspects of the invention provide pharmaceutical compositions containing the Eg5 and VEGF dsRNAs and a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of the Eg5 gene and the VEGF gene respectively, and methods of using the pharmaceutical compositions to treat diseases caused by expression of the Eg5 and VEGF genes.
I. DEFINITIONS
For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.
“G,” “C,” “A” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively. “T” and “dT” are used interchangeably herein and refer to a deoxyribonucleotide wherein the nucleobase is thymine, e.g., deoxyribothymine. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences comprising such replacement moieties are embodiments of the invention.
As used herein, “Eg5” refers to the human kinesin family member 11, which is also known as KIF11, Eg5, HKSP, KSP, KNSL1 or TRIP5. Eg5 sequence can be found as NCBI GeneID:3832, HGNC ID: HGNC:6388 and RefSeq ID number:NM—004523. The terms “Eg5” and “KSP” and “Eg5/KSP are used interchangeably
As used herein, VEGF, also known as vascular permeability factor, is an angiogenic growth factor. VEGF is a homodimeric 45 kDa glycoprotein that exists in at least three different isoforms. VEGF isoforms are expressed in endothelial cells. The VEGF gene contains 8 exons that express a 189-amino acid protein isoform. A 165-amino acid isoform lacks the residues encoded by exon 6, whereas a 121-amino acid isoform lacks the residues encoded by exons 6 and 7. VEGF145 is an isoform predicted to contain 145 amino acids and to lack exon 7. VEGF can act on endothelial cells by binding to an endothelial tyrosine kinase receptor, such as Flt-1 (VEGFR-1) or KDR/flk-1 (VEGFR-2). VEGFR-2 is expressed in endothelial cells and is involved in endothelial cell differentiation and vasculogenesis. A third receptor, VEGFR-3, has been implicated in lymphogenesis.
The various isoforms have different biologic activities and clinical implications. For example, VEGF145 induces angiogenesis and like VEGF189 (but unlike VEGF165) VEGF 145 binds efficiently to the extracellular matrix by a mechanism that is not dependent on extracellular matrix-associated heparin sulfates. VEGF displays activity as an endothelial cell mitogen and chemoattractant in vitro and induces vascular permeability and angiogenesis in vivo. VEGF is secreted by a wide variety of cancer cell types and promotes the growth of tumors by inducing the development of tumor-associated vasculature. Inhibition of VEGF function has been shown to limit both the growth of primary experimental tumors as well as the incidence of metastases in immunocompromised mice. Various dsRNAs directed to VEGF are described in co-pending U.S. Ser. Nos. 11/078,073 and 11/340,080, which are hereby incorporated by reference in their entirety.
As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of the Eg5/KSP and/or VEGF gene, including mRNA that is a product of RNA processing of a primary transcription product.
As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.
As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.
This includes base-pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence to the oligonucleotide or polynucleotide comprising the second nucleotide sequence over the entire length of the first and second nucleotide sequence. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 4, 3 or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as “fully complementary” for the purposes of the invention.
“Complementary” sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs includes, but not limited to, G:U Wobble or Hoogstein base pairing.
The terms “complementary”, “fully complementary” and “substantially complementary” herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as will be understood from the context of their use.
As used herein, a polynucleotide which is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide which is substantially complementary to a contiguous portion of the mRNA of interest (e.g., encoding Eg5/KSP and/or VEGF) including a 5′ UTR, an open reading frame (ORF), or a 3′ UTR. For example, a polynucleotide is complementary to at least a part of a Eg5 mRNA if the sequence is substantially complementary to a non-interrupted portion of a mRNA encoding Eg5.
The term “double-stranded RNA” or “dsRNA”, as used herein, refers to a duplex structure comprising two anti-parallel and substantially complementary, as defined above, nucleic acid strands. The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′ end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop”. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′ end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker”. The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, a dsRNA may comprise one or more nucleotide overhangs. In general, the majority of nucleotides of each strand are ribonucleotides, but as described in detail herein, each or both strands can also include at least one non-ribonucleotide, e.g., a deoxyribonucleotide and/or a modified nucleotide. In addition, as used in this specification, “dsRNA” may include chemical modifications to ribonucleotides, including substantial modifications at multiple nucleotides and including all types of modifications disclosed herein or known in the art. Any such modifications, as used in an siRNA type molecule, are encompassed by “dsRNA” for the purposes of this specification and claims.
As used herein, a “nucleotide overhang” refers to the unpaired nucleotide or nucleotides that protrude from the duplex structure of a dsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-end of the other strand, or vice versa. “Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the dsRNA, i.e., no nucleotide overhang. A “blunt ended” dsRNA is a dsRNA that is double-stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule. In some embodiments the dsRNA can have a nucleotide overhang at one end of the duplex and a blunt end at the other end.
The term “antisense strand” refers to the strand of a dsRNA which includes a region that is substantially complementary to a target sequence. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches may be in the internal or terminal regions of the molecule. Generally the most tolerated mismatches are in the terminal regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.
The term “sense strand,” as used herein, refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand.
As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle. A SNALP represents a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as an iRNA agent or a plasmid from which an iRNA agent is transcribed.
“Introducing into a cell”, when referring to a dsRNA, means facilitating uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of dsRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; a dsRNA may also be “introduced into a cell”, wherein the cell is part of a living organism. In such instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, dsRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection.
The terms “silence” and “inhibit the expression of” “down-regulate the expression of,” “suppress the expression of” and the like in as far as they refer to the Eg5 and/or VEGF gene, herein refer to the at least partial suppression of the expression of the Eg5 gene, as manifested by a reduction of the amount of Eg5 mRNA and/or VEGF mRNA which may be isolated from a first cell or group of cells in which the Eg5 and/or VEGF gene is transcribed and which has or have been treated such that the expression of the Eg5 and/or VEGF gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition is usually expressed in terms of




  
    
      
        
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Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to Eg5 and/or VEGF gene expression, e.g. the amount of protein encoded by the Eg5 and/or VEGF gene which is produced by a cell, or the number of cells displaying a certain phenotype, e.g. apoptosis. In principle, target gene silencing can be determined in any cell expressing the target, either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference is needed in order to determine whether a given dsRNA inhibits the expression of the Eg5 gene by a certain degree and therefore is encompassed by the instant invention, the assay provided in the Examples below shall serve as such reference.
For example, in certain instances, expression of the Eg5 gene (or VEGF gene) is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of the double-stranded oligonucleotide of the invention. In some embodiments, the Eg5 and/or VEGF gene is suppressed by at least about 60%, 70%, or 80% by administration of the double-stranded oligonucleotide of the invention. In other embodiments, the Eg5 and/or VEGF gene is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide of the invention. The Tables and Example below provides values for inhibition of expression using various Eg5 and/or VEGF dsRNA molecules at various concentrations.
As used herein in the context of Eg5 expression (or VEGF expression), the terms “treat”, “treatment”, and the like, refer to relief from or alleviation of pathological processes mediated by Eg5 and/or VEGF expression. In the context of the present invention insofar as it relates to any of the other conditions recited herein below (other than pathological processes mediated by Eg5 and/or VEGF expression), the terms “treat”, “treatment”, and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition, such as the slowing and progression of hepatic carcinoma.
As used herein, the phrases “therapeutically effective amount” and “prophylactically effective amount” refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of pathological processes mediated by Eg5 and/or VEGF expression or an overt symptom of pathological processes mediated by Eg5 and/or VEGF expression. The specific amount that is therapeutically effective can be readily determined by ordinary medical practitioner, and may vary depending on factors known in the art, such as, e.g. the type of pathological processes mediated by Eg5 and/or VEGF expression, the patient's history and age, the stage of pathological processes mediated by Eg5 and/or VEGF expression, and the administration of other anti-pathological processes mediated by Eg5 and/or VEGF expression agents.
As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of a dsRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 25% reduction in that parameter.
The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. As described in more detail below, such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.
As used herein, a “transformed cell” is a cell into which a vector has been introduced from which a dsRNA molecule may be expressed.
II. DOUBLE-STRANDED RIBONUCLEIC ACID (DSRNA)
As described in more detail below, the invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of the Eg5 and/or VEGF gene in a cell or mammal, wherein the dsRNA comprises an antisense strand comprising a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of the Eg5 and/or VEGF gene, and wherein the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and wherein said dsRNA, upon contact with a cell expressing said Eg5 and/or VEGF gene, inhibits the expression of said Eg5 and/or VEGF gene.
The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.
The dsRNA comprises two strands that are sufficiently complementary to hybridize to form a duplex structure. One strand of the dsRNA (the antisense strand) comprises a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of the Eg5 and/or VEGF gene, the other strand (the sense strand) comprises a region which is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 base pairs in length. In other embodiments the duplex structure is 25-30 base pairs in length.
In one embodiment the duplex is 19 base pairs in length. In another embodiment the duplex is 21 base pairs in length. When two different siRNAs are used in combination, the duplex lengths can be identical or can differ. For example, a composition can include a first dsRNA targeted to Eg5 with a duplex length of 19 base pairs and a second dsRNA targeted to VEGF with a duplex length of 21 base pairs.
Similarly, the region of complementarity to the target sequence is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 nucleotides in length. In other embodiments the region of complementarity is 25-30 nucleotides in length.
In one embodiment the region of complementarity is 19 nucleotides in length. In another embodiment the region of complementarity is 21 nucleotides in length. When two different siRNAs are used in combination, the region of complementarity can be identical or can differ. For example, a composition can include a first dsRNA targeted to Eg5 with a region of complementarity of 19 nucleotides and a second dsRNA targeted to VEGF with a region of complementarity of 21 nucleotides.
Each strand of the dsRNA of invention is generally between 15 and 30, or between 18 and 25, or 18, 19, 20, 21, 22, 23, or 24 nucleotides in length. In other embodiments, each is strand is 25-30 base pairs in length. Each strand of the duplex can be the same length or of different lengths. When two different siRNAs are used in combination, the lengths of each strand of each siRNA can be identical or can differ. For example, a composition can include a dsRNA targeted to Eg5 with a sense strand of 21 nucleotides and an antisense strand of 21 nucleotides, and a second dsRNA targeted to VEGF with a sense strand of 21 nucleotides and an antisense strand of 23 nucleotides.
The dsRNA of the invention can include one or more single-stranded overhang(s) of one or more nucleotides. In one embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. In another embodiment, the antisense strand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ end and the 5′ end over the sense strand. In further embodiments, the sense strand of the dsRNA has 1-nucleotides overhangs each at the 3′ end and the 5′ end over the antisense strand.
A dsRNAs having at least one nucleotide overhang can have unexpectedly superior inhibitory properties than the blunt-ended counterpart. In some embodiments the presence of only one nucleotide overhang strengthens the interference activity of the dsRNA, without affecting its overall stability. A dsRNA having only one overhang has proven particularly stable and effective in vivo, as well as in a variety of cells, cell culture mediums, blood, and serum. Generally, the single-stranded overhang is located at the 3′-terminal end of the antisense strand or, alternatively, at the 3′-terminal end of the sense strand. The dsRNA can also have a blunt end, generally located at the 5′-end of the antisense strand. Such dsRNAs can have improved stability and inhibitory activity, thus allowing administration at low dosages, i.e., less than 5 mg/kg body weight of the recipient per day. Generally, the antisense strand of the dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.
As described in more detail herein, the composition of the invention includes a first dsRNA targeting Eg5 and a second dsRNA targeting VEGF. The first and second dsRNA can have the same overhang architecture, e.g., number of nucleotide overhangs on each strand, or each dsRNA can have a different architecture. In one embodiment, the first dsRNA targeting Eg5 includes a 2 nucleotide overhang at the 3′ end of each strand and the second dsRNA targeting VEGF includes a 2 nucleotide overhang on the 3′ end of the antisense strand and a blunt end at the 5′ end of the antisense strand (e.g., the 3′ end of the sense strand).
In one embodiment, the Eg5 gene targeted by the dsRNA of the invention is the human Eg5 gene. In one embodiment, the antisense strand of the dsRNA targeting Eg5 comprises at least 15 contiguous nucleotides of one of the antisense sequences of Table 1-3. In specific embodiments, the first sequence of the dsRNA is selected from one of the sense strands of Tables 1-3 and the second sequence is selected from the group consisting of the antisense sequences of Tables 1-3. Alternative antisense agents that target elsewhere in the target sequence provided in Tables 1-3 can readily be determined using the target sequence and the flanking Eg5 sequence. In some embodiments the dsRNA targeted to Eg5 will comprise at least two nucleotide sequence selected from the groups of sequences provided in Tables 1-3. One of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of the Eg5 gene. As such, the dsRNA will comprises two oligonucleotides, wherein one oligonucleotide is described as the sense strand in Tables 1-3 and the second oligonucleotide is described as the antisense strand in Tables 1-3
In embodiments using a second dsRNA targeting VEGF, such agents are exemplified in the Examples, Tables 4a and 4b, and in co-pending U.S. Ser. Nos. 11/078,073 and 11/340,080, herein incorporated by reference. In one embodiment the dsRNA targeting VEGF has an antisense strand complementary to at least 15 contiguous nucleotides of the VEGF target sequences described in Table 4a. In other embodiments, the dsRNA targeting VEGF comprises one of the antisense sequences of Table 4b, or one of the sense sequences of Table 4b, or comprises one of the duplexes (sense and antisense strands) of Table 4b.
The skilled person is well aware that dsRNAs comprising a duplex structure of between 20 and 23, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer dsRNAs can be effective as well. In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Tables 1-3, the dsRNAs of the invention can comprise at least one strand of a length of minimally 21 nt. It can be reasonably expected that shorter dsRNAs comprising one of the sequences of Tables 1-3 minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs comprising a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Tables 1-3, and differing in their ability to inhibit the expression of the Eg5 gene in a FACS assay as described herein below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated by the invention. Further dsRNAs that cleave within the target sequence provided in Tables 1-3 can readily be made using the Eg5 sequence and the target sequence provided. Additional dsRNA targeting VEGF can be designed in a similar matter using the sequences disclosed in Tables 4a and 4b, the Examples and co-pending U.S. Ser. Nos. 11/078,073 and 11/340,080, herein incorporated by reference.
In addition, the RNAi agents provided in Tables 1-3 identify a site in the Eg5 mRNA that is susceptible to RNAi based cleavage. As such the present invention further includes RNAi agents, e.g., dsRNA, that target within the sequence targeted by one of the agents of the present invention. As used herein a second RNAi agent is said to target within the sequence of a first RNAi agent if the second RNAi agent cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first RNAi agent. Such a second agent will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Tables 1-3 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in the Eg5 gene. For example, the last 15 nucleotides of SEQ ID NO: 1 combined with the next 6 nucleotides from the target Eg5 gene produces a single strand agent of 21 nucleotides that is based on one of the sequences provided in Tables 1-3. Additional RNAi agents, e.g., dsRNA, targeting VEGF can be designed in a similar matter using the sequences disclosed in Tables 4a and 4b, the Examples and co-pending U.S. Ser. Nos. 11/078,073 and 11/340,080, herein incorporated by reference.
The dsRNA of the invention can contain one or more mismatches to the target sequence. In a preferred embodiment, the dsRNA of the invention contains no more than 3 mismatches. If the antisense strand of the dsRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of complementarity. If the antisense strand of the dsRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or 1 nucleotide from either the 5′ or 3′ end of the region of complementarity. For example, for a 23 nucleotide dsRNA strand which is complementary to a region of the Eg5 gene, the dsRNA generally does not contain any mismatch within the central 13 nucleotides. The methods described within the invention can be used to determine whether a dsRNA containing a mismatch to a target sequence is effective in inhibiting the expression of the Eg5 gene. Consideration of the efficacy of dsRNAs with mismatches in inhibiting expression of the Eg5 gene is important, especially if the particular region of complementarity in the Eg5 gene is known to have polymorphic sequence variation within the population.
Modifications
In yet another embodiment, the dsRNA is chemically modified to enhance stability. The nucleic acids of the invention may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry”, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Specific examples of preferred dsRNA compounds useful in this invention include dsRNAs containing modified backbones or no natural internucleoside linkages. As defined in this specification, dsRNAs having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified dsRNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
Preferred modified dsRNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.
Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is herein incorporated by reference
Preferred modified dsRNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or ore or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, each of which is herein incorporated by reference.
In other preferred dsRNA mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an dsRNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an dsRNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.
Most preferred embodiments of the invention are dsRNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH2—NH—CH2—, —CH2—N(CH3)—O—CH2—[known as a methylene (methylimino) or MMI backbone], —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2— and —N(CH3)—CH2—CH2—[wherein the native phosphodiester backbone is represented as —O—P—O—CH2—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. Also preferred are dsRNAs having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
Modified dsRNAs may also contain one or more substituted sugar moieties. Preferred dsRNAs comprise one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O—, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl.
Particularly preferred are O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. Other preferred dsRNAs comprise one of the following at the 2′ position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an dsRNA, or a group for improving the pharmacodynamic properties of an dsRNA, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxy-alkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH2—O—CH2—N(CH2)2, also described in examples herein below.
Other preferred modifications include 2′-methoxy (2′-OCH3), 2′-aminopropoxy (2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions on the dsRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. DsRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.
DsRNAs may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosine's, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., DsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.
Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941, each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference.
Conjugates
Another modification of the dsRNAs of the invention involves chemically linking to the dsRNA one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the dsRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 199, 86, 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994 4 1053-1060), a thioether, e.g., beryl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).
Representative U.S. patents that teach the preparation of such dsRNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporated by reference.
It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an dsRNA. The present invention also includes dsRNA compounds which are chimeric compounds. “Chimeric” dsRNA compounds or “chimeras,” in the context of this invention, are dsRNA compounds, particularly dsRNAs, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an dsRNA compound. These dsRNAs typically contain at least one region wherein the dsRNA is modified so as to confer upon the dsRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the dsRNA may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of dsRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter dsRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
In certain instances, the dsRNA may be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to dsRNAs in order to enhance the activity, cellular distribution or cellular uptake of the dsRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such dsRNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of dsRNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the dsRNA still bound to the solid support or following cleavage of the dsRNA in solution phase. Purification of the dsRNA conjugate by HPLC typically affords the pure conjugate.
In some cases, a ligand can be multifunctional and/or a dsRNA can be conjugated to more than one ligand. For example, the dsRNA can be conjugated to one ligand for improved uptake and to a second ligand for improved release.
Vector Encoded RNAi Agents
In another aspect of the invention, Eg5 and VEGF specific dsRNA molecules that are expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be incorporated and inherited as a transgene integrated into the host genome. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).
The individual strands of a dsRNA can be transcribed by promoters on two separate expression vectors and co-transfected into a target cell. Alternatively each individual strand of the dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In a preferred embodiment, a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.
The recombinant dsRNA expression vectors are generally DNA plasmids or viral vectors. dsRNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus (for a review, see Muzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129)); adenovirus (see, for example, Berkner, et al., BioTechniques (1998) 6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld et al. (1992), Cell 68:143-155)); or alphavirus as well as others known in the art. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see, e.g., Eglitis, et al., Science (1985) 230:1395-1398; Danos and Mulligan, Proc. Natl. Acad. Sci. USA (1998) 85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al., 1990, Proc. Natl. Acad. Sci. USA 87:61416145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; van Beusechem. et al., 1992, Proc. Natl. Acad. Sci. USA 89:7640-19; Kay et al., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573). Recombinant retroviral vectors capable of transducing and expressing genes inserted into the genome of a cell can be produced by transfecting the recombinant retroviral genome into suitable packaging cell lines such as PA317 and Psi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al., 1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviral vectors can be used to infect a wide variety of cells and tissues in susceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al., 1992, J. Infectious Disease, 166:769), and also have the advantage of not requiring mitotically active cells for infection.
Any viral vector capable of accepting the coding sequences for the dsRNA molecule(s) to be expressed can be used, for example vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g., lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like. The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate.
For example, lentiviral vectors of the invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors of the invention can be made to target different cells by engineering the vectors to express different capsid protein serotypes. For example, an AAV vector expressing a serotype 2 capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can be replaced by a serotype 5 capsid gene to produce an AAV 2/5 vector. Techniques for constructing AAV vectors which express different capsid protein serotypes are within the skill in the art; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.
Selection of recombinant viral vectors suitable for use in the invention, methods for inserting nucleic acid sequences for expressing the dsRNA into the vector, and methods of delivering the viral vector to the cells of interest are within the skill in the art. See, for example, Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988), Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14; Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et al., Nat. Genet. 33: 401-406, the entire disclosures of which are herein incorporated by reference.
Preferred viral vectors are those derived from AV and AAV. In a particularly preferred embodiment, the dsRNA of the invention is expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter.
A suitable AV vector for expressing the dsRNA of the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.
Suitable AAV vectors for expressing the dsRNA of the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.
The promoter driving dsRNA expression in either a DNA plasmid or viral vector of the invention may be a eukaryotic RNA polymerase I (e.g. ribosomal RNA promoter), RNA polymerase II (e.g. CMV early promoter or actin promoter or U1 snRNA promoter) or generally RNA polymerase III promoter (e.g. U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter, for example the T7 promoter, provided the expression plasmid also encodes T7 RNA polymerase required for transcription from a T7 promoter. The promoter can also direct transgene expression to the pancreas (see, e.g., the insulin regulatory sequence for pancreas (Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).
In addition, expression of the transgene can be precisely regulated, for example, by using an inducible regulatory sequence and expression systems such as a regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of transgene expression in cells or in mammals include regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the dsRNA transgene.
Generally, recombinant vectors capable of expressing dsRNA molecules are delivered as described below, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of dsRNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the dsRNAs bind to target RNA and modulate its function or expression. Delivery of dsRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.
dsRNA expression DNA plasmids are typically transfected into target cells as a complex with cationic lipid carriers (e.g. Oligofectamine) or non-cationic lipid-based carriers (e.g. Transit-TKOTM). Multiple lipid transfections for dsRNA-mediated knockdowns targeting different regions of a single EG5 gene (or VEGF gene) or multiple Eg5 genes (or VEGF genes) over a period of a week or more are also contemplated by the invention. Successful introduction of the vectors of the invention into host cells can be monitored using various known methods. For example, transient transfection. can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of ex vivo cells can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.
The Eg5 specific dsRNA molecules and VEGF specific dsRNA molecules can also be inserted into vectors and used as gene therapy vectors for human patients. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
Pharmaceutical Compositions Containing dsRNA
In one embodiment, the invention provides pharmaceutical compositions containing a dsRNA, as described herein, and a pharmaceutically acceptable carrier and methods of administering the same. The pharmaceutical composition containing the dsRNA is useful for treating a disease or disorder associated with the expression or activity of a Eg5/KSP and/or VEGF gene, such as pathological processes mediated by Eg5/KSP and/or VEGF expression, e.g., liver cancer. Such pharmaceutical compositions are formulated based on the mode of delivery.
Dosage
The pharmaceutical compositions featured herein are administered in dosages sufficient to inhibit expression of EG5/KSP and/or VEGF genes. In general, a suitable dose of dsRNA will be in the range of 0.01 to 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 to 50 mg per kilogram body weight per day. For example, the dsRNA can be administered at 0.01 mg/kg, 0.05 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 3 mg/kg, 5.0 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg per single dose.
The pharmaceutical composition can be administered once daily, or the dsRNA may be administered as two, three, or more sub-doses at appropriate intervals throughout the day. The effect of a single dose on EG5/KSP AND/OR VEGF levels is long lasting, such that subsequent doses are administered at not more than 7 day intervals, or at not more than 1, 2, 3, or 4 week intervals.
In some embodiments the dsRNA is administered using continuous infusion or delivery through a controlled release formulation. In that case, the dsRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the dsRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.
The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual dsRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.
Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as pathological processes mediated by EG5/KSP AND/OR VEGFexpression. Such models are used for in vivo testing of dsRNA, as well as for determining a therapeutically effective dose. A suitable mouse model is, for example, a mouse containing a plasmid expressing human EG5/KSP AND/OR VEGF. Another suitable mouse model is a transgenic mouse carrying a transgene that expresses human EG5/KSP AND/OR VEGF.
Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred.
The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
In addition to their administration, as discussed above, the dsRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by target gene expression. In any event, the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.
Administration
The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical, pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, and subdermal, oral or parenteral, e.g., subcutaneous.
Typically, when treating a mammal with hyperlipidemia, the dsRNA molecules are administered systemically via parental means. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intraparenchymal, intrathecal or intraventricular, administration. For example, dsRNAs, conjugated or unconjugate or formulated with or without liposomes, can be administered intravenously to a patient. For such, a dsRNA molecule can be formulated into compositions such as sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions in liquid or solid oil bases. Such solutions also can contain buffers, diluents, and other suitable additives. For parenteral, intrathecal, or intraventricular administration, a dsRNA molecule can be formulated into compositions such as sterile aqueous solutions, which also can contain buffers, diluents, and other suitable additives (e.g., penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers). Formulations are described in more detail herein.
The dsRNA can be delivered in a manner to target a particular tissue, such as the liver (e.g., the hepatocytes of the liver).
Formulations
The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. In one aspect are formulations that target the liver when treating hepatic disorders such as hyperlipidemia.
In addition, dsRNA that target the EG5/KSP AND/OR VEGFgene can be formulated into compositions containing the dsRNA admixed, encapsulated, conjugated, or otherwise associated with other molecules, molecular structures, or mixtures of nucleic acids. For example, a composition containing one or more dsRNA agents that target the Eg5/KSP and/or VEGFgene can contain other therapeutic agents such as other cancer therapeutics or one or more dsRNA compounds that target non-EG5/KSP AND/OR VEGFgenes.
Oral, Parenteral, Topical, and Biologic Formulations
Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, U.S. patent publication. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.
Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Suitable topical formulations include those in which the dsRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). DsRNAs featured in the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, dsRNAs may be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C1-10 alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference. In addition, dsRNA molecules can be administered to a mammal as biologic or abiologic means as described in, for example, U.S. Pat. No. 6,271,359. Abiologic delivery can be accomplished by a variety of methods including, without limitation, (1) loading liposomes with a dsRNA acid molecule provided herein and (2) complexing a dsRNA molecule with lipids or liposomes to form nucleic acid-lipid or nucleic acid-liposome complexes. The liposome can be composed of cationic and neutral lipids commonly used to transfect cells in vitro. Cationic lipids can complex (e.g., charge-associate) with negatively charged nucleic acids to form liposomes. Examples of cationic liposomes include, without limitation, lipofectin, lipofectamine, lipofectace, and DOTAP. Procedures for forming liposomes are well known in the art. Liposome compositions can be formed, for example, from phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, or dioleoyl phosphatidylethanolamine. Numerous lipophilic agents are commercially available, including LipofectinTM (Invitrogen/Life Technologies, Carlsbad, Calif.) and EffecteneTM (Qiagen, Valencia, Calif.). In addition, systemic delivery methods can be optimized using commercially available cationic lipids such as DDAB or DOTAP, each of which can be mixed with a neutral lipid such as DOPE or cholesterol. In some cases, liposomes such as those described by Templeton et al. (Nature Biotechnology, 15: 647-652 (1997)) can be used. In other embodiments, polycations such as polyethyleneimine can be used to achieve delivery in vivo and ex vivo (Boletta et al., J. Am. Soc. Nephrol. 7: 1728 (1996)). Additional information regarding the use of liposomes to deliver nucleic acids can be found in U.S. Pat. No. 6,271,359, PCT Publication WO 96/40964 and Morrissey, D. et al. 2005. Nat. Biotechnol. 23(8):1002-7.
Biologic delivery can be accomplished by a variety of methods including, without limitation, the use of viral vectors. For example, viral vectors (e.g., adenovirus and herpesvirus vectors) can be used to deliver dsRNA molecules to liver cells. Standard molecular biology techniques can be used to introduce one or more of the dsRNAs provided herein into one of the many different viral vectors previously developed to deliver nucleic acid to cells. These resulting viral vectors can be used to deliver the one or more dsRNAs to cells by, for example, infection.
Characterization of Formulated dsRNAs
Formulations prepared by either the in-line mixing or extrusion-free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total siRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated siRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-X100. The total siRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” siRNA content (as measured by the signal in the absence of surfactant) from the total siRNA content. Percent entrapped siRNA is typically >85%. For SNALP formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.
Liposomal Formulations
There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.
Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.
In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.
Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.
Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.
Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.
Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis
Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).
Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).
One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g., as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).
Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising NovasomeTM I (glyceryl dilaurate/cholesterol/po-lyoxyethylene-10-stearyl ether) and NovasomeTM II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).
Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside GM1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).
Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside GM1, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside GM1 or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphat-idylcholine are disclosed in WO 97/13499 (Lim et al).
Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C1215G, that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.
A number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising dsRNAs targeted to the raf gene.
Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.
Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.
If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.
If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.
If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.
The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
SNALPs
In one embodiment, a dsRNA featured in the invention is fully encapsulated in the lipid formulation to form a SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle. As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle, including SPLP. As used herein, the term “SPLP” refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. SNALPs and SPLPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs and SPLPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). SPLPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.
In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1.
The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, or a mixture thereof. The cationic lipid may comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.
In another embodiment, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. provisional patent application No. 61/107,998 filed on Oct. 23, 2008, which is herein incorporated by reference.
In one embodiment, the lipid-siRNA particle includes 40% 2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0±20 nm and a 0.027 siRNA/Lipid Ratio.
The non-cationic lipid may be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid may be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.
The conjugated lipid that inhibits aggregation of particles may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci2), a PEG-dimyristyloxypropyl (Ci4), a PEG-dipalmityloxypropyl (Ci6), or a PEG-distearyloxypropyl (C]8). The conjugated lipid that prevents aggregation of particles may be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.
In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.
LNP01
In one embodiment, the lipidoid ND98.4HCl (MW 1487) (Formula 1), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-siRNA nanoparticles (i.e., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous siRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-siRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.




LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.
Emulsions
The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.
Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).
Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.
A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.
Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.
The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.
In one embodiment of the present invention, the compositions of dsRNAs and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).
The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.
Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or dsRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of dsRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of dsRNAs and nucleic acids.
Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the dsRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.
Penetration Enhancers
In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly dsRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.
Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.
Surfactants: In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of dsRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).
Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C1-10 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carryier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).
Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).
Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of dsRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).
Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of dsRNAs through the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).
Agents that enhance uptake of dsRNAs at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs.
Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.
Carriers
dsRNAs of the present invention can be formulated in a pharmaceutically acceptable carrier or diluent. A “pharmaceutically acceptable carrier” (also referred to herein as an “excipient”) is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle. Pharmaceutically acceptable carriers can be liquid or solid, and can be selected with the planned manner of administration in mind so as to provide for the desired bulk, consistency, and other pertinent transport and chemical properties. Typical pharmaceutically acceptable carriers include, by way of example and not limitation: water; saline solution; binding agents (e.g., polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, gelatin, or calcium sulfate); lubricants (e.g., starch, polyethylene glycol, or sodium acetate); disintegrates (e.g., starch or sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate).
Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The co-administration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extra-circulatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is co-administered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.
Excipients
In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).
Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.
Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
Other Components
The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
Combination Therapy
In one aspect, a composition of the invention can be used in combination therapy. The term “combination therapy” includes the administration of the subject compounds in further combination with other biologically active ingredients (such as, but not limited to, a second and different antineoplastic agent) and non-drug therapies (such as, but not limited to, surgery or radiation treatment). For instance, the compounds of the invention can be used in combination with other pharmaceutically active compounds, preferably compounds that are able to enhance the effect of the compounds of the invention. The compounds of the invention can be administered simultaneously (as a single preparation or separate preparation) or sequentially to the other drug therapy. In general, a combination therapy envisions administration of two or more drugs during a single cycle or course of therapy.
In one aspect of the invention, the subject compounds may be administered in combination with one or more separate agents that modulate protein kinases involved in various disease states. Examples of such kinases may include, but are not limited to: serine/threonine specific kinases, receptor tyrosine specific kinases and non-receptor tyrosine specific kinases. Serine/threonine kinases include mitogen activated protein kinases (MAPK), meiosis specific kinase (MEK), RAF and aurora kinase. Examples of receptor kinase families include epidermal growth factor receptor (EGFR) (e.g., HER2/neu, HER3, HER4, ErbB, ErbB2, ErbB3, ErbB4, Xmrk, DER, Let23); fibroblast growth factor (FGF) receptor (e.g. FGF-R1, GFF-R2/BEK/CEK3, FGF-R3/CEK2, FGF-R4/TKF, KGF-R); hepatocyte growth/scatter factor receptor (HGFR) (e.g., MET, RON, SEA, SEX); insulin receptor (e.g. IGFI-R); Eph (e.g. CEK5, CEK8, EBK, ECK, EEK, EHK-I, EHK-2, ELK, EPH, ERK, HEK, MDK2, MDK5, SEK); AxI (e.g. Mer/Nyk, Rse); RET; and platelet-derived growth factor receptor (PDGFR) (e.g. PDGFα-R, PDGβ-R, CSF1-R/FMS, SCF-R/C-KIT, VEGF-R/FLT, NEK/FLK1, FLT3/FLK2/STK-1). Non-receptor tyrosine kinase families include, but are not limited to, BCR-ABL (e.g. p43abl, ARG); BTK (e.g. ITK/EMT, TEC); CSK, FAK, FPS, JAK, SRC, BMX, FER, CDK and SYK.
In another aspect of the invention, the subject compounds may be administered in combination with one or more agents that modulate non-kinase biological targets or processes. Such targets include histone deacetylases (HDAC), DNA methyltransferase (DNMT), heat shock proteins (e.g., HSP90), and proteosomes.
In one embodiment, subject compounds may be combined with antineoplastic agents (e.g. small molecules, monoclonal antibodies, antisense RNA, and fusion proteins) that inhibit one or more biological targets such as Zolinza, Tarceva, Iressa, Tykerb, Gleevec, Sutent, Sprycel, Nexavar, Sorafenib, CNF2024, RG108, BMS387032, Affmitak, Avastin, Herceptin, Erbitux, AG24322, PD325901, ZD6474, PD 184322, Obatodax, ABT737 and AEE788. Such combinations may enhance therapeutic efficacy over efficacy achieved by any of the agents alone and may prevent or delay the appearance of resistant mutational variants.
In certain preferred embodiments, the compounds of the invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents encompass a wide range of therapeutic treatments in the field of oncology. These agents are administered at various stages of the disease for the purposes of shrinking tumors, destroying remaining cancer cells left over after surgery, inducing remission, maintaining remission and/or alleviating symptoms relating to the cancer or its treatment. Examples of such agents include, but are not limited to, alkylating agents such as mustard gas derivatives (Mechlorethamine, cylophosphamide, chlorambucil, melphalan, ifosfamide), ethylenimines (thiotepa, hexamethylmelanine), Alkylsulfonates (Busulfan), Hydrazines and Triazines (Altretamine, Procarbazine, Dacarbazine and Temozolomide), Nitrosoureas (Carmustine, Lomustine and Streptozocin), Ifosfamide and metal salts (Carboplatin, Cisplatin, and Oxaliplatin); plant alkaloids such as Podophyllotoxins (Etoposide and Tenisopide), Taxanes (Paclitaxel and Docetaxel), Vinca alkaloids (Vincristine, Vinblastine, Vindesine and Vinorelbine), and Camptothecan analogs (Irinotecan and Topotecan); anti-tumor antibiotics such as Chromomycins (Dactinomycin and Plicamycin), Anthracyclines (Doxorubicin, Daunorubicin, Epirubicin, Mitoxantrone, Valrubicin and Idarubicin), and miscellaneous antibiotics such as Mitomycin, Actinomycin and Bleomycin; anti-metabolites such as folic acid antagonists (Methotrexate, Pemetrexed, Raltitrexed, Aminopterin), pyrimidine antagonists (5-Fluorouracil, Floxuridine, Cytarabine, Capecitabine, and Gemcitabine), purine antagonists (6-Mercaptopurine and 6-Thioguanine) and adenosine deaminase inhibitors (Cladribine, Fludarabine, Mercaptopurine, Clofarabine, Thioguanine, Nelarabine and Pentostatin); topoisomerase inhibitors such as topoisomerase I inhibitors (Ironotecan, topotecan) and topoisomerase II inhibitors (Amsacrine, etoposide, etoposide phosphate, teniposide); monoclonal antibodies (Alemtuzumab, Gemtuzumab ozogamicin, Rituximab, Trastuzumab, Ibritumomab Tioxetan, Cetuximab, Panitumumab, Tositumomab, Bevacizumab); and miscellaneous anti-neoplasties such as ribonucleotide reductase inhibitors (Hydroxyurea); adrenocortical steroid inhibitor (Mitotane); enzymes (Asparaginase and Pegaspargase); anti-microtubule agents (Estramustine); and retinoids (Bexarotene, Isotretinoin, Tretinoin (ATRA). In certain preferred embodiments, the compounds of the invention are administered in combination with a chemoprotective agent. Chemoprotective agents act to protect the body or minimize the side effects of chemotherapy. Examples of such agents include, but are not limited to, amfostine, mesna, and dexrazoxane.
In one aspect of the invention, the subject compounds are administered in combination with radiation therapy. Radiation is commonly delivered internally (implantation of radioactive material near cancer site) or externally from a machine that employs photon (x-ray or gamma-ray) or particle radiation. Where the combination therapy further comprises radiation treatment, the radiation treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the combination of the therapeutic agents and radiation treatment is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the radiation treatment is temporally removed from the administration of the therapeutic agents, perhaps by days or even weeks.
It will be appreciated that compounds of the invention can be used in combination with an immunotherapeutic agent. One form of immunotherapy is the generation of an active systemic tumor-specific immune response of host origin by administering a vaccine composition at a site distant from the tumor. Various types of vaccines have been proposed, including isolated tumor-antigen vaccines and anti-idiotype vaccines. Another approach is to use tumor cells from the subject to be treated, or a derivative of such cells (reviewed by Schirrmacher et al. (1995) J. Cancer Res. Clin. Oncol. 121:487). In U.S. Pat. No. 5,484,596, Hanna Jr. et al. claim a method for treating a resectable carcinoma to prevent recurrence or metastases, comprising surgically removing the tumor, dispersing the cells with collagenase, irradiating the cells, and vaccinating the patient with at least three consecutive doses of about 107 cells.
It will be appreciated that the compounds of the invention may advantageously be used in conjunction with one or more adjunctive therapeutic agents. Examples of suitable agents for adjunctive therapy include steroids, such as corticosteroids (amcinonide, betamethasone, betamethasone dipropionate, betamethasone valerate, budesonide, clobetasol, clobetasol acetate, clobetasol butyrate, clobetasol 17-propionate, cortisone, deflazacort, desoximetasone, diflucortolone valerate, dexamethasone, dexamethasone sodium phosphate, desonide, furoate, fluocinonide, fluocinolone acetonide, halcinonide, hydrocortisone, hydrocortisone butyrate, hydrocortisone sodium succinate, hydrocortisone valerate, methyl prednisolone, mometasone, prednicarbate, prednisolone, triamcinolone, triamcinolone acetonide, and halobetasol proprionate); a 5HTi agonist, such as a triptan (e.g. sumatriptan or naratriptan); an adenosine A1 agonist; an EP ligand; an NMDA modulator, such as a glycine antagonist; a sodium channel blocker (e.g. lamotrigine); a substance P antagonist (e.g. an NKi antagonist); a cannabinoid; acetaminophen or phenacetin; a 5-lipoxygenase inhibitor; a leukotriene receptor antagonist; a DMARD (e.g. methotrexate); gabapentin and related compounds; a tricyclic antidepressant (e.g. amitryptilline); a neurone stabilizing antiepileptic drug; a mono-aminergic uptake inhibitor (e.g. venlafaxine); a matrix metalloproteinase inhibitor; a nitric oxide synthase (NOS) inhibitor, such as an iNOS or an nNOS inhibitor; an inhibitor of the release, or action, of tumour necrosis factor α; an antibody therapy, such as a monoclonal antibody therapy; an antiviral agent, such as a nucleoside inhibitor (e.g. lamivudine) or an immune system modulator (e.g. interferon); an opioid analgesic; a local anaesthetic; a stimulant, including caffeine; an H2-antagonist (e.g. ranitidine); a proton pump inhibitor (e.g. omeprazole); an antacid (e.g. aluminium or magnesium hydroxide; an antiflatulent (e.g. simethicone); a decongestant (e.g. phenylephrine, phenylpropanolamine, pseudoephedrine, oxymetazoline, epinephrine, naphazoline, xylometazoline, propylhexedrine, or levo-desoxyephedrine); an antitussive (e.g. codeine, hydrocodone, carmiphen, carbetapentane, or dextramethorphan); a diuretic; or a sedating or non-sedating antihistamine.
The compounds of the invention can be co-administered with siRNA that target other genes. For example, a compound of the invention can be co-administered with an siRNA targeted to a c-Myc gene. In one example, AD-12115 can be co-administered with a c-Myc siRNA. Examples of c-Myc targeted siRNAs are disclosed in U.S. patent application Ser. No. 12/373,039 which is herein incorporated by reference.
Methods for Treating Diseases Caused by Expression of the Eg5 and VEGF Genes
The invention relates in particular to the use of a composition containing at least two dsRNAs, one targeting an Eg5 gene, and one targeting a VEGF gene, for the treatment of a cancer, such as liver cancer, e.g., for inhibiting tumor growth and tumor metastasis. For example, a composition, such as pharmaceutical composition, may be used for the treatment of solid tumors, like intrahepatic tumors such as may occur in cancers of the liver. A composition containing a dsRNA targeting Eg5 and a dsRNA targeting VEGF may also be used to treat other tumors and cancers, such as breast cancer, lung cancer, head and neck cancer, brain cancer, abdominal cancer, colon cancer, colorectal cancer, esophagus cancer, gastrointestinal cancer, glioma, tongue cancer, neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, Wilm's tumor, multiple myeloma and for the treatment of skin cancer, like melanoma, for the treatment of lymphomas and blood cancer. The invention further relates to the use of a composition containing an Eg5 dsRNA and a VEGF dsRNA for inhibiting accumulation of ascites fluid and pleural effusion in different types of cancer, e.g., liver cancer, breast cancer, lung cancer, head cancer, neck cancer, brain cancer, abdominal cancer, colon cancer, colorectal cancer, esophagus cancer, gastrointestinal cancer, glioma, tongue cancer, neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, Wilm's tumor, multiple myeloma, skin cancer, melanoma, lymphomas and blood cancer. Owing to the inhibitory effects on Eg5 and VEGF expression, a composition according to the invention or a pharmaceutical composition prepared therefrom can enhance the quality of life.
In one embodiment, a patient having a tumor associated with AFP expression, or a tumor secreting AFP, e.g., a hepatoma or teratoma, is treated. In certain embodiments, the patient has a malignant teratoma, an endodermal sinus tumor (yolk sac carcinoma), a neuroblastoma, a hepatoblastoma, a heptocellular carcinoma, testicular cancer or ovarian cancer.
The invention furthermore relates to the use of a dsRNA or a pharmaceutical composition thereof, e.g., for treating cancer or for preventing tumor metastasis, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating cancer and/or for preventing tumor metastasis. Preference is given to a combination with radiation therapy and chemotherapeutic agents, such as cisplatin, cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen.
The invention can also be practiced by including with a specific RNAi agent, in combination with another anti-cancer chemotherapeutic agent, such as any conventional chemotherapeutic agent. The combination of a specific binding agent with such other agents can potentiate the chemotherapeutic protocol. Numerous chemotherapeutic protocols will present themselves in the mind of the skilled practitioner as being capable of incorporation into the method of the invention. Any chemotherapeutic agent can be used, including alkylating agents, antimetabolites, hormones and antagonists, radioisotopes, as well as natural products. For example, the compound of the invention can be administered with antibiotics such as doxorubicin and other anthracycline analogs, nitrogen mustards such as cyclophosphamide, pyrimidine analogs such as 5-fluorouracil, cisplatin, hydroxyurea, taxol and its natural and synthetic derivatives, and the like. As another example, in the case of mixed tumors, such as adenocarcinoma of the breast, where the tumors include gonadotropin-dependent and gonadotropin-independent cells, the compound can be administered in conjunction with leuprolide or goserelin (synthetic peptide analogs of LH-RH). Other antineoplastic protocols include the use of a tetracycline compound with another treatment modality, e.g., surgery, radiation, etc., also referred to herein as “adjunct antineoplastic modalities.” Thus, the method of the invention can be employed with such conventional regimens with the benefit of reducing side effects and enhancing efficacy.
Methods for Inhibiting Expression of the Eg5 Gene and the VEGF Gene
In yet another aspect, the invention provides a method for inhibiting the expression of the Eg5 gene and the VEGF gene in a mammal. The method includes administering a composition featured in the invention to the mammal such that expression of the target Eg5 gene and the target VEGF gene is silenced.
In one embodiment, a method for inhibiting Eg5 gene expression and VEGF gene expression includes administering a composition containing two different dsRNA molecules, one having a nucleotide sequence that is complementary to at least a part of an RNA transcript of the Eg5 gene and the other having a nucleotide sequence that is complementary to at least a part of an RNA transcript of the VEGF gene of the mammal to be treated. When the organism to be treated is a mammal such as a human, the composition may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In preferred embodiments, the compositions are administered by intravenous infusion or injection.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
EXAMPLES
Example 1
dsRNA Synthesis
Source of Reagents
Where the source of a reagent is not specifically given herein, such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.
siRNA Synthesis
For screening of dsRNA, single-stranded RNAs were produced by solid phase synthesis on a scale of 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass (CPG, 500 Å, Proligo Biochemie GmbH, Hamburg, Germany) as solid support. RNA and RNA containing 2′-O-methyl nucleotides were generated by solid phase synthesis employing the corresponding phosphoramidites and 2′-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH, Hamburg, Germany). These building blocks were incorporated at selected sites within the sequence of the oligoribonucleotide chain using standard nucleoside phosphoramidite chemistry such as described in Current protocols in nucleic acid chemistry, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioate linkages were introduced by replacement of the iodine oxidizer solution with a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) in acetonitrile (1%). Further ancillary reagents were obtained from Mallinckrodt Baker (Griesheim, Germany).
Deprotection and purification of the crude oligoribonucleotides by anion exchange HPLC were carried out according to established procedures. Yields and concentrations were determined by UV absorption of a solution of the respective RNA at a wavelength of 260 nm using a spectral photometer (DU 640B, Beckman Coulter GmbH, Unterschleiβheim, Germany). Double stranded RNA was generated by mixing an equimolar solution of complementary strands in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3 minutes and cooled to room temperature over a period of 3-4 hours. The annealed RNA solution was stored at −20° C. until use.
Conjugates
The following is a prophetic description of the synthesis of 3′-cholesterol-conjugated siRNAs (herein referred to as -Chol-3′), an appropriately modified solid support was used for RNA synthesis. The modified solid support was prepared as follows:
Diethyl-2-azabutane-1,4-dicarboxylate AA




