RNA inteference mediated inhibition of hepatitis C virus (HVC) gene expression using short interfering nucleic acid (siNA)

FIELD OF THE INVENTION

The present invention relates to compounds, compositions, and methods for the study, diagnosis, and treatment of traits, diseases and conditions that respond to the modulation of hepatitis C virus (HCV) gene expression and/or activity. The present invention is also directed to compounds, compositions, and methods relating to traits, diseases and conditions that respond to the modulation of expression and/or activity of genes involved in hepatitis C virus (HCV) gene expression pathways or other cellular processes that mediate the maintenance or development of such traits, diseases and conditions. Specifically, the invention relates to double stranded nucleic acid molecules including small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of mediating RNA interference (RNAi) against hepatitis C virus (HCV) gene expression, including cocktails of such small nucleic acid molecules and lipid nanoparticle (LNP) formulations of such small nucleic acid molecules. The present invention also relates to small nucleic acid molecules, such as siNA, siRNA, and others that can inhibit the function of endogenous RNA molecules, such as endogenous micro-RNA (miRNA) (e.g, miRNA inhibitors) or endogenous short interfering RNA (siRNA), (e.g., siRNA inhibitors) or that can inhibit the function of RISC (e.g., RISC inhibitors), to modulate hepatitis C virus (HCV) gene expression by interfering with the regulatory function of such endogenous RNAs or proteins associated with such endogenous RNAs (e.g., RISC), including cocktails of such small nucleic acid molecules and lipid nanoparticle (LNP) formulations of such small nucleic acid molecules. Such small nucleic acid molecules and formulations are useful, for example, in providing compositions to prevent, inhibit, or reduce HCV infection, liver failure, hepatocellular carcinoma, cirrhosis, and/or other disease states associated with HCV infection in a subject or organism.

BACKGROUND OF THE INVENTION

The following is a discussion of relevant art pertaining to RNAi. The discussion is provided only for understanding of the invention that follows. The summary is not an admission that any of the work described below is prior art to the claimed invention.

RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Fire et al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286, 950-951; Lin et al., 1999, Nature, 402, 128-129; Sharp, 1999, genes & Dev., 13:139-141; and Strauss, 1999, Science, 286, 886). The corresponding process in plants (Heifetz et al., International PCT Publication No. WO 99/61631) is commonly referred to as post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes and is commonly shared by diverse flora and phyla (Fire et al., 1999, Trends genet., 15, 358). Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or from the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA or viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response through a mechanism that has yet to be fully characterized. This mechanism appears to be different from other known mechanisms involving double stranded RNA-specific ribonucleases, such as the interferon response that results from dsRNA-mediated activation of protein kinase PKR and 2′,5′-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L (see for example U.S. Pat. Nos. 6,107,094; 5,898,031; Clemens et al., 1997, J. Interferon & Cytokine Res., 17, 503-524; Adah et al., 2001, Curr. Med. Chem., 8, 1189).

The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer (Bass, 2000, Cell, 101, 235; Zamore et al., 2000, Cell, 101, 25-33; Hammond et al., 2000, Nature, 404, 293). Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Bass, 2000, Cell, 101, 235; Berstein et al., 2001, Nature, 409, 363). Short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes (Zamore et al., 2000, Cell, 101, 25-33; Elbashir et al., 2001, genes Dev., 15, 188). Dicer has also been implicated in the excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001, Science, 293, 834). The RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir et al., 2001, genes Dev., 15, 188).

RNAi has been studied in a variety of systems. Fire et al., 1998, Nature, 391, 806, were the first to observe RNAi in C. elegans. Bahramian and Zarbl, 1999, Molecular and Cellular Biology, 19, 274-283 and Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated by dsRNA in mammalian systems. Hammond et al., 2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001, Nature, 411, 494 and Tuschl et al., International PCT Publication No. WO 01/75164, describe RNAi induced by introduction of duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells. Recent work in Drosophila embryonic lysates (Elbashir et al., 2001, EMBO J, 20, 6877 and Tuschl et al., International PCT Publication No. WO 01/75164) has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies have shown that 21-nucleotide siRNA duplexes are most active when containing 3′-terminal dinucleotide overhangs. Furthermore, complete substitution of one or both siRNA strands with 2′-deoxy (2′-H) or 2′-O-methyl nucleotides abolishes RNAi activity, whereas substitution of the 3′-terminal siRNA overhang nucleotides with 2′-deoxy nucleotides (2′-H) was shown to be tolerated. Single mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity. In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5′-end of the siRNA guide sequence rather than the 3′-end of the guide sequence (Elbashir et al., 2001, EMBO J., 20, 6877). Other studies have indicated that a 5′-phosphate on the target-complementary strand of a siRNA duplex is required for siRNA activity and that ATP is utilized to maintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309).

Studies have shown that replacing the 3′-terminal nucleotide overhanging segments of a 21-mer siRNA duplex having two-nucleotide 3′-overhangs with deoxyribonucleotides does not have an adverse effect on RNAi activity. Replacing up to four nucleotides on each end of the siRNA with deoxyribonucleotides has been reported to be well tolerated, whereas complete substitution with deoxyribonucleotides results in no RNAi activity (Elbashir et al., 2001, EMBO J., 20, 6877 and Tuschl et al., International PCT Publication No. WO 01/75164). In addition, Elbashir et al., supra, also report that substitution of siRNA with 2′-O-methyl nucleotides completely abolishes RNAi activity. Li et al., International PCT Publication No. WO 00/44914, and Beach et al., International PCT Publication No. WO 01/68836 preliminarily suggest that siRNA may include modifications to either the phosphate-sugar backbone or the nucleoside to include at least one of a nitrogen or sulfur heteroatom, however, neither application postulates to what extent such modifications would be tolerated in siRNA molecules, nor provides any further guidance or examples of such modified siRNA. Kreutzer et al., Canadian Patent Application No. 2,359,180, also describe certain chemical modifications for use in dsRNA constructs in order to counteract activation of double-stranded RNA-dependent protein kinase PKR, specifically 2′-amino or 2′-O-methyl nucleotides, and nucleotides containing a 2′-O or 4′-C methylene bridge. However, Kreutzer et al. similarly fails to provide examples or guidance as to what extent these modifications would be tolerated in dsRNA molecules.

Parrish et al., 2000, Molecular Cell, 6, 1077-1087, tested certain chemical modifications targeting the unc-22 gene in C. elegans using long (>25 nt) siRNA transcripts. The authors describe the introduction of thiophosphate residues into these siRNA transcripts by incorporating thiophosphate nucleotide analogs with T7 and T3 RNA polymerase and observed that RNAs with two phosphorothioate modified bases also had substantial decreases in effectiveness as RNAi. Further, Parrish et al. reported that phosphorothioate modification of more than two residues greatly destabilized the RNAs in vitro such that interference activities could not be assayed. Id. at 1081. The authors also tested certain modifications at the 2′-position of the nucleotide sugar in the long siRNA transcripts and found that substituting deoxynucleotides for ribonucleotides produced a substantial decrease in interference activity, especially in the case of Uridine to Thymidine and/or Cytidine to deoxy-Cytidine substitutions. Id. In addition, the authors tested certain base modifications, including substituting, in sense and antisense strands of the siRNA, 4-thiouracil, 5-bromouracil, 5-iodouracil, and 3-(aminoallyl)uracil for uracil, and inosine for guanosine. Whereas 4-thiouracil and 5-bromouracil substitution appeared to be tolerated, Parrish reported that inosine produced a substantial decrease in interference activity when incorporated in either strand. Parrish also reported that incorporation of 5-iodouracil and 3-(aminoallyl)uracil in the antisense strand resulted in a substantial decrease in RNAi activity as well.