A 4.7 M aqueous solution of sodium hydroxide (50 mL) was added into a stirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g, 0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole) was added and the mixture was stirred at room temperature until completion of the reaction was ascertained by TLC. After 19 h the solution was partitioned with dichloromethane (3×100 mL). The organic layer was dried with anhydrous sodium sulfate, filtered and evaporated. The residue was distilled to afford AA (28.8 g, 61%).
3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionic acid ethyl ester AB




Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was dissolved in dichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde (3.25 g, 3.99 mL, 25.83 mmol) was added to the solution at 0° C. It was then followed by the addition of Diethyl-azabutane-1,4-dicarboxylate (5 g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). The solution was brought to room temperature and stirred further for 6 h. Completion of the reaction was ascertained by TLC. The reaction mixture was concentrated under vacuum and ethyl acetate was added to precipitate diisopropyl urea. The suspension was filtered. The filtrate was washed with 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. The combined organic layer was dried over sodium sulfate and concentrated to give the crude product which was purified by column chromatography (50% EtOAC/Hexanes) to yield 11.87 g (88%) of AB.
3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethyl ester AC




3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionic acid ethyl ester AB (11.5 g, 21.3 mmol) was dissolved in 20% piperidine in dimethylformamide at 0° C. The solution was continued stirring for 1 h. The reaction mixture was concentrated under vacuum, water was added to the residue, and the product was extracted with ethyl acetate. The crude product was purified by conversion into its hydrochloride salt.
3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionic acid ethyl ester AD




The hydrochloride salt of 3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethyl ester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane. The suspension was cooled to 0° C. on ice. To the suspension diisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) was added. To the resulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) was added. The reaction mixture was stirred overnight. The reaction mixture was diluted with dichloromethane and washed with 10% hydrochloric acid. The product was purified by flash chromatography (10.3 g, 92%).
1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylic acid ethyl ester AE




Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of dry toluene. The mixture was cooled to 0° C. on ice and 5 g (6.6 mmol) of diester AD was added slowly with stirring within 20 mins. The temperature was kept below 5° C. during the addition. The stirring was continued for 30 mins at 0° C. and 1 mL of glacial acetic acid was added, immediately followed by 4 g of NaH2 PO4.H2O in 40 mL of water The resultant mixture was extracted twice with 100 mL of dichloromethane each and the combined organic extracts were washed twice with 10 mL of phosphate buffer each, dried, and evaporated to dryness. The residue was dissolved in 60 mL of toluene, cooled to 0° C. and extracted with three 50 mL portions of cold pH 9.5 carbonate buffer. The aqueous extracts were adjusted to pH 3 with phosphoric acid, and extracted with five 40 mL portions of chloroform which were combined, dried and evaporated to dryness. The residue was purified by column chromatography using 25% ethylacetate/hexane to afford 1.9 g of b-ketoester (39%).
[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AF




Methanol (2 mL) was added dropwise over a period of 1 h to a refluxing mixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride (0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring was continued at reflux temperature for 1 h. After cooling to room temperature, 1 N HCl (12.5 mL) was added, the mixture was extracted with ethylacetate (3×40 mL). The combined ethylacetate layer was dried over anhydrous sodium sulfate and concentrated under vacuum to yield the product which was purified by column chromatography (10% MeOH/CHCl3) (89%).
(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AG




Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2×5 mL) in vacuo. Anhydrous pyridine (10 mL) and 4,4′-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added with stirring. The reaction was carried out at room temperature overnight. The reaction was quenched by the addition of methanol. The reaction mixture was concentrated under vacuum and to the residue dichloromethane (50 mL) was added. The organic layer was washed with 1M aqueous sodium bicarbonate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residual pyridine was removed by evaporating with toluene. The crude product was purified by column chromatography (2% MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl3) (1.75 g, 95%).
Succinic acid mono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl 2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl) ester AH




Compound AG (1.0 g, 1.05 mmol) was mixed with succinic anhydride (0.150 g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40° C. overnight. The mixture was dissolved in anhydrous dichloroethane (3 mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) was added and the solution was stirred at room temperature under argon atmosphere for 16 h. It was then diluted with dichloromethane (40 mL) and washed with ice cold aqueous citric acid (5 wt %, 30 mL) and water (2×20 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The residue was used as such for the next step.
Cholesterol Derivatised CPG AI




Succinate AH (0.254 g, 0.242 mmol) was dissolved in a mixture of dichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296 g, 0.242 mmol) in acetonitrile (1.25 mL), 2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) in acetonitrile/dichloroethane (3:1, 1.25 mL) were added successively. To the resulting solution triphenylphosphine (0.064 g, 0.242 mmol) in acetonitrile (0.6 ml) was added. The reaction mixture turned bright orange in color. The solution was agitated briefly using a wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM) was added. The suspension was agitated for 2 h. The CPG was filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups were masked using acetic anhydride/pyridine. The achieved loading of the CPG was measured by taking UV measurement (37 mM/g).
The synthesis of siRNAs bearing a 5′-12-dodecanoic acid bisdecylamide group (herein referred to as “5′-C32-”) or a 5′-cholesteryl derivative group (herein referred to as “5′-Chol-”) was performed as described in WO 2004/065601, except that, for the cholesteryl derivative, the oxidation step was performed using the Beaucage reagent in order to introduce a phosphorothioate linkage at the 5′-end of the nucleic acid oligomer.
dsRNA Targeting the Eg5 Gene
Initial Screening Set
siRNA design was carried out to identify siRNAs targeting Eg5 (also known as KIF11, HSKP, KNSL1 and TRIP5). Human mRNA sequences to Eg5, RefSeq ID number:NM—004523, was used.
siRNA duplexes cross-reactive to human and mouse Eg5 were designed. Twenty-four duplexes were synthesized for screening. (Table 1a). A second screening set was defined with 266 siRNAs targeting human Eg5, as well as its rhesus monkey ortholog (Table 2a). An expanded screening set was selected with 328 siRNA targeting human Eg5, with no necessity to hit any Eg5 mRNA of other species (Table 3a).
The sequences for human and a partial rhesus Eg5 mRNAs were downloaded from NCBI Nucleotide database and the human sequence was further on used as reference sequence (Human EG5:NM—004523.2, 4908 bp, and Rhesus EG5: XM—001087644.1, 878 bp (only 5′ part of human EG5)
For the Tables: Key: A,G,C,U-ribonucleotides: T-deoxythymidine: u,c-2′-O-methyl nucleotides: s-phosphorothioate linkage.







TABLE 1a







Sequences of Eg5/KSP dsRNA duplexes














position









in human
SEQ

SEQ

SEQ




Eg5/KSP
ID
sequence of 23mer
ID
sense sequence
ID
antisense sequence
duplex


sequence
NO:
target site
NO:
(5′-3′)
No:
(5′-3′)
name

















385-407
1244
ACCGAAGUGUUGUUUGUC
 1
cGAAGuGuuGuuuGuccA
 2
UUGGAcAAAcAAcACUUCG
AL-DP-




CAAUU

ATsT

TsT
6226


 


347-369
1245
UAUGGUGUUUGGAGCAUC
 3
uGGuGuuuGGAGcAucuA
 4
GuAGAUGCUCcAAAcACcA
AL-DP-




VACUA

cTsT

TsT
6227


 


1078-1100
1246
AAUCUAAACUAACUAGAA
 5
ucuAAAcuAAcuAGAAuc
 6
GGAUUCuAGUuAGUUuAGA
AL-DP-




UCCUC

cTsT

TsT
6228


 


1067-1089
1247
UCCUUAUCGAGAAUCUAA
 7
cuuAucGAGAAucuAAAc
 8
AGUUuAGAUUCUCGAuAAG
AL-DP-




ACUAA

uTsT

TsT
6229


 


374-396
1248
GAUUGAUGUUUACCGAAG
 9
uuGAuGuuuAccGAAGuG
10
AcACUUCGGuAAAcAUcAA
AL-DP-




UGUUG

uTsT

TsT
6230


 


205-227
1249
UGGUGAGAUGCAGACCAU
11
GuGAGAuGcAGAccAuuu
12
uAAAUGGUCUGcAUCUcAC
AL-DP-




UUAAU

ATsT

TsT
6231


 


1176-1198
1250
ACUCUGAGUACAUUGGAA
13
ucuGAGuAcAuuGGAAuA
14
AuAUUCcAAUGuACUcAGA
AL-DP-




UAUGC

uTsT

TsT
6232


 


386-408
1251
CCGAAGUGUUGUUUGUCC
15
GAAGuGuuGuuuGuccAA
16
AUUGGAcAAAcAAcACUUC
AL-DP-




AAUUC

uTsT

TsT
6233


 


416-438
1252
AGUUAUUAUGGGCUAUAA
17
uuAuuAuGGGcuAuAAuu
18
cAAUuAuAGCCcAuAAuAA
AL-DP-




UUGCA

GTsT

TsT
6234


 


485-507
1253
GGAAGGUGAAAGGUCACC
19
AAGGuGAAAGGucAccuA
20
UuAGGUGACCUUUcACCUU
AL-DP-




UAAUG

ATsT

TsT
6235


 


476-498
1254
UUUUACAAUGGAAGGUGA
21
uuAcAAuGGAAGGuGAAA
22
CUUUcACCUUCcAUUGuAA
AL-DP-




AAGGU

GTsT

TsT
6236


 


486-508
1255
GAAGGUGAAAGGUCACCU
23
AGGuGAAAGGucAccuAA
24
AUuAGGUGACCUUUcACCU
AL-DP-




AAUGA

uTsT

TsT
6237


 


487-509
1256
AAGGUGAAAGGUCACCUA
25
GGuGAAAGGucAccuAAu
26
cAUuAGGUGACCUUUcACC
AL-DP-




AUGAA

GTsT

TsT
6238


 


1066-1088
1257
UUCCUUAUCGAGAAUCUA
27
ccuuAucGAGAAucuAAA
28
GUUuAGAUUCUCGAuAAGG
AL-DP-




AACUA

cTsT

TsT
6239


 


1256-1278
1258
AGCUCUUAUUAAGGAGUA
29
cucuuAuuAAGGAGuAuA
30
GuAuACUCCUuAAuAAGAG
AL-DP-




UACGG

cTsT

TsT
6240


 


2329-2351
1259
CAGAGAGAUUCUGUGCUU
31
GAGAGAuucuGuGcuuuG
32
CcAAAGcAcAGAAUCUCUC
AL-DP-




UGGAG

GTsT

TsT
6241


 


1077-1099
1260
GAAUCUAAACUAACUAGA
33
AucuAAAcuAAcuAGAAu
34
GAUUCuAGUuAGUUuAGAU
AL-DP-




AUCCU

cTsT

TsT
6242


 


1244-1266
1261
ACUCACCAAAAAAGCUCU
35
ucAccAAAAAAGcucuuA
36
AuAAGAGCUUUUUUGGUGA
AL-DP-




UAUUA

uTsT

TsT
6243


 


637-659
1262
AAGAGCUUUUUGAUCUUC
37
GAGcuuuuuGAucuucuu
38
uAAGAAGAUcAAAAAGCUC
AL-DP-




UUAAU

ATsT

TsT
6244


 


1117-1139
1263
GGCGUACAAGAACAUCUA
39
cGuAcAAGAAcAucuAuA
40
UuAuAGAUGUUCUUGuACG
AL-DP-




UAAUU

ATsT

TsT
6245


 


373-395
1264
AGAUUGAUGUUUACCGAA
41
AuuGAuGuuuAccGAAGu
42
cACUUCGGuAAAcAUcAAU
AL-DP-




GUGUU

GTsT

TsT
6246


 


1079-1101
1265
AUCUAAACUAACUAGAAU

cuAAAcuAAcuAGAAucc
44
AGGAUUCuAGUuAGUUuAG
AL-DP-




CCUCC

uTsT

TsT
6247


 


383-405
1266
UUACCGAAGUGUUGUUUG
45
AccGAAGuGuuGuuuGuc
46
GGAcAAAcAAcACUUCGGU
AL-DP-




UCCAA

cTsT

TsT
6248


 


200-222
1267
GGUGGUGGUGAGAUGCAG
47
uGGuGGuGAGAuGcAGAc
48
GGUCUGcAUCUcACcACcA
AL-DP-




ACCAU

cTsT

TsT
6249
















TABLE 1b







Analysis of Eg5/KSP ds duplexes












single





dose




screen @




25 nM [%
SDs 2nd screen



duplex
residual
(among



name
mRNA]
quadruplicates)















AL-DP-6226
23%
3%



AL-DP-6227
69%
10%



AL-DP-6228
33%
2%



AL-DP-6229
2%
2%



AL-DP-6230
66%
11%



AL-DP-6231
17%
1%



AL-DP-6232
9%
3%



AL-DP-6233
24%
6%



AL-DP-6234
91%
2%



AL-DP-6235
112%
4%



AL-DP-6236
69%
4%



AL-DP-6237
42%
2%



AL-DP-6238
45%
2%



AL-DP-6239
2%
1%



AL-DP-6240
48%
2%



AL-DP-6241
41%
2%



AL-DP-6242
8%
2%



AL-DP-6243
7%
1%



AL-DP-6244
6%
2%



AL-DP-6245
12%
2%



AL-DP-6246
28%
3%



AL-DP-6247
71%
4%



AL-DP-6248
5%
2%



AL-DP-6249
28%
3%

















TABLE 2a







Sequences of Eg5/ KSP dsRNA duplexes













SEQ

SEQ

SEQ




ID
sequence of 19-mer
ID

ID
antisense sequence
duplex


NO:
target site
NO.
sense sequence (5′-3′)
NO.
(5′-3′)
name
















1268
CAUACUCUAGUCGUUCCCA
 49
cAuAcucuAGucGuucccATsT
 50
UGGGAACGACuAGAGuAUGTsT
AD-12072


 


1269
AGCGCCCAUUCAAUAGUAG
 51
AGcGcccAuucAAuAGuAGTsT
 52
CuACuAUUGAAUGGGCGCUTsT
AD-12073


 


1270
GGAAAGCUAGCGCCCAUUC
 53
GGAAAGcuAGcGcccAuucTsT
 54
GAAUGGGCGCuAGCUUUCCTsT
AD-12074


 


1271
GAAAGCUAGCGCCCAUUCA
 55
GAAAGcuAGcGcccAuucATsT
 56
UGAAUGGGCGCuAGCUUUCTsT
AD-12075


 


1272
AGAAACUACGAUUGAUGGA
 57
AGAAAcuAcGAuuGAuGGATsT
 58
UCcAUcAAUCGuAGUUUCUTsT
AD-12076


 


1273
UGUUCCUUAUCGAGAAUCU
 59
uGuuccuuAucGAGAAucuTsT
 60
AGAUUCUCGAuAAGGAAcATsT
AD-12077


 


1274
CAGAUUACCUCUGCGAGCC
 61
cAGAuuAccucuGcGAGccTsT
 62
GGCUCGcAGAGGuAAUCUGTsT
AD-12078


 


1275
GCGCCCAUUCAAUAGUAGA
 63
GcGcccAuucAAuAGuAGATsT
 64
UCuACuAUUGAAUGGGCGCTsT
AD-12079


 


1276
UUGCACUAUCUUUGCGUAU
 65
uuGcAcuAucuuuGcGuAuTsT
 66
AuACGcAAAGAuAGUGcAATsT
AD-12080


 


1277
CAGAGCGGAAAGCUAGCGC
 67
cAGAGcGGAAAGcuAGcGcTsT
 68
GCGCuAGCUUUCCGCUCUGTsT
AD-12081


 


1278
AGACCUUAUUUGGUAAUCU
 69
AGAccuuAuuuGGuAAucuTsT
 70
AGAUuACcAAAuAAGGUCUTsT
AD-12082


 


1279
AUUCUCUUGGAGGGCGUAC
 71
AuucucuuGGAGGGcGuAcTsT
 72
GuACGCCCUCcAAGAGAAUTsT
AD-12083


 


1280
GGCUGGUAUAAUUCCACGU
 73
GGcuGGuAuAAuuccAcGuTsT
 74
ACGUGGAAUuAuACcAGCCTsT
AD-12084


 


1281
GCGGAAAGCUAGCGCCCAU
 75
GcGGAAAGcuAGcGcccAuTsT
 76
AUGGGCGCuAGCUUUCCGCTsT
AD-12085


 


1282
UGCACUAUCUUUGCGUAUG
 77
uGcAcuAucuuuGcGuAuGTsT
 78
cAuACGcAAAGAuAGUGcATsT
AD-12086


 


1283
GUAUAAUUCCACGUACCCU
 79
GuAuAAuuccAcGuAcccuTsT
 80
AGGGuACGUGGAAUuAuACTsT
AD-12087


 


1284
AGAAUCUAAACUAACUAGA
 81
AGAAucuAAAcuAAcuAGATsT
 82
UCuAGUuAGUUuAGAUUCUTsT
AD-12088


 


1285
AGGAGCUGAAUAGGGUUAC
 83
AGGAGcuGAAuAGGGuuAcTsT
 84
GuAACCCuAUUcAGCUCCUTsT
AD-12089


 


1286
GAAGUACAUAAGACCUUAU
 85
GAAGuAcAuAAGAccuuAuTsT
 86
AuAAGGUCUuAUGuACUUCTsT
AD-12090


 


1287
GACAGUGGCCGAUAAGAUA
 87
GAcAGuGGccGAuAAGAuATsT
 88
uAUCUuAUCGGCcACUGUCTsT
AD-12091


 


1288
AAACCACUUAGUAGUGUCC
 89
AAAccAcuuAGuAGuGuccTsT
 90
GGAcACuACuAAGUGGUUUTsT
AD-12092


 


1289
UCCCUAGACUUCCCUAUUU
 91
ucccuAGAcuucccuAuuuTsT
 92
AAAuAGGGAAGUCuAGGGATsT
AD-12093


 


1290
UAGACUUCCCUAUUUCGCU
 93
uAGAcuucccuAuuucGcuTsT
 94
AGCGAAAuAGGGAAGUCuATsT
AD-12094


 


1291
GCGUCGCAGCCAAAUUCGU
 95
GcGucGcAGccAAAuucGuTsT
 96
ACGAAUUUGGCUGCGACGCTsT
AD-12095


 


1292
AGCUAGCGCCCAUUCAAUA
 97
AGcuAGcGcccAuucAAuATsT
 98
uAUUGAAUGGGCGCuAGCUTsT
AD-12096


 


1293
GAAACUACGAUUGAUGGAG
 99
GAAAcuAcGAuuGAuGGAGTsT
100
CUCcAUcAAUCGuAGUUUCTsT
AD-12097


 


1294
CCGAUAAGAUAGAAGAUCA
101
ccGAuAAGAuAGAAGAucATsT
102
UGAUCUUCuAUCUuAUCGGTsT
AD-12098


 


1295
UAGCGCCCAUUCAAUAGUA
103
uAGcGcccAuucAAuAGuATsT
104
uACuAUUGAAUGGGCGCuATsT
AD-12099


 


1296
UUUGCGUAUGGCCAAACUG
105
uuuGcGuAuGGccAAAcuGTsT
106
cAGUUUGGCcAuACGcAAATsT
AD-12100


 


1297
CACGUACCCUUCAUCAAAU
107
cAcGuAcccuucAucAAAuTsT
108
AUUUGAUGAAGGGuACGUGTsT
AD-12101


 


1298
UCUUUGCGUAUGGCCAAAC
109
ucuuuGcGuAuGGccAAAcTsT
110
GUUUGGCcAuACGcAAAGATsT
AD-12102


 


1299
CCGAAGUGUUGUUUGUCCA
111
ccGAAGuGuuGuuuGuccATsT
112
UGGAcAAAcAAcACUUCGGTsT
AD-12103


 


1300
AGAGCGGAAAGCUAGCGCC
113
AGAGcGGAAAGcuAGcGccTsT
114
GGCGCuAGCUUUCCGCUCUTsT
AD-12104


 


1301
GCUAGCGCCCAUUCAAUAG
115
GcuAGcGcccAuucAAuAGTsT
116
CuAUUGAAUGGGCGCuAGCTsT
AD-12105


 


1302
AAGUUAGUGUACGAACUGG
117
AAGuuAGuGuAcGAAcuGGTsT
118
CcAGUUCGuAcACuAACUUTsT
AD-12106


 


1303
GUACGAACUGGAGGAUUGG
119
GuAcGAAcuGGAGGAuuGGTsT
120
CcAAUCCUCcAGUUCGuACTsT
AD-12107


 


1304
ACGAACUGGAGGAUUGGCU
121
AcGAAcuGGAGGAuuGGcuTsT
122
AGCcAAUCCUCcAGUUCGUTsT
AD-12108


 


1305
AGAUUGAUGUUUACCGAAG
123
AGAuuGAuGuuuAccGAAGTsT
124
CUUCGGuAAAcAUcAAUCUTsT
AD-12109


 


1306
UAUGGGCUAUAAUUGCACU
125
uAuGGGcuAuAAuuGcAcuTsT
126
AGUGcAAUuAuAGCCcAuATsT
AD-12110


 


1307
AUCUUUGCGUAUGGCCAAA
127
AucuuuGcGuAuGGccAAATsT
128
UUUGGCcAuACGcAAAGAUTsT
AD-12111


 


1308
ACUCUAGUCGUUCCCACUC
129
AcucuAGucGuucccAcucTsT
130
GAGUGGGAACGACuAGAGUTsT
AD-12112


 


1309
AACUACGAUUGAUGGAGAA
131
AAcuAcGAuuGAuGGAGAATsT
132
UUCUCcAUcAAUCGuAGUUTsT
AD-12113


 


1310
GAUAAGAGAGCUCGGGAAG
133
GAuAAGAGAGcucGGGAAGTsT
134
CUUCCCGAGCUCUCUuAUCTsT
AD-12114


 


1311
UCGAGAAUCUAAACUAACU
135
ucGAGAAucuAAAcuAAcuTsT
136
AGUuAGUUuAGAUUCUCGATsT
AD-12115


 


1312
AACUAACUAGAAUCCUCCA
137
AAcuAAcuAGAAuccuccATsT
138
UGGAGGAUUCuAGUuAGUUTsT
AD-12116


 


1313
GGAUCGUAAGAAGGCAGUU
139
GGAucGuAAGAAGGcAGuuTsT
140
AACUGCCUUCUuACGAUCCTsT
AD-12117


 


1314
AUCGUAAGAAGGCAGUUGA
141
AucGuAAGAAGGcAGuuGATsT
142
UcAACUGCCUUCUuACGAUTsT
AD-12118


 


1315
AGGCAGUUGACCAACACAA
143
AGGcAGuuGAccAAcAcAATsT
144
UUGUGUUGGUcAACUGCCUTsT
AD-12119


 


1316
UGGCCGAUAAGAUAGAAGA
145
uGGccGAuAAGAuAGAAGATsT
146
UCUUCuAUCUuAUCGGCcATsT
AD-12120


 


1317
UCUAAGGAUAUAGUCAACA
147
ucuAAGGAuAuAGucAAcATsT
148
UGUUGACuAuAUCCUuAGATsT
AD-12121


 


1318
ACUAAGCUUAAUUGCUUUC
149
AcuAAGcuuAAuuGcuuucTsT
150
GAAAGcAAUuAAGCUuAGUTsT
AD-12122


 


1319
GCCCAGAUCAACCUUUAAU
151
GcccAGAucAAccuuuAAuTsT
152
AUuAAAGGUUGAUCUGGGCTsT
AD-12123


 


1320
UUAAUUUGGCAGAGCGGAA
153
uuAAuuuGGcAGAGcGGAATsT
154
UUCCGCUCUGCcAAAUuAATsT
AD-12124


 


1321
UUAUCGAGAAUCUAAACUA
155
uuAucGAGAAucuAAAcuATsT
156
uAGUUuAGAUUCUCGAuAATsT
AD-12125


 


1322
CUAGCGCCCAUUCAAUAGU
157
cuAGcGcccAuucAAuAGuTsT
158
ACuAUUGAAUGGGCGCuAGTsT
AD-12126


 


1323
AAUAGUAGAAUGUGAUCCU
159
AAuAGuAGAAuGuGAuccuTsT
160
AGGAUcAcAUUCuACuAUUTsT
AD-12127


 


1324
UACGAAAAGAAGUUAGUGU
161
uAcGAAAAGAAGuuAGuGuTsT
162
AcACuAACUUCUUUUCGuATsT
AD-12128


 


1325
AGAAGUUAGUGUACGAACU
163
AGAAGuuAGuGuAcGAAcuTsT
164
AGUUCGuAcACuAACUUCUTsT
AD-12129


 


1326
ACUAAACAGAUUGAUGUUU
165
AcuAAAcAGAuuGAuGuuuTsT
166
AAAcAUcAAUCUGUUuAGUTsT
AD-12130


 


1327
CUUUGCGUAUGGCCAAACU
167
cuuuGcGuAuGGccAAAcuTsT
168
AGUUUGGCcAuACGcAAAGTsT
AD-12131


 


1328
AAUGAAGAGUAUACCUGGG
169
AAuGAAGAGuAuAccuGGGTsT
170
CCcAGGuAuACUCUUcAUUTsT
AD-12132


 


1329
AUAAUUCCACGUACCCUUC
171
AuAAuuccAcGuAcccuucTsT
172
GAAGGGuACGUGGAAUuAUTsT
AD-12133


 


1330
ACGUACCCUUCAUCAAAUU
173
AcGuAcccuucAucAAAuuTsT
174
AAUUUGAUGAAGGGuACGUTsT
AD-12134


 


1331
CGUACCCUUCAUCAAAUUU
175
cGuAcccuucAucAAAuuuTsT
176
AAAUUUGAUGAAGGGuACGTsT
AD-12135


 


1332
GUACCCUUCAUCAAAUUUU
177
GuAcccuucAucAAAuuuuTsT
178
AAAAUUUGAUGAAGGGuACTsT
AD-12136


 


1333
AACUUACUGAUAAUGGUAC
179
AAcuuAcuGAuAAuGGuAcTsT
180
GuACcAUuAUcAGuAAGUUTsT
AD-12137


 


1334
UUCAGUCAAAGUGUCUCUG
181
uucAGucAAAGuGucucuGTsT
182
cAGAGAcACUUUGACUGAATsT
AD-12138


 


1335
UUCUUAAUCCAUCAUCUGA
183
uucuuAAuccAucAucuGATsT
184
UcAGAUGAUGGAUuAAGAATsT
AD-12139


 


1336
ACAGUACACAACAAGGAUG
185
AcAGuAcAcAAcAAGGAuGTsT
186
cAUCCUUGUUGUGuACUGUTsT
AD-12140


 


1337
AAGAAACUACGAUUGAUGG
187
AAGAAAcuAcGAuuGAuGGTsT
188
CcAUcAAUCGuAGUUUCUUTsT
AD-12141


 


1338
AAACUACGAUUGAUGGAGA
189
AAAcuAcGAuuGAuGGAGATsT
190
UCUCcAUcAAUCGuAGUUUTsT
AD-12142


 


1339
UGGAGCUGUUGAUAAGAGA
191
uGGAGcuGuuGAuAAGAGATsT
192
UCUCUuAUcAAcAGCUCcATsT
AD-12143


 


1340
CUAACUAGAAUCCUCCAGG
193
cuAAcuAGAAuccuccAGGTsT
194
CCUGGAGGAUUCuAGUuAGTsT
AD-12144


 


1341
GAAUAUGCUCAUAGAGCAA
195
GAAuAuGcucAuAGAGcAATsT
196
UUGCUCuAUGAGcAuAUUCTsT
AD-12145


 


1342
AUGCUCAUAGAGCAAAGAA
197
AuGcucAuAGAGcAAAGAATsT
198
UUCUUUGCUCuAUGAGcAUTsT
AD-12146


 


1343
AAAAAUUGGUGCUGUUGAG
199
AAAAAuuGGuGcuGuuGAGTsT
200
CUcAAcAGcACcAAUUUUUTsT
AD-12147


 


1344
GAGGAGCUGAAUAGGGUUA
201
GAGGAGcuGAAuAGGGuuATsT
202
uAACCCuAUUcAGCUCCUCTsT
AD-12148


 


1345
GGAGCUGAAUAGGGUUACA
203
GGAGcuGAAuAGGGuuAcATsT
204
UGuAACCCuAUUcAGCUCCTsT
AD-12149


 


1346
GAGCUGAAUAGGGUUACAG
205
GAGcuGAAuAGGGuuAcAGTsT
206
CUGuAACCCuAUUcAGCUCTsT
AD-12150


 


1347
AGCUGAAUAGGGUUACAGA
207
AGcuGAAuAGGGuuAcAGATsT
208
UCUGuAACCCuAUUcAGCUTsT
AD-12151


 


1348
GCUGAAUAGGGUUACAGAG
209
GcuGAAuAGGGuuAcAGAGTsT
210
CUCUGuAACCCuAUUcAGCTsT
AD-12152


 


1349
CCAAACUGGAUCGUAAGAA
211
ccAAAcuGGAucGuAAGAATsT
212
UUCUuACGAUCcAGUUUGGTsT
AD-12153


 


1350
GAUCGUAAGAAGGCAGUUG
213
GAucGuAAGAAGGcAGuuGTsT
214
cAACUGCCUUCUuACGAUCTsT
AD-12154


 


1351
ACCUUAUUUGGUAAUCUGC
215
AccuuAuuuGGuAAucuGcTsT
216
GcAGAUuACcAAAuAAGGUTsT
AD-12155


 


1352
UUAGAUACCAUUACUACAG
217
uuAGAuAccAuuAcuAcAGTsT
218
CUGuAGuAAUGGuAUCuAATsT
AD-12156


 


1353
AUACCAUUACUACAGUAGC
219
AuAccAuuAcuAcAGuAGcTsT
220
GCuACUGuAGuAAUGGuAUTsT
AD-12157


 


1354
UACUACAGUAGCACUUGGA
221
uAcuAcAGuAGcAcuuGGATsT
222
UCcAAGUGCuACUGuAGuATsT
AD-12158


 


1355
AAAGUAAAACUGUACUACA
223
AAAGuAAAAcuGuAcuAcATsT
224
UGuAGuAcAGUUUuACUUUTsT
AD-12159


 


1356
CUCAAGACUGAUCUUCUAA
225
cucAAGAcuGAucuucuAATsT
226
UuAGAAGAUcAGUCUUGAGTsT
AD-12160


 


1357
UUGACAGUGGCCGAUAAGA
227
uuGAcAGuGGccGAuAAGATsT
228
UCUuAUCGGCcACUGUcAATsT
AD-12161


 


1358
UGACAGUGGCCGAUAAGAU
229
uGAcAGuGGccGAuAAGAuTsT
230
AUCUuAUCGGCcACUGUcATsT
AD-12162


 


1359
GCAAUGUGGAAACCUAACU
231
GcAAuGuGGAAAccuAAcuTsT
232
AGUuAGGUUUCcAcAUUGCTsT
AD-12163


 


1360
CCACUUAGUAGUGUCCAGG
233
ccAcuuAGuAGuGuccAGGTsT
234
CCUGGAcACuACuAAGUGGTsT
AD-12164


 


1361
AGAAGGUACAAAAUUGGUU
235
AGAAGGuAcAAAAuuGGuuTsT
236
AACcAAUUUUGuACCUUCUTsT
AD-12165


 