The use of longer dsRNA has been described. For example, Beach et al., International PCT Publication No. WO 01/68836, describes specific methods for attenuating gene expression using endogenously-derived dsRNA. Tuschl et al., International PCT Publication No. WO 01/75164, describe a Drosophila in vitro RNAi system and the use of specific siRNA molecules for certain functional genomic and certain therapeutic applications; although Tuschl, 2001, Chem. Biochem., 2, 239-245, doubts that RNAi can be used to cure genetic diseases or viral infection due to the danger of activating interferon response. Li et al., International PCT Publication No. WO 00/44914, describe the use of specific long (141 bp-488 bp) enzymatically synthesized or vector expressed dsRNAs for attenuating the expression of certain target genes. Zernicka-Goetz et al., International PCT Publication No. WO 01/36646, describe certain methods for inhibiting the expression of particular genes in mammalian cells using certain long (550 bp-714 bp), enzymatically synthesized or vector expressed dsRNA molecules. Fire et al., International PCT Publication No. WO 99/32619, describe particular methods for introducing certain long dsRNA molecules into cells for use in inhibiting gene expression in nematodes. Plaetinck et al., International PCT Publication No. WO 00/01846, describe certain methods for identifying specific genes responsible for conferring a particular phenotype in a cell using specific long dsRNA molecules. Mello et al., International PCT Publication No. WO 01/29058, describe the identification of specific genes involved in dsRNA-mediated RNAi. Pachuck et al., International PCT Publication No. WO 00/63364, describe certain long (at least 200 nucleotide) dsRNA constructs. Deschamps Depaillette et al., International PCT Publication No. WO 99/07409, describe specific compositions consisting of particular dsRNA molecules combined with certain anti-viral agents. Waterhouse et al., International PCT Publication No. 99/53050 and 1998, PNAS, 95, 13959-13964, describe certain methods for decreasing the phenotypic expression of a nucleic acid in plant cells using certain dsRNAs. Driscoll et al., International PCT Publication No. WO 01/49844, describe specific DNA expression constructs for use in facilitating gene silencing in targeted organisms.

Others have reported on various RNAi and gene-silencing systems. For example, Parrish et al., 2000, Molecular Cell, 6, 1077-1087, describe specific chemically-modified dsRNA constructs targeting the unc-22 gene of C. elegans. Grossniklaus, International PCT Publication No. WO 01/38551, describes certain methods for regulating polycomb gene expression in plants using certain dsRNAs. Churikov et al., International PCT Publication No. WO 01/42443, describe certain methods for modifying genetic characteristics of an organism using certain dsRNAs. Cogoni et al, International PCT Publication No. WO 01/53475, describe certain methods for isolating a Neurospora silencing gene and uses thereof. Reed et al., International PCT Publication No. WO 01/68836, describe certain methods for gene silencing in plants. Honer et al., International PCT Publication No. WO 01/70944, describe certain methods of drug screening using transgenic nematodes as Parkinson's Disease models using certain dsRNAs. Deak et al., International PCT Publication No. WO 01/72774, describe certain Drosophila-derived gene products that may be related to RNAi in Drosophila. Arndt et al., International PCT Publication No. WO 01/92513 describe certain methods for mediating gene suppression by using factors that enhance RNAi. Tuschl et al., International PCT Publication No. WO 02/44321, describe certain synthetic siRNA constructs. Pachuk et al., International PCT Publication No. WO 00/63364, and Satishchandran et al., International PCT Publication No. WO 01/04313, describe certain methods and compositions for inhibiting the function of certain polynucleotide sequences using certain long (over 250 bp), vector expressed dsRNAs. Echeverri et al., International PCT Publication No. WO 02/38805, describe certain C. elegans genes identified via RNAi. Kreutzer et al., International PCT Publications Nos. WO 02/055692, WO 02/055693, and EP 1144623 B1 describes certain methods for inhibiting gene expression using dsRNA. Graham et al., International PCT Publications Nos. WO 99/49029 and WO 01/70949, and AU 4037501 describe certain vector expressed siRNA molecules. Fire et al., U.S. Pat. No. 6,506,559, describe certain methods for inhibiting gene expression in vitro using certain long dsRNA (299 bp-1033 bp) constructs that mediate RNAi. Martinez et al., 2002, Cell, 110, 563-574, describe certain single stranded siRNA constructs, including certain 5′-phosphorylated single stranded siRNAs that mediate RNA interference in Hela cells. Harborth et al., 2003, Antisense & Nucleic Acid Drug Development, 13, 83-105, describe certain chemically and structurally modified siRNA molecules. Chiu and Rana, 2003, RNA, 9, 1034-1048, describe certain chemically and structurally modified siRNA molecules. Woolf et al., International PCT Publication Nos. WO 03/064626 and WO 03/064625 describe certain chemically modified dsRNA constructs. Hornung et al., 2005, Nature Medicine, 11, 263-270, describe the sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7. Judge et al., 2005, Nature Biotechnology, Published online: 20 Mar. 2005, describe the sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Yuki et al., International PCT Publication Nos. WO 05/049821 and WO 04/048566, describe certain methods for designing short interfering RNA sequences and certain short interfering RNA sequences with optimized activity. Saigo et al., US Patent Application Publication No. US20040539332, describe certain methods of designing oligo- or polynucleotide sequences, including short interfering RNA sequences, for achieving RNA interference. Tei et al., International PCT Publication No. WO 03/044188, describe certain methods for inhibiting expression of a target gene, which comprises transfecting a cell, tissue, or individual organism with a double-stranded polynucleotide comprising DNA and RNA having a substantially identical nucleotide sequence with at least a partial nucleotide sequence of the target gene.

Mattick, 2005, Science, 309, 1527-1528; Claverie, 2005, Science, 309, 1529-1530; Sethupathy et al., 2006, RNA, 12, 192-197; and Czech, 2006 NEJM, 354, 11: 1194-1195; Hutvagner et al., US 20050227256, and Tuschl et al., US 20050182005, all describe antisense molecules that can inhibit miRNA function via steric blocking and are all incorporated by reference herein in their entirety.

McCaffrey et al., 2002, Nature, 418, 38-39, describes the use of certain siRNA constructs targeting a chimeric HCV NS5B protein/luciferase transcript in mice.

Randall et al., 2003, PNAS USA, 100, 235-240, describe certain siRNA constructs targeting HCV RNA in Huh7 hepatoma cell lines.

SUMMARY OF THE INVENTION

This invention relates to compounds, compositions, and methods useful for modulating the expression of genes, such as those genes associated with the development or maintenance of HCV infection, liver failure, hepatocellular carcinoma, cirrhosis, and/or other disease states associated with HCV infection, by RNA interference (RNAi) using short interfering nucleic acid (siNA) molecules. This invention also relates to compounds, compositions, and methods useful for modulating the expression and activity of one or more genes involved in pathways of HCV gene expression and/or activity by RNA interference (RNAi) using small nucleic acid molecules. In particular, the instant invention features small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methods used to modulate the expression of HCV genes and/or other genes (e.g., cellular or host genes) involved in pathways of HCV gene expression and/or infection.

The instant invention also relates to small nucleic acid molecules, such as siNA, siRNA, and others that can inhibit the function of endogenous RNA molecules, such as endogenous micro-RNA (miRNA) (e.g, miRNA inhibitors) or endogenous short interfering RNA (siRNA), (e.g., siRNA inhibitors) or that can inhibit the function of RISC (e.g., RISC inhibitors), to modulate HCV gene expression by interfering with the regulatory function of such endogenous RNAs or proteins associated with such endogenous RNAs (e.g., RISC). Such molecules are collectively referred to herein as RNAi inhibitors.

A siNA or RNAi inhibitor of the invention can be unmodified or chemically-modified. A siNA or RNAi inhibitor of the instant invention can be chemically synthesized, expressed from a vector or enzymatically synthesized. The instant invention also features various chemically-modified synthetic short interfering nucleic acid (siNA) molecules capable of modulating target gene expression or activity in cells by RNA interference (RNAi). The instant invention also features various chemically-modified synthetic short nucleic acid (siNA) molecules capable of modulating RNAi activity in cells by interacting with miRNA, siRNA, or RISC, and hence down regulating or inhibiting RNA interference (RNAi), translational inhibition, or transcriptional silencing in a cell or organism. The use of chemically-modified siNA and/or RNAi inhibitors improves various properties of native siNA molecules and/or RNAi inhibitors through increased resistance to nuclease degradation in vivo and/or through improved cellular uptake. Further, contrary to earlier published studies, siNA molecules of the invention having multiple chemical modifications, including fully modified siNA, retains its RNAi activity. Therefore, Applicant teaches herein chemically modified siRNA (generally referred to herein as siNA) that retains or improves upon the activity of native siRNA. The siNA molecules of the instant invention provide useful reagents and methods for a variety of therapeutic, prophylactic, veterinary, diagnostic, target validation, genomic discovery, genetic engineering, and pharmacogenomic applications.