1362
UGGUUUGACUAAGCUUAAU
237
uGGuuuGAcuAAGcuuAAuTsT
238
AUuAAGCUuAGUcAAACcATsT
AD-12166


 


1363
GGUUUGACUAAGCUUAAUU
239
GGuuuGAcuAAGcuuAAuuTsT
240
AAUuAAGCUuAGUcAAACCTsT
AD-12167


 


1364
UCUAAGUCAAGAGCCAUCU
241
ucuAAGucAAGAGccAucuTsT
242
AGAUGGCUCUUGACUuAGATsT
AD-12168


 


1365
UCAUCCCUAUAGUUCACUU
243
ucAucccuAuAGuucAcuuTsT
244
AAGUGAACuAuAGGGAUGATsT
AD-12169


 


1366
CAUCCCUAUAGUUCACUUU
245
cAucccuAuAGuucAcuuuTsT
246
AAAGUGAACuAuAGGGAUGTsT
AD-12170


 


1367
CCCUAGACUUCCCUAUUUC
247
cccuAGAcuucccuAuuucTsT
248
GAAAuAGGGAAGUCuAGGGTsT
AD-12171


 


1368
AGACUUCCCUAUUUCGCUU
249
AGAcuucccuAuuucGcuuTsT
250
AAGCGAAAuAGGGAAGUCUTsT
AD-12172


 


1369
UCACCAAACCAUUUGUAGA
251
ucAccAAAccAuuuGuAGATsT
252
UCuAcAAAUGGUUUGGUGATsT
AD-12173


 


1370
UCCUUUAAGAGGCCUAACU
253
uccuuuAAGAGGccuAAcuTsT
254
AGUuAGGCCUCUuAAAGGATsT
AD-12174


 


1371
UUUAAGAGGCCUAACUCAU
255
uuuAAGAGGccuAAcucAuTsT
256
AUGAGUuAGGCCUCUuAAATsT
AD-12175


 


1372
UUAAGAGGCCUAACUCAUU
257
uuAAGAGGccuAAcucAuuTsT
258
AAUGAGUuAGGCCUCUuAATsT
AD-12176


 


1373
GGCCUAACUCAUUCACCCU
259
GGccuAAcucAuucAcccuTsT
260
AGGGUGAAUGAGUuAGGCCTsT
AD-12177


 


1374
UGGUAUUUUUGAUCUGGCA
261
uGGuAuuuuuGAucuGGcATsT
262
UGCcAGAUcAAAAAuACcATsT
AD-12178


 


1375
AGUUUAGUGUGUAAAGUUU
263
AGuuuAGuGuGuAAAGuuuTsT
264
AAACUUuAcAcACuAAACUTsT
AD-12179


 


1376
GCCAAAUUCGUCUGCGAAG
265
GccAAAuucGucuGcGAAGTsT
266
CUUCGcAGACGAAUUUGGCTsT
AD-12180


 


1377
AAUUCGUCUGCGAAGAAGA
267
AAuucGucuGcGAAGAAGATsT
268
UCUUCUUCGcAGACGAAUUTsT
AD-12181


 


1378
UGAAAGGUCACCUAAUGAA
269
uGAAAGGucAccuAAuGAATsT
270
UUcAUuAGGUGACCUUUcATsT
AD-12182


 


1379
CAGACCAUUUAAUUUGGCA
271
cAGAccAuuuAAuuuGGcATsT
272
UGCcAAAUuAAAUGGUCUGTsT
AD-12183


 


1380
AGACCAUUUAAUUUGGCAG
273
AGAccAuuuAAuuuGGcAGTsT
274
CUGCcAAAUuAAAUGGUCUTsT
AD-12184


 


1381
AGUUAUUAUGGGCUAUAAU
275
AGuuAuuAuGGGcuAuAAuTsT
276
AUuAuAGCCcAuAAuAACUTsT
AD-12185


 


1382
GCUGGUAUAAUUCCACGUA
277
GcuGGuAuAAuuccAcGuATsT
278
uACGUGGAAUuAuACcAGCTsT
AD-12186


 


1383
AUUUAAUUUGGCAGAGCGG
279
AuuuAAuuuGGcAGAGcGGTsT
280
CCGCUCUGCcAAAUuAAAUTsT
AD-12187


 


1384
UUUAAUUUGGCAGAGCGGA
281
uuuAAuuuGGcAGAGcGGATsT
282
UCCGCUCUGCcAAAUuAAATsT
AD-12188


 


1385
UUUGGCAGAGCGGAAAGCU
283
uuuGGcAGAGcGGAAAGcuTsT
284
AGCUUUCCGCUCUGCcAAATsT
AD-12189


 


1386
UUUUACAAUGGAAGGUGAA
285
uuuuAcAAuGGAAGGuGAATsT
286
UUcACCUUCcAUUGuAAAATsT
AD-12190


 


1387
AAUGGAAGGUGAAAGGUCA
287
AAuGGAAGGuGAAAGGucATsT
288
UGACCUUUcACCUUCcAUUTsT
AD-12191


 


1388
UGAGAUGCAGACCAUUUAA
289
uGAGAuGcAGAccAuuuAATsT
290
UuAAAUGGUCUGcAUCUcATsT
AD-12192


 


1389
UCGCAGCCAAAUUCGUCUG
291
ucGcAGccAAAuucGucuGTsT
292
cAGACGAAUUUGGCUGCGATsT
AD-12193


 


1390
GGCUAUAAUUGCACUAUCU
293
GGcuAuAAuuGcAcuAucuTsT
294
AGAuAGUGcAAUuAuAGCCTsT
AD-12194


 


1391
AUUGACAGUGGCCGAUAAG
295
AuuGAcAGuGGccGAuAAGTsT
296
CUuAUCGGCcACUGUcAAUTsT
AD-12195


 


1392
CUAGACUUCCCUAUUUCGC
297
cuAGAcuucccuAuuucGcTsT
298
GCGAAAuAGGGAAGUCuAGTsT
AD-12196


 


1393
ACUAUCUUUGCGUAUGGCC
299
AcuAucuuuGcGuAuGGccTsT
300
GGCcAuACGcAAAGAuAGUTsT
AD-12197


 


1394
AUACUCUAGUCGUUCCCAC
301
AuAcucuAGucGuucccAcTsT
302
GUGGGAACGACuAGAGuAUTsT
AD-12198


 


1395
AAAGAAACUACGAUUGAUG
303
AAAGAAAcuAcGAuuGAuGTsT
304
cAUcAAUCGuAGUUUCUUUTsT
AD-12199


 


1396
GCCUUGAUUUUUUGGCGGG
305
GccuuGAuuuuuuGGcGGGTsT
306
CCCGCcAAAAAAUcAAGGCTsT
AD-12200


 


1397
CGCCCAUUCAAUAGUAGAA
307
cGcccAuucAAuAGuAGAATsT
308
UUCuACuAUUGAAUGGGCGTsT
AD-12201


 


1398
CCUUAUUUGGUAAUCUGCU
309
ccuuAuuuGGuAAucuGcuTsT
310
AGcAGAUuACcAAAuAAGGTsT
AD-12202


 


1399
AGAGACAAUUCCGGAUGUG
311
AGAGAcAAuuccGGAuGuGTsT
312
cAcAUCCGGAAUUGUCUCUTsT
AD-12203


 


1400
UGACUUUGAUAGCUAAAUU
313
uGAcuuuGAuAGcuAAAuuTsT
314
AAUUuAGCuAUcAAAGUcATsT
AD-12204


 


1401
UGGCAGAGCGGAAAGCUAG
315
uGGcAGAGcGGAAAGcuAGTsT
316
CuAGCUUUCCGCUCUGCcATsT
AD-12205


 


1402
GAGCGGAAAGCUAGCGCCC
317
GAGcGGAAAGcuAGcGcccTsT
318
GGGCGCuAGCUUUCCGCUCTsT
AD-12206


 


1403
AAAGAAGUUAGUGUACGAA
319
AAAGAAGuuAGuGuAcGAATsT
320
UUCGuAcACuAACUUCUUUTsT
AD-12207


 


1404
AUUGCACUAUCUUUGCGUA
321
AuuGcAcuAucuuuGcGuATsT
322
uACGcAAAGAuAGUGcAAUTsT
AD-12208


 


1405
GGUAUAAUUCCACGUACCC
323
GGuAuAAuuccAcGuAcccTsT
324
GGGuACGUGGAAUuAuACCTsT
AD-12209


 


1406
UACUCUAGUCGUUCCCACU
325
uAcucuAGucGuucccAcuTsT
326
AGUGGGAACGACuAGAGuATsT
AD-12210


 


1407
UAUGAAAGAAACUACGAUU
327
uAuGAAAGAAAcuAcGAuuTsT
328
AAUCGuAGUUUCUUUcAuATsT
AD-12211


 


1408
AUGCUAGAAGUACAUAAGA
329
AuGcuAGAAGuAcAuAAGATsT
330
UCUuAUGuACUUCuAGcAUTsT
AD-12212


 


1409
AAGUACAUAAGACCUUAUU
331
AAGuAcAuAAGAccuuAuuTsT
332
AAuAAGGUCUuAUGuACUUTsT
AD-12213


 


1410
ACAGCCUGAGCUGUUAAUG
333
AcAGccuGAGcuGuuAAuGTsT
334
cAUuAAcAGCUcAGGCUGUTsT
AD-12214


 


1411
AAAGAAGAGACAAUUCCGG
335
AAAGAAGAGAcAAuuccGGTsT
336
CCGGAAUUGUCUCUUCUUUTsT
AD-12215


 


1412
CACACUGGAGAGGUCUAAA
337
cAcAcuGGAGAGGucuAAATsT
338
UUuAGACCUCUCcAGUGUGTsT
AD-12216


 


1413
CACUGGAGAGGUCUAAAGU
339
cAcuGGAGAGGucuAAAGuTsT
340
ACUUuAGACCUCUCcAGUGTsT
AD-12217


 


1414
ACUGGAGAGGUCUAAAGUG
341
AcuGGAGAGGucuAAAGuGTsT
342
cACUUuAGACCUCUCcAGUTsT
AD-12218


 


1415
CGUCGCAGCCAAAUUCGUC
343
cGucGcAGccAAAuucGucTsT
344
GACGAAUUUGGCUGCGACGTsT
AD-12219


 


1416
GAAGGCAGUUGACCAACAC
345
GAAGGcAGuuGAccAAcAcTsT
346
GUGUUGGUcAACUGCCUUCTsT
AD-12220


 


1417
CAUUCACCCUGACAGAGUU
347
cAuucAcccuGAcAGAGuuTsT
348
AACUCUGUcAGGGUGAAUGTsT
AD-12221


 


1418
AAGAGGCCUAACUCAUUCA
349
AAGAGGccuAAcucAuucATsT
350
UGAAUGAGUuAGGCCUCUUTsT
AD-12222


 


1419
GAGACAAUUCCGGAUGUGG
351
GAGAcAAuuccGGAuGuGGTsT
352
CcAcAUCCGGAAUUGUCUCTsT
AD-12223


 


1420
UUCCGGAUGUGGAUGUAGA
353
uuccGGAuGuGGAuGuAGATsT
354
UCuAcAUCcAcAUCCGGAATsT
AD-12224


 


1421
AAGCUAGCGCCCAUUCAAU
355
AAGcuAGcGcccAuucAAuTsT
356
AUUGAAUGGGCGCuAGCUUTsT
AD-12225


 


1422
GAAGUUAGUGUACGAACUG
357
GAAGuuAGuGuAcGAAcuGTsT
358
cAGUUCGuAcACuAACUUCTsT
AD-12226


 


1423
UAUAAUUCCACGUACCCUU
359
uAuAAuuccAcGuAcccuuTsT
360
AAGGGuACGUGGAAUuAuATsT
AD-12227


 


1424
ACAGUGGCCGAUAAGAUAG
361
AcAGuGGccGAuAAGAuAGTsT
362
CuAUCUuAUCGGCcACUGUTsT
AD-12228


 


1425
UCUGUCAUCCCUAUAGUUC
363
ucuGucAucccuAuAGuucTsT
364
GAACuAuAGGGAUGAcAGATsT
AD-12229


 


1426
UUCUUGCUAUGACUUGUGU
365
uucuuGcuAuGAcuuGuGuTsT
366
AcAcAAGUcAuAGcAAGAATsT
AD-12230


 


1427
GUAAGAAGGCAGUUGACCA
367
GuAAGAAGGcAGuuGAccATsT
368
UGGUcAACUGCCUUCUuACTsT
AD-12231


 


1428
CAUUGACAGUGGCCGAUAA
369
cAuuGAcAGuGGccGAuAATsT
370
UuAUCGGCcACUGUcAAUGTsT
AD-12232


 


1429
AGAAACCACUUAGUAGUGU
371
AGAAAccAcuuAGuAGuGuTsT
372
AcACuACuAAGUGGUUUCUTsT
AD-12233


 


1430
GGAUUGUUCAUCAAUUGGC
373
GGAuuGuucAucAAuuGGcTsT
374
GCcAAUUGAUGAAcAAUCCTsT
AD-12234


 


1431
UAAGAGGCCUAACUCAUUC
375
uAAGAGGccuAAcucAuucTsT
376
GAAUGAGUuAGGCCUCUuATsT
AD-12235


 


1432
AGUUAGUGUACGAACUGGA
377
AGuuAGuGuAcGAAcuGGATsT
378
UCcAGUUCGuAcACuAACUTsT
AD-12236


 


1433
AGUACAUAAGACCUUAUUU
379
AGuAcAuAAGAccuuAuuuTsT
380
AAAuAAGGUCUuAUGuACUTsT
AD-12237


 


1434
UGAGCCUUGUGUAUAGAUU
381
uGAGccuuGuGuAuAGAuuTsT
382
AAUCuAuAcAcAAGGCUcATsT
AD-12238


 


1435
CCUUUAAGAGGCCUAACUC
383
ccuuuAAGAGGccuAAcucTsT
384
GAGUuAGGCCUCUuAAAGGTsT
AD-12239


 


1436
ACCACUUAGUAGUGUCCAG
385
AccAcuuAGuAGuGuccAGTsT
386
CUGGAcACuACuAAGUGGUTsT
AD-12240


 


1437
GAAACUUCCAAUUAUGUCU
387
GAAAcuuccAAuuAuGucuTsT
388
AGAcAuAAUUGGAAGUUUCTsT
AD-12241


 


1438
UGCAUACUCUAGUCGUUCC
389
uGcAuAcucuAGucGuuccTsT
390
GGAACGACuAGAGuAUGcATsT
AD-12242


 


1439
AGAAGGCAGUUGACCAACA
391
AGAAGGcAGuuGAccAAcATsT
392
UGUUGGUcAACUGCCUUCUTsT
AD-12243


 


1440
GUACAUAAGACCUUAUUUG
393
GuAcAuAAGAccuuAuuuGTsT
394
cAAAuAAGGUCUuAUGuACTsT
AD-12244


 


1441
UAUAAUUGCACUAUCUUUG
395
uAuAAuuGcAcuAucuuuGTsT
396
cAAAGAuAGUGcAAUuAuATsT
AD-12245


 


1442
UCUCUGUUACAAUACAUAU
397
ucucuGuuAcAAuAcAuAuTsT
398
AuAUGuAUUGuAAcAGAGATsT
AD-12246


 


1443
UAUGCUCAUAGAGCAAAGA
399
uAuGcucAuAGAGcAAAGATsT
400
UCUUUGCUCuAUGAGcAuATsT
AD-12247


 


1444
UGUUGUUUGUCCAAUUCUG
401
uGuuGuuuGuccAAuucuGTsT
402
cAGAAUUGGAcAAAcAAcATsT
AD-12248


 


1445
ACUAACUAGAAUCCUCCAG
403
AcuAAcuAGAAuccuccAGTsT
404
CUGGAGGAUUCuAGUuAGUTsT
AD-12249


 


1446
UGUGGUGUCUAUACUGAAA
405
uGuGGuGucuAuAcuGAAATsT
406
UUUcAGuAuAGAcACcAcATsT
AD-12250


 


1447
UAUUAUGGGAGACCACCCA
407
uAuuAuGGGAGAccAcccATsT
408
UGGGUGGUCUCCcAuAAuATsT
AD-12251


 


1448
AAGGAUGAAGUCUAUCAAA
409
AAGGAuGAAGucuAucAAATsT
410
UUUGAuAGACUUcAUCCUUTsT
AD-12252


 


1449
UUGAUAAGAGAGCUCGGGA
411
uuGAuAAGAGAGcucGGGATsT
412
UCCCGAGCUCUCUuAUcAATsT
AD-12253


 


1450
AUGUUCCUUAUCGAGAAUC
413
AuGuuccuuAucGAGAAucTsT
414
GAUUCUCGAuAAGGAAcAUTsT
AD-12254


 


1451
GGAAUAUGCUCAUAGAGCA
415
GGAAuAuGcucAuAGAGcATsT
416
UGCUCuAUGAGcAuAUUCCTsT
AD-12255


 


1452
CCAUUCCAAACUGGAUCGU
417
ccAuuccAAAcuGGAucGuTsT
418
ACGAUCcAGUUUGGAAUGGTsT
AD-12256


 


1453
GGCAGUUGACCAACACAAU
419
GGcAGuuGAccAAcAcAAuTsT
420
AUUGUGUUGGUcAACUGCCTsT
AD-12257


 


1454
CAUGCUAGAAGUACAUAAG
421
cAuGcuAGAAGuAcAuAAGTsT
422
CUuAUGuACUUCuAGcAUGTsT
AD-12258


 


1455
CUAGAAGUACAUAAGACCU
423
cuAGAAGuAcAuAAGAccuTsT
424
AGGUCUuAUGuACUUCuAGTsT
AD-12259


 


1456
UUGGAUCUCUCACAUCUAU
425
uuGGAucucucAcAucuAuTsT
426
AuAGAUGUGAGAGAUCcAATsT
AD-12260


 


1457
AACUGUGGUGUCUAUACUG
427
AAcuGuGGuGucuAuAcuGTsT
428
cAGuAuAGAcACcAcAGUUTsT
AD-12261


 


1458
UCAUUGACAGUGGCCGAUA
429
ucAuuGAcAGuGGccGAuATsT
430
uAUCGGCcACUGUcAAUGATsT
AD-12262


 


1459
AUAAAGCAGACCCAUUCCC
431
AuAAAGcAGAcccAuucccTsT
432
GGGAAUGGGUCUGCUUuAUTsT
AD-12263


 


1460
ACAGAAACCACUUAGUAGU
433
AcAGAAAccAcuuAGuAGuTsT
434
ACuACuAAGUGGUUUCUGUTsT
AD-12264


 


1461
GAAACCACUUAGUAGUGUC
435
GAAAccAcuuAGuAGuGucTsT
436
GAcACuACuAAGUGGUUUCTsT
AD-12265


 


1462
AAAUCUAAGGAUAUAGUCA
437
AAAucuAAGGAuAuAGucATsT
438
UGACuAuAUCCUuAGAUUUTsT
AD-12266


 


1463
UUAUUUAUACCCAUCAACA
439
uuAuuuAuAcccAucAAcATsT
440
UGUUGAUGGGuAuAAAuAATsT
AD-12267


 


1464
ACAGAGGCAUUAACACACU
441
AcAGAGGcAuuAAcAcAcuTsT
442
AGUGUGUuAAUGCCUCUGUTsT
AD-12268


 


1465
ACACACUGGAGAGGUCUAA
443
AcAcAcuGGAGAGGucuAATsT
444
UuAGACCUCUCcAGUGUGUTsT
AD-12269


 


1466
ACACUGGAGAGGUCUAAAG
445
AcAcuGGAGAGGucuAAAGTsT
446
CUUuAGACCUCUCcAGUGUTsT
AD-12270


 


1467
CGAGCCCAGAUCAACCUUU
447
cGAGcccAGAucAAccuuuTsT
448
AAAGGUUGAUCUGGGCUCGTsT
AD-12271


 


1468
UCCCUAUUUCGCUUUCUCC
449
ucccuAuuucGcuuucuccTsT
450
GGAGAAAGCGAAAuAGGGATsT
AD-12272


 


1469
UCUAAAAUCACUGUCAACA
451
ucuAAAAucAcuGucAAcATsT
452
UGUUGAcAGUGAUUUuAGATsT
AD-12273


 


1470
AGCCAAAUUCGUCUGCGAA
453
AGccAAAuucGucuGcGAATsT
454
UUCGcAGACGAAUUUGGCUTsT
AD-12274


 


1471
CCCAUUCAAUAGUAGAAUG
455
cccAuucAAuAGuAGAAuGTsT
456
cAUUCuACuAUUGAAUGGGTsT
AD-12275


 


1472
GAUGAAUGCAUACUCUAGU
457
GAuGAAuGcAuAcucuAGuTsT
458
ACuAGAGuAUGcAUUcAUCTsT
AD-12276


 


1473
CUCAUGUUCCUUAUCGAGA
459
cucAuGuuccuuAucGAGATsT
460
UCUCGAuAAGGAAcAUGAGTsT
AD-12277


 


1474
GAGAAUCUAAACUAACUAG
461
GAGAAucuAAAcuAAcuAGTsT
462
CuAGUuAGUUuAGAUUCUCTsT
AD-12278


 


1475
UAGAAGUACAUAAGACCUU
463
uAGAAGuAcAuAAGAccuuTsT
464
AAGGUCUuAUGuACUUCuATsT
AD-12279


 


1476
CAGCCUGAGCUGUUAAUGA
465
cAGccuGAGcuGuliAAuGATsT
466
UcAUuAAcAGCUcAGGCUGTsT
AD-12280


 


1477
AAGAAGAGACAAUUCCGGA
467
AAGAAGAGAcAAuuccGGATsT
468
UCCGGAAUUGUCUCUUCUUTsT
AD-12281


 


1478
UGCUGGUGUGGAUUGUUCA
469
uGcuGGuGuGGAuuGuucATsT
470
UGAAcAAUCcAcACcAGcATsT
AD-12282


 


1479
AAAUUCGUCUGCGAAGAAG
471
AAAuucGucuGcGAAGAAGTsT
472
CUUCUUCGcAGACGAAUUUTsT
AD-12283


 


1480
UUUCUGGAAGUUGAGAUGU
473
uuucuGGAAGuuGAGAuGuTsT
474
AcAUCUcAACUUCcAGAAATsT
AD-12284


 


1481
UACUAAACAGAUUGAUGUU
475
uAcuAAAcAGAuuGAuGuuTsT
476
AAcAUcAAUCUGUUuAGuATsT
AD-12285


 


1482
GAUUGAUGUUUACCGAAGU
477
GAuuGAuGuuuAccGAAGuTsT
478
ACUUCGGuAAAcAUcAAUCTsT
AD-12286


 


1483
GCACUAUCUUUGCGUAUGG
479
GcAcuAucuuuGcGuAuGGTsT
480
CcAuACGcAAAGAuAGUGCTsT
AD-12287


 


1484
UGGUAUAAUUCCACGUACC
481
uGGuAuAAuuccAcGuAccTsT
482
GGuACGUGGAAUuAuACcATsT
AD-12288


 


1485
AGCAAGCUGCUUAACACAG
483
AGcAAGcuGcuuAAcAcAGTsT
484
CUGUGUuAAGcAGCUUGCUTsT
AD-12289


 


1486
CAGAAACCACUUAGUAGUG
485
cAGAAAccAcuuAGuAGuGTsT
486
cACuACuAAGUGGUUUCUGTsT
AD-12290


 


1487
AACUUAUUGGAGGUUGUAA
487
AAcuuAuuGGAGGuuGuAATsT
488
UuAcAACCUCcAAuAAGUUTsT
AD-12291


 


1488
CUGGAGAGGUCUAAAGUGG
489
cuGGAGAGGucuAAAGuGGTsT
490
CcACUUuAGACCUCUCcAGTsT
AD-12292


 


1489
AAAAAAGAUAUAAGGCAGU
491
AAAAAAGAuAuAAGGcAGuTsT
492
ACUGCCUuAuAUCUUUUUUTsT
AD-12293


 


1490
GAAUUUUGAUAUCUACCCA
493
GAAuuuuGAuAucuAcccATsT
494
UGGGuAGAuAUcAAAAUUCTsT
AD-12294


 


1491
GUAUUUUUGAUCUGGCAAC
495
GuAuuuuuGAucuGGcAAcTsT
496
GUUGCcAGAUcAAAAAuACTsT
AD-12295


 


1492
AGGAUCCCUUGGCUGGUAU
497
AGGAucccuuGGcuGGuAuTsT
498
AuACcAGCcAAGGGAUCCUTsT
AD-12296


 


1493
GGAUCCCUUGGCUGGUAUA
499
GGAucccuuGGcuGGuAuATsT
500
uAuACcAGCcAAGGGAUCCTsT
AD-12297


 


1494
CAAUAGUAGAAUGUGAUCC
501
cAAuAGuAGAAuGuGAuccTsT
502
GGAUcAcAUUCuACuAUUGTsT
AD-12298


 


1495
GCUAUAAUUGCACUAUCUU
503
GcuAuAAuuGcAcuAucuuTsT
504
AAGAuAGUGcAAUuAuAGCTsT
AD-12299


 


1496
UACCCUUCAUCAAAUUUUU
505
uAcccuucAucAAAuuuuuTsT
506
AAAAAUUUGAUGAAGGGuATsT
AD-12300


 


1497
AGAACAUAUUGAAUAAGCC
507
AGAAcAuAuuGAAuAAGccTsT
508
GGCUuAUUcAAuAUGUUCUTsT
AD-12301


 


1498
AAAUUGGUGCUGUUGAGGA
509
AAAuuGGuGcuGuuGAGGATsT
510
UCCUcAAcAGcACcAAUUUTsT
AD-12302


 


1499
UGAAUAGGGUUACAGAGUU
511
uGAAuAGGGuuAcAGAGuuTsT
512
AACUCUGuAACCCuAUUcATsT
AD-12303


 


1500
AAGAACUUGAAACCACUCA
513
AAGAAcuuGAAAccAcucATsT
514
UGAGUGGUUUcAAGUUCUUTsT
AD-12304


 


1501
AAUAAAGCAGACCCAUUCC
515
AAuAAAGcAGAcccAuuccTsT
516
GGAAUGGGUCUGCUUuAUUTsT
AD-12305


 


1502
AUACCCAUCAACACUGGUA
517
AuAcccAucAAcAcuGGuATsT
518
uACcAGUGUUGAUGGGuAUTsT
AD-12306


 


1503
UGGAUUGUUCAUCAAUUGG
519
uGGAuuGuucAucAAuuGGTsT
520
CcAAUUGAUGAAcAAUCcATsT
AD-12307


 


1504
UGGAGAGGUCUAAAGUGGA
521
uGGAGAGGucuAAAGuGGATsT
522
UCcACUUuAGACCUCUCcATsT
AD-12308


 


1505
GUCAUCCCUAUAGUUCACU
523
GucAucccuAuAGuucAcuTsT
524
AGUGAACuAuAGGGAUGACTsT
AD-12309


 


1506
AUAAUGGCUAUAAUUUCUC
525
AuAAuGGcuAuAAuuucucTsT
526
GAGAAAUuAuAGCcAUuAUTsT
AD-12310


 


1507
AUCCCUUGGCUGGUAUAAU
527
AucccuuGGcuGGuAuAAuTsT
528
AUuAuACcAGCcAAGGGAUTsT
AD-12311


 


1508
GGGCUAUAAUUGCACUAUC
529
GGGcuAuAAuuGcAcuAucTsT
530
GAuAGUGcAAUuAuAGCCCTsT
AD-12312


 


1509
GAUUCUCUUGGAGGGCGUA
531
GAuucucuuGGAGGGcGuATsT
532
uACGCCCUCcAAGAGAAUCTsT
AD-12313


 


1510
GCAUCUCUCAAUCUUGAGG
533
GcAucucucAAucuuGAGGTsT
534
CCUcAAGAUUGAGAGAUGCTsT
AD-12314


 


1511
CAGCAGAAAUCUAAGGAUA
535
cAGcAGAAAucuAAGGAuATsT
536
uAUCCUuAGAUUUCUGCUGTsT
AD-12315


 


1512
GUCAAGAGCCAUCUGUAGA
537
GucAAGAGccAucuGuAGATsT
538
UCuAcAGAUGGCUCUUGACTsT
AD-12316


 


1513
AAACAGAGGCAUUAACACA
539
AAAcAGAGGcAuuAAcAcATsT
540
UGUGUuAAUGCCUCUGUUUTsT
AD-12317


 


1514
AGCCCAGAUCAACCUUUAA
541
AGcccAGAucAAccuuuAATsT
542
UuAAAGGUUGAUCUGGGCUTsT
AD-12318


 


1515
UAUUUUUGAUCUGGCAACC
543
uAuuuuuGAucuGGcAAccTsT
544
GGUUGCcAGAUcAAAAAuATsT
AD-12319


 


1516
UGUUUGGAGCAUCUACUAA
545
uGuuuGGAGcAucuAcuAATsT
546
UuAGuAGAUGCUCcAAAcATsT
AD-12320


 


1517
GAAAUUACAGUACACAACA
547
GAAAuuAcAGuAcAcAAcATsT
548
UGUUGUGuACUGuAAUUUCTsT
AD-12321


 


1518
ACUUGACCAGUGUAAAUCU
549
AcuuGAccAGuGuAAAucuTsT
550
AGAUUuAcACUGGUcAAGUTsT
AD-12322


 


1519
ACCAGUGUAAAUCUGACCU
551
AccAGuGuAAAucuGAccuTsT
552
AGGUcAGAUUuAcACUGGUTsT
AD-12323


 


1520
AGAACAAUCAUUAGCAGCA
553
AGAAcAAucAuuAGcAGcATsT
554
UGCUGCuAAUGAUUGUUCUTsT
AD-12324


 


1521
CAAUGUGGAAACCUAACUG
555
cAAuGuGGAAAccuAAcuGTsT
556
cAGUuAGGUUUCcAcAUUGTsT
AD-12325


 


1522
ACCAAGAAGGUACAAAAUU
557
AccAAGAAGGuAcAAAAuuTsT
558
AAUUUUGuACCUUCUUGGUTsT
AD-12326


 


1523
GGUACAAAAUUGGUUGAAG
559
GGuAcAAAAuuGGuuGAAGTsT
560
CUUcAACcAAUUUUGuACCTsT
AD-12327


 


1524
GGUGUGGAUUGUUCAUCAA
561
GGuGuGGAuuGuucAucAATsT
562
UUGAUGAAcAAUCcAcACCTsT
AD-12328


 


1525
AGAGUUCACAAAAAGCCCA
563
AGAGuucAcAAAAAGcccATsT
564
UGGGCUUUUUGUGAACUCUTsT
AD-12329


 


1526
UGAUAGCUAAAUUAAACCA
565
uGAuAGcuAAAuuAAAccATsT
566
UGGUUuAAUUuAGCuAUcATsT
AD-12330


 


1527
AAUAAGCCUGAAGUGAAUC
567
AAuAAGccuGAAGuGAAucTsT
568
GAUUcACUUcAGGCUuAUUTsT
AD-12331


 


1528
CAGUUGACCAACACAAUGC
569
cAGuuGAccAAcAcAAuGcTsT
570
GcAUUGUGUUGGUcAACUGTsT
AD-12332


 


1529
UGGUGUGGAUUGUUCAUCA
571
uGGuGuGGAuuGuucAucATsT
572
UGAUGAAcAAUCcAcACcATsT
AD-12333


 


1530
AUUCACCCUGACAGAGUUC
573
AuucAcccuGAcAGAGuucTsT
574
GAACUCUGUcAGGGUGAAUTsT
AD-12334


 


1531
UAAGACCUUAUUUGGUAAU
575
uAAGAccuuAuuuGGuAAuTsT
576
AUuACcAAAuAAGGUCUuATsT
AD-12335


 


1532
AAGCAAUGUGGAAACCUAA
577
AAGcAAuGuGGAAAccuAATsT
578
UuAGGUUUCcAcAUUGCUUTsT
AD-12336


 


1533
UCUGAAACUGGAUAUCCCA
579
ucuGAAAcuGGAuAucccATsT
580
UGGGAuAUCcAGUUUcAGATsT
AD-12337
















TABLE 2b







Analysis of Eg5/KSP dsRNA duplexes














1st 

2nd 






single

single






dose

dose

3rd




screen @
SDs 1st
screen @
SDs 2nd 
single
SDs 3rd 


Eg5/KSP
50 nM [%
screen
25 nM [%
screen
dose
screen


duplex
resudual
(among
resudual
(among
screen
(among


name
mRNA]
quadruplicates)
mRNA]
quadruplicates)
@ 25 nM
quadruplicates)
















AD-12072
65%
2%
82%
5%




AD-12073
84%
1%
61%
6%




AD-12074
51%
3%
36%
9%




AD-12075
56%
4%
36%
4%




AD-12076
21%
4%
13%
3%




AD-12077
11%
2%
 6%
1%




AD-12078
22%
3%
 9%
2%




AD-12079
22%
10% 
15%
7%




AD-12080
68%
4%
52%
13% 




AD-12081
34%
8%
35%
24% 




AD-12082
20%
2%
92%
5%




AD-12083
85%
6%
63%
10% 




AD-12084
18%
6%
17%
4%




AD-12085
13%
4%
12%
4%




AD-12086
26%
5%
17%
3%




AD-12087
95%
4%
80%
4%




AD-12088
29%
6%
29%
2%




AD-12089
69%
5%
64%
7%




AD-12090
46%
15% 
34%
5%




AD-12091
16%
6%
17%
3%




AD-12092
82%
26% 
63%
5%




AD-12093
84%
4%
70%
4%




AD-12094
46%
3%
34%
1%




AD-12095
14%
2%
13%
1%




AD-12096
26%
11% 
17%
1%




AD-12097
23%
2%
21%
1%




AD-12098
41%
14% 
17%
3%




AD-12099
57%
2%
48%
6%




AD-12100
101% 
11% 
98%
8%




AD-12101
46%
7%
32%
2%




AD-12102
96%
17% 
88%
18% 




AD-12103
19%
5%
20%
2%




AD-12104
40%
8%
24%
2%




AD-12105
39%
2%
36%
10% 




AD-12106
87%
6%
79%
19% 




AD-12107
29%
2%
32%
16% 




AD-12108
38%
4%
39%
8%




AD-12109
49%
3%
44%
10% 




AD-12110
85%
5%
80%
14% 




AD-12111
64%
6%
71%
18% 




AD-12112
48%
4%
41%
5%




AD-12113
13%
0%
14%
3%




AD-12114
32%
6%
16%
4%




AD-12115
8%
4%
 7%
5%




AD-12116
74%
5%
61%
7%




AD-12117
21%
4%
20%
2%




AD-12118
44%
4%
42%
6%




AD-12119
37%
4%
24%
3%




AD-12120
22%
2%
15%
4%




AD-12121
32%
1%
22%
2%




AD-12122
36%
16% 
19%
5%




AD-12123
28%
1%
16%





AD-12124
28%
2%
16%





AD-12125
15%
1%
14%





AD-12126
51%
22% 
27%





AD-12127
54%
4%
42%
9%




AD-12128
29%
1%
20%
2%




AD-12129
22%
3%
19%
3%




AD-12130
53%
6%
42%
7%




AD-12131
28%
5%
22%
3%




AD-12132
88%
2%
90%
18% 




AD-12133
34%
2%
26%
6%




AD-12134
18%
3%
14%
2%




AD-12135
50%
6%
37%
4%




AD-12136
42%
19% 
22%
2%




AD-12137
85%
12% 
92%
4%




AD-12138
47%
6%
49%
1%




AD-12139
80%
5%
72%
4%




AD-12140
97%
22% 
67%
9%




AD-12141
120% 
4%
107% 
10% 




AD-12142
55%
8%
33%
4%




AD-12143
64%
34% 
19%
2%




AD-12144
58%
29% 
17%
2%




AD-12145
27%
8%
18%
2%




AD-12146
19%
20% 
15%
1%




AD-12147
29%
9%
35%
3%




AD-12148
30%
3%
56%
5%




AD-12149
8%
2%
12%
3%




AD-12150
31%
2%
31%
7%




AD-12151
 9%
5%
14%
2%




AD-12152
 3%
3%
23%
3%




AD-12153
20%
6%
34%
4%




AD-12154
24%
7%
44%
3%




AD-12155
33%
6%
53%
11% 




AD-12156
35%
5%
40%
5%




AD-12157
 8%
3%
23%
4%




AD-12158
13%
2%
22%
5%




AD-12159
34%
6%
46%
5%




AD-12160
19%
3%
31%
4%




AD-12161
88%
4%
83%
7%




AD-12162
26%
7%
32%
7%




AD-12163
55%
9%
40%
3%




AD-12164


21%
3%




AD-12165
30%
3%
41%
4%




AD-12166
 9%
10% 
22%
9%




AD-12167
26%
3%
30%
2%




AD-12168
54%
4%
59%
20%




AD-12169
41%
4%
51%
16%




AD-12170
43%
4%
52%
20%




AD-12171
67%
3%
73%
25%




AD-12172
53%
15% 
37%
2%




AD-12173
39%
0%
39%
0%




AD-12174
41%
5%
27%
0%




AD-12175
29%
0%
38%
14% 




AD-12176
43%
2%
56%
25% 




AD-12177
68%
6%
74%
30% 




AD-12178
41%
4%
41%
6%




AD-12179
53%
5%
44%
5%




AD-12180
16%
2%
13%
4%




AD-12181
19%
3%
14%
2%




AD-12182
16%
4%
18%
8%




AD-12183
26%
3%
19%
4%




AD-12184
54%
2%
77%
8%




AD-12185
8%
1%
 9%
1%




AD-12186
36%
3%
41%
6%




AD-12187
34%
17% 
27%
1%




AD-12188
30%
3%
27%
4%




AD-12189
51%
4%
48%
5%




AD-12190
33%
2%
26%
4%




AD-12191
20%
2%
13%
0%




AD-12192
21%
1%
23%
10% 




AD-12193
64%
8%
98%
6%




AD-12194
 8%
2%
15%
4%




AD-12195
34%
2%
48%
3%




AD-12196
34%
2%
51%
3%




AD-12197
75%
4%
93%
6%




AD-12198
55%
5%
48%
2%




AD-12199
102%
6%
118% 
9%




AD-12200
75%
6%
60%
12% 




AD-12201
42%
3%
16%
4%




AD-12202
29%
4%
 9%
3%




AD-12203
114% 
14% 
89%
20% 




AD-12204
64%
7%
26%
5%




AD-12205
66%
12% 
35%
4%




AD-12206
46%
3%
32%
12% 




AD-12207
57%
5%
40%
6%




AD-12208
30%
8%
10%
5%




AD-12209
101% 
6%
102% 
23% 




AD-12210
38%
11%
27%
14% 




AD-12211
16%
6%
10%
5%




AD-12212
59%
8%
65%
5%




AD-12213
24%
9%
12%
2%




AD-12214
67%
14% 
70%
12% 




AD-12215
29%
13% 
13%
4%




AD-12216
36%
4%
13%
1%




AD-12217
36%
9%
11%
2%




AD-12218
35%
5%
17%
3%




AD-12219
41%
9%
14%
1%




AD-12220
37%
5%
23%
3%




AD-12221
58%
7%
39%
6%




AD-12222
74%
9%
53%
3%




AD-12223
74%
10%
67%
7%




AD-12224
24%
2%
11%
2%




AD-12225
75%
5%
76%
14% 




AD-12226
45%
8%
40%
3%




AD-12227
61%
6%
47%
5%




AD-12228
28%
3%
25%
5%




AD-12229
54%
13% 
37%
6%




AD-12230
70%
17% 
65%
4%




AD-12231
32%
12% 
22%
6%




AD-12232
30%
3%
17%
2%




AD-12233
38%
2%
32%
3%




AD-12234
90%
5%
95%
7%




AD-12235
57%
7%
46%
3%




AD-12236
34%
8%
16%
2%




AD-12237
42%
9%
32%
8%




AD-12238
42%
6%
34%
6%




AD-12239
42%
3%
40%
4%




AD-12240
47%
6%
36%
5%




AD-12241
69%
5%
70%
8%




AD-12242
61%
2%
47%
3%




AD-12243
26%
7%
15%
1%




AD-12244
25%
6%
15%
1%




AD-12245
65%
6%
83%
13% 




AD-12246
29%
7%
31%
6%




AD-12247
57%
13% 
50%
3%




AD-12248
36%
8%
20%
3%
15%
 7%


AD-12249
44%
3%
70%
11% 
103%
34%


AD-12250
47%
5%
18%
5%
17%
 4%


AD-12251
121% 
28% 
35%
8%
60%
42%


AD-12252
94%
19% 
 8%
3%
 5%
 3%


AD-12253
94%
33% 
42%
8%
49%
27%


AD-12254
101% 
58% 
70%
5%
80%
32%


AD-12255
163% 
27% 
28%
6%
36%
10%


AD-12256
112% 
62% 
18%
3%
 9%
 4%


AD-12257
10%
4%
 9%
2%
 6%
 2%


AD-12258
27%
9%
18%
3%
20%
 6%


AD-12259
20%
5%
12%
2%
13%
 5%


AD-12260
22%
7%
81%
7%
65%
13%


AD-12261
122% 
11% 
66%
7%
80%
22%


AD-12262
97%
30% 
33%
6%
44%
18%


AD-12263
177% 
57% 
85%
11% 
84%
15%


AD-12264
37%
6%
10%
1%
10%
 4%


AD-12265
40%
8%
17%
1%
20%
10%


AD-12266
33%
9%
 9%
1%
 8%
 4%


AD-12267
34%
13% 
11%
1%
 6%
 2%


AD-12268
34%
6%
11%
1%
 9%
 2%


AD-12269
54%
6%
33%
4%
29%
 7%


AD-12270
52%
5%
29%
4%
27%
 6%


AD-12271
53%
7%
27%
3%
19%
 6%


AD-12272
85%
15% 
57%
7%
51%
16%


AD-12273
36%
6%
26%
2%
30%
 5%


AD-12274
75%
21% 
40%
2%
50%
19%


AD-12275
29%
9%
 8%
1%
 8%
 4%


AD-12276
45%
19% 
15%
2%
16%
12%


AD-12277
58%
17% 
32%
2%
55%
14%


AD-12278
120% 
35% 
96%
10% 
124% 
38%


AD-12279
47%
29% 
17%
1%
12%
 4%


AD-12280
 2%
0%
 3%
1%




AD-12281
 2%
0%
 5%
2%




AD-12282
 3%
0%
25%
5%




AD-12283
 3%
1%
35%
4%




AD-12284
 5%
2%
49%
8%




AD-12285
 7%
7%
21%
26%




AD-12286
28%
34% 
12%
7%




AD-12287
40%
21% 
51%
23% 




AD-12288
26%
7%
155% 
146% 




AD-12289
43%
21% 
220% 
131% 




AD-12290
 2%
1%
81%
23% 




AD-12291
 4%
1%
70%
3%




AD-12292
 2%
1%
6%
2%




AD-12293
 4%
2%
36%
3%




AD-12294
10%
6%
38%
3%




AD-12295
29%
31% 
37%
3%




AD-12296
82%
4%
89%
2%




AD-12297
75%
3%
65%
2%




AD-12298
73%
4%
60%
3%




AD-12299
76%
4%
66%
4%




AD-12300
36%
4%
15%
1%




AD-12301
33%
4%
18%
2%




AD-12302
66%
5%
65%
3%




AD-12303
35%
6%
17%
2%




AD-12304
70%
8%
70%
6%




AD-12305
63%
8%
80%
7%




AD-12306
23%
6%
20%
3%




AD-12307
78%
10% 
58%
5%




AD-12308
27%
8%
15%
2%




AD-12309
58%
11% 
42%
3%




AD-12310
106% 
23% 
80%
2%




AD-12311
73%
12% 
60%
2%




AD-12312
39%
3%
36%
3%




AD-12313
64%
9%
49%
6%




AD-12314
28%
7%
14%
6%




AD-12315
31%
7%
13%
2%




AD-12316
42%
5%
14%
2%




AD-12317
34%
9%
15%
5%




AD-12318
46%
4%
28%
4%




AD-12319
77%
3%
56%
4%




AD-12320
55%
7%
41%
3%




AD-12321
21%
3%
10%
2%




AD-12322
27%
8%
30%
12% 




AD-12323
26%
7%
35%
18% 




AD-12324
27%
8%
27%
14% 




AD-12325
32%
12% 
32%
22% 




AD-12326
42%
22% 
45%
41% 




AD-12327
36%
14% 
37%
32% 




AD-12328
45%
2%
31%
3%




AD-12329
61%
4%
34%
3%




AD-12330
63%
5%
38%
4%




AD-12331
50%
2%
26%
5%




AD-12332
80%
4%
51%
7%




AD-12333
34%
6%
12%
2%




AD-12334
27%
2%
18%
3%




AD-12335
84%
6%
60%
7%




AD-12336
45%
4%
36%
4%




AD-12337
30%
7%
19%
2%
















TABLE 3







Sequences and analysis of Eg5/KSP dsRNA duplexes


















single








dose
SDs







screen
2nd







@ 25 nM
screen



SEQ

SEQ

[%
(among



ID
Antisense sequence
ID
duplex
residual
quadru-


Sense sequence (5′-3′)
NO.
(5′-3′)
NO.
name
mRNA]
plicates)





ccAuuAcuAcAGuAGcAcuTsT
 582
AGUGCuACUGuAGuAAUGGTsT
 583
AD-14085
 19%
 1%


 


AucuGGcAAccAuAuuucuTsT
 584
AGAAAuAUGGUUGCcAGAUTsT
 585
AD-14086
 38%
 1%


 


GAuAGcuAAAuuAAAccAATsT
 586
UUGGUUuAAUUuAGCuAUCTsT
 587
AD-14087
 75%
10%


 


AGAuAccAuuAcuAcAGuATsT
 588
uACUGuAGuAAUGGuAUCUTsT
 589
AD-14088
 22%
 8%


 


GAuuGuucAucAAuuGGcGTsT
 590
CGCcAAUUGAUGAAcAAUCTsT
 591
AD-14089
 70%
12%


 


GcuuucuccucGGcucAcuTsT
 592
AGuGAGCCGAGGAGAAAGCTsT
 593
AD-14090
 79%
11%


 


GGAGGAuuGGcuGAcAAGATsT
 594
UCUUGUcAGCcAAUCCUCCTsT
 595
AD-14091
 29%
 3%


 


uAAuGAAGAGuAuAccuGGTsT
 596
CcAGGuAuACUCUUcAUuATsT
 597
AD-14092
 23%
 2%


 


uuucAccAAAccAuuuGuATsT
 598
uAcAAAUGGUUUGGUGAAATsT
 599
AD-14093
 60%
 2%


 


cuuAuuAAGGAGuAuAcGGTsT
 600
CCGuAuACUCCUuAAuAAGTsT
 601
AD-14094
 11%
 3%


 


GAAAucAGAuGGAcGuAAGTsT
 602
CUuACGUCcAUCUGAUUUCTsT
 603
AD-14095
 10%
 2%


 


cAGAuGucAGcAuAAGcGATsT
 604
UCGCUuAUGCUGAcAUCUGTsT
 605
AD-14096
 27%
 2%


 


AucuAAcccuAGuuGuAucTsT
 606
GAuAcAACuAGGGUuAGAUTsT
 607
AD-14097
 45%
 6%


 


AAGAGcuuGuuAAAAucGGTsT
 608
CCGAUUUuAAcAAGCUCUUTsT
 609
AD-14098
 50%
10%


 


uuAAGGAGuAuAcGGAGGATsT
 610
UCCUCCGuAuACUCCUuAATsT
 611
AD-14099
 12%
 4%


 


uuGcAAuGuAAAuAcGuAuTsT
 612
AuACGuAUUuAcAUUGcAATsT
 613
AD-14100
 49%
 7%


 


ucuAAcccuAGuuGuAuccTsT
 614
GGAuAcAACuAGGGUuAGATsT
 615
AD-14101
 36%
 1%


 


cAuGuAucuuuuucucGAuTsT
 616
AUCGAGAAAAAGAuAcAUGTsT
 617
AD-14102
 49%
 3%


 


GAuGucAGcAuAAGcGAuGTsT
 618
cAUCGCUuAUGCUGAcAUCTsT
 619
AD-14103
 74%
 5%


 


ucccAAcAGGuAcGAcAccTsT
 620
GGUGUCGuACCUGUUGGGATsT
 621
AD-14104
 27%
 3%


 


uGcucAcGAuGAGuuuAGuTsT
 622
ACuAAACUcAUCGUGAGcATsT
 623
AD-14105
 34%
 4%


 


AGAGcuuGuuAAAAucGGATsT
 624
UCCGAUUUuAAcAAGCUCUTsT
 625
AD-14106
  9%
 2%


 


GcGuAcAAGAAcAucuAuATsT
 626
uAuAGAUGUUCUUGuACGCTsT
 627
AD-14107
  5%
 1%


 


GAGGuuGuAAGccAAuGuuTsT
 628
AAcAUUGGCUuAcAACCUCTsT
 629
AD-14108
 15%
 1%


 


AAcAGGuAcGAcAccAcAGTsT
 630
CUGUGGUGUCGuACCUGUUTsT
 631
AD-14109
 91%
 2%


 


AAcccuAGuuGuAucccucTsT
 632
GAGGGAuAcAACuAGGGUUTsT
 633
AD-14110
 66%
 5%


 


GcAuAAGcGAuGGAuAAuATsT
 634
uAUuAUCcAUCGCUuAUGCTsT
 635
AD-14111
 33%
 3%


 


AAGcGAuGGAuAAuAccuATsT
 636
uAGGuAUuAUCcAUCGCUUTsT
 637
AD-14112
 51%
 3%


 


uGAuccuGuAcGAAAAGAATsT
 638
UUCUUUUCGuAcAGGAUcATsT
 639
AD-14113
 22%
 3%


 


AAAAcAuuGGccGuucuGGTsT
 640
CcAGAACGGCcAAUGUUUUTsT
 641
AD-14114
117%
 8%


 


cuuGGAGGGcGuAcAAGAATsT
 642
UUCUUGuACGCCCUCcAAGTsT
 643
AD-14115
 50%
 8%


 


GGcGuAcAAGAAcAucuAuTsT
 644
AuAGAUGUUCUUGuACGCCTsT
 645
AD-14116
 14%
 3%


 


AcucuGAGuAcAuuGGAAuTsT
 646
AUUCcAAUGuACUcAGAGUTsT
 647
AD-14117
 12%
 4%


 


uuAuuAAGGAGuAuAcGGATsT
 648
UCCGuAuACUCCUuAAuAATsT
 649
AD-14118
 26%
 4%


 


uAAGGAGuAuAcGGAGGAGTsT
 650
CUCCUCCGuAuACUCCUuATsT
 651
AD-14119
 24%
 5%


 


AAAucAAuAGucAAcuAAATsT
 652
UUuAGUUGACuAUUGAUUUTsT
 653
AD-14120
  8%
 1%


 


AAucAAuAGucAAcuAAAGTsT
 654
CUUuAGUUGACuAUUGAUUTsT
 655
AD-14121
 24%
 2%


 


uucucAGuAuAcuGuGuAATsT
 656
UuAcAcAGuAuACUGAGAATsT
 657
AD-14122
 10%
 1%


 


uGuGAAAcAcucuGAuAAATsT
 658
UUuAUcAGAGUGUUUcAcATsT
 659
AD-14123
  8%
 1%


 


AGAuGuGAAucucuGAAcATsT
 660
UGUUcAGAGAUUcAcAUCUTsT
 661
AD-14124
  9%
 2%


 


AGGuuGuAAGccAAuGuuGTsT
 662
cAAcAUUGGCUuAcAACCUTsT
 663
AD-14125
114%
 6%


 


uGAGAAAucAGAuGGAcGuTsT
 664
ACGUCcAUCUGAUUUCUcATsT
 665
AD-14126
  9%
 1%


 


AGAAAucAGAuGGAcGuAATsT
 666
UuACGUCcAUCUGAUUUCUTsT
 667
AD-14127
 57%
 6%


 


AuAucccAAcAGGuAcGAcTsT
 668
GUCGuACCUGUUGGGAuAUTsT
 669
AD-14128
104%
 6%


 


cccAAcAGGuAcGAcAccATsT
 670
UGGUGUCGuACCUGUUGGGTsT
 671
AD-14129
 21%
 2%


 


AGuAuAcuGAAGAAccucuTsT
 672
AGAGGUUCUUcAGuAuACUTsT
 673
AD-14130
 57%
 6%


 


AuAuAuAucAGccGGGcGcTsT
 674
GCGCCCGGCUGAuAuAuAUTsT
 675
AD-14131
 93%
 6%


 


AAucuAAcccuAGuuGuAuTsT
 676
AuAcAACuAGGGUuAGAUUTsT
 677
AD-14132
 75%
 8%


 


cuAAcccuAGuuGuAucccTsT
 678
GGGAuAcAACuAGGGUuAGTsT
 679
AD-14133
 66%
 4%


 


cuAGuuGuAucccuccuuuTsT
 680
AAAGGAGGGAuAcAACuAGTsT
 681
AD-14134
 44%
 6%


 


AGAcAucuGAcuAAuGGcuTsT
 682
AGCcAUuAGUcAGAUGUCUTsT
 683
AD-14135
 55%
 6%


 


GAAGcucAcAAuGAuuuAATsT
 684
UuAAAUcAUUGUGAGCUUCTsT
 685
AD-14136
 29%
 3%


 


AcAuGuAucuuuuucucGATsT
 686
UCGAGAAAAAGAuAcAUGUTsT
 687
AD-14137
 40%
 3%


 


ucGAuucAAAucuuAAcccTsT
 688
GGGUuAAGAUUUGAAUCGATsT
 689
AD-14138
 39%
 5%


 


ucuuAAcccuuAGGAcucuTsT
 690
AGAGUCCuAAGGGUuAAGATsT
 691
AD-14139
 71%
11%


 


GcucAcGAuGAGuuuAGuGTsT
 692
cACuAAACUcAUCGUGAGCTsT
 693
AD-14140
 43%
15%


 


cAuAAGcGAuGGAuAAuAcTsT
 694
GuAUuAUCcAUCGCUuAUGTsT
 695
AD-14141
 33%
 6%


 


AuAAGcGAuGGAuAAuAccTsT
 696
GGuAUuAUCcAUCGCUuAUTsT
 697
AD-14142
 51%
14%


 


ccuAAuAAAcuGcccucAGTsT
 698
CUGAGGGcAGUUuAUuAGGTsT
 699
AD-14143
 42%
 1%


 


ucGGAAAGuuGAAcuuGGuTsT
 700
ACcAAGUUcAACUUUCCGATsT
 701
AD-14144
  4%
 4%


 


GAAAAcAuuGGccGuucuGTsT
 702
cAGAACGGCcAAUGUUUUCTsT
 703
AD-14145
 92%
 5%


 


AAGAcuGAucuucuAAGuuTsT
 704
AACUuAGAAGAUcAGUCUUTsT
 705
AD-14146
 13%
 2%


 


GAGcuuGuuAAAAucGGAATsT
 706
UUCCGAUUUuAAcAAGCUCTsT
 707
AD-14147
  8%
 1%


 


AcAuuGGccGuucuGGAGcTsT
 708
GCUCcAGAACGGCcAAUGUTsT
 709
AD-14148
 80%
 7%


 


AAGAAcAucuAuAAuuGcATsT
 710
UGcAAUuAuAGAUGUUCUUTsT
 711
AD-14149
 44%
 7%


 


AAAuGuGucuAcucAuGuuTsT
 712
AAcAUGAGuAGAcAcAUUUTsT
 713
AD-14150
 32%
29%


 


uGucuAcucAuGuuucucATsT
 714
UGAGAAAcAUGAGuAGAcATsT
 715
AD-14151
 75%
11%


 


GuAuAcuGuGuAAcAAucuTsT
 716
AGAUUGUuAcAcAGuAuACTsT
 717
AD-14152
  8%
 5%


 


uAuAcuGuGuAAcAAucuATsT
 718
uAGAUUGUuAcAcAGuAuATsT
 719
AD-14153
 17%
11%


 


cuuAGuAGuGuccAGGAAATsT
 720
UUUCCUGGAcACuACuAAGTsT
 721
AD-14154
 16%
 4%


 


ucAGAuGGAcGuAAGGcAGTsT
 722
CUGCCUuACGUCcAUCUGATsT
 723
AD-14155
 11%
 1%


 


AGAuAAAuuGAuAGcAcAATsT
 724
UUGUGCuAUcAAUUuAUCUTsT
 725
AD-14156
 10%
 1%


 


cAAcAGGuAcGAcAccAcATsT
 726
UGUGGUGUCGuACCUGUUGTsT
 727
AD-14157
 29%
 3%


 


uGcAAuGuAAAuAcGuAuuTsT
 728
AAuACGuAUUuAcAUUGcATsT
 729
AD-14158
 51%
 3%


 


AGucAGAAuuuuAucuAGATsT
 730
UCuAGAuAAAAUUCUGACUTsT
 731
AD-14159
 53%
 5%


 


cuAGAAAucuuuuAAcAccTsT
 732
GGUGUuAAAAGAUUUCuAGTsT
 733
AD-14160
 40%
 3%


 


AAuAAAucuAAcccuAGuuTsT
 734
AACuAGGGUuAGAUUuAUUTsT
 735
AD-14161
 83%
 7%


 


AAuuuucuGcucAcGAuGATsT
 736
UcAUCGUGAGcAGAAAAUUTsT
 737
AD-14162
 44%
 6%


 


GcccucAGuAAAuccAuGGTsT
 738
CcAUGGAUUuACUGAGGGCTsT
 739
AD-14163
 57%
 3%


 


AcGuuuAAAAcGAGAucuuTsT
 740
AAGAUCUCGUUUuAAACGUTsT
 741
AD-14164
  4%
 1%


 


AGGAGAuAGAAcGuuuAAATsT
 742
UUuAAACGUUCuAUCUCCUTsT
 743
AD-14165
 11%
 1%


 


GAccGucAuGGcGucGcAGTsT
 744
CUGCGACGCcAUGACGGUCTsT
 745
AD-14166
 90%
 5%


 


AccGucAuGGcGucGcAGcTsT
 746
GCUGCGACGCcAUGACGGUTsT
 747
AD-14167
 49%
 1%


 


GAAcGuuuAAAAcGAGAucTsT
 748
GAUCUCGUUUuAAACGUUCTsT
 749
AD-14168
 12%
 2%


 


uuGAGcuuAAcAuAGGuAATsT
 750
UuACCuAUGUuAAGCUcAATsT
 751
AD-14169
 66%
 4%


 


AcuAAAuuGAucucGuAGATsT
 752
UCuACGAGAUcAAUUuAGUTsT
 753
AD-14170
 52%
 6%


 


ucGuAGAAuuAucuuAAuATsT
 754
uAUuAAGAuAAUUCuACGATsT
 755
AD-14171
 42%
 4%


 


GGAGAuAGAAcGuuuAAAATsT
 756
UUUuAAACGUUCuAUCUCCTsT
 757
AD-14172
  3%
 1%


 


AcAAcuuAuuGGAGGuuGuTsT
 758
AcAACCUCcAAuAAGUUGUTsT
 759
AD-14173
 29%
 2%


 


uGAGcuuAAcAuAGGuAAATsT
 760
UUuACCuAUGUuAAGCUcATsT
 761
AD-14174
 69%
 2%


 


AucucGuAGAAuuAucuuATsT
 762
uAAGAuAAUUCuACGAGAUTsT
 763
AD-14175
 53%
 3%


 


cuGcGuGcAGucGGuccucTsT
 764
GAGGACCGACUGcACGcAGTsT
 765
AD-14176
111%
 4%


 


cAcGcAGcGcccGAGAGuATsT
 766
uACUCUCGGGCGCUGCGUGTsT
 767
AD-14177
 87%
 6%


 


AGuAccAGGGAGAcuccGGTsT
 768
CCGGAGUCUCCCUGGuACUTsT
 769
AD-14178
 59%
 2%


 


AcGGAGGAGAuAGAAcGuuTsT
 770
AACGUUCuAUCUCCUCCGUTsT
 771
AD-14179
  9%
 2%


 


AGAAcGuuuAAAAcGAGAuTsT
 772
AUCUCGUUUuAAACGUUCUTsT
 773
AD-14180
 43%
 2%


 


AAcGuuuAAAAcGAGAucuTsT
 774
AGAUCUCGUUUuAAACGUUTsT
 775
AD-14181
 70%
10%


 


AGcuuGAGcuuAAcAuAGGTsT
 776
CCuAUGUuAAGCUcAAGCUTsT
 777
AD-14182
100%
 7%


 


AGcuuAAcAuAGGuAAAuATsT
 778
uAUUuACCuAUGUuAAGCUTsT
 779
AD-14183
 60%
 5%


 


uAGAGcuAcAAAAccuAucTsT
 780
GAuAGGUUUUGuAGCUCuATsT
 781
AD-14184
129%
 6%


 


uAGuuGuAucccuccuuuATsT
 782
uAAAGGAGGGAuAcAACuATsT
 783
AD-14185
 62%
 4%


 


AccAcccAGAcAucuGAcuTsT
 784
AGUcAGAUGUCUGGGUGGUTsT
 785
AD-14186
 42%
 3%


 


AGAAAcuAAAuuGAucucGTsT
 786
CGAGAUcAAUUuAGUUUCUTsT
 787
AD-14187
123%
12%


 


ucucGuAGAAuuAucuuAATsT
 788
UuAAGAuAAUUCuACGAGATsT
 789
AD-14188
 38%
 2%


 


cAAcuuAuuGGAGGuuGuATsT
 790
uAcAACCUCcAAuAAGUUGTsT
 791
AD-14189
 13%
 1%


 


uuGuAucccuccuuuAAGuTsT
 792
ACUuAAAGGAGGGAuAcAATsT
 793
AD-14190
 59%
 3%


 


ucAcAAcuuAuuGGAGGuuTsT
 794
AACCUCcAAuAAGUUGUGATsT
 795
AD-14191
 93%
 3%


 


AGAAcuGuAcucuucucAGTsT
 796
CUGAGAAGAGuAcAGUUCUTsT
 797
AD-14192
 45%
 5%


 


GAGcuuAAcAuAGGuAAAuTsT
 798
AUUuACCuAUGUuAAGCUCTsT
 799
AD-14193
 57%
 3%


 


cAccAAcAucuGuccuuAGTsT
 800
CuAAGGAcAGAUGUUGGUGTsT
 801
AD-14194
 51%
 4%


 


AAAGcccAcuuuAGAGuAuTsT
 802
AuACUCuAAAGUGGGCUUUTsT
 803
AD-14195
 77%
 5%


 


AAGcccAcuuuAGAGuAuATsT
 804
uAuACUCuAAAGUGGGCUUTsT
 805
AD-14196
 42%
 6%


 


GAccuuAuuuGGuAAucuGTsT
 806
cAGAUuACcAAAuAAGGUCTsT
 807
AD-14197
 15%
 2%


 


GAuuAAuGuAcucAAGAcuTsT
 808
AGUCUUGAGuAcAUuAAUCTsT
 809
AD-14198
 12%
 2%


 


cuuuAAGAGGccuAAcucATsT
 810
UGAGUuAGGCCUCUuAAAGTsT
 811
AD-14199
 18%
 2%


 


uuAAAccAAAcccuAuuGATsT
 812
UcAAuAGGGUUUGGUUuAATsT
 813
AD-14200
 72%
 9%


 


ucuGuuGGAGAucuAuAAuTsT
 814
AUuAuAGAUCUCcAAcAGATsT
 815
AD-14201
  9%
 3%


 


cuGAuGuuucuGAGAGAcuTsT
 816
AGUCUCUcAGAAAcAUcAGTsT
 817
AD-14202
 25%
 3%


 


GcAuAcucuAGucGuucccTsT
 818
GGGAACGACuAGAGuAUGCTsT
 819
AD-14203
 21%
 1%


 


GuuccuuAucGAGAAucuATsT
 820
uAGAUUCUCGAuAAGGAACTsT
 821
AD-14204
  4%
 2%


 


GcAcuuGGAucucucAcAuTsT
 822
AUGUGAGAGAUCcAAGUGCTsT
 823
AD-14205
  5%
 1%


 


AAAAAAGGAAcuAGAuGGcTsT
 824
GCcAUCuAGUUCCUUUUUUTsT
 825
AD-14206
 79%
 6%


 


AGAGcAGAuuAccucuGcGTsT
 826
CGcAGAGGuAAUCUGCUCUTsT
 827
AD-14207
 55%
 2%


 


AGcAGAuuAccucuGcGAGTsT
 828
CUCGcAGAGGuAAUCUGCUTsT
 829
AD-14208
100%
 4%


 


cccuGAcAGAGuucAcAAATsT
 830
UUUGUGAACUCUGUcAGGGTsT
 831
AD-14209
 34%
 3%


 


GuuuAccGAAGuGuuGuuuTsT
 832
AAAcAAcACUUCGGuAAACTsT
 833
AD-14210
 13%
 2%


 


uuAcAGuAcAcAAcAAGGATsT
 834
UCCUUGUUGUGuACUGuAATsT
 835
AD-14211
  9%
 1%


 


AcuGGAucGuAAGAAGGcATsT
 836
UGCCUUCUuACGAUCcAGUTsT
 837
AD-14212
 20%
 3%


 


GAGcAGAuuAccucuGcGATsT
 838
UCGcAGAGGuAAUCUGCUCTsT
 839
AD-14213
 48%
 5%


 


AAAAGAAGuuAGuGuAcGATsT
 840
UCGuAcACuAACUUCUUUUTsT
 841
AD-14214
 28%
18%


 


GAccAuuuAAuuuGGcAGATsT
 842
UCUGCcAAAUuAAAUGGUCTsT
 843
AD-14215
132%
 0%


 


GAGAGGAGuGAuAAuuAAATsT
 844
UUuAAUuAUcACUCCUCUCTsT
 845
AD-14216
  3%
 0%


 


cuGGAGGAuuGGcuGAcAATsT
 846
UUGUcAGCcAAUCCUCcAGTsT
 847
AD-14217
 19%
 1%


 


cucuAGucGuucccAcucATsT
 848
UGAGUGGGAACGACuAGAGTsT
 849
AD-14218
 67%
 8%


 


GAuAccAuuAcuAcAGuAGTsT
 850
CuACUGuAGuAAUGGuAUCTsT
 851
AD-14219
 76%
 4%


 


uucGucuGcGAAGAAGAAATsT
 852
UUUCUUCUUCGcAGACGAATsT
 853
AD-14220
 33%
 8%


 


GAAAAGAAGuuAGuGuAcGTsT
 854
CGuAcACuAACUUCUUUUCTsT
 855
AD-14221
 25%
 2%


 


uGAuGuuuAccGAAGuGuuTsT
 856
AAcACUUCGGuAAAcAUcATsT
 857
AD-14222
  7%
 2%


 


uGuuuGuccAAuucuGGAuTsT
 858
AUCcAGAAUUGGAcAAAcATsT
 859
AD-14223
 19%
 2%


 


AuGAAGAGuAuAccuGGGATsT
 860
UCCcAGGuAuACUCUUcAUTsT
 861
AD-14224
 13%
 1%


 


GcuAcucuGAuGAAuGcAuTsT
 862
AUGcAUUcAUcAGAGuAGCTsT
 863
AD-14225
 15%
 2%


 


GcccuuGuAGAAAGAAcAcTsT
 864
GUGUUCUUUCuAcAAGGGCTsT
 865
AD-14226
 11%
 0%


 


ucAuGuuccuuAucGAGAATsT
 866
UUCUCGAuAAGGAAcAUGATsT
 867
AD-14227
  5%
 1%


 


GAAuAGGGuuAcAGAGuuGTsT
 868
cAACUCUGuAACCCuAUUCTsT
 869
AD-14228
 34%
 3%


 


cAAAcuGGAucGuAAGAAGTsT
 870
CUUCUuACGAUCcAGUUUGTsT
 871
AD-14229
 15%
 2%


 


cuuAuuuGGuAAucuGcuGTsT
 872
cAGcAGAUuACcAAAuAAGTsT
 873
AD-14230
 20%
 1%


 


AGcAAuGuGGAAAccuAAcTsT
 874
GUuAGGUUUCcAcAUUGCUTsT
 875
AD-14231
 18%
 1%


 


AcAAuAAAGcAGAcccAuuTsT
 876
AAUGGGUCUGCUUuAUUGUTsT
 877
AD-14232
 21%
 1%


 


AAccAcuuAGuAGuGuccATsT
 878
UGGAcACuACuAAGUGGUUTsT
 879
AD-14233
106%
12%


 


AGucAAGAGccAucuGuAGTsT
 880
CuAcAGAUGGCUCUUGACUTsT
 881
AD-14234
 35%
 3%


 


cucccuAGAcuucccuAuuTsT
 882
AAuAGGGAAGUCuAGGGAGTsT
 883
AD-14235
 48%
 4%


 


AuAGcuAAAuuAAAccAAATsT
 884
UUUGGUUuAAUUuAGCuAUTsT
 885
AD-14236
 23%
 3%


 


uGGcuGGuAuAAuuccAcGTsT
 886
CGUGGAAUuAuACcAGCcATsT
 887
AD-14237
 79%
 9%


 


uuAuuuGGuAAucuGcuGuTsT
 888
AcAGcAGAUuACcAAAuAATsT
 889
AD-14238
 92%
 7%


 


AAcuAGAuGGcuuucucAGTsT
 890
CUGAGAAAGCcAUCuAGUUTsT
 891
AD-14239
 20%
 2%


 


ucAuGGcGucGcAGccAAATsT
 892
UUUGGCUGCGACGCcAUGATsT
 893
AD-14240
 71%
 6%


 


AcuGGAGGAuuGGcuGAcATsT
 894
UGUcAGCcAAUCCUCcAGUTsT
 895
AD-14241
 14%
 1%


 


cuAuAAuuGcAcuAucuuuTsT
 896
AAAGAuAGUGcAAUuAuAGTsT
 897
AD-14242
 11%
 2%


 


AAAGGucAccuAAuGAAGATsT
 898
UCUUcAUuAGGUGACCUUUTsT
 899
AD-14243
 11%
 1%


 


AuGAAuGcAuAcucuAGucTsT
 900
GACuAGAGuAUGcAUUcAUTsT
 901
AD-14244
 15%
 2%


 


AAcAuAuuGAAuAAGccuGTsT
 902
cAGGCUuAUUcAAuAUGUUTsT
 903
AD-14245
 50%
 7%


 


AAGAAGGcAGuuGAccAAcTsT
 904
GUUGGUcAACUGCCUUCUUTsT
 905
AD-14246
 57%
 5%


 


GAuAcuAAAAGAAcAAucATsT
 906
UGAUUGUUCUUUuAGuAUCTsT
 907
AD-14247
  9%
 3%


 


AuAcuGAAAAucAAuAGucTsT
 908
GACuAUUGAUUUUcAGuAUTsT
 909
AD-14248
 39%
 4%


 


AAAAAGGAAcuAGAuGGcuTsT
 910
AGCcAUCuAGUUCCUUUUUTsT
 911
AD-14249
 64%
 2%


 


GAAcuAGAuGGcuuucucATsT
 912
UGAGAAAGCcAUCuAGUUCTsT
 913
AD-14250
 18%
 2%


 


GAAAccuAAcuGAAGAccuTsT
 914
AGGUCUUcAGUuAGGUUUCTsT
 915
AD-14251
 56%
 6%


 


uAcccAucAAcAcuGGuAATsT
 916
UuACcAGUGUUGAUGGGuATsT
 917
AD-14252
 48%
 6%


 


AuuuuGAuAucuAcccAuuTsT
 918
AAUGGGuAGAuAUcAAAAUTsT
 919
AD-14253
 39%
 5%


 


AucccuAuAGuucAcuuuGTsT
 920
cAAAGUGAACuAuAGGGAUTsT
 921
AD-14254
 44%
 8%


 


AuGGGcuAuAAuuGcAcuATsT
 922
uAGUGcAAUuAuAGCCcAUTsT
 923
AD-14255
108%
 8%


 


AGAuuAccucuGcGAGcccTsT
 924
GGGCUCGcAGAGGuAAUCUTsT
 925
AD-14256
108%
 6%


 


uAAuuccAcGuAcccuucATsT
 926
UGAAGGGuACGUGGAAUuATsT
 927
AD-14257
 23%
 2%


 


GucGuucccAcucAGuuuuTsT
 928
AAAACuGAGuGGGAACGACTsT
 929
AD-14258
 21%
 3%


 


AAAucAAucccuGuuGAcuTsT
 930
AGUcAAcAGGGAUUGAUUUTsT
 931
AD-14259
 19%
 2%


 


ucAuAGAGcAAAGAAcAuATsT
 932
uAUGUUCUUUGCUCuAUGATsT
 933
AD-14260
 10%
 1%


 


uuAcuAcAGuAGcAcuuGGTsT
 934
CcAAGUGCuACUGuAGuAATsT
 935
AD-14261
 76%
 3%


 


AuGuGGAAAccuAAcuGAATsT
 936
UUcAGUuAGGUUUCcAcAUTsT
 937
AD-14262
 13%
 2%


 


uGuGGAAAccuAAcuGAAGTsT
 938
CUUcAGUuAGGUUUCcAcATsT
 939
AD-14263
 14%
 2%


 


ucuuccuuAAAuGAAAGGGTsT
 940
CCCUUUcAUUuAAGGAAGATsT
 941
AD-14264
 65%
 3%


 


uGAAGAAccucuAAGucAATsT
 942
UUGACUuAGAGGUUCUUcATsT
 943
AD-14265
 13%
 1%


 


AGAGGucuAAAGuGGAAGATsT
 944
UCUUCcACUUuAGACCUCUTsT
 945
AD-14266
 18%
 3%


 


AuAucuAcccAuuuuucuGTsT
 946
cAGAAAAAUGGGuAGAuAUTsT
 947
AD-14267
 50%
 9%


 


uAAGccuGAAGuGAAucAGTsT
 948
CUGAUUcACUUcAGGCUuATsT
 949
AD-14268
 13%
 3%


 


AGAuGcAGAccAuuuAAuuTsT
 950
AAUuAAAUGGUCUGcAUCUTsT
 951
AD-14269
 19%
 4%


 


AGuGuuGuuuGuccAAuucTsT
 952
GAAUUGGAcAAAcAAcACUTsT
 953
AD-14270
 11%
 2%


 


cuAuAAuGAAGAGcuuuuuTsT
 954
AAAAAGCUCUUcAUuAuAGTsT
 955
AD-14271
 11%
 1%


 


AGAGGAGuGAuAAuuAAAGTsT
 956
CUUuAAUuAUcACUCCUCUTsT
 957
AD-14272
  7%
 1%


 


uuucucuGuuAcAAuAcAuTsT
 958
AUGuAUUGuAAcAGAGAAATsT
 959
AD-14273
 14%
 2%


 


AAcAucuAuAAuuGcAAcATsT
 960
UGUUGcAAUuAuAGAUGUUTsT
 961
AD-14274
 73%
 4%


 


uGcuAGAAGuAcAuAAGAcTsT
 962
GUCUuAUGuACUUCuAGcATsT
 963
AD-14275
 10%
 1%


 


AAuGuAcucAAGAcuGAucTsT
 964
GAUcAGUCUUGAGuAcAUUTsT
 965
AD-14276
 89%
 2%


 


GuAcucAAGAcuGAucuucTsT
 966
GAAGAUcAGUCUUGAGuACTsT
 967
AD-14277
  7%
 1%


 


cAcucuGAuAAAcucAAuGTsT
 968
cAUUGAGUUuAUcAGAGUGTsT
 969
AD-14278
 12%
 1%


 


AAGAGcAGAuuAccucuGcTsT
 970
GcAGAGGuAAUCUGCUCUUTsT
 971
AD-14279
104%
 3%


 


ucuGcGAGcccAGAucAAcTsT
 972
GUUGAUCUGGGCUCGcAGATsT
 973
AD-14280
 21%
 2%


 


AAcuuGAGccuuGuGuAuATsT
 974
uAuAcAcAAGGCUcAAGUUTsT
 975
AD-14281
 43%
 3%


 


GAAuAuAuAuAucAGccGGTsT
 976
CCGGCUGAuAuAuAuAUUCTsT
 977
AD-14282
 45%
 6%


 


uGucAucccuAuAGuucAcTsT
 978
GUGAACuAuAGGGAUGAcATsT
 979
AD-14283
 35%
 5%


 


GAucuGGcAAccAuAuuucTsT
 980
GAAAuAUGGUUGCcAGAUCTsT
 981
AD-14284
 58%
 3%


 


uGGcAAccAuAuuucuGGATsT
 982
UCcAGAAAuAUGGUUGCcATsT
 983
AD-14285
 48%
 3%


 


GAuGuuuAccGAAGuGuuGTsT
 984
cAAcACUUCGGuAAAcAUCTsT
 985
AD-14286
 49%
 3%


 


uuccuuAucGAGAAucuAATsT
 986
UuAGAUUCUCGAuAAGGAATsT
 987
AD-14287
  6%
 1%


 


AGcuuAAuuGcuuucuGGATsT
 988
UCcAGAAAGcAAUuAAGCUTsT
 989
AD-14288
 50%
 2%


 


uuGcuAuuAuGGGAGAccATsT
 990
UGGUCUCCcAuAAuAGcAATsT
 991
AD-14289
 48%
 1%


 


GucAuGGcGucGcAGccAATsT
 992
UUGGCUGCGACGCcAUGACTsT
 993
AD-14290
112%
 7%


 


uAAuuGcAcuAucuuuGcGTsT
 994
CGcAAAGAuAGUGcAAUuATsT
 995
AD-14291
 77%
 2%


 


cuAucuuuGcGuAuGGccATsT
 996
UGGCcAuACGcAAAGAuAGTsT
 997
AD-14292
 80%
 6%


 


ucccuAuAGuucAcuuuGuTsT
 998
AcAAAGUGAACuAuAGGGATsT
 999
AD-14293
 58%
 2%


 


ucAAccuuuAAuucAcuuGTsT
1000
cAAGUGAAUuAAAGGUUGATsT
1001
AD-14294
 77%
 2%


 