In one embodiment, the invention features one or more siNA molecules and/or RNAi inhibitors and methods that independently or in combination modulate the expression of HCV and HCV related host target genes encoding proteins, such as proteins that are associated with the maintenance or development of HCV infection, liver failure, hepatocellular carcinoma, and cirrhosis, such as genes encoding sequences comprising those sequences referred to by GenBank Accession Nos. shown in Table I, referred to herein generally as HCV. The description below of the various aspects and embodiments of the invention is provided with reference to exemplary hepatitis C virus (HCV) genes, generally referred to herein as HCV. However, such reference is meant to be exemplary only and the various aspects and embodiments of the invention are also directed to other genes that express alternate HCV genes, such as mutant HCV genes, splice variants of HCV genes, and genes encoding different strains of HCV, as well as as cellular targets for HCV, such as those described herein and also referred to by GenBank Accession Nos. herein and in U.S. Provisional Patent Application No. 60/363,124, U.S. Ser. No. 10/923,536, and PCT/US03/05028 all of which are incorporated by reference herein, referred to herein generally as “target” sequences. The various aspects and embodiments are also directed to other genes involved in HCV pathways, including genes that encode cellular proteins involved in the maintenance and/or development of HCV infection, liver failure, hepatocellular carcinoma, and cirrhosis or other genes that express other proteins associated with HCV infection, such as cellular proteins that are utilized in the HCV life-cycle. Such additional genes can be analyzed for target sites using the methods described herein for HCV. Thus, the inhibition and the effects of such inhibition of the other genes can be performed as described herein. In other words, the term “HCV” as it is defined herein below and recited in the described embodiments, is meant to encompass genes associated with the development and/or maintenance of HCV infection, such as genes which encode HCV polypeptides, including polypeptides of different strains of HCV, regulatory polynucleotides (e.g., miRNAs and siRNAs), mutant HCV genes, and splice variants of HCV genes, as well as cellular genes involved in HCV pathways of gene expression, replication, and/or HCV activity. Also, the term “HCV” as it is defined herein below and recited in the described embodiments, is meant to encompass HCV viral gene products and cellular gene products involved in HCV infection, such as those described herein. Thus, each of the embodiments described herein with reference to the term “HCV” are applicable to all of the virus, cellular and viral protein, peptide, polypeptide, and/or polynucleotide molecules covered by the term “HCV”, as that term is defined herein. Comprehensively, such gene targets are also referred to herein generally as “target” sequences.

In one embodiment, the invention features a composition comprising two or more different siNA molecules and/or RNAi inhibitors of the invention (e.g., siNA, duplex forming siNA, or multifunctional siNA or any combination thereof) targeting different polynucleotide targets, such as different regions of HCV RNA (e.g., two different target sites herein), different viral strains (e.g., HCV strains, or HIV and HCV, HCV and HBV etc.), or different viral and cellular targets (e.g., a HCV target and a cellular target). Such pools of siNA molecules can prevent or overcome viral resistance or otherwise provide increased therapeutic effect.

In one embodiment, the invention features siNA molecules having RNAi specificity for the HCV minus strand, for example, Genbank Accession No. HPCK1S1, Hepatitis C virus (strain HCV-1b, clone HCV-K1-S1), complete genome; Genbank Accession No. D50483, 9410 nt.

In one embodiment, the invention features a pool of two or more different siNA molecules of the invention (e.g., siNA, duplex forming siNA, or multifunctional siNA or any combination thereof) that have specificity for different HCV polynucleotide targets, such as different regions of target HCV RNA or DNA (e.g., two different target sites herein or any combination of targets or host/pathway targets) or both coding and non-coding targets, wherein the pool comprises siNA molecules targeting about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different targets.

In one embodiment, the invention features one or more siNA molecules and methods that independently or in combination modulate the expression of genes representing cellular targets for HCV infection, such as cellular receptors, cell surface molecules, cellular enzymes, cellular transcription factors, and/or cytokines, second messengers, and cellular accessory molecules including, but not limited to, La antigen (see for example Costa-Mattioli et al., 2004, Mol Cell Biol., 24, 6861-70, e.g., Genbank Accession No. NM_—003142); FAS (e.g., Genbank Accession No. NM_—000043) or FAS ligand (e.g., Genbank Accession No. NM_—000639); interferon regulatory factors (IRFs; e.g., Genbank Accession No. AF082503.1); cellular PKR protein kinase (e.g., Genbank Accession No. XM_—002661.7); human eukaryotic initiation factors 2B (elF2Bgamma; e.g., Genbank Accession No. AF256223, and/or elF2gamma; e.g., Genbank Accession No. NM_—006874.1); human DEAD Box protein (DDX3; e.g., Genbank Accession No. XM_—018021.2); and cellular proteins that bind to the poly(U) tract of the HCV 3′-UTR, such as polypyrimidine tract-binding protein (e.g., Genbank Accession Nos. NM_—031991.1 and XM_—042972.3). Such cellular targets are also referred to herein generally as HCV targets, and specifically as “host target” or “host targets”.

Due to the high sequence variability of the HCV genome, selection of siNA molecules for broad therapeutic applications likely involve the conserved regions of the HCV genome. In one embodiment, the present invention relates to siNA molecules that target the conserved regions of the HCV genome. Examples of conserved regions of the HCV genome include, but are not limited to, the 5′-Non Coding Region (NCR, also referred to as the 5′-untranslated region, UTR), the 5′-end of the core protein coding region, and the 3′-NCR. HCV genomic RNA contains an internal ribosome entry site (IRES) in the 5′-NCR which mediates translation independently of a 5′-cap structure (Wang et al., 1993, J. Virol., 67, 3338-44). The full-length sequence of the HCV RNA genome is heterologous among clinically isolated subtypes, of which there are at least fifteen (Simmonds, 1995, Hepatology, 21, 570-583), however, the 5′-NCR sequence of HCV is highly conserved across all known subtypes, most likely to preserve the shared IRES mechanism (Okamoto et al., 1991, J. General Virol., 72, 2697-2704). Therefore, a siNA molecule can be designed to target the different isolates of HCV by targeting a conserved region, such as the 5′ NCR sequence. siNA molecules designed to target conserved regions of various HCV isolates enable efficient inhibition of HCV replication in diverse patient populations and ensure the effectiveness of the siNA molecules against HCV quasi species which evolve due to mutations in the non-conserved regions of the HCV genome. As described, a single siNA molecule can be targeted against all isolates of HCV by designing the siNA molecule to interact with conserved nucleotide sequences of HCV (e.g., sequences that are expected to be present in the RNA of various HCV isolates).

In one embodiment, the invention features a double stranded nucleic acid molecule, such as an siNA molecule, where one of the strands comprises nucleotide sequence having complementarity to a predetermined nucleotide sequence in a HCV target nucleic acid molecule, or a portion thereof. The predetermined nucleotide sequence can be a nucleotide target sequence, such as a sequence described herein or known in the art. In another embodiment, the predetermined nucleotide sequence is a HCV target sequence or pathway/host target sequence as is known in the art.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a target RNA, wherein said siNA molecule comprises about 15 to about 28 base pairs.

In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a target RNA via RNA interference (RNAi), wherein the double stranded siNA molecule comprises a first strand and a second strand, each strand of the siNA molecule is about 18 to about 28 (e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28) nucleotides in length, the first strand of the siNA molecule comprises nucleotide sequence having sufficient complementarity to the target RNA for the siNA molecule to direct cleavage of the target RNA via RNA interference, and the second strand of said siNA molecule comprises nucleotide sequence that is complementary to the first strand. In one specific embodiment, for example, each strand of the siNA molecule is about 18 to about 27 nucleotides in length.

In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a target RNA via RNA interference (RNAi), wherein the double stranded siNA molecule comprises a first strand and a second strand, each strand of the siNA molecule is about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) nucleotides in length, the first strand of the siNA molecule comprises nucleotide sequence having sufficient complementarity to the target RNA for the siNA molecule to direct cleavage of the target RNA via RNA interference, and the second strand of said siNA molecule comprises nucleotide sequence that is complementary to the first strand.

In one embodiment, the invention features a siNA molecule that down-regulates expression of a target gene or that directs cleavage of a target RNA, for example, wherein the target gene or RNA comprises protein encoding sequence. In one embodiment, the invention features a siNA molecule that down-regulates expression of a target gene or that directs cleavage of a target RNA, for example, wherein the target gene or RNA comprises non-coding sequence or regulatory elements involved in target gene expression (e.g., non-coding RNA, miRNA, stRNA etc.).

In one embodiment, a siNA of the invention is used to inhibit the expression of HCV target genes or a HCV target gene family (e.g., different HCV strains), wherein the genes or gene family sequences share sequence homology. Such homologous sequences can be identified as is known in the art, for example using sequence alignments. siNA molecules can be designed to target such homologous sequences, for example using perfectly complementary sequences or by incorporating non-canonical base pairs, for example mismatches and/or wobble base pairs, that can provide additional target sequences. In instances where mismatches are identified, non-canonical base pairs (for example, mismatches and/or wobble bases) can be used to generate siNA molecules that target more than one gene sequence. In a non-limiting example, non-canonical base pairs such as UU and CC base pairs are used to generate siNA molecules that are capable of targeting sequences for differing polynucleotide targets that share sequence homology. As such, one advantage of using siNAs of the invention is that a single siNA can be designed to include nucleic acid sequence that is complementary to the nucleotide sequence that is conserved between the homologous genes. In this approach, a single siNA can be used to inhibit expression of more than one gene instead of using more than one siNA molecule to target the different genes.