GGcAAccAuAuuucuGGAATsT
1002
UUCcAGAAAuAUGGUUGCCTsT
1003
AD-14295
 62%
 2%


 


AuGuAcucAAGAcuGAucuTsT
1004
AGAUcAGUCUUGAGuAcAUTsT
1005
AD-14296
 59%
 4%


 


GcAGAccAuuuAAuuuGGcTsT
1006
GCcAAAUuAAAUGGUCUGCTsT
1007
AD-14297
 37%
 1%


 


ucuGAGAGAcuAcAGAuGuTsT
1008
AcAUCUGuAGUCUCUcAGATsT
1009
AD-14298
 21%
 1%


 


uGcucAuAGAGcAAAGAAcTsT
1010
GUUCUUUGCUCuAUGAGcATsT
1011
AD-14299
  6%
 1%


 


AcAuAAGAccuuAuuuGGuTsT
1012
ACcAAAuAAGGUCUuAUGUTsT
1013
AD-14300
 17%
 2%


 


uuuGuGcuGAuucuGAuGGTsT
1014
CcAUcAGAAUcAGcAcAAATsT
1015
AD-14301
 97%
 6%


 


ccAucAAcAcuGGuAAGAATsT
1016
UUCUuACcAGUGUUGAUGGTsT
1017
AD-14302
 13%
 1%


 


AGAcAAuuccGGAuGuGGATsT
1018
UCcAcAUCCGGAAUUGUCUTsT
1019
AD-14303
 13%
 3%


 


GAAcuuGAGccuuGuGuAuTsT
1020
AuAcAcAAGGCUcAAGUUCTsT
1021
AD-14304
 38%
 2%


 


uAAuuuGGcAGAGcGGAAATsT
1022
UUUCCGCUCUGCcAAAUuATsT
1023
AD-14305
 14%
 2%


 


uGGAuGAAGuuAuuAuGGGTsT
1024
CCcAuAAuAACUUcAUCcATsT
1025
AD-14306
 22%
 4%


 


AucuAcAuGAAcuAcAAGATsT
1026
UCUUGuAGUUcAUGuAGAUTsT
1027
AD-14307
 26%
 6%


 


GGuAuuuuuGAucuGGcAATsT
1028
UUGCcAGAUcAAAAAuACCTsT
1029
AD-14308
 62%
 8%


 


cuAAuGAAGAGuAuAccuGTsT
1030
cAGGuAuACUCUUcAUuAGTsT
1031
AD-14309
 52%
 5%


 


uuuGAGAAAcuuAcuGAuATsT
1032
uAUcAGuAAGUUUCUcAAATsT
1033
AD-14310
 32%
 3%


 


cGAuAAGAuAGAAGAucAATsT
1034
UUGAUCUUCuAUCUuAUCGTsT
1035
AD-14311
 23%
 2%


 


cuGGcAAccAuAuuucuGGTsT
1036
CcAGAAAuAUGGUUGCcAGTsT
1037
AD-14312
 49%
 6%


 


uAGAuAccAuuAcuAcAGuTsT
1038
ACUGuAGuAAUGGuAUCuATsT
1039
AD-14313
 69%
 4%


 


GuAuuAAAuuGGGuuucAuTsT
1040
AUGAAACCcAAUUuAAuACTsT
1041
AD-14314
 52%
 3%


 


AAGAccuuAuuuGGuAAucTsT
1042
GAUuACcAAAuAAGGUCUUTsT
1043
AD-14315
 66%
 4%


 


GcuGuuGAuAAGAGAGcucTsT
1044
GAGCUCUCUuAUcAAcAGCTsT
1045
AD-14316
 19%
 4%


 


uAcucAuGuuucucAGAuuTsT
1046
AAUCUGAGAAAcAUGAGuATsT
1047
AD-14317
 16%
 5%


 


cAGAuGGAcGuAAGGcAGcTsT
1048
GCUGCCUuACGUCcAUCUGTsT
1049
AD-14318
 52%
11%


 


uAucccAAcAGGuAcGAcATsT
1050
UGUCGuACCUGUUGGGAuATsT
1051
AD-14319
 28%
11%


 


cAuuGcuAuuAuGGGAGAcTsT
1052
GUCUCCcAuAAuAGcAAUGTsT
1053
AD-14320
 52%
10%


 


cccucAGuAAAuccAuGGuTsT
1054
ACcAUGGAUUuACUGAGGGTsT
1055
AD-14321
 53%
 6%


 


GGucAuuAcuGcccuuGuATsT
1056
uAcAAGGGcAGuAAUGACCTsT
1057
AD-14322
 20%
 2%


 


AAccAcucAAAAAcAuuuGTsT
1058
cAAAUGUUUUUGAGUGGUUTsT
1059
AD-14323
116%
 6%


 


uuuGcAAGuuAAuGAAucuTsT
1060
AGAUUcAUuAACUUGcAAATsT
1061
AD-14324
 14%
 2%


 


uuAuuuucAGuAGucAGAATsT
1062
UUCUGACuACUGAAAAuAATsT
1063
AD-14325
 50%
 2%


 


uuuucucGAuucAAAucuuTsT
1064
AAGAUUuGAAUCGAGAAAATsT
1065
AD-14326
 47%
 3%


 


GuAcGAAAAGAAGuuAGuGTsT
1066
cACuAACUUCUUUUCGuACTsT
1067
AD-14327
 18%
 2%


 


uuuAAAAcGAGAucuuGcuTsT
1068
AGcAAGAUCUCGUUUuAAATsT
1069
AD-14328
 19%
 1%


 


GAAuuGAuuAAuGuAcucATsT
1070
UGAGuAcAUuAAUcAAUUCTsT
1071
AD-14329
 94%
10%


 


GAuGGAcGuAAGGcAGcucTsT
1072
GAGCUGCCUuACGUCcAUCTsT
1073
AD-14330
 60%
 4%


 


cAucuGAcuAAuGGcucuGTsT
1074
cAGAGCcAUuAGUcAGAUGTsT
1075
AD-14331
 54%
 7%


 


GuGAuccuGuAcGAAAAGATsT
1076
UCUUUUCGuAcAGGAUcACTsT
1077
AD-14332
 22%
 4%


 


AGcucuuAuuAAGGAGuAuTsT
1078
AuACUCCUuAAuAAGAGCUTsT
1079
AD-14333
 70%
10%


 


GcucuuAuuAAGGAGuAuATsT
1080
uAuACUCCUuAAuAAGAGCTsT
1081
AD-14334
 18%
 3%


 


ucuuAuuAAGGAGuAuAcGTsT
1082
CGuAuACUCCUuAAuAAGATsT
1083
AD-14335
 38%
 6%


 


uAuuAAGGAGuAuAcGGAGTsT
1084
CUCCGuAuACUCCUuAAuATsT
1085
AD-14336
 16%
 3%


 


cuGcAGcccGuGAGAAAAATsT
1086
UUUUUCUcACGGGCUGcAGTsT
1087
AD-14337
 65%
 4%


 


ucAAGAcuGAucuucuAAGTsT
1088
CUuAGAAGAUcAGUCUUGATsT
1089
AD-14338
 18%
 0%


 


cuucuAAGuucAcuGGAAATsT
1090
UUUCcAGUGAACUuAGAAGTsT
1091
AD-14339
 20%
 4%


 


uGcAAGuuAAuGAAucuuuTsT
1092
AAAGAUUcAUuAACUUGcATsT
1093
AD-14340
 24%
 1%


 


AAucuAAGGAuAuAGucAATsT
1094
UUGACuAuAUCCUuAGAUUTsT
1095
AD-14341
 27%
 3%


 


AucucuGAAcAcAAGAAcATsT
1096
UGUUCUUGUGUUcAGAGAUTsT
1097
AD-14342
 13%
 1%


 


uucuGAAcAGuGGGuAucuTsT
1098
AGAuACCcACUGUUcAGAATsT
1099
AD-14343
 19%
 1%


 


AGuuAuuuAuAcccAucAATsT
1100
UUGAUGGGuAuAAAuAACUTsT
1101
AD-14344
 23%
 2%


 


AuGcuAAAcuGuucAGAAATsT
1102
UUUCUGAAcAGUUuAGcAUTsT
1103
AD-14345
 21%
 4%


 


cuAcAGAGcAcuuGGuuAcTsT
1104
GuAACcAAGUGCUCUGuAGTsT
1105
AD-14346
 18%
 2%


 


uAuAuAucAGccGGGcGcGTsT
1106
CGCGCCCGGCUGAuAuAuATsT
1107
AD-14347
 67%
 2%


 


AuGuAAAuAcGuAuuucuATsT
1108
uAGAAAuACGuAUUuAcAUTsT
1109
AD-14348
 39%
 3%


 


uuuuucucGAuucAAAucuTsT
1110
AGAUUuGAAUCGAGAAAAATsT
1111
AD-14349
 83%
 6%


 


AAucuuAAcccuuAGGAcuTsT
1112
AGUCCuAAGGGUuAAGAUUTsT
1113
AD-14350
 54%
 2%


 


ccuuAGGAcucuGGuAuuuTsT
1114
AAAuACcAGAGUCCuAAGGTsT
1115
AD-14351
 57%
 8%


 


AAuAAAcuGcccucAGuAATsT
1116
UuACUGAGGGcAGUUuAUUTsT
1117
AD-14352
 82%
 3%


 


GAuccuGuAcGAAAAGAAGTsT
1118
CUUCUUUUCGuAcAGGAUCTsT
1119
AD-14353
  2%
 1%


 


AAuGuGAuccuGuAcGAAATsT
1120
UUUCGuAcAGGAUcAcAUUTsT
1121
AD-14354
 18%
11%


 


GuGAAAAcAuuGGccGuucTsT
1122
GAACGGCcAAUGUUUUcACTsT
1123
AD-14355
  2%
 1%


 


cuuGAGGAAAcucuGAGuATsT
1124
uACUcAGAGUUUCCUcAAGTsT
1125
AD-14356
  8%
 2%


 


cGuuuAAAAcGAGAucuuGTsT
1126
cAAGAUCUCGUUUuAAACGTsT
1127
AD-14357
  6%
 3%


 


uuAAAAcGAGAucuuGcuGTsT
1128
cAGcAAGAUCUCGUUUuAATsT
1129
AD-14358
 98%
17%


 


AAAGAuGuAucuGGucuccTsT
1130
GGAGACcAGAuAcAUCUUUTsT
1131
AD-14359
 10%
 1%


 


cAGAAAAuGuGucuAcucATsT
1132
UGAGuAGAcAcAUUUUCUGTsT
1133
AD-14360
  6%
 4%


 


cAGGAAuuGAuuAAuGuAcTsT
1134
GuAcAUuAAUcAAUUCCUGTsT
1135
AD-14361
 30%
 5%


 


AGucAAcuAAAGcAuAuuuTsT
1136
AAAuAUGCUUuAGUUGACUTsT
1137
AD-14362
 28%
 2%


 


uGuGuAAcAAucuAcAuGATsT
1138
UcAUGuAGAUUGUuAcAcATsT
1139
AD-14363
 60%
 6%


 


AuAccAuuuGuuccuuGGuTsT
1140
ACcAAGGAAcAAAUGGuAUTsT
1141
AD-14364
 12%
 9%


 


GcAGAAAucuAAGGAuAuATsT
1142
uAuAUCCUuAGAUUUCUGCTsT
1143
AD-14365
  5%
 2%


 


uGGcuucucAcAGGAAcucTsT
1144
GAGUUCCUGUGAGAAGCcATsT
1145
AD-14366
 28%
 5%


 


GAGAuGuGAAucucuGAAcTsT
1146
GUUcAGAGAUUcAcAUCUCTsT
1147
AD-14367
 42%
 4%


 


uGuAAGccAAuGuuGuGAGTsT
1148
CUcAcAAcAUUGGCUuAcATsT
1149
AD-14368
 93%
12%


 


AGccAAuGuuGuGAGGcuuTsT
1150
AAGCCUcAcAAcAUUGGCUTsT
1151
AD-14369
 65%
 4%


 


uuGuGAGGcuucAAGuucATsT
1152
UGAACUUGAAGCCUcAcAATsT
1153
AD-14370
  5%
 2%


 


AGGcAGcucAuGAGAAAcATsT
1154
UGUUUCUcAUGAGCUGCCUTsT
1155
AD-14371
 54%
 5%


 


AuAAAuuGAuAGcAcAAAATsT
1156
UUUUGUGCuAUcAAUUuAUTsT
1157
AD-14372
  4%
 1%


 


AcAAAAucuAGAAcuuAAuTsT
1158
AUuAAGUUCuAGAUUUUGUTsT
1159
AD-14373
  5%
 1%


 


GAuAucccAAcAGGuAcGATsT
1160
UCGuACCUGUUGGGAuAUCTsT
1161
AD-14374
 92%
 6%


 


AAGuuAuuuAuAcccAucATsT
1162
UGAUGGGuAuAAAuAACUUTsT
1163
AD-14375
 76%
 4%


 


uGuAAAuAcGuAuuucuAGTsT
1164
CuAGAAAuACGuAUUuAcATsT
1165
AD-14376
 70%
 5%


 


ucuAGuuuucAuAuAAAGuTsT
1166
ACUUuAuAUGAAAACuAGATsT
1167
AD-14377
 48%
 4%


 


AuAAAGuAGuucuuuuAuATsT
1168
uAuAAAAGAACuACUUuAUTsT
1169
AD-14378
 48%
 3%


 


ccAuuuGuAGAGcuAcAAATsT
1170
UUUGuAGCUCuAcAAAUGGTsT
1171
AD-14379
 44%
 5%


 


uAuuuucAGuAGucAGAAuTsT
1172
AUUCUGACuACUGAAAAuATsT
1173
AD-14380
 35%
16%


 


AAAucuAAcccuAGuuGuATsT
1174
uAcAACuAGGGUuAGAUUUTsT
1175
AD-14381
 44%
 5%


 


cuuuAGAGuAuAcAuuGcuTsT
1176
AGcAAUGuAuACUCuAAAGTsT
1177
AD-14382
 28%
 1%


 


AucuGAcuAAuGGcucuGuTsT
1178
AcAGAGCcAUuAGUcAGAUTsT
1179
AD-14383
 55%
11%


 


cAcAAuGAuuuAAGGAcuGTsT
1180
cAGUCCUuAAAUcAUUGUGTsT
1181
AD-14384
 48%
 9%


 


ucuuuuucucGAuucAAAuTsT
1182
AUUuGAAUCGAGAAAAAGATsT
1183
AD-14385
 36%
 2%


 


cuuuuucucGAuucAAAucTsT
1184
GAUUuGAAUCGAGAAAAAGTsT
1185
AD-14386
 41%
 7%


 


AuuuucuGcucAcGAuGAGTsT
1186
CUcAUCGUGAGcAGAAAAUTsT
1187
AD-14387
 38%
 3%


 


uuucuGcucAcGAuGAGuuTsT
1188
AACUcAUCGUGAGcAGAAATsT
1189
AD-14388
 50%
 4%


 


AGAGcuAcAAAAccuAuccTsT
1190
GGAuAGGUUUUGuAGCUCUTsT
1191
AD-14389
 98%
 6%


 


GAGccAAAGGuAcAccAcuTsT
1192
AGUGGUGuACCUUUGGCUCTsT
1193
AD-14390
 43%
 8%


 


GccAAAGGuAcAccAcuAcTsT
1194
GuAGUGGUGuACCUUUGGCTsT
1195
AD-14391
 48%
 4%


 


GAAcuGuAcucuucucAGcTsT
1196
GCUGAGAAGAGuAcAGUUCTsT
1197
AD-14392
 44%
 3%


 


AGGuAAAuAucAccAAcAuTsT
1198
AUGUUGGUGAuAUUuACCUTsT
1199
AD-14393
 37%
 2%


 


AGcuAcAAAAccuAuccuuTsT
1200
AAGGAuAGGUUUUGuAGCUTsT
1201
AD-14394
114%
 7%


 


uGuGAAAGcAuuuAAuuccTsT
1202
GGAAUuAAAUGCUUUcAcATsT
1203
AD-14395
 55%
 4%


 


GcccAcuuuAGAGuAuAcATsT
1204
UGuAuACUCuAAAGUGGGCTsT
1205
AD-14396
 49%
 5%


 


uGuGccAcAcuccAAGAccTsT
1206
GGUCUUGGAGUGUGGcAcATsT
1207
AD-14397
 71%
 6%


 


AAAcuAAAuuGAucucGuATsT
1208
uACGAGAUcAAUUuAGUUUTsT
1209
AD-14398
 81%
 7%


 


uGAucucGuAGAAuuAucuTsT
1210
AGAuAAUUCuACGAGAUcATsT
1211
AD-14399
 38%
 4%


 


GcGuGcAGucGGuccuccATsT
1212
UGGAGGACCGACUGcACGCTsT
1213
AD-14400
106%
 8%


 


AAAGuuuAGAGAcAucuGATsT
1214
UcAGAUGUCUCuAAACUUUTsT
1215
AD-14401
 47%
 3%


 


cAGAAGGAAuAuGuAcAAATsT
1216
UUUGuAcAuAUUCCUUCUGTsT
1217
AD-14402
 31%
 1%


 


cGcccGAGAGuAccAGGGATsT
1218
UCCCUGGuACUCUCGGGCGTsT
1219
AD-14403
105%
 4%


 


cGGAGGAGAuAGAAcGuuuTsT
1220
AAACGUUCuAUCUCCUCCGTsT
1221
AD-14404
  3%
 1%


 


AGAuAGAAcGuuuAAAAcGTsT
1222
CGUUUuAAACGUUCuAUCUTsT
1223
AD-14405
 15%
 1%


 


GGAAcAGGAAcuucAcAAcTsT
1224
GUuGuGAAGUUCCuGUUCCTsT
1225
AD-14406
 44%
 5%


 


GuGAGccAAAGGuAcAccATsT
1226
UGGUGuACCUUUGGCUcACTsT
1227
AD-14407
 41%
 4%


 


AuccucccuAGAcuucccuTsT
1228
AGGGAAGUCuAGGGAGGAUTsT
1229
AD-14408
104%
 3%


 


cAcAcuccAAGAccuGuGcTsT
1230
GcAcAGGUCUUGGAGUGUGTsT
1231
AD-14409
 67%
 4%


 


AcAGAAGGAAuAuGuAcAATsT
1232
UUGuAcAuAUUCCUUCUGUTsT
1233
AD-14410
 22%
 1%


 


uuAGAGAcAucuGAcuuuGTsT
1234
cAAAGUcAGAUGUCUCuAATsT
1235
AD-14411
 29%
 3%


 


AAuuGAucucGuAGAAuuATsT
1236
uAAUUCuACGAGAUcAAUUTsT
1237
AD-14412
 31%
 4%









dsRNA Targeting the VEGF Gene
Four hundred target sequences were identified within exons 1-5 of the VEGF-A121 mRNA sequence. reference transcript is: NM—003376.








(SEQ ID NO: 1539)



  1 augaacuuuc ugcugucuug ggugcauugg agccuugccu ugcugcucua ccuccaccau



 


 61 gccaaguggu cccaggcugc acccauggca gaaggaggag ggcagaauca ucacgaagug


 


121 gugaaguuca uggaugucua ucagcgcagc uacugccauc caaucgagac ccugguggac


 


181 aucuuccagg aguacccuga ugagaucgag uacaucuuca agccauccug ugugccccug


 


241 augcgaugcg ggggcugcug caaugacgag ggccuggagu gugugcccac ugaggagucc


 


301 aacaucacca ugcagauuau gcggaucaaa ccucaccaag gccagcacau aggagagaug


 


361 agcuuccuac agcacaacaa augugaaugc agaccaaaga aagauagagc aagacaagaa


 


421 aaaugugaca agccgaggcg guga






Table 4a includes the identified target sequences. Corresponding siRNAs targeting these sequences were subjected to a bioinformatics screen.
To ensure that the sequences were specific to VEGF sequence and not to sequences from any other genes, the target sequences were checked against the sequences in Genbank using the BLAST search engine provided by NCBI. The use of the BLAST algorithm is described in Altschul et al., J. Mol. Biol. 215:403, 1990; and Altschul and Gish, Meth. Enzymol. 266:460, 1996.
siRNAs were also prioritized for their ability to cross react with monkey, rat and human VEGF sequences.
Of these 400 potential target sequences 80 were selected for analysis by experimental screening in order to identify a small number of lead candidates. A total of 114 siRNA molecules were designed for these 80 target sequences 114 (Table 4b).







TABLE 4a







Target sequences in VEGF-121











SEQ
position
TARGET SEQUENCE IN



ID
in VEGF-
VEGF121 mRNA



NO:
121 ORF
5′ to 3′







1540
  1
AUGAACUUUCUGCUGUCUUGGGU



 



1541
  2
UGAACUUUCUGCUGUCUUGGGUG



 



1542
  3
GAACUUUCUGCUGUCUUGGGUGC



 



1543
  4
AACUUUCUGCUGUCUUGGGUGCA



 



1544
  5
ACUUUCUGCUGUCUUGGGUGCAU



 



1545
  6
CUUUCUGCUGUCUUGGGUGCAUU



 



1546
  7
UUUCUGCUGUCUUGGGUGCAUUG



 



1547
  8
UUCUGCUGUCUUGGGUGCAUUGG



 



1548
  9
UCUGCUGUCUUGGGUGCAUUGGA



 



1549
 10
CUGCUGUCUUGGGUGCAUUGGAG



 



1550
 11
UGCUGUCUUGGGUGCAUUGGAGC



 



1551
 12
GCUGUCUUGGGUGCAUUGGAGCC



 



1552
 13
CUGUCUUGGGUGCAUUGGAGCCU



 



1553
 14
UGUCUUGGGUGCAUUGGAGCCUU



 



1554
 15
GUCUUGGGUGCAUUGGAGCCUUG



 



1555
 16
UCUUGGGUGCAUUGGAGCCUUGC



 



1556
 17
CUUGGGUGCAUUGGAGCCUUGCC



 



1557
 18
UUGGGUGCAUUGGAGCCUUGCCU



 



1558
 19
UGGGUGCAUUGGAGCCUUGCCUU



 



1559
 20
GGGUGCAUUGGAGCCUUGCCUUG



 



1560
 21
GGUGCAUUGGAGCCUUGCCUUGC



 



1561
 22
GUGCAUUGGAGCCUUGCCUUGCU



 



1562
 23
UGCAUUGGAGCCUUGCCUUGCUG



 



1563
 24
GCAUUGGAGCCUUGCCUUGCUGC



 



1564
 25
CAUUGGAGCCUUGCCUUGCUGCU



 



1565
 26
AUUGGAGCCUUGCCUUGCUGCUC



 



1566
 27
UUGGAGCCUUGCCUUGCUGCUCU



 



1567
 28
UGGAGCCUUGCCUUGCUGCUCUA



 



1568
 29
GGAGCCUUGCCUUGCUGCUCUAC



 



1569
 30
GAGCCUUGCCUUGCUGCUCUACC



 



1570
 31
AGCCUUGCCUUGCUGCUCUACCU



 



1571
 32
GCCUUGCCUUGCUGCUCUACCUC



 



1572
 33
CCUUGCCUUGCUGCUCUACCUCC



 



1573
 34
CUUGCCUUGCUGCUCUACCUCCA



 



1574
 35
UUGCCUUGCUGCUCUACCUCCAC



 



1575
 36
UGCCUUGCUGCUCUACCUCCACC



 



1576
 37
GCCUUGCUGCUCUACCUCCACCA



 



1577
 38
CCUUGCUGCUCUACCUCCACCAU



 



1578
 39
CUUGCUGCUCUACCUCCACCAUG



 



1579
 40
UUGCUGCUCUACCUCCACCAUGC



 



1580
 41
UGCUGCUCUACCUCCACCAUGCC



 



1581
 42
GCUGCUCUACCUCCACCAUGCCA



 



1582
 43
CUGCUCUACCUCCACCAUGCCAA



 



1583
 44
UGCUCUACCUCCACCAUGCCAAG



 



1584
 45
GCUCUACCUCCACCAUGCCAAGU



 



1585
 46
CUCUACCUCCACCAUGCCAAGUG



 



1586
 47
UCUACCUCCACCAUGCCAAGUGG



 



1587
 48
CUACCUCCACCAUGCCAAGUGGU



 



1588
 49
UACCUCCACCAUGCCAAGUGGUC



 



1589
 50
ACCUCCACCAUGCCAAGUGGUCC



 



1590
 51
CCUCCACCAUGCCAAGUGGUCCC



 



1591
 52
CUCCACCAUGCCAAGUGGUCCCA



 



1592
 53
UCCACCAUGCCAAGUGGUCCCAG



 



1593
 54
CCACCAUGCCAAGUGGUCCCAGG



 



1594
 55
CACCAUGCCAAGUGGUCCCAGGC



 



1595
 56
ACCAUGCCAAGUGGUCCCAGGCU



 



1596
 57
CCAUGCCAAGUGGUCCCAGGCUG



 



1597
 58
CAUGCCAAGUGGUCCCAGGCUGC



 



1598
 59
AUGCCAAGUGGUCCCAGGCUGCA



 



1599
 60
UGCCAAGUGGUCCCAGGCUGCAC



 



1600
 61
GCCAAGUGGUCCCAGGCUGCACC



 



1601
 62
CCAAGUGGUCCCAGGCUGCACCC



 



1602
 63
CAAGUGGUCCCAGGCUGCACCCA



 



1603
 64
AAGUGGUCCCAGGCUGCACCCAU



 



1604
 65
AGUGGUCCCAGGCUGCACCCAUG



 



1605
 66
GUGGUCCCAGGCUGCACCCAUGG



 



1606
 67
UGGUCCCAGGCUGCACCCAUGGC



 



1607
 68
GGUCCCAGGCUGCACCCAUGGCA



 



1608
 69
GUCCCAGGCUGCACCCAUGGCAG



 



1609
 70
UCCCAGGCUGCACCCAUGGCAGA



 



1610
 71
CCCAGGCUGCACCCAUGGCAGAA



 



1611
 72
CCAGGCUGCACCCAUGGCAGAAG



 



1612
 73
CAGGCUGCACCCAUGGCAGAAGG



 



1613
 74
AGGCUGCACCCAUGGCAGAAGGA



 



1614
 75
GGCUGCACCCAUGGCAGAAGGAG



 



1615
 76
GCUGCACCCAUGGCAGAAGGAGG



 



1616
 77
CUGCACCCAUGGCAGAAGGAGGA



 



1617
 78
UGCACCCAUGGCAGAAGGAGGAG



 



1618
 79
GCACCCAUGGCAGAAGGAGGAGG



 



1619
 80
CACCCAUGGCAGAAGGAGGAGGG



 



1620
 81
ACCCAUGGCAGAAGGAGGAGGGC



 



1621
 82
CCCAUGGCAGAAGGAGGAGGGCA



 



1622
 83
CCAUGGCAGAAGGAGGAGGGCAG



 



1623
 84
CAUGGCAGAAGGAGGAGGGCAGA



 



1624
 85
AUGGCAGAAGGAGGAGGGCAGAA



 



1625
 86
UGGCAGAAGGAGGAGGGCAGAAU



 



1626
 87
GGCAGAAGGAGGAGGGCAGAAUC



 



1627
 88
GCAGAAGGAGGAGGGCAGAAUCA



 



1628
 89
CAGAAGGAGGAGGGCAGAAUCAU



 



1629
 90
AGAAGGAGGAGGGCAGAAUCAUC



 



1630
 91
GAAGGAGGAGGGCAGAAUCAUCA



 



1631
 92
AAGGAGGAGGGCAGAAUCAUCAC



 



1632
 93
AGGAGGAGGGCAGAAUCAUCACG



 



1633
 94
GGAGGAGGGCAGAAUCAUCACGA



 



1634
 95
GAGGAGGGCAGAAUCAUCACGAA



 



1635
 96
AGGAGGGCAGAAUCAUCACGAAG



 



1636
 97
GGAGGGCAGAAUCAUCACGAAGU



 



1637
 98
GAGGGCAGAAUCAUCACGAAGUG



 



1638
 99
AGGGCAGAAUCAUCACGAAGUGG



 



1639
100
GGGCAGAAUCAUCACGAAGUGGU



 



1640
101
GGCAGAAUCAUCACGAAGUGGUG



 



1641
102
GCAGAAUCAUCACGAAGUGGUGA



 



1642
103
CAGAAUCAUCACGAAGUGGUGAA



 



1643
104
AGAAUCAUCACGAAGUGGUGAAG



 



1644
105
GAAUCAUCACGAAGUGGUGAAGU



 



1645
106
AAUCAUCACGAAGUGGUGAAGUU



 



1646
107
AUCAUCACGAAGUGGUGAAGUUC



 



1647
108
UCAUCACGAAGUGGUGAAGUUCA



 



1648
109
CAUCACGAAGUGGUGAAGUUCAU



 



1649
110
AUCACGAAGUGGUGAAGUUCAUG



 



1650
111
UCACGAAGUGGUGAAGUUCAUGG



 



1651
112
CACGAAGUGGUGAAGUUCAUGGA



 



1652
113
ACGAAGUGGUGAAGUUCAUGGAU



 



1653
114
CGAAGUGGUGAAGUUCAUGGAUG



 



1654
115
GAAGUGGUGAAGUUCAUGGAUGU



 



1655
116
AAGUGGUGAAGUUCAUGGAUGUC



 



1656
117
AGUGGUGAAGUUCAUGGAUGUCU



 



1657
118
GUGGUGAAGUUCAUGGAUGUCUA



 



1658
119
UGGUGAAGUUCAUGGAUGUCUAU



 



1659
120
GGUGAAGUUCAUGGAUGUCUAUC



 



1660
121
GUGAAGUUCAUGGAUGUCUAUCA



 



1661
122
UGAAGUUCAUGGAUGUCUAUCAG



 



1662
123
GAAGUUCAUGGAUGUCUAUCAGC



 



1663
124
AAGUUCAUGGAUGUCUAUCAGCG



 



1664
125
AGUUCAUGGAUGUCUAUCAGCGC



 



1665
126
GUUCAUGGAUGUCUAUCAGCGCA



 



1666
127
UUCAUGGAUGUCUAUCAGCGCAG



 



1667
128
UCAUGGAUGUCUAUCAGCGCAGC



 



1668
129
CAUGGAUGUCUAUCAGCGCAGCU



 



1669
130
AUGGAUGUCUAUCAGCGCAGCUA



 



1670
131
UGGAUGUCUAUCAGCGCAGCUAC



 



1671
132
GGAUGUCUAUCAGCGCAGCUACU



 



1672
133
GAUGUCUAUCAGCGCAGCUACUG



 



1673
134
AUGUCUAUCAGCGCAGCUACUGC



 



1674
135
UGUCUAUCAGCGCAGCUACUGCC



 



1675
136
GUCUAUCAGCGCAGCUACUGCCA



 



1676
137
UCUAUCAGCGCAGCUACUGCCAU



 



1677
138
CUAUCAGCGCAGCUACUGCCAUC



 



1678
139
UAUCAGCGCAGCUACUGCCAUCC



 



1679
140
AUCAGCGCAGCUACUGCCAUCCA



 



1680
141
UCAGCGCAGCUACUGCCAUCCAA



 



1681
142
CAGCGCAGCUACUGCCAUCCAAU



 



1682
143
AGCGCAGCUACUGCCAUCCAAUC



 



1683
144
GCGCAGCUACUGCCAUCCAAUCG



 



1684
145
CGCAGCUACUGCCAUCCAAUCGA



 



1685
146
GCAGCUACUGCCAUCCAAUCGAG



 



1686
147
CAGCUACUGCCAUCCAAUCGAGA



 



1687
148
AGCUACUGCCAUCCAAUCGAGAC



 



1688
149
GCUACUGCCAUCCAAUCGAGACC



 



1689
150
CUACUGCCAUCCAAUCGAGACCC



 



1690
151
UACUGCCAUCCAAUCGAGACCCU



 



1691
152
ACUGCCAUCCAAUCGAGACCCUG



 



1692
153
CUGCCAUCCAAUCGAGACCCUGG



 



1693
154
UGCCAUCCAAUCGAGACCCUGGU



 



1694
155
GCCAUCCAAUCGAGACCCUGGUG



 



1695
156
CCAUCCAAUCGAGACCCUGGUGG



 



1696
157
CAUCCAAUCGAGACCCUGGUGGA



 



1697
158
AUCCAAUCGAGACCCUGGUGGAC



 



1698
159
UCCAAUCGAGACCCUGGUGGACA



 



1699
160
CCAAUCGAGACCCUGGUGGACAU



 



1700
161
CAAUCGAGACCCUGGUGGACAUC



 



1701
162
AAUCGAGACCCUGGUGGACAUCU



 



1702
163
AUCGAGACCCUGGUGGACAUCUU



 



1703
164
UCGAGACCCUGGUGGACAUCUUC



 



1704
165
CGAGACCCUGGUGGACAUCUUCC



 



1705
166
GAGACCCUGGUGGACAUCUUCCA



 



1706
167
AGACCCUGGUGGACAUCUUCCAG



 



1707
168
GACCCUGGUGGACAUCUUCCAGG



 



1708
169
ACCCUGGUGGACAUCUUCCAGGA



 



1709
170
CCCUGGUGGACAUCUUCCAGGAG



 



1710
171
CCUGGUGGACAUCUUCCAGGAGU



 



1711
172
CUGGUGGACAUCUUCCAGGAGUA



 



1712
173
UGGUGGACAUCUUCCAGGAGUAC



 



1713
174
GGUGGACAUCUUCCAGGAGUACC



 



1714
175
GUGGACAUCUUCCAGGAGUACCC



 



1715
176
UGGACAUCUUCCAGGAGUACCCU



 



1716
177
GGACAUCUUCCAGGAGUACCCUG



 



1717
178
GACAUCUUCCAGGAGUACCCUGA



 



1718
179
ACAUCUUCCAGGAGUACCCUGAU



 



1719
180
CAUCUUCCAGGAGUACCCUGAUG



 



1720
181
AUCUUCCAGGAGUACCCUGAUGA



 



1721
182
UCUUCCAGGAGUACCCUGAUGAG



 



1722
183
CUUCCAGGAGUACCCUGAUGAGA



 



1723
184
UUCCAGGAGUACCCUGAUGAGAU



 



1724
185
UCCAGGAGUACCCUGAUGAGAUC



 



1725
186
CCAGGAGUACCCUGAUGAGAUCG



 



1726
187
CAGGAGUACCCUGAUGAGAUCGA



 



1727
188
AGGAGUACCCUGAUGAGAUCGAG



 



1728
189
GGAGUACCCUGAUGAGAUCGAGU



 



1729
190
GAGUACCCUGAUGAGAUCGAGUA



 



1730
191
AGUACCCUGAUGAGAUCGAGUAC



 



1731
192
GUACCCUGAUGAGAUCGAGUACA



 



1732
193
UACCCUGAUGAGAUCGAGUACAU



 



1733
194
ACCCUGAUGAGAUCGAGUACAUC



 



1734
195
CCCUGAUGAGAUCGAGUACAUCU



 



1735
196
CCUGAUGAGAUCGAGUACAUCUU



 



1736
197
CUGAUGAGAUCGAGUACAUCUUC



 



1737
198
UGAUGAGAUCGAGUACAUCUUCA



 



1738
199
GAUGAGAUCGAGUACAUCUUCAA



 



1739
200
AUGAGAUCGAGUACAUCUUCAAG



 



1740
201
UGAGAUCGAGUACAUCUUCAAGC



 



1741
202
GAGAUCGAGUACAUCUUCAAGCC



 



1742
203
AGAUCGAGUACAUCUUCAAGCCA



 



1743
204
GAUCGAGUACAUCUUCAAGCCAU



 



1744
205
AUCGAGUACAUCUUCAAGCCAUC



 



1745
206
UCGAGUACAUCUUCAAGCCAUCC



 



1746
207
CGAGUACAUCUUCAAGCCAUCCU



 



1747
208
GAGUACAUCUUCAAGCCAUCCUG



 



1748
209
AGUACAUCUUCAAGCCAUCCUGU



 



1749
210
GUACAUCUUCAAGCCAUCCUGUG



 



1750
211
UACAUCUUCAAGCCAUCCUGUGU



 



1751
212
ACAUCUUCAAGCCAUCCUGUGUG



 



1752
213
CAUCUUCAAGCCAUCCUGUGUGC



 



1753
214
AUCUUCAAGCCAUCCUGUGUGCC



 



1754
215
UCUUCAAGCCAUCCUGUGUGCCC



 



1755
216
CUUCAAGCCAUCCUGUGUGCCCC



 



1756
217
UUCAAGCCAUCCUGUGUGCCCCU



 



1757
218
UCAAGCCAUCCUGUGUGCCCCUG



 



1758
219
CAAGCCAUCCUGUGUGCCCCUGA



 



1759
220
AAGCCAUCCUGUGUGCCCCUGAU



 



1760
221
AGCCAUCCUGUGUGCCCCUGAUG



 



1761
222
GCCAUCCUGUGUGCCCCUGAUGC



 



1762
223
CCAUCCUGUGUGCCCCUGAUGCG



 



1763
224
CAUCCUGUGUGCCCCUGAUGCGA



 



1764
225
AUCCUGUGUGCCCCUGAUGCGAU



 



1765
226
UCCUGUGUGCCCCUGAUGCGAUG



 



1766
227
CCUGUGUGCCCCUGAUGCGAUGC



 



1767
228
CUGUGUGCCCCUGAUGCGAUGCG



 



1768
229
UGUGUGCCCCUGAUGCGAUGCGG



 



1769
230
GUGUGCCCCUGAUGCGAUGCGGG



 



1770
231
UGUGCCCCUGAUGCGAUGCGGGG



 



1771
232
GUGCCCCUGAUGCGAUGCGGGGG



 



1772
233
UGCCCCUGAUGCGAUGCGGGGGC



 



1773
234
GCCCCUGAUGCGAUGCGGGGGCU



 



1774
235
CCCCUGAUGCGAUGCGGGGGCUG



 



1775
236
CCCUGAUGCGAUGCGGGGGCUGC



 



1776
237
CCUGAUGCGAUGCGGGGGCUGCU



 



1777
238
CUGAUGCGAUGCGGGGGCUGCUG



 



1778
239
UGAUGCGAUGCGGGGGCUGCUGC



 



1779
240
GAUGCGAUGCGGGGGCUGCUGCA



 



1780
241
AUGCGAUGCGGGGGCUGCUGCAA



 



1781
242
UGCGAUGCGGGGGCUGCUGCAAU



 



1782
243
GCGAUGCGGGGGCUGCUGCAAUG



 



1783
244
CGAUGCGGGGGCUGCUGCAAUGA



 



1784
245
GAUGCGGGGGCUGCUGCAAUGAC



 



1785
246
AUGCGGGGGCUGCUGCAAUGACG



 



1786
247
UGCGGGGGCUGCUGCAAUGACGA



 



1787
248
GCGGGGGCUGCUGCAAUGACGAG



 



1788
249
CGGGGGCUGCUGCAAUGACGAGG



 



1789
250
GGGGGCUGCUGCAAUGACGAGGG



 



1790
251
GGGGCUGCUGCAAUGACGAGGGC



 



1791
252
GGGCUGCUGCAAUGACGAGGGCC



 



1792
253
GGCUGCUGCAAUGACGAGGGCCU



 



1793
254
GCUGCUGCAAUGACGAGGGCCUG



 



1794
255
CUGCUGCAAUGACGAGGGCCUGG



 



1795
256
UGCUGCAAUGACGAGGGCCUGGA



 



1796
257
GCUGCAAUGACGAGGGCCUGGAG



 



1797
258
CUGCAAUGACGAGGGCCUGGAGU



 



1798
259
UGCAAUGACGAGGGCCUGGAGUG



 



1799
260
GCAAUGACGAGGGCCUGGAGUGU



 



1800
261
CAAUGACGAGGGCCUGGAGUGUG



 



1801
262
AAUGACGAGGGCCUGGAGUGUGU



 



1802
263
AUGACGAGGGCCUGGAGUGUGUG



 



1803
264
UGACGAGGGCCUGGAGUGUGUGC



 



1804
265
GACGAGGGCCUGGAGUGUGUGCC



 



1805
266
ACGAGGGCCUGGAGUGUGUGCCC



 



1806
267
CGAGGGCCUGGAGUGUGUGCCCA



 



1807
268
GAGGGCCUGGAGUGUGUGCCCAC



 



1808
269
AGGGCCUGGAGUGUGUGCCCACU



 



1809
270
GGGCCUGGAGUGUGUGCCCACUG



 



1810
271
GGCCUGGAGUGUGUGCCCACUGA



 



1811
272
GCCUGGAGUGUGUGCCCACUGAG



 



1812
273
CCUGGAGUGUGUGCCCACUGAGG



 



1813
274
CUGGAGUGUGUGCCCACUGAGGA



 



1814
275
UGGAGUGUGUGCCCACUGAGGAG



 



1815
276
GGAGUGUGUGCCCACUGAGGAGU



 



1816
277
GAGUGUGUGCCCACUGAGGAGUC



 



1817
278
AGUGUGUGCCCACUGAGGAGUCC



 



1818
279
GUGUGUGCCCACUGAGGAGUCCA



 



1819
280
UGUGUGCCCACUGAGGAGUCCAA



 



1820
281
GUGUGCCCACUGAGGAGUCCAAC



 



1821
282
UGUGCCCACUGAGGAGUCCAACA



 



1822
283
GUGCCCACUGAGGAGUCCAACAU



 



1823
284
UGCCCACUGAGGAGUCCAACAUC



 



1824
285
GCCCACUGAGGAGUCCAACAUCA



 



1825
286
CCCACUGAGGAGUCCAACAUCAC



 



1826
287
CCACUGAGGAGUCCAACAUCACC



 



1827
288
CACUGAGGAGUCCAACAUCACCA



 



1828
289
ACUGAGGAGUCCAACAUCACCAU



 



1829
290
CUGAGGAGUCCAACAUCACCAUG



 



1830
291
UGAGGAGUCCAACAUCACCAUGC



 



1831
292
GAGGAGUCCAACAUCACCAUGCA



 



1832
293
AGGAGUCCAACAUCACCAUGCAG



 



1833
294
GGAGUCCAACAUCACCAUGCAGA



 



1834
295
GAGUCCAACAUCACCAUGCAGAU



 



1835
296
AGUCCAACAUCACCAUGCAGAUU



 



1836
297
GUCCAACAUCACCAUGCAGAUUA



 



1837
298
UCCAACAUCACCAUGCAGAUUAU



 



1838
299
CCAACAUCACCAUGCAGAUUAUG



 



1839
300
CAACAUCACCAUGCAGAUUAUGC



 



1840
301
AACAUCACCAUGCAGAUUAUGCG



 



1841
302
ACAUCACCAUGCAGAUUAUGCGG



 



1842
303
CAUCACCAUGCAGAUUAUGCGGA



 



1843
304
AUCACCAUGCAGAUUAUGCGGAU



 



1844
305
UCACCAUGCAGAUUAUGCGGAUC



 



1845
306
CACCAUGCAGAUUAUGCGGAUCA



 



1846
307
ACCAUGCAGAUUAUGCGGAUCAA



 



1847
308
CCAUGCAGAUUAUGCGGAUCAAA



 



1848
309
CAUGCAGAUUAUGCGGAUCAAAC



 



1849
310
AUGCAGAUUAUGCGGAUCAAACC



 



1850
311
UGCAGAUUAUGCGGAUCAAACCU



 



1851
312
GCAGAUUAUGCGGAUCAAACCUC



 



1852
313
CAGAUUAUGCGGAUCAAACCUCA



 



1853
314
AGAUUAUGCGGAUCAAACCUCAC



 



1854
315
GAUUAUGCGGAUCAAACCUCACC



 



1855
316
AUUAUGCGGAUCAAACCUCACCA



 



1856
317
UUAUGCGGAUCAAACCUCACCAA



 



1857
318
UAUGCGGAUCAAACCUCACCAAG



 



1858
319
AUGCGGAUCAAACCUCACCAAGG



 



1859
320
UGCGGAUCAAACCUCACCAAGGC



 



1860
321
GCGGAUCAAACCUCACCAAGGCC



 



1861
322
CGGAUCAAACCUCACCAAGGCCA



 



1862
323
GGAUCAAACCUCACCAAGGCCAG



 



1863
324
GAUCAAACCUCACCAAGGCCAGC



 



1864
325
AUCAAACCUCACCAAGGCCAGCA



 



1865
326
UCAAACCUCACCAAGGCCAGCAC



 



1866
327
CAAACCUCACCAAGGCCAGCACA



 



1867
328
AAACCUCACCAAGGCCAGCACAU



 



1868
329
AACCUCACCAAGGCCAGCACAUA



 



1869
330
ACCUCACCAAGGCCAGCACAUAG



 



1870
331
CCUCACCAAGGCCAGCACAUAGG



 



1871
332
CUCACCAAGGCCAGCACAUAGGA



 



1872
333
UCACCAAGGCCAGCACAUAGGAG



 



1873
334
CACCAAGGCCAGCACAUAGGAGA



 



1874
335
ACCAAGGCCAGCACAUAGGAGAG



 



1875
336
CCAAGGCCAGCACAUAGGAGAGA



 



1876
337
CAAGGCCAGCACAUAGGAGAGAU



 



1877
338
AAGGCCAGCACAUAGGAGAGAUG



 



1878
339
AGGCCAGCACAUAGGAGAGAUGA



 



1879
340
GGCCAGCACAUAGGAGAGAUGAG



 



1880
341
GCCAGCACAUAGGAGAGAUGAGC



 



1881
342
CCAGCACAUAGGAGAGAUGAGCU



 



1882
343
CAGCACAUAGGAGAGAUGAGCUU



 



1883
344
AGCACAUAGGAGAGAUGAGCUUC



 



1884
345
GCACAUAGGAGAGAUGAGCUUCC



 



1885
346
CACAUAGGAGAGAUGAGCUUCCU



 



1886
347
ACAUAGGAGAGAUGAGCUUCCUA



 



1887
348
CAUAGGAGAGAUGAGCUUCCUAC



 



1888
349
AUAGGAGAGAUGAGCUUCCUACA



 



1889
350
UAGGAGAGAUGAGCUUCCUACAG



 



1890
351
AGGAGAGAUGAGCUUCCUACAGC



 



1891
352
GGAGAGAUGAGCUUCCUACAGCA



 



1892
353
GAGAGAUGAGCUUCCUACAGCAC



 



1893
354
AGAGAUGAGCUUCCUACAGCACA



 



1894
355
GAGAUGAGCUUCCUACAGCACAA



 



1895
356
AGAUGAGCUUCCUACAGCACAAC



 



1896
357
GAUGAGCUUCCUACAGCACAACA



 



1897
358
AUGAGCUUCCUACAGCACAACAA



 



1898
359
UGAGCUUCCUACAGCACAACAAA



 



1899
360
GAGCUUCCUACAGCACAACAAAU



 



1900
361
AGCUUCCUACAGCACAACAAAUG



 



1901
362
GCUUCCUACAGCACAACAAAUGU



 



1902
363
CUUCCUACAGCACAACAAAUGUG



 



1903
364
UUCCUACAGCACAACAAAUGUGA



 



1904
365
UCCUACAGCACAACAAAUGUGAA



 



1905
366
CCUACAGCACAACAAAUGUGAAU



 



1906
367
CUACAGCACAACAAAUGUGAAUG



 



1907
368
UACAGCACAACAAAUGUGAAUGC



 



1908
369
ACAGCACAACAAAUGUGAAUGCA



 



1909
370
CAGCACAACAAAUGUGAAUGCAG



 



1910
371
AGCACAACAAAUGUGAAUGCAGA



 



1911
372
GCACAACAAAUGUGAAUGCAGAC



 



1912
373
CACAACAAAUGUGAAUGCAGACC



 



1913
374
ACAACAAAUGUGAAUGCAGACCA



 



1914
375
CAACAAAUGUGAAUGCAGACCAA



 



1915
376
AACAAAUGUGAAUGCAGACCAAA



 



1916
377
ACAAAUGUGAAUGCAGACCAAAG



 



1917
378
CAAAUGUGAAUGCAGACCAAAGA



 



1918
379
AAAUGUGAAUGCAGACCAAAGAA



 



1919
380
AAUGUGAAUGCAGACCAAAGAAA



 



1920
381
AUGUGAAUGCAGACCAAAGAAAG



 



1921
382
UGUGAAUGCAGACCAAAGAAAGA



 



1922
383
GUGAAUGCAGACCAAAGAAAGAU



 



1923
384
UGAAUGCAGACCAAAGAAAGAUA



 



1924
385
GAAUGCAGACCAAAGAAAGAUAG



 



1925
386
AAUGCAGACCAAAGAAAGAUAGA



 



1926
387
AUGCAGACCAAAGAAAGAUAGAG



 



1927
388
UGCAGACCAAAGAAAGAUAGAGC



 



1928
389
GCAGACCAAAGAAAGAUAGAGCA



 



1929
390
CAGACCAAAGAAAGAUAGAGCAA



 



1930
391
AGACCAAAGAAAGAUAGAGCAAG



 



1931
392
GACCAAAGAAAGAUAGAGCAAGA



 



1932
393
ACCAAAGAAAGAUAGAGCAAGAC



 



1933
394
CCAAAGAAAGAUAGAGCAAGACA



 



1934
395
CAAAGAAAGAUAGAGCAAGACAA



 



1935
396
AAAGAAAGAUAGAGCAAGACAAG



 



1936
397
AAGAAAGAUAGAGCAAGACAAGA



 



1937
398
AGAAAGAUAGAGCAAGACAAGAA



 



1938
399
GAAAGAUAGAGCAAGACAAGAAA



 



1939
400
AAAGAUAGAGCAAGACAAGAAAA

















TABLE 4b







VEGF targeted duplexes













position
SEQ



SEQ



in
ID
Target sequence


ID



ORF
NO:
(5′-3′)
Duplex ID
Strand
NO:
Strand Sequences





  1
2184
AUGAACUUUCUGCUGUCUUGGGU
AL-DP-4043
S
1940
5 GAACUUUCUGCUGUCUUGGGU 3






AS
1941
3 UACUUGAAAGACGACAGAACCCA 5


 


 22
2185
GUGCAUUGGAGCCUUGCCUUGCU
AL-DP-4077
S
1942
5 GCAUUGGAGCCUUGCCUUGCU 3






AS
1943
3 CACGUAACCUCGGAACGGAACGA 5


 


 47
2186
UCUACCUCCACCAUGCCAAGUGG
AL-DP-4021
S
1944
5 UACCUCCACCAUGCCAAGUTT 3






AS
1945
3 TTAUGGAGGUGGUACGGUUCA 5


 


 48
2187
CUACCUCCACCAUGCCAAGUGGU
AL-DP-4109
S
1946
5 ACCUCCACCAUGCCAAGUGT T 3






AS
1947
3 TTUGGAGGUGGUACGGUUCAC 5


 


 50
2188
ACCUCCACCAUGCCAAGUGGUCC
AL-DP-4006
S
1948
5 CUCCACCAUGCCAAGUGGUCC 3






AS
1949
3 UGGAGGUGGUACGGUUCACCAGG 5





AL-DP-4083
S
1950
5 CUCCACCAUGCCAAGUGGUTT 3






AS
1951
3 TTGAGGUGGUACGGUUCACCA 5


 


 51
2189
CCUCCACCAUGCCAAGUGGUCCC
AL-DP-4047
S
1952
5 UCCACCAUGCCAAGUGGUCCC 3






AS
1953
3 GGAGGUGGUACGGUUCACCAGGG 5





AL-DP-4017
S
1954
5 UCCACCAUGCCAAGUGGUCTT 3






AS
1955
3 TTAGGUGGUACGGUUCACCAG 5


 


 52
2190
CUCCACCAUGCCAAGUGGUCCCA
AL-DP-4048
S
1956
5 CCACCAUGCCAAGUGGUCCCA 3






AS
1957
3 GAGGUGGUACGGUUCACCAGGGU 5





AL-DP-4103
S
1958
5 CCACCAUGCCAAGUGGUCCTT 3






AS
1959
3 TTGGUGGUACGGUUCACCAGG 5


 


 53
2191
UCCACCAUGCCAAGUGGUCCCAG
AL-DP-4035
S
1960
5 CACCAUGCCAAGUGGUCCCAG 3






AS
1961
3 AGGUGGUACGGUUCACCAGGGUC 5





AL-DP-4018
S
1962
5 CACCAUGCCAAGUGGUCCCTT 3






AS
1963
3 TTGUGGUACGGUUCACCAGGG 5


 


 54
2192
CCACCAUGCCAAGUGGUCCCAGG
AL-DP-4036
S
1964
5 ACCAUGCCAAGUGGUCCCAGG 3






AS
1965
3 GGUGGUACGGUUCACCAGGGUCC 5





AL-DP-4084
S
1966
5 ACCAUGCCAAGUGGUCCCATT 3






AS
1967
3 TTUGGUACGGUUCACCAGGGU 5


 


 55
2193
CACCAUGCCAAGUGGUCCCAGGC
AL-DP-4093
S
1968
5 CCAUGCCAAGUGGUCCCAGGC 3






AS
1969
3 GUGGUACGGUUCACCAGGGUCCG 5





AL-DP-4085
S
1970
5 CCAUGCCAAGUGGUCCCAGTT 3






AS
1971
3 TTGGUACGGUUCACCAGGGUC 5


 


 56
2194
ACCAUGCCAAGUGGUCCCAGGCU
AL-DP-4037
S
1972
5 CAUGCCAAGUGGUCCCAGGCU 3






AS
1973
3 UGGUACGGUUCACCAGGGUCCGA 5





AL-DP-4054
S
1974
5 CAUGCCAAGUGGUCCCAGGTT 3






AS
1975
3 TTGUACGGUUCACCAGGGUCC 5


 


 57
2195
CCAUGCCAAGUGGUCCCAGGCUG
AL-DP-4038
S
1976
5 AUGCCAAGUGGUCCCAGGCUG 3






AS
1977
3 GGUACGGUUCACCAGGGUCCGAC 5





AL-DP-4086
S
1978
5 AUGCCAAGUGGUCCCAGGCTT 3






AS
1979
3 TTUACGGUUCACCAGGGUCCG 5


 


 58
2196
CAUGCCAAGUGGUCCCAGGCUGC
AL-DP-4049
S
1980
5 UGCCAAGUGGUCCCAGGCUGC 3






AS
1981
3 GUACGGUUCACCAGGGUCCGACG 5





AL-DP-4087
S
1982
5 UGCCAAGUGGUCCCAGGCUTT 3






AS
1983
3 TTACGGUUCACCAGGGUCCGA 5


 


 59
2197
AUGCCAAGUGGUCCCAGGCUGCA
AL-DP-4001
S
1984
5 GCCAAGUGGUCCCAGGCUGCA 3






AS
1985
3 UACGGUUCACCAGGGUCCGACGU 5





AL-DP-4052
A
1986
5 GCCAAGUGGUCCCAGGCUGTT 3






AS
1987
3 TTCGGUUCACCAGGGUCCGAC 5


 


 60
2198
UGCCAAGUGGUCCCAGGCUGCAC
AL-DP-4007
S
1988
5 CCAAGUGGUCCCAGGCUGCAC 3






AS
1989
3 ACGGUUCACCAGGGUCCGACGUG 5





AL-DP-4088
S
1990
5 CCAAGUGGUCCCAGGCUGCTT 3






AS
1991
3 TTGGUUCACCAGGGUCCGACG 5


 


 61
2199
GCCAAGUGGUCCCAGGCUGCACC
AL-DP-4070
S
1992
5 CAAGUGGUCCCAGGCUGCACC 3






AS
1993
3 CGGUUCACCAGGGUCCGACGUGG 5





AL-DP-4055
S
1994
5 CAAGUGGUCCCAGGCUGCATT 3






AS
1995
3 TTGUUCACCAGGGUCCGACGU 5


 


 62
2200
CCAAGUGGUCCCAGGCUGCACCC
AL-DP-4071
S
1996
5 AAGUGGUCCCAGGCUGCACCC 3






AS
1997
3 GGUUCACCAGGGUCCGACGUGGG 5





AL-DP-4056
S
1998
5 AAGUGGUCCCAGGCUGCACTT 3






AS
1999
3 TTUUCACCAGGGUCCGACGUG 5


 


 63
2201
CAAGUGGUCCCAGGCUGCACCCA
AL-DP-4072
S
2000
5 AGUGGUCCCAGGCUGCACCCA 3






AS
2001
3 GUUCACCAGGGUCCGACGUGGGU 5





AL-DP-4057
S
2002
5 AGUGGUCCCAGGCUGCACCTT 3






AS
2003
3 TTUCACCAGGGUCCGACGUGG 5


 


 64
2202
AAGUGGUCCCAGGCUGCACCCAU
AL-DP-4066
S
2004
5 GUGGUCCCAGGCUGCACCCTT 3






AS
2005
3 TTCACCAGGGUCCGACGUGGG 5


 


 99
2203
AGGGCAGAAUCAUCACGAAGUGG
AL-DP-4022
S
2006
5 GGCAGAAUCAUCACGAAGUTT 3






AS
2007
3 TTCCGUCUUAGUAGUGCUUCA 5


 


100
2204
GGGCAGAAUCAUCACGAAGUGGU
AL-DP-4023
S
2008
5 GCAGAAUCAUCACGAAGUGTT 3






AS
2009
3 TTCGUCUUAGUAGUGCUUCAC 5


 


101
2205
GGCAGAAUCAUCACGAAGUGGUG
AL-DP-4024
S
2010
5 CAGAAUCAUCACGAAGUGGTT 3






AS
2011
3 TTGUCUUAGUAGUGCUUCACC 5


 


102
2206
GCAGAAUCAUCACGAAGUGGUGA
AL-DP-4076
S
2012
5 AGAAUCAUCACGAAGUGGUGA 3






AS
2013
3 CGUCUUAGUAGUGCUUCACCACU 5





AL-DP-4019
S
2014
5 AGAAUCAUCACGAAGUGGUTT 3






AS
2015
3 TTUCUUAGUAGUGCUUCACCA 5


 


103
2207
CAGAAUCAUCACGAAGUGGUGAA
AL-DP-4025
S
2016
5 GAAUCAUCACGAAGUGGUGTT 3






AS
2017
3 TTCUUAGUAGUGCUUCACCAC 5


 


104
2208
AGAAUCAUCACGAAGUGGUGAAG
AL-DP-4110
S
2018
5 AAUCAUCACGAAGUGGUGATT 3






AS
2019
3 TTUUAGUAGUGCUUCACCACU 5


 


105
2209
GAAUCAUCACGAAGUGGUGAAGU
AL-DP-4068
S
2020
5 AUCAUCACGAAGUGGUGAATT 3






AS
2021
3 TTUAGUAGUGCUUCACCACUU 5


 


113
2210
ACGAAGUGGUGAAGUUCAUGGAU
AL-DP-4078
S
2022
5 GAAGUGGUGAAGUUCAUGGAU 3






AS
2023
3 UGCUUCACCACUUCAAGUACCUA 5


 


121
2211
GUGAAGUUCAUGGAUGUCUAUCA
AL-DP-4080
S
2024
5 GAAGUUCAUGGAUGUCUAUCA 3






AS
2025
3 CACUUCAAGUACCUACAGAUAGU 5


 


129
2212
CAUGGAUGUCUAUCAGCGCAGCU
AL-DP-4111
S
2026
5 UGGAUGUCUAUCAGCGCAGTT 3






AS
2027
3 TTACCUACAGAUAGUCGCGUC 5


 


130
2213
AUGGAUGUCUAUCAGCGCAGCUA
AL-DP-4041
S
2028
5 GGAUGUCUAUCAGCGCAGCUA 3






AS
2029
3 UACCUACAGAUAGUCGCGUCGAU 5





AL-DP-4062
S
2030
5 GGAUGUCUAUCAGCGCAGCTT 3






AS
2031
3 TTCCUACAGAUAGUCGCGUCG 5


 


131
2214
UGGAUGUCUAUCAGCGCAGCUAC
AL-DP-4069
S
2032
5 GAUGUCUAUCAGCGCAGCUTT 3






AS
2033
3 TTCUACAGAUAGUCGCGUCGA 5


 


132
2215
GGAUGUCUAUCAGCGCAGCUACU
AL-DP-4112
S
2034
5 AUGUCUAUCAGCGCAGCUATT 3






AS
2035
3 TTUACAGAUAGUCGCGUCGAU 5


 


133
2216
GAUGUCUAUCAGCGCAGCUACUG
AL-DP-4026
S
2036
5 UGUCUAUCAGCGCAGCUACTT 3






AS
2037
3 TTACAGAUAGUCGCGUCGAUG 5


 


134
2217
AUGUCUAUCAGCGCAGCUACUGC
AL-DP-4095
S
2038
5 GUCUAUCAGCGCAGCUACUGC 3






AS
2039
3 UACAGAUAGUCGCGUCGAUGACG 5





AL-DP-4020
S
2040
5 GUCUAUCAGCGCAGCUACUTT 3






AS
2041
3 TTCAGAUAGUCGCGUCGAUGA 5


 


135
2218
UGUCUAUCAGCGCAGCUACUGCC
AL-DP-4027
S
2042
5 UCUAUCAGCGCAGCUACUGTT 3






AS
2043
3 TTAGAUAGUCGCGUCGAUGAC 5


 


144
2219
GCGCAGCUACUGCCAUCCAAUCG
AL-DP-4081
S
2044
5 GCAGCUACUGCCAUCCAAUCG 3






AS
2045
3 CGCGUCGAUGACGGUAGGUUAGC 5


 


146
2220
GCAGCUACUGCCAUCCAAUCGAG
AL-DP-4098
S
2046
5 AGCUACUGCCAUCCAAUCGAG 3






AS
2047
3 CGUCGAUGACGGUAGGUUAGCUC 5


 


149
2221
GCUACUGCCAUCCAAUCGAGACC
AL-DP-4028
S
2048
5 UACUGCCAUCCAAUCGAGATT 3






AS
2049
3 TTAUGACGGUAGGUUAGCUCU 5


 


150
2222
CUACUGCCAUCCAAUCGAGACCC
AL-DP-4029
S
2050
5 ACUGCCAUCCAAUCGAGACTT 3






AS
2051
3 TTUGACGGUAGGUUAGCUCUG 5


 


151
2223
UACUGCCAUCCAAUCGAGACCCU
AL-DP-4030
S
2052
5 CUGCCAUCCAAUCGAGACCTT 3






AS
2053
3 TTGACGGUAGGUUAGCUCUGG 5


 


152
2224
ACUGCCAUCCAAUCGAGACCCUG
AL-DP-4031
S
2054
5 UGCCAUCCAAUCGAGACCCTT 3






AS
2055
3 TTACGGUAGGUUAGCUCUGGG 5


 


166
2225
GAGACCCUGGUGGACAUCUUCCA
AL-DP-4008
S
2056
5 GACCCUGGUGGACAUCUUCCA 3






AS
2057
3 CUCUGGGACCACCUGUAGAAGGU 5





AL-DP-4058
S
2058
5 GACCCUGGUGGACAUCUUCTT 3






AS
2059
3 TTCUGGGACCACCUGUAGAAG 5


 


167
2226
AGACCCUGGUGGACAUCUUCCAG
AL-DP-4009
S
2060
5 ACCCUGGUGGACAUCUUCCAG 3






AS
2061
3 UCUGGGACCACCUGUAGAAGGUC 5





AL-DP-4059
S
2062
5 ACCCUGGUGGACAUCUUCCTT 3






AS
2063
3 TTUGGGACCACCUGUAGAAGG 5


 


168
2227
GACCCUGGUGGACAUCUUCCAGG
AL-DP-4010
S
2064
5 CCCUGGUGGACAUCUUCCAGG 3






AS
2065
3 CUGGGACCACCUGUAGAAGGUCC 5





AL-DP-4060
S
2066
5 CCCUGGUGGACAUCUUCCATT 3






AS
2067
3 TTGGGACCACCUGUAGAAGGU 5


 


169
2228
ACCCUGGUGGACAUCUUCCAGGA
AL-DP-4073
S
2068
5 CCUGGUGGACAUCUUCCAGGA 3






AS
2069
3 UGGGACCACCUGUAGAAGGUCCU 5





AL-DP-4104
S
2070
5 CCUGGUGGACAUCUUCCAGTT 3






AS
2071
3 TTGGACCACCUGUAGAAGGUC 5


 


170
2229
CCCUGGUGGACAUCUUCCAGGAG
AL-DP-4011
S
2072
5 CUGGUGGACAUCUUCCAGGAG 3






AS
2073
3 GGGACCACCUGUAGAAGGUCCUC 5





AL-DP-4089
S
2074
5 CUGGUGGACAUCUUCCAGGTT 3






AS
2075
3 TTGACCACCUGUAGAAGGUCC 5


 


171
2230
CCUGGUGGACAUCUUCCAGGAGU
AL-DP-4074
S
2076
5 UGGUGGACAUCUUCCAGGAGU 3






AS
2077
3 GGACCACCUGUAGAAGGUCCUCA 5





AL-DP-4090
S
2078
5 UGGUGGACAUCUUCCAGGATT 3






AS
2079
3 TTACCACCUGUAGAAGGUCCU 5


 


172
2231
CUGGUGGACAUCUUCCAGGAGUA
AL-DP-4039
S
2080
5 GGUGGACAUCUUCCAGGAGUA 3






AS
2081
3 GACCACCUGUAGAAGGUCCUCAU 5





AL-DP-4091
S
2082
5 GGUGGACAUCUUCCAGGAGTT 3






AS
2083
3 TTCCACCUGUAGAAGGUCCUC 5


 


175
2232
GUGGACAUCUUCCAGGAGUACCC
AL-DP-4003
S
2084
5 GGACAUCUUCCAGGAGUACCC 3






AS
2085
3 CCUGUAGAAGGUCCUCAUGGG 5





AL-DP-4116
S
2086
5 GGACAUCUUCCAGGAGUACCC 3






AS
2087
3 CCUGUAGAAGGUCCUCAUGGG 5





AL-DP-4015
S
2088
5 GGACAUCUUCCAGGAGUACTT 3






AS
2089
3 TTCCUGUAGAAGGUCCUCAUG 5





AL-DP-4120
S
2090
5 GGACAUCUUCCAGGAGUAC 3






AS
2091
3 CCUGUAGAAGGUCCUCAUG 5


 


179
2233
ACAUCUUCCAGGAGUACCCUGAU
AL-DP-4099
S
2092
5 AUCUUCCAGGAGUACCCUGAU 3






AS
2093
3 UGUAGAAGGUCCUCAUGGGACUA 5


 


191
2234
AGUACCCUGAUGAGAUCGAGUAC
AL-DP-4032
S
2094
5 UACCCUGAUGAGAUCGAGUTT 3






AS
2095
3 TTAUGGGACUACUCUAGCUCA 5


 


192
2235
GUACCCUGAUGAGAUCGAGUACA
AL-DP-4042
S
2096
5 ACCCUGAUGAGAUCGAGUACA 3






AS
2097
3 CAUGGGACUACUCUAGCUCAUGU 5





AL-DP-4063
S
2098
5 ACCCUGAUGAGAUCGAGUAT T 3






AS
2099
3 TTUGGGACUACUCUAGCUCAU 5


 


209
2236
AGUACAUCUUCAAGCCAUCCUGU
AL-DP-4064
S
2100
5 UACAUCUUCAAGCCAUCCUTT 3






AS
2101
3 TTAUGUAGAAGUUCGGUAGGA 5


 


260
2237
GCAAUGACGAGGGCCUGGAGUGU
AL-DP-4044
S
2102
5 AAUGACGAGGGCCUGGAGUGU 3






AS
2103
3 CGUUACUGCUCCCGGACCUCACA 5


 


263
2238
AUGACGAGGGCCUGGAGUGUGUG
AL-DP-4045
S
2104
5 GACGAGGGCCUGGAGUGUGUG 3






AS
2105
3 UACUGCUCCCGGACCUCACACAC 5


 


279
2239
GUGUGUGCCCACUGAGGAGUCCA
AL-DP-4046
S
2106
5 GUGUGCCCACUGAGGAGUCCA 3






AS
2107
3 CACACACGGGUGACUCCUCAGGU 5


 


281
2240
GUGUGCCCACUGAGGAGUCCAAC
AL-DP-4096
S
2108
5 GUGCCCACUGAGGAGUCCAAC 3






AS
2109
3 CACACGGGUGACUCCUCAGGUUG 5


 


283
2241
GUGCCCACUGAGGAGUCCAACAU
AL-DP-4040
S
2110
5 GCCCACUGAGGAGUCCAACAU 3






AS
2111
3 CACGGGUGACUCCUCAGGUUGUA 5


 


289
2242
ACUGAGGAGUCCAACAUCACCAU
AL-DP-4065
S
2112
5 UGAGGAGUCCAACAUCACCTT 3






AS
2113
3 TTACUCCUCAGGUUGUAGUGG 5


 


302
2243
ACAUCACCAUGCAGAUUAUGCGG
AL-DP-4100
S
2114
5 AUCACCAUGCAGAUUAUGCGG 3






AS
2115
3 UGUAGUGGUACGUCUAAUACGCC 5


 


305
2244
UCACCAUGCAGAUUAUGCGGAUC
AL-DP-4033
S
2116
5 ACCAUGCAGAUUAUGCGGATT 3






AS
2117
3 TTUGGUACGUCUAAUACGCCU 5


 


310
2245
AUGCAGAUUAUGCGGAUCAAACC
AL-DP-4101
S
2118
5 GCAGAUUAUGCGGAUCAAACC 3






AS
2119
3 UACGUCUAAUACGCCUAGUUUGG 5


 


312
2246
GCAGAUUAUGCGGAUCAAACCUC
AL-DP-4102
S
2120
5 AGAUUAUGCGGAUCAAACCUC 3






AS
2121
3 CGUCUAAUACGCCUAGUUUGGAG 5


 


315
2247
GAUUAUGCGGAUCAAACCUCACC
AL-DP-4034
S
2122
5 UUAUGCGGAUCAAACCUCATT 3






AS
2123
3 TTAAUACGCCUAGUUUGGAGU 5


 


316
2248
AUUAUGCGGAUCAAACCUCACCA
AL-DP-4113
S
2124
5 UAUGCGGAUCAAACCUCACTT 3






AS
2125
3 TTAUACGCCUAGUUUGGAGUG 5


 


317
2249
UUAUGCGGAUCAAACCUCACCAA
AL-DP-4114
S
2126
5 AUGCGGAUCAAACCUCACCTT 3






AS
2127
3 TTUACGCCUAGUUUGGAGUGG 5


 


319
2250
AUGCGGAUCAAACCUCACCAAGG
AL-DP-4002
S
2128
5 GCGGAUCAAACCUCACCAAGG 3






AS
2129
3 UACGCCUAGUUUGGAGUGGUUCC 5





AL-DP-4115
S
2130
5 GCGGAUCAAACCUCACCAA 3






AS
2131
3 CGCCUAGUUUGGAGUGGUU 5





AL-DP-4014
S
2132
5 GCGGAUCAAACCUCACCAATT 3






AS
2133
3 TTCGCCUAGUUUGGAGUGGUU 5





AL-DP-4119
S
2134
5 GCGGAUCAAACCUCACCAA 3






AS
2135
3 CGCCUAGUUUGGAGUGGUU 5


 


321
2251
GCGGAUCAAACCUCACCAAGGCC
AL-DP-4013
S
2136
5 GGAUCAAACCUCACCAAGGCC 3






AS
2137
3 CGCCUAGUUUGGAGUGGUUCCGG 5


 


341
2252
GCCAGCACAUAGGAGAGAUGAGC
AL-DP-4075
S
2138
5 CAGCACAUAGGAGAGAUGAGC 3






AS
2139
3 CGGUCGUGUAUCCUCUCUACUCG 5





AL-DP-4105
S
2140
5 CAGCACAUAGGAGAGAUGATT 3






AS
2141
3 TTGUCGUGUAUCCUCUCUACU 5


 


342
2253
CCAGCACAUAGGAGAGAUGAGCU
AL-DP-4050
S
2142
5 AGCACAUAGGAGAGAUGAGCU 3






AS
2143
3 GGUCGUGUAUCCUCUCUACUCGA 5





AL-DP-4106
S
2144
5 AGCACAUAGGAGAGAUGAGT T 3






AS
2145
3 TTUCGUGUAUCCUCUCUACUC 5


 


343
2254
CAGCACAUAGGAGAGAUGAGCUU
AL-DP-4094
S
2146
5 GCACAUAGGAGAGAUGAGCUU 3






AS
2147
3 GUCGUGUAUCCUCUCUACUCGAA 5





AL-DP-4118
S
2148
5 GCACAUAGGAGAGAUGAGCUU 3






AS
2149
3 CGUGUAUCCUCUCUACUCGAA 5





AL-DP-4107
S
2150
5 GCACAUAGGAGAGAUGAGCTT 3






AS
2151
3 TTCGUGUAUCCUCUCUACUCG 5





AL-DP-4122
S
2152
5 GCACAUAGGAGAGAUGAGC 3






AS
2153
3 CGUGUAUCCUCUCUACUCG 5


 


344
2255
AGCACAUAGGAGAGAUGAGCUUC
AL-DP-4012
S
2154
5 CACAUAGGAGAGAUGAGCUUC 3






AS
2155
3 UCGUGUAUCCUCUCUACUCGAAG 5





AL-DP-4108
S
2156
5 CACAUAGGAGAGAUGAGCUTT 3






AS
2157
3 TTGUGUAUCCUCUCUACUCGA 5


 


346
2256
CACAUAGGAGAGAUGAGCUUCCU
AL-DP-4051
S
2158
5 CAUAGGAGAGAUGAGCUUCCU 3






AS
2159
3 GUGUAUCCUCUCUACUCGAAGGA 5





AL-DP-4061
S
2160
5 CAUAGGAGAGAUGAGCUUCTT 3






AS
2161
3 TTGUAUCCUCUCUACUCGAAG 5


 


349
2257
AUAGGAGAGAUGAGCUUCCUACA
AL-DP-4082
S
2162
5 AGGAGAGAUGAGCUUCCUACA 3






AS
2163
3 UAUCCUCUCUACUCGAAGGAUGU 5


 


369
2258
ACAGCACAACAAAUGUGAAUGCA
AL-DP-4079
S
2164
5 AGCACAACAAAUGUGAAUGCA 3






AS
2165
3 UGUCGUGUUGUUUACACUUACGU 5


 


372
2259
GCACAACAAAUGUGAAUGCAGAC
AL-DP-4097
S
2166
5 ACAACAAAUGUGAAUGCAGAC 3






AS
2167
3 CGUGUUGUUUACACUUACGUCUG 5


 


379
2260
AAAUGUGAAUGCAGACCAAAGAA
AL-DP-4067
S
2168
5 AUGUGAAUGCAGACCAAAGTT 3






AS
2169
3 TTUACACUUACGUCUGGUUUC 5


 


380
2261
AAUGUGAAUGCAGACCAAAGAAA
AL-DP-4092
S
2170
5 UGUGAAUGCAGACCAAAGATT 3






AS
2171
3 TTACACUUACGUCUGGUUUCU 5


 


381
2262
AUGUGAAUGCAGACCAAAGAAAG
AL-DP-4004
S
2172
5 GUGAAUGCAGACCAAAGAAAG 3






AS
2173
3 UACACUUACGUCUGGUUUCUUUC 5





AL-DP-4117
S
2174
5 GUGAAUGCAGACCAAAGAAAG 3






AS
2175
3 CACUUACGUCUGGUUUCUUUC 5





AL-DP-4016
S
2176
5 GUGAAUGCAGACCAAAGAATT 3






AS
2177
3 TTCACUUACGUCUGGUUUCUU 5





AL-DP-4121
S
2178
5 GUGAAUGCAGACCAAAGAA 3






AS
2179
3 CACUUACGUCUGGUUUCUU 5


 


383
2263
GUGAAUGCAGACCAAAGAAAGAU
AL-DP-4005
S
2180
5 GAAUGCAGACCAAAGAAAGAU 3






AS
2181
3 CACUUACGUCUGGUUUCUUUCUA 5





AL-DP-4053
S
2182
5 GAAUGCAGACCAAAGAAAGTT 3






AS
2183
3 TTCUUACGUCUGGUUUCUUUC 5





Strand:


S = sense,


AS = Antisense






Example 2
Eg5 siRNA In Vitro Screening Via Cell Proliferation
As silencing of Eg5 has been shown to cause mitotic arrest (Weil, D, et al [2002] Biotechniques 33: 1244-8), a cell viability assay was used for siRNA activity screening. HeLa cells (14000 per well [Screens 1 and 3] or 10000 per well [Screen2])) were seeded in 96-well plates and simultaneously transfected with Lipofectamine 2000 (Invitrogen) at a final siRNA concentration in the well of 30 nM and at final concentrations of 50 nM (1st screen) and 25 nM (2nd screen). A subset of duplexes was tested at 25 nM in a third screen (Table 5).
Seventy-two hours post-transfection, cell proliferation was assayed the addition of WST-1 reagent (Roche) to the culture medium, and subsequent absorbance measurement at 450 nm. The absorbance value for control (non-transfected) cells was considered 100 percent, and absorbances for the siRNA transfected wells were compared to the control value. Assays were performed in sextuplicate for each of three screens. A subset of the siRNAs was further tested at a range of siRNA concentrations. Assays were performed in HeLa cells (14000 per well; method same as above, Table 5).







TABLE 5







Effects of Eg5 targeted duplexes on cell viability at 25 nM.