In one embodiment, the invention features a siNA molecule having RNAi activity against target RNA (e.g., coding or non-coding RNA), wherein the siNA molecule comprises a sequence complementary to any RNA sequence, such as those sequences having GenBank Accession Nos. herein (see for example Table I) and those shown in PCT/US03/05028, U.S. Provisional Patent Application No. 60/363,124, and/or U.S. Ser. No. 10/923,536, all of which are incorporated by reference herein. In another embodiment, the invention features a siNA molecule having RNAi activity against target RNA, wherein the siNA molecule comprises a sequence complementary to an RNA having variant encoding sequence, for example other mutant genes known in the art to be associated with the maintenance and/or development of diseases, traits, disorders, and/or conditions described herein or otherwise known in the art. Chemical modifications as shown in Table IV or otherwise described herein can be applied to any siNA construct of the invention. In another embodiment, a siNA molecule of the invention includes a nucleotide sequence that can interact with nucleotide sequence of a target gene and thereby mediate silencing of target gene expression, for example, wherein the siNA mediates regulation of target gene expression by cellular processes that modulate the chromatin structure or methylation patterns of the target gene and prevent transcription of the target gene.

In one embodiment, siNA molecules of the invention are used to down regulate or inhibit the expression of proteins arising from haplotype polymorphisms that are associated with a trait, disease or condition in a subject or organism. Analysis of genes, or protein or RNA levels can be used to identify subjects with such polymorphisms or those subjects who are at risk of developing traits, conditions, or diseases described herein. These subjects are amenable to treatment, for example, treatment with siNA molecules of the invention and any other composition useful in treating diseases related to target gene expression. As such, analysis of protein or RNA levels can be used to determine treatment type and the course of therapy in treating a subject. Monitoring of protein or RNA levels can be used to predict treatment outcome and to determine the efficacy of compounds and compositions that modulate the level and/or activity of certain proteins associated with a trait, disorder, condition, or disease.

In one embodiment of the invention a siNA molecule comprises an antisense strand comprising a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof encoding a target protein. The siNA further comprises a sense strand, wherein said sense strand comprises a nucleotide sequence of a target gene or a portion thereof.

In another embodiment, a siNA molecule comprises an antisense region comprising a nucleotide sequence that is complementary to a nucleotide sequence encoding a target protein or a portion thereof. The siNA molecule further comprises a sense region, wherein said sense region comprises a nucleotide sequence of a target gene or a portion thereof.

In another embodiment, the invention features a siNA molecule comprising nucleotide sequence, for example, nucleotide sequence in the antisense region of the siNA molecule that is complementary to a nucleotide sequence or portion of sequence of a target gene. In another embodiment, the invention features a siNA molecule comprising a region, for example, the antisense region of the siNA construct, complementary to a sequence comprising a target gene sequence or a portion thereof.

In one embodiment, the sense region or sense strand of a siNA molecule of the invention is complementary to that portion of the antisense region or antisense strand of the siNA molecule that is complementary to a target polynucleotide sequence.

In yet another embodiment, the invention features a siNA molecule comprising a sequence, for example, the antisense sequence of the siNA construct, complementary to a sequence or portion of sequence comprising sequence represented by GenBank Accession Nos. shown in Table I and in PCT/US03/05028, U.S. Provisional Patent Application No. 60/363,124, and/or U.S. Ser. No. 10/923,536, all of which are incorporated by reference herein. Chemical modifications in Tables III and IV and otherwise described herein can be applied to any siNA construct of the invention. LNP formulations described in Table VI can be applied to any siNA molecule or combination of siNA molecules herein.

In one embodiment of the invention a siNA molecule comprises an antisense strand having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein the antisense strand is complementary to a target RNA sequence or a portion thereof, and wherein said siNA further comprises a sense strand having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, and wherein said sense strand and said antisense strand are distinct nucleotide sequences where at least about 15 nucleotides in each strand are complementary to the other strand.

In one embodiment, a siNA molecule of the invention (e.g., a double stranded nucleic acid molecule) comprises an antisense (guide) strand having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that are complementary to a target RNA sequence or a portion thereof. In one embodiment, at least 15 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) of a target RNA sequence are complementary to the antisense (guide) strand of a siNA molecule of the invention.

In one embodiment, a siNA molecule of the invention (e.g., a double stranded nucleic acid molecule) comprises a sense (passenger) strand having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that comprise sequence of a target RNA or a portion thereof. In one embodiment, at least 15 nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides of a target RNA sequence comprise the sense (passenger) strand of a siNA molecule of the invention.

In another embodiment of the invention a siNA molecule of the invention comprises an antisense region having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein the antisense region is complementary to a target DNA sequence, and wherein said siNA further comprises a sense region having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein said sense region and said antisense region are comprised in a linear molecule where the sense region comprises at least about 15 nucleotides that are complementary to the antisense region.

In one embodiment, a siNA molecule of the invention has RNAi activity that modulates expression of RNA encoded by a HCV gene. Because HCV genes can share some degree of sequence homology with each other, siNA molecules can be designed to target a class of HCV genes (e.g., a class of different HCV strains) or alternately specific HCV genes (e.g., escape mutants, resistant strains, or other polymorphic variants) by selecting sequences that are either shared amongst different HCV targets or alternatively that are unique for a specific HCV target. Therefore, in one embodiment, the siNA molecule can be designed to target conserved regions of HCV RNA sequences having homology among several HCV gene variants so as to target a class of HCV genes with one siNA molecule. Accordingly, in one embodiment, the siNA molecule of the invention modulates the expression of one or more HCV stains in a subject or organism. In another embodiment, the siNA molecule can be designed to target a sequence that is unique to a specific HCV RNA sequence (e.g., a single HCV strain or HCV single nucleotide polymorphism (SNP)) due to the high degree of specificity that the siNA molecule requires to mediate RNAi activity.

In one embodiment, nucleic acid molecules of the invention that act as mediators of the RNA interference gene silencing response are double-stranded nucleic acid molecules. In another embodiment, the siNA molecules of the invention consist of duplex nucleic acid molecules containing about 15 to about 30 base pairs between oligonucleotides comprising about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In yet another embodiment, siNA molecules of the invention comprise duplex nucleic acid molecules with overhanging ends of about 1 to about 3 (e.g., about 1, 2, or 3) nucleotides, for example, about 21-nucleotide duplexes with about 19 base pairs and 3′-terminal mononucleotide, dinucleotide, or trinucleotide overhangs. In yet another embodiment, siNA molecules of the invention comprise duplex nucleic acid molecules with blunt ends, where both ends are blunt, or alternatively, where one of the ends is blunt.

In one embodiment, a double stranded nucleic acid (e.g., siNA) molecule comprises nucleotide or non-nucleotide overhangs. By “overhang” is meant a terminal portion of the nucleotide sequence that is not base paired between the two strands of a double stranded nucleic acid molecule (see for example FIG. 6). In one embodiment, a double stranded nucleic acid molecule of the invention can comprise nucleotide or non-nucleotide overhangs at the 3′-end of one or both strands of the double stranded nucleic acid molecule. For example, a double stranded nucleic acid molecule of the invention can comprise a nucleotide or non-nucleotide overhang at the 3′-end of the guide strand or antisense strand/region, the 3′-end of the passenger strand or sense strand/region, or both the guide strand or antisense strand/region and the passenger strand or sense strand/region of the double stranded nucleic acid molecule. In another embodiment, the nucleotide overhang portion of a double stranded nucleic acid (siNA) molecule of the invention comprises 2′-O-methyl, 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-deoxy-2′-fluoroarabino (FANA), 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, universal base, acyclic, or 5-C-methyl nucleotides. In another embodiment, the non-nucleotide overhang portion of a double stranded nucleic acid (siNA) molecule of the invention comprises glyceryl, abasic, or inverted deoxy abasic non-nucleotides.

In one embodiment, the nucleotides comprising the overhang portions of a double stranded nucleic acid (e.g., siNA) molecule of the invention correspond to the nucleotides comprising the target polynucleotide sequence of the siNA molecule. Accordingly, in such embodiments, the nucleotides comprising the overhang portion of a siNA molecule of the invention comprise sequence based on the target polynucleotide sequence in which nucleotides comprising the overhang portion of the guide strand or antisense strand/region of a siNA molecule of the invention can be complementary to nucleotides in the target polynucleotide sequence and nucleotides comprising the overhang portion of the passenger strand or sense strand/region of a siNA molecule of the invention can comprise the nucleotides in the target polynucleotide sequence. Such nucleotide overhangs comprise sequence that would result from Dicer processing of a native dsRNA into siRNA.

In one embodiment, the nucleotides comprising the overhang portion of a double stranded nucleic acid (e.g., siNA) molecule of the invention are complementary to the target polynucleotide sequence and are optionally chemically modified as described herein. As such, in one embodiment, the nucleotides comprising the overhang portion of the guide strand or antisense strand/region of a siNA molecule of the invention can be complementary to nucleotides in the target polynucleotide sequence, i.e. those nucleotide positions in the target polynucleotide sequence that are complementary to the nucleotide positions of the overhang nucleotides in the guide strand or antisense strand/region of a siNA molecule. In another embodiment, the nucleotides comprising the overhang portion of the passenger strand or sense strand/region of a siNA molecule of the invention can comprise the nucleotides in the target polynucleotide sequence, i.e. those nucleotide positions in the target polynucleotide sequence that correspond to same the nucleotide positions of the overhang nucleotides in the passenger strand or sense strand/region of a siNA molecule. In one embodiment, the overhang comprises a two nucleotide (e.g., 3′-GA; 3′-GU; 3′-GG; 3′GC; 3′-CA; 3′-CU; 3′-CG; 3′CC; 3′-UA; 3′-UU; 3′-UG; 3′UC; 3′-AA; 3′-AU; 3′-AG; 3′-AC; 3′-TA; 3′-TU; 3′-TG; 3′-TC; 3′-AT; 3′-UT; 3′-GT; 3′-CT) overhang that is complementary to a portion of the target polynucleotide sequence. In one embodiment, the overhang comprises a two nucleotide (e.g., 3′-GA; 3′-GU; 3′-GG; 3′GC; 3′-CA; 3′-CU; 3′-CG; 3′CC; 3′-UA; 3′-UU; 3′-UG; 3′UC; 3′-AA; 3′-AU; 3′-AG; 3′-AC; 3′-TA; 3′-TU; 3′-TG; 3′-TC; 3′-AT; 3′-UT; 3′-GT; 3′-CT) overhang that is not complementary to a portion of the target polynucleotide sequence.

In another embodiment, the overhang nucleotides of a siNA molecule of the invention are 2′-O-methyl nucleotides, 2′-deoxy-2′-fluoroarabino, and/or 2′-deoxy-2′-fluoro nucleotides. In another embodiment, the overhang nucleotides of a siNA molecule of the invention are 2′-O-methyl nucleotides in the event the overhang nucleotides are purine nucleotides and/or 2′-deoxy-2′-fluoro nucleotides or 2′-deoxy-2′-fluoroarabino nucleotides in the event the overhang nucleotides are pyrimidines nucleotides. In another embodiment, the purine nucleotide (when present) in an overhang of siNA molecule of the invention is 2′-O-methyl nucleotides. In another embodiment, the pyrimidine nucleotide (when present) in an overhang of siNA molecule of the invention are 2′-deoxy-2′-fluoro or 2′-deoxy-2′-fluoroarabino nucleotides.

In one embodiment, the nucleotides comprising the overhang portion of a double stranded nucleic acid (e.g., siNA) molecule of the invention are not complementary to the target polynucleotide sequence and are optionally chemically modified as described herein. In one embodiment, the overhang comprises a 3′-UU overhang that is not complementary to a portion of the target polynucleotide sequence. In another embodiment, the nucleotides comprising the overhanging portion of a siNA molecule of the invention are 2′-O-methyl nucleotides, 2′-deoxy-2′-fluoroarabino and/or 2′-deoxy-2′-fluoro nucleotides.

In one embodiment, the double stranded nucleic molecule (e.g. siNA) of the invention comprises a two or three nucleotide overhang, wherein the nucleotides in the overhang are the same or different. In one embodiment, the double stranded nucleic molecule (e.g. siNA) of the invention comprises a two or three nucleotide overhang, wherein the nucleotides in the overhang are the same or different and wherein one or more nucleotides in the overhang are chemically modified at the base, sugar and/or phosphate backbone.

In one embodiment, the invention features one or more chemically-modified siNA constructs having specificity for target nucleic acid molecules, such as DNA, or RNA encoding a protein or non-coding RNA associated with the expression of target genes. In one embodiment, the invention features a RNA based siNA molecule (e.g., a siNA comprising 2′-OH nucleotides) having specificity for nucleic acid molecules that includes one or more chemical modifications described herein. Non-limiting examples of such chemical modifications include without limitation phosphorothioate internucleotide linkages, 2′-deoxyribonucleotides, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, 4′-thio ribonucleotides, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides (see for example U.S. Ser. No. 10/981,966 filed Nov. 5, 2004, incorporated by reference herein), “universal base” nucleotides, “acyclic” nucleotides, 5-C-methyl nucleotides, 2′-deoxy-2′-fluoroarabino (FANA, see for example Dowler et al., 2006, Nucleic Acids Research, 34, 1669-1675) and terminal glyceryl and/or inverted deoxy abasic residue incorporation. These chemical modifications, when used in various siNA constructs, (e.g., RNA based siNA constructs), are shown to preserve RNAi activity in cells while at the same time, dramatically increasing the serum stability of these compounds.

In one embodiment, a siNA molecule of the invention comprises chemical modifications described herein (e.g., 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, 4′-thio ribonucleotides, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides, LNA) at the internal positions of the siNA molecule. By “internal position” is meant the base paired positions of a siNA duplex.

In one embodiment, a siNA molecule of the invention comprises modified nucleotides while maintaining the ability to mediate RNAi. The modified nucleotides can be used to improve in vitro or in vivo characteristics such as stability, activity, toxicity, immune response, and/or bioavailability. For example, a siNA molecule of the invention can comprise modified nucleotides as a percentage of the total number of nucleotides present in the siNA molecule. As such, a siNA molecule of the invention can generally comprise about 5% to about 100% modified nucleotides (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides). For example, in one embodiment, between about 5% to about 100% (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides) of the nucleotide positions in a siNA molecule of the invention comprise a nucleic acid sugar modification, such as a 2′-sugar modification, e.g., 2′-O-methyl nucleotides, 2′-deoxy-2′-fluoro nucleotides, 2′-deoxy-2′-fluoroarabino, 2′-O-methoxyethyl nucleotides, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides, or 2′-deoxy nucleotides. In another embodiment, between about 5% to about 100% (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides) of the nucleotide positions in a siNA molecule of the invention comprise a nucleic acid base modification, such as inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), or propyne modifications. In another embodiment, between about 5% to about 100% (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides) of the nucleotide positions in a siNA molecule of the invention comprise a nucleic acid backbone modification, such as a backbone modification having Formula I herein. In another embodiment, between about 5% to about 100% (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides) of the nucleotide positions in a siNA molecule of the invention comprise a nucleic acid sugar, base, or backbone modification or any combination thereof (e.g., any combination of nucleic acid sugar, base, backbone or non-nucleotide modifications herein). In one embodiment, a siNA molecule of the invention comprises at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides. The actual percentage of modified nucleotides present in a given siNA molecule will depend on the total number of nucleotides present in the siNA. If the siNA molecule is single stranded, the percent modification can be based upon the total number of nucleotides present in the single stranded siNA molecules. Likewise, if the siNA molecule is double stranded, the percent modification can be based upon the total number of nucleotides present in the sense strand, antisense strand, or both the sense and antisense strands.

A siNA molecule of the invention can comprise modified nucleotides at various locations within the siNA molecule. In one embodiment, a double stranded siNA molecule of the invention comprises modified nucleotides at internal base paired positions within the siNA duplex. For example, internal positions can comprise positions from about 3 to about 19 nucleotides from the 5′-end of either sense or antisense strand or region of a 21 nucleotide siNA duplex having 19 base pairs and two nucleotide 3′-overhangs. In another embodiment, a double stranded siNA molecule of the invention comprises modified nucleotides at non-base paired or overhang regions of the siNA molecule. By “non-base paired” is meant, the nucleotides are not base paired between the sense strand or sense region and the antisense strand or antisense region or the siNA molecule. The overhang nucleotides can be complementary or base paired to a corresponding target polynucleotide sequence (see for example FIG. 6C). For example, overhang positions can comprise positions from about 20 to about 21 nucleotides from the 5′-end of either sense or antisense strand or region of a 21 nucleotide siNA duplex having 19 base pairs and two nucleotide 3′-overhangs. In another embodiment, a double stranded siNA molecule of the invention comprises modified nucleotides at terminal positions of the siNA molecule. For example, such terminal regions include the 3′-position, 5′-position, for both 3′ and 5′-positions of the sense and/or antisense strand or region of the siNA molecule. In another embodiment, a double stranded siNA molecule of the invention comprises modified nucleotides at base-paired or internal positions, non-base paired or overhang regions, and/or terminal regions, or any combination thereof.