Relative absorbance at 450 nm

















Screen

Screen

Screen




I

II

III



Duplex
mean
sd
Mean
sd
mean
Sd
















AL-DP-6226
20
10
28
11
43
9


AL-DP-6227
66
27
96
41
108
33


AL-DP-6228
56
28
76
22
78
18


AL-DP-6229
17
3
31
9
48
13


AL-DP-6230
48
8
75
11
73
7


AL-DP-6231
8
1
21
4
41
10


AL-DP-6232
16
2
37
7
52
14


AL-DP-6233
31
9
37
6
49
12


AL-DP-6234
103
40
141
29
164
45


AL-DP-6235
107
34
140
27
195
75


AL-DP-6236
48
12
54
12
56
12


AL-DP-6237
73
14
108
18
154
37


AL-DP-6238
64
9
103
10
105
24


AL-DP-6239
9
1
20
4
31
11


AL-DP-6240
99
7
139
16
194
43


AL-DP-6241
43
9
54
12
66
19


AL-DP-6242
6
1
15
7
36
8


AL-DP-6243
7
2
19
5
33
13


AL-DP-6244
7
2
19
3
37
13


AL-DP-6245
25
4
45
10
58
9


AL-DP-6246
34
8
65
10
66
13


AL-DP-6247
53
6
78
14
105
20


AL-DP-6248
7
0
22
7
39
12


AL-DP-6249
36
8
48
13
61
7









The nine siRNA duplexes that showed the greatest growth inhibition in Table 5 were re-tested at a range of siRNA concentrations in HeLa cells. The siRNA concentrations tested were 100 nM, 33.3 nM, 11.1 nM, 3.70 nM, 1.23 nM, 0.41 nM, 0.14 nM and 0.046 nM. Assays were performed in sextuplicate, and the concentration of each siRNA resulting in fifty percent inhibition of cell proliferation (IC50) was calculated. This dose-response analysis was performed between two and four times for each duplex. Mean IC50 values (nM) are given in Table 6.







TABLE 6







IC50 of siRNA: cell proliferation in HeLa cells










Duplex
Mean IC50














AL-DP-6226
15.5



AL-DP-6229
3.4



AL-DP-6231
4.2



AL-DP-6232
17.5



AL-DP-6239
4.4



AL-DP-6242
5.2



AL-DP-6243
2.6



AL-DP-6244
8.3



AL-DP-6248
1.9










Example 3
Eg5 siRNA in Vitro Screening Via mRNA Inhibition
Directly before transfection, HeLa S3 (ATCC-Number: CCL-2.2, LCG Promochem GmbH, Wesel, Germany) cells were seeded at 1.5×104 cells/well on 96-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) in 75 μl of growth medium (Ham's F12, 10% fetal calf serum, 100 u penicillin/100 μg/ml streptomycin, all from Bookroom AG, Berlin, Germany). Transfections were performed in quadruplicates. For each well 0.5 μl Lipofectamine-2000 (Invitrogen GmbH, Karlsruhe, Germany) were mixed with 12 μl Opti-MEM (Invitrogen) and incubated for 15 min at room temperature. For the siRNA concentration being 50 nM in the 100 μl transfection volume, 1 μl of a 5 μM siRNA were mixed with 11.5 μl Opti-MEM per well, combined with the Lipofectamine-2000-Opti-MEM mixture and again incubated for 15 minutes at room temperature. siRNA-Lipofectamine-2000-complexes were applied completely (25 μl each per well) to the cells and cells were incubated for 24 h at 37° C. and 5% CO2 in a humidified incubator (Heroes GmbH, Hanau). The single dose screen was done once at 50 nM and at 25 nM, respectively.
Cells were harvested by applying 50 μl of lysis mixture (content of the QuantiGene bDNA-kit from Genospectra, Fremont, USA) to each well containing 100 μl of growth medium and were lysed at 53° C. for 30 min. Afterwards, 50 μl of the lists were incubated with probesets specific to human Eg5 and human GAPDH and proceeded according to the manufacturer's protocol for QuantiGene. In the end chemoluminescence was measured in a Victor2-Light (Perkin Elmer, Wiesbaden, Germany) as RLUs (relative light units) and values obtained with the hEg5 probeset were normalized to the respective GAPDH values for each well. Values obtained with siRNAs directed against Eg5 were related to the value obtained with an unspecific siRNA (directed against HCV) which was set to 100% (Tables 1b, 2b and 3b).
Effective siRNAs from the screen were further characterized by dose response curves.
Transfections of dose response curves were performed at the following concentrations: 100 nM, 16.7 nM, 2.8 nM, 0.46 nM, 77 picoM, 12.8 picoM, 2.1 picoM, 0.35 picoM, 59.5 fM, 9.9 fM and mock (no siRNA) and diluted with Opti-MEM to a final concentration of 12.5 μl according to the above protocol. Data analysis was performed by using the Microsoft Excel add-in software XL-fit 4.2 (IDBS, Guildford, Surrey, UK) and applying the dose response model number 205 (Tables 1b, 2b and 3b).
The lead siRNA AD 12115 was additionally analyzed by applying the WST-proliferation assay from Roche (as previously described).
A subset of 34 duplexes from Table 2 that showed greatest activity was assayed by transfection in HeLa cells at final concentrations ranging from 100 nM to 10 fM. Transfections were performed in quadruplicate. Two dose-response assays were performed for each duplex. The concentration giving 20% (IC20), 50% (IC50) and 80% (IC80) reduction of KSP mRNA was calculated for each duplex (Table 7).







TABLE 7







Dose response mRNA inhibition of Eg5/KSP duplexes in HeLa cells


Concentrations given in pM














IC20s

IC50s

IC80s



Duplex
1st
2nd
1st
2nd
1st
2nd


name
screen
screen
screen 
screen
screen 
screen
















AD12077
1.19
0.80
6.14
10.16
38.63
76.16


AD12078
25.43
25.43
156.18
156.18
ND
ND


AD12085
9.08
1.24
40.57
8.52
257.68
81.26


AD12095
1.03
0.97
9.84
4.94
90.31
60.47


AD12113
4.00
5.94
17.18
28.14
490.83
441.30


AD12115
0.60
0.41
3.79
3.39
23.45
23.45


AD12125
31.21
22.02
184.28
166.15 
896.85
1008.11


AD12134
2.59
5.51
17.87
22.00
116.36
107.03


AD12149
0.72
0.50
4.51
3.91
30.29
40.89


AD12151
0.53
6.84
4.27
10.72
22.88
43.01


AD12152
155.45 
7.56
867.36
66.69
13165.27
ND


AD12157
0.30
26.23
14.60
92.08 
14399.22
693.31


AD12166
0.20
0.93
3.71
3.86
46.28
20.59


AD12180
28.85
28.85
101.06 
101.06
847.21 
847.21


AD12185
2.60
0.42
15.55
13.91
109.80 
120.63


AD12194
2.08
1.11
5.37
5.09
53.03
30.92


AD12211
5.27
4.52
11.73
18.93 
26.74
191.07


AD12257
4.56
5.20
21.68
22.75
124.69
135.82


AD12280
2.37
4.53
6.89
20.23
64.80
104.82


AD12281
8.81
8.65
19.68
42.89
119.01
356.08


AD12282
7.71
456.42
20.09
558.00
ND
ND


AD12285
ND
1.28
57.30
7.31
261.79
42.53


AD12292
40.23 
12.00
929.11
109.10
ND
ND


AD12252
0.02
18.63
6.35
68.24 
138.09
404.91


AD12275
25.76
25.04
123.89 
133.10
1054.54
776.25


AD12266
4.85
7.80
10.00
32.94
41.67
162.65


AD12267
1.39
1.21
12.00
4.67
283.03 
51.12


AD12264
0.92
2.07
8.56
15.12 
56.36
196.78


AD12268
2.29
3.67
22.16
25.64 
258.27
150.84


AD12279
1.11
28.54
23.19
96.87 
327.28 
607.27


AD12256
7.20
33.52
46.49
138.04
775.54 
1076.76


AD12259
2.16
8.31
8.96
40.12 
50.05
219.42


AD12276
19.49
6.14
89.60
59.60 
672.51 
736.72


AD12321
4.67
4.91
24.88
19.43
139.50
89.49





(ND—not determined)






Example 4
Silencing of Liver Eg5/KSP in Juvenile Rats Following Single-Bolus Administration of LNP01 Formulated siRNA
From birth until approximately 23 days of age, Eg5/KSP expression can be detected in the growing rat liver. Target silencing with a formulated Eg5/KSP siRNA was evaluated in juvenile rats using duplex AD-6248.










KSP Duplex Tested










Duplex
Tar-




ID
get
Sense
Antisense





AD6248
KSP
AccGAAGuGuuGuuu
GGAcAAAcAAcACUUC




GuccTsT
GGUTsT




(SEQ ID NO: 1238)
(SEQ ID NO: 1239)









Methods
Dosing of Animals.
Male, juvenile Sprague-Dawley rats (19 days old) were administered single doses of lipidoid (“LNP01”) formulated siRNA via tail vein injection. Groups often animals received doses of 10 milligrams per kilogram (mg/kg) bodyweight of either AD6248 or an unspecific siRNA. Dose level refers to the amount of siRNA duplex administered in the formulation. A third group received phosphate-buffered saline. Animals were sacrificed two days after siRNA administration. Livers were dissected, flash frozen in liquid Nitrogen and pulverized into powders.
mRNA Measurements.
Levels of Eg5/KSP mRNA were measured in livers from all treatment groups. Samples of each liver powder (approximately ten milligrams) were homogenized in tissue lysis buffer containing proteinase K. Levels of Eg5/KSP and GAPDH mRNA were measured in triplicate for each sample using the Quantigene branched DNA assay (GenoSpectra). Mean values for Eg5/KSP were normalized to mean GAPDH values for each sample. Group means were determined and normalized to the PBS group for each experiment.
Statistical Analysis.
Significance was determined by ANOVA followed by the Tukey post-hoc test.
Results
Data Summary
Mean values (±standard deviation) for Eg5/KSP mRNA are given. Statistical significance (p value) versus the PBS group is shown (ns, not significant [p>0.05]).







TABLE 8







Experiment 1










KSP/GAPDH
p value
















PBS

1.0 ± 0.47




AD6248
10 mg/kg
0.47 ± 0.12 
<0.001



unspec
10 mg/kg
1.0 ± 0.26
ns










A statistically significant reduction in liver Eg5/KSP mRNA was obtained following treatment with formulated AD6248 at a dose of 10 mg/kg.
Example 5
Silencing of Rat Liver VEGF Following Intravenous Infusion of LNP01 Formulated VSP
A “lipidoid” formulation comprising an equimolar mixture of two siRNAs was administered to rats. As used herein, VSP refers to a composition having two siRNAs, one directed to Eg5/KSP and one directed to VEGF. For this experiment the duplex AD3133 directed towards VEGF and AD12115 directed towards Eg5/KSP were used. Since Eg5/KSP expression is nearly undetectable in the adult rat liver, only VEGF levels were measured following siRNA treatment.
siRNA Duplexes Administered (VSP)















Duplex
Tar-




ID
get
Sense
Antisense





AD12115
Eg5/
ucGAGAAucuAAA
AGUuAGUUuAGAU



KSP
cuAAcuTsT
UCUCGATsT




(SEQ ID NO:
(SEQ ID NO:




1240)
1241)


 


AD3133
VEGF
GcAcAuAGGAGA
AAGCUcAUCUCU




GAuGAGCUsU
CCuAuGuGCusG




(SEQ ID NO:
(SEQ ID NO:




1242)
1243)





Key


A,G,C,U-ribonucleotides;


c,u-2′-O-Me ribonucleotides;


s-phosphorothioate.






Unmodified versions of each strand and the targets for each siRNA are as follows















Eg5/
unmod sense
5′ UCGAGAAUCUAAACUAACUTT 3′
SEQ ID NO: 1534


KSP
unmod antisense
3′ TTAGUCCUUAGAUUUGAUUGA 5′
SEQ ID NO: 1535



target
5′ UCGAGAAUCUAAACUAACU 3′
SEQ ID NO: 1311


 


VEGF
unmod sense
5′ GCACAUAGGAGAGAUGAGCUU 3′
SEQ ID NO: 1536



unmod antisense
3′ GUCGUGUAUCCUCUCUACUCGAA 5′
SEQ ID NO: 1537



target
5′ GCACAUAGGAGAGAUGAGCUU 3′
SEQ ID NO: 1538









Methods

Dosing of animals. Adult, female Sprague-Dawley rats were administered lipidoid (“LNP01”) formulated siRNA by a two-hour infusion into the femoral vein. Groups of four animals received doses of 5, 10 and 15 milligrams per kilogram (mg/kg) bodyweight of formulated siRNA. Dose level refers to the total amount of siRNA duplex administered in the formulation. A fourth group received phosphate-buffered saline. Animals were sacrificed 72 hours after the end of the siRNA infusion. Livers were dissected, flash frozen in liquid Nitrogen and pulverized into powders.
Formulation Procedure
The lipidoid ND98.4HCl (MW 1487) (Formula 1, above), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) were used to prepare lipid-siRNA nanoparticles. Stock solutions of each in ethanol were prepared: ND98, 133 mg/mL; Cholesterol, 25 mg/mL, PEG-Ceramide C16, 100 mg/mL. ND98, Cholesterol, and PEG-Ceramide C16 stock solutions were then combined in a 42:48:10 molar ratio. Combined lipid solution was mixed rapidly with aqueous siRNA (in sodium acetate pH 5) such that the final ethanol concentration was 35-45% and the final sodium acetate concentration was 100-300 mM. Lipid-siRNA nanoparticles formed spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture was in some cases extruded through a polycarbonate membrane (100 nm cut-off) using a thermobarrel extruder (Lipex Extruder, Northern Lipids, Inc). In other cases, the extrusion step was omitted. Ethanol removal and simultaneous buffer exchange was accomplished by either dialysis or tangential flow filtration. Buffer was exchanged to phosphate buffered saline (PBS) pH 7.2.
Characterization of Formulations
Formulations prepared by either the standard or extrusion-free method are characterized in a similar manner. Formulations are first characterized by visual inspection.
They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles are measured by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be 20-300 nm, and ideally, 40-100 nm in size. The particle size distribution should be unimodal. The total siRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated siRNA is incubated with the RNA-binding dye Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, 0.5% Triton-X100. The total siRNA in the formulation is determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” siRNA content (as measured by the signal in the absence of surfactant) from the total siRNA content. Percent entrapped siRNA is typically >85%. For SNALP formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The preferred range is about at least 50 nm to about at least 110 nm, preferably about at least 60 nm to about at least 100 nm, most preferably about at least 80 nm to about at least 90 nm. In one example, each of the particle size comprises at least about 1:1 ratio of Eg5 dsRNA to VEGF dsRNA.
mRNA Measurements.
Samples of each liver powder (approximately ten milligrams) were homogenized in tissue lysis buffer containing proteinase K. Levels of VEGF and GAPDH mRNA were measured in triplicate for each sample using the Quantigene branched DNA assay (GenoSpectra). Mean values for VEGF were normalized to mean GAPDH values for each sample. Group means were determined and normalized to the PBS group for each experiment.
Protein Measurements.
Samples of each liver powder (approximately 60 milligrams) were homogenized in 1 ml RIPA buffer. Total protein concentrations were determined using the Micro BCA protein assay kit (Pierce). Samples of total protein from each animal was used to determine VEGF protein levels using a VEGF ELISA assay (R&D systems). Group means were determined and normalized to the PBS group for each experiment.
Statistical Analysis.
Significance was determined by ANOVA followed by the Tukey post-hoc test
Results
Data Summary
Mean values (±standard deviation) for mRNA (VEGF/GAPDH) and protein (rel. VEGF) are shown for each treatment group. Statistical significance (p value) versus the PBS group for each experiment is shown.












TABLE 9







VEGF/GAPDH
p value
rel VEGF
p value




















PBS
 1.0 ± 0.17

 1.0 ± 0.17



 5 mg/kg
0.74 ± 0.12
<0.05
0.23 ± 0.03
<0.001


10 mg/kg
0.65 ± 0.12
<0.005
0.22 ± 0.03
<0.001


15 mg/kg
0.49 ± 0.17
<0.001
0.20 ± 0.04
<0.001









Statistically significant reductions in liver VEGF mRNA and protein were measured at all three siRNA dose levels.
Example 6
Assessment of VSP SNALP in Mouse Models of Human Hepatic Tumors
These studies utilized a VSP siRNA cocktail containing dsRNAs targeting KSP/Eg5 and dsRNAs targeting VEGF. As used herein, VSP refers to a composition having two siRNAs, one directed to Eg5/KSP and one directed to VEGF. For this experiment the duplexes AD3133 (directed towards VEGF) and AD12115 (directed towards Eg5/KSP) were used. The siRNA cocktail was formulated in SNALPs.
The maximum study size utilized 20-25 mice. To test the efficacy of the siRNA SNALP cocktail to treat liver cancer, 1×10^6 tumor cells were injected directly into the left lateral lobe of test mice. The incisions were closed by sutures, and the mice allowed to recover for 2-5 hours. The mice were fully recovered within 48-72 hours. The SNALP siRNA treatment was initiated 8-11 days after tumor seeding.
The SNALP formulations utilized were (i) VSP (KSP+VEGF siRNA cocktail (1:1 molar ratio)); (ii) KSP (KSP+Luc siRNA cocktail); and (iii) VEGF (VEGF+Luc siRNA cocktail). All formulations contained equal amounts (mg) of each active siRNA. All mice received a total siRNA/lipid dose, and each cocktail was formulated into 1:57 cDMA SNALP (1.4% PEG-cDMA; 57.1% DLinDMA; 7.1% DPPC; and 34.3% cholesterol), 6:1 lipid:drug using original citrate buffer conditions.
Human Hep3B Study A: Anti-Tumor Activity of VSP-SNALP
Human Hepatoma Hep3B tumors were established in scid/beige mice by intrahepatic seeding. Group A (n=6) animals were administered PBS; Group B (n=6) animals were administered VSP SNALP; Group C (n=5) animals were administered KSP/Luc SNALP; and Group D (n=5) animals were administered VEGF/Luc SNALP.
SNALP treatment was initiated eight days after tumor seeding. The SNALP was dosed at 3 mg/kg total siRNA, twice weekly (Monday and Thursday), for a total of six doses (cumulative 18 mg/kg siRNA). The final dose was administered at day 25, and the terminal endpoint was at day 27.
Tumor burden was assayed by (a) body weight; (b) liver weight; (c) visual inspection+photography at day 27; (d) human-specific mRNA analysis; and (e) blood alpha-fetoprotein levels measured at day 27.
Table 10 below illustrates the results of visual scoring of tumor burden measured in the seeded (left lateral) liver lobe. Score: “−”=no visible tumor; “+”=evidence of tumor tissue at injection site; “++”=Discrete tumor nodule protruding from liver lobe; “+++”=large tumor protruding on both sides of liver lobe; “++++”=large tumor, multiple nodules throughout liver lobe.










TABLE 10







Mouse
Tumor Burden




















Group A: PBS, day 27
1
++++




2
++++




3
++




4
+++




5
++++




6
++++



Group B: VSP
1
+



(VEGF + KSP/Eg5, d. 27
2
−




3
−




4
−




5
++




6
−



Group C: KSP
1
+



(Luc + KSP), d. 27
2
++




3
−




4
+




5
++



Group D: VEGF
1
++++



(Luc + VEGF), d. 27
2
−




3
++++




4
+++




5
++++










Liver weights, as percentage of body weight, are shown in FIG. 1.
Body weights are shown in FIGS. 2A-2D.
From this study, the following conclusions were made. (1) VSP SNALP demonstrated potent anti-tumor effects in Hep3B 1H model; (2) the anti-tumor activity of the VSP cocktail appeared largely associated with the KSP component; (3) anti-KSP activity was confirmed by single dose histological analysis; and (4) VEGF siRNA showed no measurable effect on inhibition of tumor growth in this model.
Human Hep3B Study B: Prolonged Survival with VSP Treatment
In a second Hep3B study, human hepatoma Hep3B tumors were established by intrahepatic seeding into scid/beige mice. These mice were deficient for lymphocytes and natural killer (NK) cells, which is the minimal scope for immune-mediated anti-tumor effects. Group A (n=6) mice were untreated; Group B (n=6) mice were administered luciferase (luc) 1955 SNALP (Lot No. AP10-02); and Group C (n=7) mice were administered VSP SNALP (Lot No. AP10-01). SNALP was 1:57 cDMA SNALP, and 6:1 lipid:drug.
SNALP treatment was initiated eight days after tumor seeding. SNALP was dosed at 3 mg/kg siRNA, twice weekly (Mondays and Thursdays), for a total of six doses (cumulative 18 mg/kg siRNA). The final dose was delivered at day 25, and the terminal endpoint of the study was at day 27.
Tumor burden was assayed by (1) body weight; (2) visual inspection+photography at day 27; (3) human-specific mRNA analysis; and (4) blood alpha-fetoprotein measured at day 27.
Body weights were measured at each day of dosing (days 8, 11, 14, 18, 21, and 25) and on the day of sacrifice (FIG. 3).










TABLE 11








Tumor Burden




by macroscopic



Mouse
observation




















Group A: untreated,
A1R
++



day 27
A1G
++++




A1W
−




A2R
++++




A2G
+++




A2W
++++



Group B:
B1R
++++



1955 Luc SNALP, day 27
B1G
++++




B1W
+++




B2R
++




B2G
+++




B2W
++++



Group C:
C1R
−



VSP SNALP, day 27
C1G
−




C1B
−




C1W
+




C2R
+




C2G
+




C2W
−










Score: “−”=no visible tumor; “+”=evidence of tumor tissue at injection site; “++”=Discrete tumor nodule protruding from liver lobe; “+++”=large tumor protruding on both sides of liver lobe; “++++”=large tumor, multiple nodules throughout liver lobe.
The correlation between body weights and tumor burden are shown in FIGS. 4, 5 and 6.
A single dose of VSP SNALP (2 mg/kg) to Hep3B mice also resulted in the formation of mitotic spindles in liver tissue samples examined by histological staining.
Tumor burden was quantified by quantitative RT-PCR (pRT-PCR) (Taqman). Human GAPDH was normalized to mouse GAPDH via species-specific Taqman assays. Tumor score as shown by macroscopic observation in the table above correlated with GADPH levels (FIG. 7A).
Serum ELISA was performed to measure alpha-fetoprotein (AFP) secreted by the tumor. As described below, if levels of AFP go down after treatment, the tumor is not growing. Treatment with VSP lowered AFP levels in some animals compared to treatment with controls (FIG. 7B).
Human HepB3 Study C:
In a third study, human HCC cells (HepB3) were injected directly into the liver of SCID/beige mice, and treatment was initiated 20 days later. Group A animals were administered PBS; Group B animals were administered 4 mg/kg Luc-1955 SNALP; Group C animals were administered 4 mg/kg SNALP-VSP; Group D animals were administered 2 mg/kg SNALP-VSP; and Group E animals were administered 1 mg/kg SNALP-VSP. Treatment was with a single intravenous (iv) dose, and mice were sacrificed 24 hr. later.
Tumor burden and target silencing was assayed by qRT-PCR (Taqman). Tumor score was also measured visually as described above, and the results are shown in the following table. hGAPDH levels, as shown in FIG. 8, correlates with macroscopic tumor score as shown in the table below.










TABLE 12








Tumor Burden




by macroscopic



Mouse
observation




















Group A: PBS
A2
+++




A3
+++




A4
+++



Group B: 4 mg/kg Luc-
B1
+



1955 SNALP
B2
+++




B3
+++




B4
+++



Group C: 4 mg/kg
C1
++



SNALP-VSP
C2
++




C3
++




C4
+++



Group D: 2 mg/kg
D1
++



SNALP-VSP
D2
+




D3
+




D4
++



Group E: 1 mg/kg
E1
+++



SNALP-VSP
E2
+




E3
++




E4
+







Score:



“+” = variable tumor take/some small tumors;



“++” = Discrete tumor nodule protruding from liver lobe;



“+++” = large tumor protruding on both sides of liver lobe






Human (tumor-derived) KSP silencing was assayed by Taqman analysis and the results are shown in FIG. 10. hKSP expression was normalized to hGAPDH. About 80% tumor KSP silencing was observed at 4 mg/kg SNALP-VSP, and efficacy was evident at 1 mg/kg. The clear bars in FIG. 9 represent the results from small (low GAPDH) tumors.
Human (tumor-derived) VEGF silencing was assayed by Taqman analysis and the results are shown in FIG. 10. hVEGF expression was normalized to hGAPDH. About 60% tumor VEGF silencing was observed at 4 mg/kg SNALP-VSP, and efficacy was evident at 1 mg/kg. The clear bars in FIG. 10 represent the results from small (low GAPDH) tumors.
Mouse (liver-derived) VEGF silencing was assayed by Taqman analysis and the results are shown in FIG. 11A. mVEGF expression was normalized to hGAPDH. About 50% liver VEGF silencing was observed at 4 mg/kg SNALP-VSP, and efficacy was evident at 1 mg/kg.
Human HepB3 Study D: Contribution of Each dsRNA to Tumor Growth
In a fourth study, human HCC cells (HepB3) were injected directly into the liver of SCID/beige mice, and treatment was initiated 8 days later. Treatment was with intravenous (iv) bolus injections, twice weekly, for a total of six does. The final dose was administered at day 25, and the terminal endpoint was at day 27.
Tumor burden was assayed by gross histology, human-specific mRNA analysis (hGAPDH qPCR), and blood alpha-fetoprotein levels (serum AFP via ELISA).
In Study 1, Group A was treated with PBS, Group B was treated with SNALP-KSP+Luc (3 mg/kg), Group C was treated with SNALP-VEGF+Luc (3 mg/kg), and Group D was treated with ALN-VSP02 (3 mg/kg).
In Study 2, Group A was treated with PBS; Group B was treated with SNALP-KSP+Luc (1 mg/kg), Group C was treated with ALN-VSP02 (1 mg/kg).
Both GAPDH mRNA levels and serum AFP levels were shown to decrease after treatment with ALN-VSP02 (FIG. 11B).
Histology Studies:
Human hepatoma Hep3B tumors were established by intrahepatic seeding in mice. SNALP treatment was initiated 20 days after tumor seeding. Tumor-bearing mice (three per group) were treated with a single intravenous (IV) dose of (i) VSP SNALP or (ii) control (Luc) SNALP at 2 mg/kg total siRNA.
Liver/tumor samples were collected for conventional H&E histology 24 hours after single SNALP administration.
Large macroscopic tumor nodules (5-10 mm) were evident at necroscopy.
Effect of ALN-VSP in Hep3B Mice:
ALN-VSP (a cocktail of KSP dsRNA and VEGF dsRNA) treatment reduced tumor burden and expression of tumor-derived KSP and VEGF. GAPDH mRNA levels, a measure of tumor burden, were also observed to decline following administration of ALN-VSP dsRNA (see FIGS. 12A-12C). A decrease in tumor burden by visual macroscopic observation was also evident following administration of ALN-VSP.
A single IV bolus injection of ALN-VSP also resulted in mitotic spindle formation that was clearly detected in liver tissue samples from Hep3B mice. This observation indicated cell cycle arrest.
Example 7
Survival of SNALP-VSP Animals Versus SNALP-Luc Treated Animals
To test the effect of siRNA SNALP on survival rates of cancer subjects, tumors were established by intrahepatic seeding in mice and the mice were treated with SNALP-siRNA. These studies utilized a VSP siRNA cocktail containing dsRNAs targeting KSP/Eg5 and VEGF. Control was dsRNA targeting Luc. The siRNA cocktail was formulated in SNALPs.
Tumor cells (Human Hepatoma Hep3B, 1×10^6) were injected directly into the left lateral lobe of scid/beige mice. These mice were deficient for lymphocytes and natural killer (NK) cells, which is the minimal scope for immune-mediated anti-tumor effects. The incisions were closed by sutures, and the mice allowed to recover for 2-5 hours. The mice were fully recovered within 48-72 hours.
All mice received a total siRNA/lipid intravenous (iv) dose, and each cocktail was formulated into 1:57 cDMA SNALP (1.4% PEG-cDMA; 57.1% DLinDMA; 7.1% DPPC; and 34.3% cholesterol), 6:1 lipid:drug using original citrate buffer conditions.
siRNA-SNALP treatment was initiated on the day indicated below (18 or 26 days) after tumor seeding. siRNA-SNALP were administered twice a week for three weeks after 18 or 26 day is at a dose of 4 mg/kg. Survival was monitored and animals were euthanized based on humane surrogate endpoints (e.g., animal body weight, abdominal distension/discoloration, and overall health).
The survival data for treatment initiated 18 days after tumor seeing is summarized in Table 13, Table 14, and FIG. 13A.







TABLE 13







Kaplan-Meier (survival) data (% Surviving)










SNALP-
SNALP-


Day
Luc
VSP












18
100%
100%


22
100%
100%


25
100%
100%


27
100%
100%


28
100%
100%


28
86%
100%


29
86%
100%


32
86%
100%


33
86%
100%


33
43%
100%


35
43%
100%


36
43%
100%


36
29%
100%


38
29%
100%


38
14%
100%


38
14%
88%


40
14%
88%


43
14%
88%


45
14%
88%


49
14%
88%


51
14%
88%


51
14%
50%


53
14%
50%


53
14%
25%


55
14%
25%


57
14%
25%


57
0%
0%
















TABLE 14







Survival in days, for each animal.











Treatment




Animal
group
Survival













1
SNALP-Luc
28
days


2
SNALP-Luc
33
days


3
SNALP-Luc
33
days


4
SNALP-Luc
33
days


5
SNALP-Luc
36
days


6
SNALP-Luc
38
days


7
SNALP-Luc
57
days


8
SNALP-VSP
38
days


9
SNALP-VSP
51
days


10
SNALP-VSP
51
days


11
SNALP-VSP
51
days


12
SNALP-VSP
53
days


13
SNALP-VSP
53
days


14
SNALP-VSP
57
days


15
SNALP-VSP
57
days









FIG. 13A shows the mean survival of SNALP-VSP animals and SNALP-Luc treated animals versus days after tumor seeding. The mean survival of SNALP-VSP animals was extended by approximately 15 days versus SNALP-Luc treated animals.







TABLE 15







Serum alpha fetoprotein (AFP) concentration, for each animal, at a


time pre-treatment and at end of treatment (concentration in μg/ml)











End of



pre-Rx
Rx














1
SNALP-Luc
30.858
454.454


2
SNALP-Luc
10.088
202.082


3
SNALP-Luc
23.736
648.952


4
SNALP-Luc
1.696
13.308


5
SNALP-Luc
4.778
338.688


6
SNALP-Luc
15.004
826.972


7
SNALP-Luc
11.036
245.01


8
SNALP-VSP
37.514
182.35


9
SNALP-VSP
91.516
248.06


10
SNALP-VSP
25.448
243.13


11
SNALP-VSP
24.862
45.514


12
SNALP-VSP
57.774
149.352


13
SNALP-VSP
12.446
78.724


14
SNALP-VSP
2.912
9.61


15
SNALP-VSP
4.516
11.524









Tumor burden was monitored using serum AFP levels during the course of the experiment. Alpha-fetoprotein (AFP) is a major plasma protein produced by the yolk sac and the liver during fetal life. The protein is thought to be the fetal counterpart of serum albumin, and human AFP and albumin gene are present in tandem in the same transcriptional orientation on chromosome 4. AFP is found in monomeric as well as dimeric and trimeric forms, and binds copper, nickel, fatty acids and bilirubin. AFP levels decrease gradually after birth, reaching adult levels by 8-12 months. Normal adult AFP levels are low, but detectable. AFP has no known function in normal adults and AFP expression in adults is often associated with a subset of tumors such as hepatoma and teratoma. AFP is a tumor marker used to monitor testicular cancer, ovarian cancer, and malignant teratoma. Principle tumors that secrete AFP include endodermal sinus tumor (yolk sac carcinoma), neuroblastoma, hepatoblastoma, and heptocellular carcinoma. In patients with AFP-secreting tumors, serum levels of AFP often correlate with tumor size. Serum levels are useful in assessing response to treatment. Typically, if levels of AFP go down after treatment, the tumor is not growing. A temporary increase in AFP immediately following chemotherapy may indicate not that the tumor is growing but rather that it is shrinking (and releasing AFP as the tumor cells die). Resection is usually associated with a fall in serum levels. As shown in FIG. 14, tumor burden in SNALP-VSP treated animals was significantly reduced.
The experiment was repeated with SNALP-siRNA treatment at 26, 29, 32 35, 39, and 42 days after implantation. The data is shown in FIG. 13B. The mean survival of SNALP-VSP animals was extended by approximately 15 days versus SNALP-Luc treated animals by approximately 19 days, or 38%.
Example 8
Induction of Mono-asters in Established Tumors
Inhibition of KSP in dividing cells leads to the formation of mono asters that are readily observable in histological sections. To determine whether mono aster formation occurred in SNALP-VSP treated tumors, tumor bearing animals (three weeks after Hep3B cell implantation) were administered 2 mg/kg SNALP-VSP via tail vein injection. Control animals received 2 mg/kg SNALP-Luc. Twenty four hours later, animals were sacrificed, and tumor bearing liver lobes were processed for histological analysis. Representative images of H&E stained tissue sections are shown in FIG. 15. Extensive mono aster formation was evident in ALN VSP02 treated (A), but not SNALP-Luc treated (B), tumors. In the latter, normal mitotic figures were evident. The generation of mono asters is a characteristic feature of KSP inhibition and provides further evidence that SNALP-VSP has significant activity in established liver tumors.
Example 9
Manufacturing Process and Product Specification of ALN-VSP02 (SNALP-VSP)
ALN-VSP02 product contains 2 mg/mL of drug substance ALN-VSPDS01 formulated in a sterile lipid particle formulation (referred to as SNALP) for IV administration via infusion. Drug substance ALN-VSPDS01 consists of two siRNAs (ALN-12115 targeting KSP and ALN-3133 targeting VEGF) in an equimolar ratio. The drug product is packaged in mL glass vials with a fill volume of 5 mL.
The following terminology is used herein:















Drug

Single Strand



Substance
siRNA Duplexes
Intermediates









ALN-VSPDS01
ALN-12115*
Sense: A-19562





Antisense: A-19563




ALN-3133**
Sense: A-3981





Antisense: A-3982







*Alternate names = AD-12115, AD12115;



**Alternate names = AD-3133, AD3133






9.1 Preparation of drug substance ALN-VSPDS01
The two siRNA components of drug substance ALN-VSPDS01, ALN-12115 and ALN-3133, are chemically synthesized using commercially available synthesizers and raw materials. The manufacturing process consists of synthesizing the two single strand oligonucleotides of each duplex (A 19562 sense and A 19563 antisense of ALN 12115 and A 3981 sense and A 3982 antisense of ALN 3133) by conventional solid phase oligonucleotide synthesis using phosphoramidite chemistry and 5′ O dimethoxytriphenylmethyl (DMT) protecting group with the 2′ hydroxyl protected with tert butyldimethylsilyl (TBDMS) or the 2′ hydroxyl replaced with a 2′ methoxy group (2′ OMe). Assembly of an oligonucleotide chain by the phosphoramidite method on a solid support such as controlled pore glass or polystyrene. The cycle consists of 5′ deprotection, coupling, oxidation, and capping. Each coupling reaction is carried out by activation of the appropriately protected ribo, 2′ OMe, or deoxyribonucleoside amidite using 5 (ethylthio) 1H tetrazole reagent followed by the coupling of the free 5′ hydroxyl group of a support immobilized protected nucleoside or oligonucleotide. After the appropriate number of cycles, the final 5′ protecting group is removed by acid treatment. The crude oligonucleotide is cleaved from the solid support by aqueous methylamine treatment with concomitant removal of the cyanoethyl protecting group as well as nucleobase protecting groups. The 2′ O TBDMS group is then cleaved using a hydrogen fluoride containing reagent to yield the crude oligoribonucleotide, which is purified using strong anion exchange high performance liquid chromatography (HPLC) followed by desalting using ultrafiltration. The purified single strands are analyzed to confirm the correct molecular weight, the molecular sequence, impurity profile and oligonucleotide content, prior to annealing into the duplexes. The annealed duplex intermediates ALN 12115 and ALN 3133 are either lyophilized and stored at 20° C. or mixed in 1:1 molar ratio and the solution is lyophilized to yield drug substance ALN VSPDS01. If the duplex intermediates were stored as dry powder, they are redissolved in water before mixing. The equimolar ratio is achieved by monitoring the mixing process by an HPLC method.
The manufacturing process flow diagram is shown in FIG. 16.
Example specifications are shown in Table 16a.
The results of up to 12 month stability testing for ALN-VSPDS01 drug substance are shown in Tables 16c. The assay methods were chosen to assess physical property (appearance, pH, moisture), purity (by SEC and denaturing anion exchange chromatography) and potency (by denaturing anion exchange chromatography [AX-HPLC]).







TABLE 16a







Example specifications for ALN-VSPDS01









Test
Method
Acceptance Criteria





Appearance
Visual
White to off-white powder


Identity,
Duplex AX-HPLC
Duplex retention times are


ALN-VSPDS01

consistent with those of


ALN-3133

reference standards


ALN-12115


Identity,
MS
Molecular weight of single


ALN-VSPDS01

strands are within the




following ranges:




A-3981: 6869-6873 Da




A-3982: 7305-7309 Da




A-19562: 6762-6766 Da




A-19563: 6675-6679 Da


Sodium counter ion
Flame AAS or
Report data


(% w/w on
ICP-OES


anhydrous


basis)


ALN-VSPDS01
Denaturing
90-110%


assay
AX-HPLC


Purity of
SEC
≧90.0 area %


ALN-VSPDS01


Single strand purity,
Denaturing
Report data


ALN-VSPDS01
AX-HPLC
Report area % for




total impurities


siRNA molar ratio
Duplex AX-HPLC
1.0 ± 0.1


Moisture content
Karl Fischer
≦15%



titration


Residual solvents
HS-Capillary GC


Acetonitrile

≦410 ppm


Ethanol

≦5000 ppm


Isopropanol

≦5000 ppm


pH of 1% solution
USP <791>
Report data


Heavy metals
ICP-MS
Report data


As, Cd, Cu, Cr,


Fe, Ni, Pb, Sn


Bacterial endotoxins
USP <85>
≦0.5 EU/mg


Bioburden
Modified USP <61>
<100 CFU/g
















TABLE 16b







Stability of drug substance








Lot No.: A05M07001N
Study Storage Conditions: −20° C. (Storage Condition)












Acceptance
Results














Test
Method
Criteria
Initial
1 Month
3 Months
6 Months 
12 Months





Appearance
Visual
White to off-
Pass
Pass
Pass
Pass
Pass




white 









powder







pH
USP <791>
Report data
6.7
6.4
6.6
6.4
6.8


Moisture
Karl Fischer
≦15%
 3.6*
6.7
6.2
5.6
5.0


content
titration








(% w/w)









Purity (area









%)
SEC
≧90.0 area %
95
95
94
92
95


A-3981









(sense)
Denatur-
Report data
24
23
23
23
23


(area %)
ing AX-









HPLC








A-3982
Denatur-
Report data
23
23
23
23
24


(antisense)
ing AX-








(area %)
HPLC








A-19562
Denatur-
Report data
22
21
21
21
21


(sense)
ing AX-








(area %)
HPLC








A-19563
Denatur-
Report data
23
22
22
22
22


(antisense)
ing AX-








(area %)
HPLC









9.2 Preparation of drug product ALN-VSP02 (SNALP-VSP)
ALN VSP02, is a sterile formulation of the two siRNAs (in a 1:1 molar ratio) with lipid excipients in isotonic buffer. The lipid excipients associate with the two siRNAs, protect them from degradation in the circulatory system, and aid in their delivery to the target tissue. The specific lipid excipients and the quantitative proportion of each (shown in Table 17) have been selected through an iterative series of experiments comparing the physicochemical properties, stability, pharmacodynamics, pharmacokinetics, toxicity and product manufacturability of numerous different formulations. The excipient DLinDMA is a titratable aminolipid that is positively charged at low pH, such as that found in the endosome of mammalian cells, but relatively uncharged at the more neutral pH of whole blood. This feature facilitates the efficient encapsulation of the negatively charged siRNAs at low pH, preventing formation of empty particles, yet allows for adjustment (reduction) of the particle charge by replacing the formulation buffer with a more neutral storage buffer prior to use. Cholesterol and the neutral lipid DPPC are incorporated in order to provide physicochemical stability to the particles. The polyethyleneglycol lipid conjugate PEG2000 C DMA aids drug product stability, and provides optimum circulation time for the proposed use. ALN VSP02 lipid particles have a mean diameter of approximately 80-90 nm with low polydispersity values. A representative cryo transmission electron microscope (cryo TEM) image is shown in FIG. 17. At neutral pH, the particles are essentially uncharged, with Zeta Potential values of less than 6 mV. There is no evidence of empty (non loaded) particles based on the manufacturing process.