One aspect of the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a target gene or that directs cleavage of a target RNA. In one embodiment, the double stranded siNA molecule comprises one or more chemical modifications and each strand of the double-stranded siNA is about 21 nucleotides long. In one embodiment, the double-stranded siNA molecule does not contain any ribonucleotides. In another embodiment, the double-stranded siNA molecule comprises one or more ribonucleotides. In one embodiment, each strand of the double-stranded siNA molecule independently comprises about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein each strand comprises about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that are complementary to the nucleotides of the other strand. In one embodiment, one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof of the target gene, and the second strand of the double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence of the target gene or a portion thereof.

In another embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a target gene or that directs cleavage of a target RNA, comprising an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of the target gene or a portion thereof, and a sense region, wherein the sense region comprises a nucleotide sequence substantially similar to the nucleotide sequence of the target gene or a portion thereof. In one embodiment, the antisense region and the sense region independently comprise about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein the antisense region comprises about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that are complementary to nucleotides of the sense region.

In one embodiment, a siNA molecule of the invention comprises blunt ends, i.e., ends that do not include any overhanging nucleotides. For example, a siNA molecule comprising modifications described herein (e.g., comprising nucleotides having Formulae I-VII or siNA constructs comprising “Stab 00”-“Stab 36” or “Stab 3F”-“Stab 36F” (Table IV) or any combination thereof) and/or any length described herein can comprise blunt ends or ends with no overhanging nucleotides.

In one embodiment, any siNA molecule of the invention can comprise one or more blunt ends, i.e. where a blunt end does not have any overhanging nucleotides. In one embodiment, the blunt ended siNA molecule has a number of base pairs equal to the number of nucleotides present in each strand of the siNA molecule. In another embodiment, the siNA molecule comprises one blunt end, for example wherein the 5′-end of the antisense strand and the 3′-end of the sense strand do not have any overhanging nucleotides. In another example, the siNA molecule comprises one blunt end, for example, wherein the 3′-end of the antisense strand and the 5′-end of the sense strand do not have any overhanging nucleotides. In another example, a siNA molecule comprises two blunt ends, for example, wherein the 3′-end of the antisense strand and the 5′-end of the sense strand as well as the 5′-end of the antisense strand and 3′-end of the sense strand do not have any overhanging nucleotides. A blunt ended siNA molecule can comprise, for example, from about 15 to about 30 nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides). Other nucleotides present in a blunt ended siNA molecule can comprise, for example, mismatches, bulges, loops, or wobble base pairs to modulate the activity of the siNA molecule to mediate RNA interference.

By “blunt ends” is meant symmetric termini or termini of a double stranded siNA molecule having no overhanging nucleotides. The two strands of a double stranded siNA molecule align with each other without over-hanging nucleotides at the termini. For example, a blunt ended siNA construct comprises terminal nucleotides that are complementary between the sense and antisense regions of the siNA molecule.

In one embodiment, a double stranded nucleic acid molecule (e.g., siNA) molecule of the invention comprises ribonucleotides at positions that maintain or enhance RNAi activity. In one embodiment, ribonucleotides are present in the sense strand or sense region of the siNA molecule, which can provide for RNAi activity by allowing cleavage of the sense strand or sense region by an enzyme within the RISC (e.g., ribonucleotides present at the position of passenger strand, sense strand, or sense region cleavage, such as position 9 of the passenger strand of a 19 base-pair duplex, which is cleaved in the RISC by AGO2 enzyme, see, for example, Matranga et al., 2005, Cell, 123:1-114 and Rand et al., 2005, Cell, 123:621-629). In another embodiment, one or more (for example 1, 2, 3, 4 or 5) nucleotides at the 5′-end of the guide strand or guide region (also known as antisense strand or antisense region) of the siNA molecule are ribonucleotides.

In one embodiment, a double stranded nucleic acid molecule (e.g., siNA) molecule of the invention comprises one or more ribonucleotides at positions within the passenger strand or passenger region (also known as the sense strand or sense region) that allows cleavage of the passenger strand or passenger region by an enzyme in the RISC complex, (e.g., ribonucleotides present at the position of passenger strand, such as position 9 of the passenger strand of a 19 base-pair duplex that is cleaved in the RISC, see, for example, Matranga et al., 2005, Cell, 123:1-114 and Rand et al., 2005, Cell, 123:621-629).

In one embodiment, a siNA molecule of the invention contains at least 2, 3, 4, 5, or more chemical modifications that can be the same of different. In one embodiment, a siNA molecule of the invention contains at least 2, 3, 4, 5, or more different chemical modifications.

In one embodiment, a siNA molecule of the invention is a double-stranded short interfering nucleic acid (siNA), wherein the double stranded nucleic acid molecule comprises about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) of the nucleotide positions in each strand of the siNA molecule comprises a chemical modification. In another embodiment, the siNA contains at least 2, 3, 4, 5, or more different chemical modifications.

In one embodiment, the invention features double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a target gene or that directs cleavage of a target RNA, wherein the siNA molecule comprises about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein each strand of the siNA molecule comprises one or more chemical modifications. In one embodiment, each strand of the double stranded siNA molecule comprises at least two (e.g., 2, 3, 4, 5, or more) different chemical modifications, e.g., different nucleotide sugar, base, or backbone modifications. In another embodiment, one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of a target gene or a portion thereof, and the second strand of the double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence or a portion thereof of the target gene. In another embodiment, one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of a target gene or portion thereof, and the second strand of the double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence or portion thereof of the target gene. In another embodiment, each strand of the siNA molecule comprises about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, and each strand comprises at least about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that are complementary to the nucleotides of the other strand. The target gene can comprise, for example, sequences referred to herein or incorporated herein by reference. The gene can comprise, for example, sequences referred to by GenBank Accession number herein.

In one embodiment, each strand of a double stranded siNA molecule of the invention comprises a different pattern of chemical modifications, such as any “Stab 00”-“Stab 36” or “Stab 3F”-“Stab 36F” (Table IV) modification patterns herein or any combination thereof. Non-limiting examples of sense and antisense strands of such siNA molecules having various modification patterns are shown in Table III and FIGS. 4 and 5.

In one embodiment, a siNA molecule of the invention comprises no ribonucleotides. In another embodiment, a siNA molecule of the invention comprises one or more ribonucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more ribonucleotides).

In one embodiment, a siNA molecule of the invention comprises an antisense region comprising a nucleotide sequence that is complementary to a nucleotide sequence of a target gene or a portion thereof, and the siNA further comprises a sense region comprising a nucleotide sequence substantially similar to the nucleotide sequence of the target gene or a portion thereof. In another embodiment, the antisense region and the sense region each comprise about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides and the antisense region comprises at least about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that are complementary to nucleotides of the sense region. In one embodiment, each strand of the double stranded siNA molecule comprises at least two (e.g., 2, 3, 4, 5, or more) different chemical modifications, e.g., different nucleotide sugar, base, or backbone modifications. The target gene can comprise, for example, sequences referred to herein or incorporated by reference herein. In another embodiment, the siNA is a double stranded nucleic acid molecule, where each of the two strands of the siNA molecule independently comprise about 15 to about 40 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23, 33, 34, 35, 36, 37, 38, 39, or 40) nucleotides, and where one of the strands of the siNA molecule comprises at least about 15 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 or more) nucleotides that are complementary to the nucleic acid sequence of the target gene or a portion thereof.

In one embodiment, a siNA molecule of the invention comprises a sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by a target gene, or a portion thereof, and the sense region comprises a nucleotide sequence that is complementary to the antisense region. In one embodiment, the siNA molecule is assembled from two separate oligonucleotide fragments, wherein one fragment comprises the sense region and the second fragment comprises the antisense region of the siNA molecule. In another embodiment, the sense region is connected to the antisense region via a linker molecule. In another embodiment, the sense region is connected to the antisense region via a linker molecule, such as a nucleotide or non-nucleotide linker. In one embodiment, each strand of the double stranded siNA molecule comprises at least two (e.g., 2, 3, 4, 5, or more) different chemical modifications, e.g., different nucleotide sugar, base, or backbone modifications. The target gene can comprise, for example, sequences referred herein or incorporated by reference herein.

In one embodiment, a siNA molecule of the invention comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) 2′-deoxy-2′-fluoro pyrimidine modifications (e.g., where one or more or all pyrimidine (e.g., U or C) positions of the siNA are modified with 2′-deoxy-2′-fluoro nucleotides). In one embodiment, the 2′-deoxy-2′-fluoro pyrimidine modifications are present in the sense strand. In one embodiment, the 2′-deoxy-2′-fluoro pyrimidine modifications are present in the antisense strand. In one embodiment, the 2′-deoxy-2′-fluoro pyrimidine modifications are present in both the sense strand and the antisense strand of the siNA molecule.

In one embodiment, a siNA molecule of the invention comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) 2′-O-methyl purine modifications (e.g., where one or more or all purine (e.g., A or G) positions of the siNA are modified with 2′-O-methyl nucleotides). In one embodiment, the 2′-O-methyl purine modifications are present in the sense strand. In one embodiment, the 2′-O-methyl purine modifications are present in the antisense strand. In one embodiment, the 2′-O-methyl purine modifications are present in both the sense strand and the antisense strand of the siNA molecule.

In one embodiment, a siNA molecule of the invention comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) 2′-deoxy purine modifications (e.g., where one or more or all purine (e.g., A or G) positions of the siNA are modified with 2′-deoxy nucleotides). In one embodiment, the 2′-deoxy purine modifications are present in the sense strand. In one embodiment, the 2′-deoxy purine modifications are present in the antisense strand. In one embodiment, the 2′-deoxy purine modifications are present in both the sense strand and the antisense strand of the siNA molecule.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a target gene or that directs cleavage of a target RNA, comprising a sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by the target gene or a portion thereof and the sense region comprises a nucleotide sequence that is complementary to the antisense region, and wherein the siNA molecule has one or more modified pyrimidine and/or purine nucleotides. In one embodiment, each strand of the double stranded siNA molecule comprises at least two (e.g., 2, 3, 4, 5, or more) different chemical modifications, e.g., different nucleotide sugar, base, or backbone modifications. In one embodiment, the pyrimidine nucleotides in the sense region are 2′-O-methylpyrimidine nucleotides or 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-deoxy purine nucleotides. In another embodiment, the pyrimidine nucleotides in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-O-methyl purine nucleotides. In another embodiment, the pyrimidine nucleotides in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-deoxy purine nucleotides. In one embodiment, the pyrimidine nucleotides in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the antisense region are 2′-O-methyl or 2′-deoxy purine nucleotides. In another embodiment of any of the above-described siNA molecules, any nucleotides present in a non-complementary region of the sense strand (e.g. overhang region) are 2′-deoxy nucleotides.

In one embodiment, the invention features a siNA molecule comprising at least one modified nucleotide, wherein the modified nucleotide is a 2′-deoxy-2′-fluoro nucleotide, 2′-deoxy-2′-fluoroarabino, 2′-O-trifluoromethyl nucleotide, 2′-O-ethyl-trifluoromethoxy nucleotide, or 2′-O-difluoromethoxy-ethoxy nucleotide or any other modified nucleoside/nucleotide described herein and in U.S. Ser. No. 10/981,966, filed Nov. 5, 2004, incorporated by reference herein. In one embodiment, the invention features a siNA molecule comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) modified nucleotides, wherein the modified nucleotide is selected from the group consisting of 2′-deoxy-2′-fluoro nucleotide, 2′-deoxy-2′-fluoroarabino, 2′-O-trifluoromethyl nucleotide, 2′-O-ethyl-trifluoromethoxy nucleotide, or 2′-O-difluoromethoxy-ethoxy nucleotide or any other modified nucleoside/nucleotide described herein and in U.S. Ser. No. 10/981,966, filed Nov. 5, 2004, incorporated by reference herein. The modified nucleotide/nucleoside can be the same or different. The siNA can be, for example, about 15 to about 40 nucleotides in length. In one embodiment, all pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro, 2′-deoxy-2′-fluoroarabino, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy, 4′-thio pyrimidine nucleotides. In one embodiment, the modified nucleotides in the siNA include at least one 2′-deoxy-2′-fluoro cytidine or 2′-deoxy-2′-fluoro uridine nucleotide. In another embodiment, the modified nucleotides in the siNA include at least one 2′-deoxy-2′-fluoro cytidine and at least one 2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all uridine nucleotides present in the siNA are 2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all cytidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro cytidine nucleotides. In one embodiment, all adenosine nucleotides present in the siNA are 2′-deoxy-2′-fluoro adenosine nucleotides. In one embodiment, all guanosine nucleotides present in the siNA are 2′-deoxy-2′-fluoro guanosine nucleotides. The siNA can further comprise at least one modified internucleotidic linkage, such as phosphorothioate linkage. In one embodiment, the 2′-deoxy-2′-fluoronucleotides are present at specifically selected locations in the siNA that are sensitive to cleavage by ribonucleases, such as locations having pyrimidine nucleotides.

In one embodiment, the invention features a method of increasing the stability of a siNA molecule against cleavage by ribonucleases comprising introducing at least one modified nucleotide into the siNA molecule, wherein the modified nucleotide is a 2′-deoxy-2′-fluoro nucleotide. In one embodiment, all pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides. In one embodiment, the modified nucleotides in the siNA include at least one 2′-deoxy-2′-fluoro cytidine or 2′-deoxy-2′-fluoro uridine nucleotide. In another embodiment, the modified nucleotides in the siNA include at least one 2′-fluoro cytidine and at least one 2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all uridine nucleotides present in the siNA are 2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all cytidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro cytidine nucleotides. In one embodiment, all adenosine nucleotides present in the siNA are 2′-deoxy-2′-fluoro adenosine nucleotides. In one embodiment, all guanosine nucleotides present in the siNA are 2′-deoxy-2′-fluoro guanosine nucleotides. The siNA can further comprise at least one modified internucleotidic linkage, such as a phosphorothioate linkage. In one embodiment, the 2′-deoxy-2′-fluoronucleotides are present at specifically selected locations in the siNA that are sensitive to cleavage by ribonucleases, such as locations having pyrimidine nucleotides.

In one embodiment, the invention features a method of increasing the stability of a siNA molecule against cleavage by ribonucleases comprising introducing at least one modified nucleotide into the siNA molecule, wherein the modified nucleotide is a 2′-deoxy-2′-fluoroarabino nucleotide. In one embodiment, all pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoroarabino pyrimidine nucleotides. In one embodiment, the modified nucleotides in the siNA include at least one 2′-deoxy-2′-fluoroarabino cytidine or 2′-deoxy-2′-fluoroarabino uridine nucleotide. In another embodiment, the modified nucleotides in the siNA include at least one 2′-fluoro cytidine and at least one 2′-deoxy-2′-fluoroarabino uridine nucleotides. In one embodiment, all uridine nucleotides present in the siNA are 2′-deoxy-2′-fluoroarabino uridine nucleotides. In one embodiment, all cytidine nucleotides present in the siNA are 2′-deoxy-2′-fluoroarabino cytidine nucleotides. In one embodiment, all adenosine nucleotides present in the siNA are 2′-deoxy-2′-fluoroarabino adenosine nucleotides. In one embodiment, all guanosine nucleotides present in the siNA are 2′-deoxy-2′-fluoroarabino guanosine nucleotides. The siNA can further comprise at least one modified internucleotidic linkage, such as a phosphorothioate linkage. In one embodiment, the 2′-deoxy-2′-fluoroarabinonucleotides are present at specifically selected locations in the siNA that are sensitive to cleavage by ribonucleases, such as locations having pyrimidine nucleotides.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a target gene or that directs cleavage of a target RNA, comprising a sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by the target gene or a portion thereof and the sense region comprises a nucleotide sequence that is complementary to the antisense region, and wherein the purine nucleotides present in the antisense region comprise 2′-deoxy-purine nucleotides. In an alternative embodiment, the purine nucleotides present in the antisense region comprise 2′-O-methyl purine nucleotides. In either of the above embodiments, the antisense region can comprise a phosphorothioate internucleotide linkage at the 3′ end of the antisense region. Alternatively, in either of the above embodiments, the antisense region can comprise a glyceryl modification at the 3′ end of the antisense region. In another embodiment of any of the above-described siNA molecules, any nucleotides present in a non-complementary region of the antisense strand (e.g. overhang region) are 2′-deoxy nucleotides.