TABLE 17







Quantitative Composition of ALN-VSP02











Proportion



Component, grade
(mg/mL)














ALN-VSPDS01, cGMP
2.0*



DLinDMA
7.3



(1,2-Dilinoleyloxy-



N,N-dimethyl-3-aminopropane),



cGMP



DPPC (R-1,2-Dipalmitoyl-sn-glycero-3-
1.1



phosphocholine), cGMP



Cholesterol, Synthetic, cGMP
2.8



PEG2000-C-DMA
0.8



(3-N-[(ω-Methoxy poly(ethylene glycol) 2000)



carbamoyl]-1,2-dimyristyloxy-propylamine),



cGMP



Phosphate Buffered Saline, cGMP
q.s.







*The 1:1 molar ratio of the two siRNAs in the drug product is maintained throughout the size distribution of the drug product particles.






Solutions of lipid (in ethanol) and ALN VSPDS01 drug substance (in aqueous buffer) are mixed and diluted to form a colloidal dispersion of siRNA lipid particles with an average particle size of approximately 80-90 nm. This dispersion is then filtered through 0.45/0.2 μm filters, concentrated, and diafiltered by Tangential Flow Filtration. After in process testing and concentration adjustment to 2.0 mg/mL, the product is sterile filtered, aseptically filled into glass vials, stoppered, capped and placed at 5±3° C. The ethanol and all aqueous buffer components are USP grade; all water used is USP Sterile Water For Injection grade. Representative ALN-VSP02 process is shown in flow diagram in FIG. 18.







TABLE 18a







Example ALN-VSP02 specifications









Test
Analytical Procedure
Acceptance Criteria





Appearance
Visual
White to off-white, homogeneous




opalescent liquid, no foreign




particles


pH
USP <791>
6.8-7.8


Osmolality
USP <785>
250-350 mOsm/kg


Identity, ALN-VSPDS01
Duplex
Retention times consistent with


ALN-3133
Anion Exchange (AX)-HPLC
those of reference standards


ALN-12115


Identity, ALN-VSPDS01
Denaturing
Retention times consistent with


A-3981
AX-HPLC
those of reference standards


A-3982


A-19562


A-19563


Lipid identity
Reversed Phase (RP)-HPLC with
Retention times consistent with


DLinDMA
Evaporative Light Scattering
those of reference standards


PEG2000-C-DMA
(ELS) detection


DPPC


Cholesterol


ALN-VSPDS01 label claim
Denaturing
1.7-2.3 mg/mL


(Strength/Potency)
AX-HPLC


Duplex molar ratio
Duplex
1.0 ± 0.1



AX-HPLC


DLinDMA content
RP-HPLC with
5.6-10.3 mg/mL



ELS detection


PEG2000-C-DMA content
RP-HPLC with
0.6-1.1 mg/mL



ELS detection


DPPC content
RP-HPLC with
0.8-1.5 mg/mL



ELS detection


Cholesterol content
RP-HPLC with
2.1-3.9 mg/mL



ELS detection


Total lipid:ALN-VSPDS01 ratio
Calculated from total lipid assay
4.9-8.1 mg/mg



and label claim for drug



substance


ALN-VSPDS01 encapsulation
Fluorometric assay
≧90.0%


Purity
Denaturing
≧80.0 area %



AX-HPLC


Impurity profile
Denaturing
Report retention times (relative to



AX-HPLC
A-19563) and area % for all peaks




≧0.20%


Residual ethanol
USP <467>
≦5000 ppm


Residual EDTA
Ion Pairing (IP)-HPLC with UV
≦2000 μg/mL



detection


Particle size Z average
Dynamic light scattering
60-120 nm


Polydispersity
Dynamic light scattering
≦0.15


Particle size distribution
Dynamic light scattering
Report data


D10


D50


D90


Particulate matter
Modified USP <788>


≧25 μm

≦300 per container


≧10 μm

≦3000 per container


Bacterial endotoxins
Modified USP <85>
≦5.0 EU/mL


Sterility
USP <71>
Pass


Volume in container
USP <1>
≧5.0 mL


Dose uniformity
USP <905>
Pass


Heavy metal analysis
Inductive Coupled Plasma Mass
Report data



Spectrometry



(ICP-MS)









9.4 Container/Closure System
The ALN VSP02 drug product is packaged in 10 mL glass vials with a fill volume of 5 mL. The container closure system is comprised of a USP/EP Type I borosilicate glass vial, a teflon faced butyl rubber stopper and an aluminum flip off cap. The drug product will be stored at 5±3° C.
9.5 Stability of Drug Product ALN-VSP02
Stability data (25° C./60% RH) are given in Table 18b and 18c.







TABLE 18b







Example ALN-VSPO2 stability at storage conditions









Study Storage Conditions: 2-8° C.


Lot No.: IC097
Results

















Acceptance

1
2
3
4
6


Test
Method
Criteria
Initial
Month
Months
Months
Months
Months





Appearance
Visual
White to off-white,
Pass
Pass
Pass
Pass
Pass
Pass




homogeneous










opalescent liquid,










no foreign particles








pH
USP
6.8-7.8
7.4
7.4
7.4
7.3
7.4
7.3



<791>























Osmolality
USP
250-350
mOsm/kg
308
307
305
306
309
305
















<791>









ALN-
Duplex
Retention times
Pass
Pass
Pass
Pass
Pass
Pass


VSPDS01
AX-HPLC
consistent with








Identity,

those of reference








ALN-3133

standards








ALN-










12115










ALN-
Denaturin
Retention times
Pass
Pass
Pass
Pass
Pass
Pass


VSPDS01
g
consistent with








Identity,
AX-HPLC
those of reference








A-3981

standards








A-3982










A-19562










A-19563










Lipid
RP-HPLC
Retention times
Pass
Pass
Pass
Pass
Pass
Pass


identity,
with ELS
consistent with








DLinDMA
Detection
those of reference








PEGr000-

standards








C-DMA










DPPC










Cholesterol
























ALN-
Denaturin
1.7-2.3
mg/mL
2.1
2.2
2.1
2.1
2.1
2.1















VSPDS01
g









strength/
AX-HPLC









potency










Duplex
Duplex
1.0 ± 0.1
1.0
1.0
1.0
1.0
1.0
1.0


molar ratio
AX-HPLC























DLinDMA
RP-HPLC
5.6-10.3 
mg/mL
9.1
9.4
9.1
9.6
9.1
9.2















content
with ELS










Detection























Cholesterol 
RP-HPLC
2.1-3.9
mg/mL
3.4
3.5
3.4
3.5
3.4
3.5















content
with ELS










Detection























DPPC
RP-HPLC
0.8-1.5
mg/mL
1.3
1.3
1.4
1.4
1.2
1.3















content
with ELS










Detection























PEG2000-
RP-HPLC
0.6-1.1
mg/mL
1.0
1.0
1.0
1.1
1.0
1.0















C-DMA
with ELS









content
Detection























Total
Calcula-
4.9-8.1
mg/mg
7.0
6.9
7.1
7.4
7.0
7.1















lipid: ALN-
tion 









VSPDS01










ratio










ALN-
Fluorometric
≧90.0%
95.9
96.5
94.4
98.1
97.8
96.4


VSPDS01
assay









encapsu-










lation










Purity
Denaturin
≧80.0%
90.7
89.6
90.8
91.3
92.4
90.8



g










AX-HPLC























Particle
Light
60-120
nm
86
87
87
87
87
87















size,
scattering









Z-average










Polydispersity
Light
≦0.15
0.02
0.03
0.02
0.03
0.03
0.03



scattering









Particle
Light
Report data (nm)
56
56
56
56
56
56


size
scattering









distribution,










D10










Particle
Light
Report data (nm)
76
77
77
77
78
77


size
scattering









distribution,










D50










Particle
Light
Report data (nm)
110
112
112
113
112
113


size
scattering









distribution,










D90










Particulate
Modified
(per container)








matter,
USP
≦300
18
NS
NS
NS
NS
3


≧25 μm
<788>
≦3000
48




11


≧10 μm
























Bacterial
USP <85>
≦5.0
EU/mL
0.50
NS
NS
NS
NS
NS















endotoxins










Sterility
USP <71>
Pass
Pass
NS
NS
NS
NS
NS
















TABLE 18c







Example ALN-VSPO2 stability at 25° C./ambient humidity













Study Storage Conditions: 25° C./ambient humidity








Lot No.: IC097
Results

















Acceptance

1
2
3
4
6


Test
Method
Criteria
Initial
Month
Months
Months
Months
Months





Appearance
Visual
White to off-white,
Pass
Pass
Pass
Pass
Pass
Pass




homogeneous










opalescent liquid, 










no foreign particles








pH
USP <791>
6.8-7.8
7.4
7.3
7.2
7.1
7.2
7.1


Osmolality
USP <785>
250-350 mOsm/kg
308
306
304
307
307
304


ALN-










VSPDS01










Identity,
Duplex
Retention times
Pass
Pass
Pass
Pass
Pass
Pass


ALN-3133
AX-HPLC
consistent with








ALN-

those of reference








12115

standards








ALN-
Denaturing
Retention times
Pass
Pass
Pass
Pass
Pass
Pass


VSPDS01
AX-HPLC
consistent with








Identity,

those of reference








A-3981

standards








A-3982










A-19562










A-19563










Lipid
RP-HPLC
Retention times
Pass
Pass
Pass
Pass
Pass
Pass


identity,
with ELS
consistent with








DLinDMA
Detection
those of reference








PEG2000-

standards








C-DMA










DPPC










Cholesterol
























ALN-
Denaturing
1.7-2.3
mg/ mL
2.1
2.1
2.0
2.0
2.0
2.0















VSPDS01
AX-HPLC









strength/










potency










Duplex
Duplex
1.0 ± 0.1
1.0
1.0
1.0
1.0
1.0
1.0


molar ratio
AX-HPLC























DLinDMA
RP-HPLC
5.6-10.3
mg/ mL
9.1
9.6
9.0
9.3
9.2
9.3















content
with ELS










Detection























Cholesterol
RP-HPLC
2.1-3.9
mg/ mL
3.4
3.5
3.4
3.5
3.4
3.5















content
with ELS










Detection























DPPC
RP-HPLC
0.8-1.5
mg/ mL
1.3
1.3
1.3
1.2
1.2
1.1















content
with ELS










Detection























PEG2000-
RP-HPLC
0.6-1.1
mg/ mL
1.0
1.0
1.0
1.1
1.0
1.0















C-DMA
with ELS









content
Detection























Total
Calculation
4.9-8.1
mg/ mg
7.0
7.3
7.4
7.6
7.4
7.5















lipid: ALN-










VSPDS01










ratio










ALN-
Fluorometri
≧90.0%
95.9
97.2
94.6
97.9
97.9
96.7


VSPDS01
c assay









encapsulation










Purity
Denaturing
≧80.0%
90.7
88.0
88.9
88.4
89.0
85.3



AX-HPLC























Particle
Light
60-120
nm
86
85
86
89
87
87















size,
scattering









Z-average










Polydispersity
Light
≦0.15
0.02
0.05
0.03
0.04
0.04
0.03



scattering









Particle
Light
Report data (nm)
56
54
56
58
56
57


size
scattering









distribution,










D10










Particle
Light
Report data (nm)
76
75
77
79
77
78


size
scattering









distribution,










D50










Particle
Light
Report data (nm)
110
110
111
116
113
113


size
scattering









distribution, 










D90










Particulate
Modified
(per container)








matter,
USP <788>
≦300
18
NS
NS
NS
NS
1


≧25 μm

≦3000
48




16


≧10 μm
























Bacterial
USP <85>
≦5.0
EU/mL
0.50
NS
NS
NS
NS
<0.50















endotoxins










Sterility
USP <71>
Pass
Pass
NS
NS
NS
NS
Pass









Example 10
In Vitro Efficacy of ALN-VSP02 in Human Cancer Cell Lines
The efficacy of ALN-VSP02 treatment in human cancer cell lines was determined via measurement of KSP mRNA, VEGF mRNA, and cell viability after treatment. IC50 (nM) values determined for KSP and VEGF in each cell line.







TABLE 19







cell lines










Cell line tested
ATCC cat number







HELA
ATCC Cat N: CCL-2



KB
ATCC Cat N: CCL-17



HEP3B
ATCC Cat N: HB-8064



SKOV-3
ATCC Cat N: HTB-77



HCT-116
ATCC Cat N: CCL-247



HT-29
ATCC Cat N: HTB-38



PC-3
ATCC Cat N: CRL-1435



A549
ATCC Cat N: CCL-185



MDA-MB-231
ATCC Cat N: HTB-26










Cells were plated in 96 well plates in complete media at day 1 to reach a density of 70% on day 2. On day 2 media was replaced with Opti-MEM reduced serum media (Invitrogen Cat N: 11058-021) and cells were transfected with either ALN-VSP02 or control SNALP-Luc with concentration range starting at 1.8 μM down to 10 μM. After 6 hours the media was changed to complete media. Three replicate plates for each cell line for each experiment was done.
Cells were harvested 24 hours after transfection. KSP levels were measured using bDNA; VEGF mRNA levels were measured using human TaqMan assay.
Viability was measured using Cell Titer Blue reagent (Promega Cat N: G8080) at 48 and/or 72 h following manufacturer's recommendations.
As shown in Table 20, nM concentrations of VSP02 are effective in reducing expression of both KSP and VEGF in multiple human cell lines. Viability of treated cells was not







TABLE 20







Results












IC50 (nM)
IC50 (nM)



Cell line
KSP
VEGF















HeLa
8.79
672



SKOV-3
142
1347



HCT116
31.6
27.5



Hep3B
1.3
14.5



HT-29
262
ND



PC3
127
ND



KB
50.6
ND



A549
201
ND



MB231
187
ND










Example 11
Anti-Tumor Efficacy of VSP SNALP Vs. Sorafenib in Established Hep3B Intrahepatic Tumors
The anti-tumor effects of multi-dosing VSP SNALP verses Sorafenib in scid/beige mice bearing established Hep3B intrahepatic tumors was studied. Sorafenib is a small molecule inhibitor of protein kinases approved for treatment of hepatic cellular carcinoma (HCC).
Tumors were established by intrahepatic seeding in scid/beige mice as described herein. Treatment was initiated 11 days post-seeding. Mice were treated with Sorafenib and a control siRNA-SNALP, Sorafenib and VSP siRNA-SNALP, or VSP siRNA-SNALP only. Control mice were treated with buffers only (DMSO for Sorafenib and PBS for siRNA-SNALP). Sorafenib was administered intraparenterally from Mon to Fri for three weeks, at mg/kg according to body weight for a total of 15 injections. Sorafenib was administered a minimum of 1 hour after SNALP injections. The siRNA-SNALPS were administered intravenously via the lateral tail vein according at 3 mg/kg based on the most recently recorded body weight (10 ml/kg) for 3 weeks (total of 6 doses) on days 1, 4, 7, 10, 14, and 17.
Mice were euthanized based on an assessment of tumor burden including progressive weight loss and clinical signs including condition, abdominal distension/discoloration and mobility.
The percent survival data are shown in FIG. 19. Co-administration of VSP siRNA-SNALP with Sorafenib increased survival proportion compared to administration of Sorafenib or VSP siRNA-SNALP alone. VSP siRNA-SNALP increased survival proportion compared to Sorafenib.
Example 12
In Vitro Efficacy of VSP Using Variants of AD-12115 and AD-3133
Two sets of duplexes targeted to Eg5/KSP and VEGF were designed and synthesized. Each set included duplexes tiling 10 nucleotides in each direction of the target sites for either AD-12115 and AD-3133.
Sequences of the target, sense strand, and antisense strand for each duplex are shown in the Table below.
Each duplex is assayed for inhibition of expression using the assays described herein.
The duplexes are administered alone and/or in combination, e.g., an Eg5/KSP dsRNA in combination with a VEGF dsRNA. In some embodiments, the dsRNA are administered in a SNALP formulation as described herein.







TABLE 21







Sequences of dsRNA targeted to VEGF and Eg5/KSP (tiling)















SEQ
Sense Strand
SEQ


Duplex
target
target sequence
ID
Antisense strand
ID


ID
gene
5′ to 3′
NO:
5′ to 3′
NO:





AD-
VEGFA
ACCAAGGCCAGCACAUAGG
2264
AccAAGGccAGcAcAuAGGTsT
2304


20447.1



CCuAUGUGCUGGCCUUGGUTsT
2305


 


AD-
VEGFA
CCAAGGCCAGCACAUAGGA
2265
ccAAGGccAGcAcAuAGGATsT
2306


20448.1



UCCuAUGUGCUGGCCUUGGTsT
2307


 


AD-
VEGFA
CCAAGGCCAGCACAUAGGA
2266
ccAAGGccAGcAcAuAGGATsT
2308


20449.1



CUCCuAUGUGCUGGCCUUGTsT
2309


 


AD-
VEGFA
AAGGCCAGCACAUAGGAGA
2267
AAGGccAGcAcAuAGGAGATsT
2310


20450.1



UCUCCuAUGUGCUGGCCUUTsT
2311


 


AD-
VEGFA
AGGCCAGCACAUAGGAGAG
2268
AGGccAGcAcAuAGGAGAGTsT
2312


20451.1



CUCUCCuAUGUGCUGGCCUTsT
2313


 


AD-
VEGFA
GGCCAGCACAUAGGAGAGA
2269
GGccAGcAcAuAGGAGAGATsT
2314


20452.1



UCUCUCCuAUGUGCUGGCCTsT
2315


 


AD-
VEGFA
GCCAGCACAUAGGAGAGAU
2270
GccAGcAcAuAGGAGAGAuTsT
2316


20453.1



AUCUCUCCuAUGUGCUGGCTsT
2317


 


AD-
VEGFA
CCAGCACAUAGGAGAGAUG
2271
ccAGcAcAuAGGAGAGAuGTsT
2318


20454.1



cAUCUCUCCuAUGUGCUGGTsT
2319


 


AD-
VEGFA
CAGCACAUAGGAGAGAUGA
2272
cAGcAcAuAGGAGAGAuGATsT
2320


20455.1



UcAUCUCUCCuAUGUGCUGTsT
2321


 


AD-
VEGFA
AGCACAUAGGAGAGAUGAG
2273
AGcAcAuAGGAGAGAuGAGTsT
2322


20456.1



CUcAUCUCUCCuAUGUGCUTsT
2323


 


AD-
VEGFA
CACAUAGGAGAGAUGAGCU
2274
cAcAuAGGAGAGAuGAGcuTsT
2324


20457.1



AGCUcAUCUCUCCuAUGUGTsT
2325


 


AD-
VEGFA
ACAUAGGAGAGAUGAGCUU
2275
AcAuAGGAGAGAuGAGcuuTsT
2326


20458.1



AAGCUcAUCUCUCCuAUGUTsT
2327


 


AD-
VEGFA
CAUAGGAGAGAUGAGCUUC
2276
cAuAGGAGAGAuGAGcuucTsT
2328


20459.1



GAAGCUcAUCUCUCCuAUGTsT
2329


 


AD-
VEGFA
AUAGGAGAGAUGAGCUUCC
2277
AuAGGAGAGAuGAGcuuccTsT
2330


20460.1



GGAAGCUcAUCUCUCCuAUTsT
2331


 


AD-
VEGFA
UAGGAGAGAUGAGCUUCCU
2278
uAGGAGAGAuGAGcuuccuTsT
2332


20461.1



AGGAAGCUcAUCUCUCCuATsT
2333


 


AD-
VEGFA
AGGAGAGAUGAGCUUCCUA
2279
AGGAGAGAuGAGcuuccuATsT
2334


20462.1



uAGGAAGCUcAUCUCUCCUTsT
2335


 


AD-
VEGFA
GGAGAGAUGAGCUUCCUAC
2280
GGAGAGAuGAGcuuccuAcTsT
2336


20463.1



GuAGGAAGCUcAUCUCUCCTsT
2337


 


AD-
VEGFA
GAGAGAUGAGCUUCCUACA
2281
GAGAGAuGAGcuuccuAcATsT
2338


20464.1



UGuAGGAAGCUcAUCUCUCTsT
2339


 


AD-
VEGFA
AGAGAUGAGCUUCCUACAG
2282
AGAGAuGAGcuuccuAcAGTsT
2340


20465.1



CUGuAGGAAGCUcAUCUCUTsT
2341


 


AD-
VEGFA
GAGAUGAGCUUCCUACAGC
2283
GAGAuGAGcuuccuAcAGcTsT
2342


20466.1



GCUGuAGGAAGCUcAUCUCTsT
2343


 


AD-
KSP
AUGUUCCUUAUCGAGAAUC
2284
AuGuuccuuAucGAGAAucTsT
2344


20467.1



GAUUCUCGAuAAGGAAcAUTsT
2345


 


AD-
KSP
UGUUCCUUAUCGAGAAUCU
2285
uGuuccuuAucGAGAAucuTsT
2346


20468.1



AGAUUCUCGAuAAGGAAcATsT
2347


 


AD-
KSP
GUUCCUUAUCGAGAAUCUA
2286
GuuccuuAucGAGAAucuATsT
2348


20469.1



uAGAUUCUCGAuAAGGAACTsT
2349


 


AD-
KSP
UUCCUUAUCGAGAAUCUAA
2287
uuccuuAucGAGAAucuAATsT
2350


20470.1



UuAGAUUCUCGAuAAGGAATsT
2351


 


AD-
KSP
UCCUUAUCGAGAAUCUAAA
2288
uccuuAucGAGAAucuAAATsT
2352


20471.1



UUuAGAUUCUCGAuAAGGATsT
2353


 


AD-
KSP
CCUUAUCGAGAAUCUAAAC
2289
ccuuAucGAGAAucuAAAcTsT
2354


20472.1



GUUuAGAUUCUCGAuAAGGTsT
2355


 


AD-
KSP
CUUAUCGAGAAUCUAAACU
2290
cuuAucGAGAAucuAAAcuTsT
2356


20473.1



AGUUuAGAUUCUCGAuAAGTsT
2357


 


AD-
KSP
UUAUCGAGAAUCUAAACUA
2291
uuAucGAGAAucuAAAcuATsT
2358


20474.1



uAGUUuAGAUUCUCGAuAATsT
2359


 


AD-
KSP
UAUCGAGAAUCUAAACUAA
2292
uAucGAGAAucuAAAcuAATsT
2360


20475.1



UuAGUUuAGAUUCUCGAuATsT
2361


 


AD-
KSP
AUCGAGAAUCUAAACUAAC
2293
AucGAGAAucuAAAcuAAcTsT
2362


20476.1



GUuAGUUuAGAUUCUCGAUTsT
2363


 


AD-
KSP
CGAGAAUCUAAACUAACUA
2294
cGAGAAucuAAAcuAAcuATsT
2364


20477.1



uAGUuAGUUuAGAUUCUCGTsT
2365


 


AD-
KSP
GAGAAUCUAAACUAACUAG
2295
GAGAAucuAAAcuAAcuAGTsT
2366


20478.1



CuAGUuAGUUuAGAUUCUCTsT
2367


 


AD-
KSP
AGAAUCUAAACUAACUAGA
2296
AGAAucuAAAcuAAcuAGATsT
2368


20479.1



UCuAGUuAGUUuAGAUUCUTsT
2369


 


AD-
KSP
GAAUCUAAACUAACUAGAA
2297
GAAucuAAAcuAAcuAGAATsT
2370


20480.1



UUCuAGUuAGUUuAGAUUCTsT
2371


 


AD-
KSP
AAUCUAAACUAACUAGAAU
2298
AAucuAAAcuAAcuAGAAuTsT
2372


20481.1



AUUCuAGUuAGUUuAGAUUTsT
2373


 


AD-
KSP
AUCUAAACUAACUAGAAUC
2299
AucuAAAcuAAcuAGAAucTsT
2374


20482.1



GAUUCuAGUuAGUUuAGAUTsT
2375


 


AD-
KSP
UCUAAACUAACUAGAAUCC
2300
ucuAAAcuAAcuAGAAuccTsT
2376


20483.1



GGAUUCuAGUuAGUUuAGATsT
2377


 


AD-
KSP
CUAAACUAACUAGAAUCCU
2301
cuAAAcuAAcuAGAAuccuTsT
2378


20484.1



AGGAUUCuAGUuAGUUuAGTsT
2379


 


AD-
KSP
UAAACUAACUAGAAUCCUC
2302
uAAAcuAAcuAGAAuccucTsT
2380


20485.1



GAGGAUUCuAGUuAGUUuATsT
2381


 


AD-
KSP
AAACUAACUAGAAUCCUCC
2303
AAAcuAAcuAGAAuccuccTsT
2382


20486.1



GGAGGAUUCuAGUuAGUUUTsT
2383









Example 13
VEGF Targeted dsRNA with a Single Blunt End
A set duplexes targeted to VEGF were designed and synthesized. The set included duplexes tiling 10 nucleotides in each direction of the target sites for AD-3133. Each duplex includes a 2 base overhang at the end corresponding to the 3′ end of the antisense strand and no overhang, e.g., a blunt end, at the end corresponding to the 5′ end of the antisense strand.
The sequences of each strand of these duplexes are shown in the following table.
Each duplex is assayed for inhibition of expression using the assays described herein. The VEGF duplexes are administered alone and/or in combination with an Eg5/KSP dsRNA (e.g., AD-12115). In some embodiments, the dsRNA are administered in a SNALP formulation as described herein.







TABLE 22







Target sequences of blunt ended


dsRNA targeted to VEGF













posi-



SEQ

tion



ID
VEGF target sequence
on VEGF


duplex ID
NO:
5′ to 3′
gene





AD-20447.1
2384
ACCAAGGCCAGCACAUAGG
1365


 


AD-20448.1
2385
CCAAGGCCAGCACAUAGGA
1366


 


AD-20449.1
2386
CAAGGCCAGCACAUAGGAG
1367


 


AD-20450.1
2387
AAGGCCAGCACAUAGGAGA
1368


 


AD-20451.1
2388
AGGCCAGCACAUAGGAGAG
1369


 


AD-20452.1
2389
GGCCAGCACAUAGGAGAGA
1370


 


AD-20453.1
2390
GCCAGCACAUAGGAGAGAU
1371


 


AD-20454.1
2391
CCAGCACAUAGGAGAGAUG
1372


 


AD-20455.1
2392
CAGCACAUAGGAGAGAUGA
1373


 


AD-20456.1
2393
AGCACAUAGGAGAGAUGAG
1374


 


AD-20457.1
2394
CACAUAGGAGAGAUGAGCU
1376


 


AD-20458.1
2395
ACAUAGGAGAGAUGAGCUU
1377


 


AD-20459.1
2396
CAUAGGAGAGAUGAGCUUC
1378


 


AD-20460.1
2397
AUAGGAGAGAUGAGCUUCC
1379


 


AD-20461.1
2398
UAGGAGAGAUGAGCUUCCU
1380


 


AD-20462.1
2399
AGGAGAGAUGAGCUUCCUA
1381


 


AD-20463.1
2400
GGAGAGAUGAGCUUCCUAC
1382


 


AD-20464.1
2401
GAGAGAUGAGCUUCCUACA
1383


 


AD-20465.1
2402
AGAGAUGAGCUUCCUACAG
1384


 


AD-20466.1
2403
GAGAUGAGCUUCCUACAGC
1385
















TABLE 23







Strand sequences of blunt ended dsRNAtargetedto VEGF













SEQ

SEQ



Sense strand
ID
Antisense strand
ID


duplex ID
(5′ to 3′)
NO:
(5′ to 3′)
NO:





AD-20447.1
ACCAAGGCCAGCACAUAGGAG
2404
CUCCUAUGUGCUGGCCUUGGUGA
2424


 


AD-20448.1
CCAAGGCCAGCACAUAGGAGA
2405
UCUCCUAUGUGCUGGCCUUGGUG
2425


 


AD-20449.1
CAAGGCCAGCACAUAGGAGAG
2406
CUCUCCUAUGUGCUGGCCUUGGU
2426


 


AD-20450.1
AAGGCCAGCACAUAGGAGAGA
2407
UCUCUCCUAUGUGCUGGCCUUGG
2427


 


AD-20451.1
AGGCCAGCACAUAGGAGAGAU
2408
AUCUCUCCUAUGUGCUGGCCUUG
2428


 


AD-20452.1
GGCCAGCACAUAGGAGAGAUG
2409
CAUCUCUCCUAUGUGCUGGCCUU
2429


 


AD-20453.1
GCCAGCACAUAGGAGAGAUGA
2410
UCAUCUCUCCUAUGUGCUGGCCU
2430


 


AD-20454.1
CCAGCACAUAGGAGAGAUGAG
2411
CUCAUCUCUCCUAUGUGCUGGCC
2431


 


AD-20455.1
CAGCACAUAGGAGAGAUGAGC
2412
GCUCAUCUCUCCUAUGUGCUGGC
2432


 


AD-20456.1
AGCACAUAGGAGAGAUGAGCU
2413
AGCUCAUCUCUCCUAUGUGCUGG
2433


 


AD-20457.1
CACAUAGGAGAGAUGAGCUUC
2414
GAAGCUCAUCUCUCCUAUGUGCU
2434


 


AD-20458.1
ACAUAGGAGAGAUGAGCUUCC
2415
GGAAGCUCAUCUCUCCUAUGUGC
2435


 


AD-20459.1
CAUAGGAGAGAUGAGCUUCCU
2416
AGGAAGCUCAUCUCUCCUAUGUG
2436


 


AD-20460.1
AUAGGAGAGAUGAGCUUCCUA
2417
UAGGAAGCUCAUCUCUCCUAUGU
2437


 


AD-20461.1
UAGGAGAGAUGAGCUUCCUAC
2418
GUAGGAAGCUCAUCUCUCCUAUG
2438


 


AD-20462.1
AGGAGAGAUGAGCUUCCUACA
2419
UGUAGGAAGCUCAUCUCUCCUAU
2439


 


AD-20463.1
GGAGAGAUGAGCUUCCUACAG
2420
CUGUAGGAAGCUCAUCUCUCCUA
2440


 


AD-20464.1
GAGAGAUGAGCUUCCUACAGC
2421
GCUGUAGGAAGCUCAUCUCUCCU
2441


 


AD-20465.1
AGAGAUGAGCUUCCUACAGCA
2422
UGCUGUAGGAAGCUCAUCUCUCC
2442


 


AD-20466.1
GAGAUGAGCUUCCUACAGCAC
2423
GUGCUGUAGGAAGCUCAUCUCUC
2443









Example 14
Inhibition of Eg5/KSP and VEGF Expression in Humans
A human subject is treated with a pharmaceutical composition, e.g., ALNVSP02, having both a SNALP formulated dsRNA targeted to a Eg5/KSP gene and a SNALP formulated dsRNA targeted to a VEGF gene to inhibit expression of the Eg5/KSP and VEGF genes.
A subject in need of treatment is selected or identified. The subject can be in need of cancer treatment, e.g., liver cancer.
At time zero, a suitable first dose of the composition is subcutaneously administered to the subject. The composition is formulated as described herein. After a period of time, the subject's condition is evaluated, e.g., by measurement of tumor growth, measuring serum AFP levels, and the like. This measurement can be accompanied by a measurement of Eg5/KSP and/or VEGF expression in said subject, and/or the products of the successful siRNA-targeting of Eg5/KSP and/or VEGF mRNA. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.
After treatment, the subject's condition is compared to the condition existing prior to the treatment, or relative to the condition of a similarly afflicted but untreated subject.
Those skilled in the art are familiar with methods and compositions in addition to those specifically set out in the present disclosure which will allow them to practice this invention to the full scope of the claims hereinafter appended.

## Claims

1. A composition comprising a first double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a human kinesin family member 11 (Eg5/KSP) gene in a cell and a second dsRNA for inhibiting expression of a human VEGF in a cell, wherein:
both said first and said second dsRNA are formulated in a stable nucleic acid lipid particle (SNALP) wherein said SNALP comprises DLinDMA, cholesterol, DPPC, and PEG2000-C-DMA;
said first dsRNA consists of a first sense strand and a first antisense strand, and said first sense strand comprises a first sequence and said first antisense strand comprises a second sequence complementary to at least 15 contiguous nucleotides of SEQ ID NO:1311 (5′-UCGAGAAUCUAAACUAACU-3′), wherein said first sequence is complementary to said second sequence and wherein said first dsRNA is between 15 and 30 base pairs in length; and
said second dsRNA consists of a second sense strand and a second antisense strand, said second sense strand comprising a third sequence and said second antisense strand comprising a fourth sequence complementary to at least 15 contiguous nucleotides of SEQ ID NO:1538 (5′-GCACAUAGGAGAGAUGAGCUU-3′), wherein said third sequence is complementary to said fourth sequence and wherein each strand is between 15 and 30 base pairs in length.

2. The composition of claim 1, wherein the first antisense strand comprises a second sequence complementary to SEQ ID NO:1311 (5′-UCGAGAAUCUAAACUAACU-3′) and the second antisense strand comprises a fourth sequence complementary to SEQ ID NO:1538 (5′-GCACAUAGGAGAGAUGAGCUU-3′).

3. The composition of claim 1, wherein the first dsRNA consists of a sense strand consisting of SEQ ID NO:1534 (5′-UCGAGAAUCUAAACUAACUTT-3′) and an antisense strand consisting of SEQ ID NO:1535 (5′-AGUUAGUUUAGAUUCUCGATT-3′) and the second dsRNA consists of a sense strand consisting of SEQ ID NO:1536 (5′-GCACAUAGGAGAGAUGAGCUU-3′), and an antisense strand consisting of SEQ ID NO:1537 (5′-AAGCUCAUCUCUCCUAUGUGCUG-3′).

4. The composition of claim 3, wherein each strand is modified as follows to include a 2′-O-methyl ribonucleotide as indicated by a lower case letter “c” or “u” and a phosphorothioate as indicated by a lower case letter “s”:
the first dsRNA consists of a sense strand consisting of
SEQ ID NO:1240 (5′-ucGAGAAucuAAAcuAAcuTsT-3′)
and an antisense strand consisting of
SEQ ID NO:1241 (5′-AGUuAGUUuAGAUUCUCGATsT);
the second dsRNA consists of a sense strand consisting of
SEQ ID NO:1242 (5′-GcAcAuAGGAGAGAuGAGCUsU-3′)
and an antisense strand consisting of
SEQ ID NO:1243 (5′-AAGCUcAUCUCUCCuAuGuGCusG-3′).

5. The composition of claim 1, 2, or 3, wherein said first and second dsRNA comprises at least one modified nucleotide.

6. The composition of claim 5, wherein said modified nucleotide is chosen from the group of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group.

7. The composition of claim 5, wherein said modified nucleotide is chosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.

8. The composition of claim 1, 2, or 3, wherein said first and second dsRNA each comprise at least one 2′-O-methyl modified ribonucleotide and at least one nucleotide comprising a 5′-phosphorothioate group.

9. The composition of claim 1 or 2, wherein each strand of each dsRNA is 19-23 bases in length.

10. The composition of claim 1 or 2, wherein each strand of each dsRNA is 21-23 bases in length.

11. The composition of claim 1 or 2, wherein each strand of the first dsRNA is 21 bases in length and the sense strand of the second dsRNA is 21 bases in length and the antisense strand of the second dsRNA is 23 bases in length.

12. The composition of claim 1, 2, 3, or 4, wherein the first and second dsRNA are present in an equimolar ratio.

13. The composition of claim 1, 2, 3, or 4, comprising the components in the proportions listed in Table 17.

14. The composition of claim 1, 2, 3, or 4, wherein said composition, upon contact with a cell expressing Eg5, inhibits expression of Eg5 by at least 40, 50, 60, 70, 80, or by at least 90%.

15. The composition of claim 1, 2, 3, or 4, wherein said composition, upon contact with a cell expressing VEGF, inhibits expression of VEGF by at least 40, 50, 60, 70, 80, or by at least 90%.

16. The composition of claim 1, 2, 3, or 4, wherein administration of said composition to a cell decreases expression of both Eg5 and VEGF in said cell.

17. The composition of claim 16, wherein the composition is administered in a nM concentration.

18. The composition of claim 1, 2, 3, or 4, wherein administration of said composition to a cell increases mono-aster formation in the cell.

19. The composition of claim 1, 2, 3, or 4, wherein administration of said composition to a mammal results in at least one effect selected from the group consisting of prevention of tumor growth, reduction in tumor growth, or prolonged survival in said mammal.

20. The composition of claim 19, wherein said effect is measured using at least one assay selected from the group consisting of determination of body weight, determination of organ weight, visual inspection, mRNA analysis, serum AFP analysis and survival monitoring.

21. The composition of claim 1, 2, 3, or 4, further comprising Sorafenib.

22. The composition of claim 1 or 2, wherein the first dsRNA contains two overhangs and the second dsRNA contains an overhang at the 3′ of the antisense and a blunt end at the 5′ end of the antisense strand.

23. A method for inhibiting the expression of Eg5/KSP and VEGF in a cell comprising administering any of the compositions of claim 1, 2, 3, or 4 to the cell.

24. A method for preventing tumor growth, reducing tumor growth, or prolonging survival in a mammal in need of treatment for cancer comprising administering the composition of claim 1, 2, 3, or 4 to said mammal.

25. The method of claim 24, wherein said mammal has liver cancer.

26. The method of claim 24, wherein said mammal is a human with liver cancer.

27. The method of claim 23, further comprising administering Sorafenib.