In one embodiment, the antisense region of a siNA molecule of the invention comprises sequence complementary to a portion of an endogenous transcript having sequence unique to a particular disease or trait related allele in a subject or organism, such as sequence comprising a single nucleotide polymorphism (SNP) associated with the disease or trait specific allele. As such, the antisense region of a siNA molecule of the invention can comprise sequence complementary to sequences that are unique to a particular allele to provide specificity in mediating selective RNAi against the disease, condition, or trait related allele.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a target gene or that directs cleavage of a target RNA, wherein the siNA molecule is assembled from two separate oligonucleotide fragments wherein one fragment comprises the sense region and the second fragment comprises the antisense region of the siNA molecule. In one embodiment, each strand of the double stranded siNA molecule is about 21 nucleotides long where about 19 nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule, wherein at least two 3′ terminal nucleotides of each fragment of the siNA molecule are not base-paired to the nucleotides of the other fragment of the siNA molecule. In another embodiment, the siNA molecule is a double stranded nucleic acid molecule, where each strand is about 19 nucleotide long and where the nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule to form at least about 15 (e.g., 15, 16, 17, 18, or 19) base pairs, wherein one or both ends of the siNA molecule are blunt ends. In one embodiment, each of the two 3′ terminal nucleotides of each fragment of the siNA molecule is a 2′-deoxy-pyrimidine nucleotide, such as a 2′-deoxy-thymidine. In one embodiment, each of the two 3′ terminal nucleotides of each fragment of the siNA molecule is a 2′-O-methylpyrimidine nucleotide, such as a 2′-O-methyl uridine, cytidine, or thymidine. In another embodiment, all nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule. In another embodiment, the siNA molecule is a double stranded nucleic acid molecule of about 19 to about 25 base pairs having a sense region and an antisense region, where about 19 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the target gene. In another embodiment, about 21 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the target gene. In any of the above embodiments, the 5′-end of the fragment comprising said antisense region can optionally include a phosphate group.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits the expression of a target RNA sequence, wherein the siNA molecule does not contain any ribonucleotides and wherein each strand of the double-stranded siNA molecule is about 15 to about 30 nucleotides. In one embodiment, the siNA molecule is 21 nucleotides in length. Examples of non-ribonucleotide containing siNA constructs are combinations of stabilization chemistries shown in Table IV in any combination of Sense/Antisense chemistries, such as Stab 7/8, Stab 7/11, Stab 8/8, Stab 18/8, Stab 18/11, Stab 12/13, Stab 7/13, Stab 18/13, Stab 7/19, Stab 8/19, Stab 18/19, Stab 7/20, Stab 8/20, Stab 18/20, Stab 7/32, Stab 8/32, or Stab 18/32 (e.g., any siNA having Stab 7, 8, 11, 12, 13, 14, 15, 17, 18, 19, 20, or 32 sense or antisense strands or any combination thereof). Herein, numeric Stab chemistries can include both 2′-fluoro and 2′-OCF3 versions of the chemistries shown in Table IV. For example, “Stab 7/8” refers to both Stab 7/8 and Stab 7F/8F etc. In one embodiment, the invention features a chemically synthesized double stranded RNA molecule that directs cleavage of a target RNA via RNA interference, wherein each strand of said RNA molecule is about 15 to about 30 nucleotides in length; one strand of the RNA molecule comprises nucleotide sequence having sufficient complementarity to the target RNA for the RNA molecule to direct cleavage of the target RNA via RNA interference; and wherein at least one strand of the RNA molecule optionally comprises one or more chemically modified nucleotides described herein, such as without limitation deoxynucleotides, 2′-O-methyl nucleotides, 2′-deoxy-2′-fluoro nucleotides, 2′-deoxy-2′-fluoroarabino, 2′-O-methoxyethyl nucleotides, 4′-thio nucleotides, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides, etc. or any combination thereof.

In one embodiment, a target RNA of the invention comprises sequence encoding a protein, such as an HCV or HCV pathway/host RNA encoding a HCV or HCV pathway/host protein.

In one embodiment, target RNA of the invention comprises non-coding RNA sequence (e.g., miRNA, snRNA, siRNA etc.), see for example Mattick, 2005, Science, 309, 1527-1528; Claverie, 2005, Science, 309, 1529-1530; Sethupathy et al., 2006, RNA, 12, 192-197; and Czech, 2006 NEJM, 354, 11: 1194-1195.

In one embodiment, the invention features a medicament comprising a siNA molecule of the invention.

In one embodiment, the invention features an active ingredient comprising a siNA molecule of the invention.

In one embodiment, the invention features the use of a double-stranded short interfering nucleic acid (siNA) molecule to inhibit, down-regulate, or reduce expression of a target gene, wherein the siNA molecule comprises one or more chemical modifications and each strand of the double-stranded siNA is independently about 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more) nucleotides long. In one embodiment, the siNA molecule of the invention is a double stranded nucleic acid molecule comprising one or more chemical modifications, where each of the two fragments of the siNA molecule independently comprise about 15 to about 40 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23, 33, 34, 35, 36, 37, 38, 39, or 40) nucleotides and where one of the strands comprises at least 15 nucleotides that are complementary to nucleotide sequence of target encoding RNA or a portion thereof. In a non-limiting example, each of the two fragments of the siNA molecule comprise about 21 nucleotides. In another embodiment, the siNA molecule is a double stranded nucleic acid molecule comprising one or more chemical modifications, where each strand is about 21 nucleotide long and where about 19 nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule, wherein at least two 3′ terminal nucleotides of each fragment of the siNA molecule are not base-paired to the nucleotides of the other fragment of the siNA molecule. In another embodiment, the siNA molecule is a double stranded nucleic acid molecule comprising one or more chemical modifications, where each strand is about 19 nucleotide long and where the nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule to form at least about 15 (e.g., 15, 16, 17, 18, or 19) base pairs, wherein one or both ends of the siNA molecule are blunt ends. In one embodiment, each of the two 3′ terminal nucleotides of each fragment of the siNA molecule is a 2′-deoxy-pyrimidine nucleotide, such as a 2′-deoxy-thymidine. In one embodiment, each of the two 3′ terminal nucleotides of each fragment of the siNA molecule is a 2′-O-methylpyrimidine nucleotide, such as a 2′-O-methyl uridine, cytidine, or thymidine. In another embodiment, all nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule. In another embodiment, the siNA molecule is a double stranded nucleic acid molecule of about 19 to about 25 base pairs having a sense region and an antisense region and comprising one or more chemical modifications, where about 19 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the target gene. In another embodiment, about 21 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the target gene. In any of the above embodiments, the 5′-end of the fragment comprising said antisense region can optionally include a phosphate group.

In one embodiment, the invention features the use of a double-stranded short interfering nucleic acid (siNA) molecule that inhibits, down-regulates, or reduces expression of a target gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of target RNA or a portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand. In one embodiment, each strand has at least two (e.g., 2, 3, 4, 5, or more) chemical modifications, which can be the same or different, such as nucleotide, sugar, base, or backbone modifications. In one embodiment, a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification. In one embodiment, a majority of the purine nucleotides present in the double-stranded siNA molecule comprises a sugar modification.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits, down-regulates, or reduces expression of a target gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of target RNA that encodes a protein or portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification. In one embodiment, each strand of the siNA molecule comprises about 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides, wherein each strand comprises at least about 15 nucleotides that are complementary to the nucleotides of the other strand. In one embodiment, the siNA molecule is assembled from two oligonucleotide fragments, wherein one fragment comprises the nucleotide sequence of the antisense strand of the siNA molecule and a second fragment comprises nucleotide sequence of the sense region of the siNA molecule. In one embodiment, the sense strand is connected to the antisense strand via a linker molecule, such as a polynucleotide linker or a non-nucleotide linker. In a further embodiment, the pyrimidine nucleotides present in the sense strand are 2′-deoxy-2′fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-deoxy purine nucleotides. In another embodiment, the pyrimidine nucleotides present in the sense strand are 2′-deoxy-2′fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-O-methyl purine nucleotides. In still another embodiment, the pyrimidine nucleotides present in the antisense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and any purine nucleotides present in the antisense strand are 2′-deoxy purine nucleotides. In another embodiment, the antisense strand comprises one or more 2′-deoxy-2′-fluoro pyrimidine nucleotides and one or more 2′-O-methyl purine nucleotides. In another embodiment, the pyrimidine nucleotides present in the antisense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and any purine nucleotides present in the antisense strand are 2′-O-methyl purine nucleotides. In a further embodiment the sense strand comprises a 3′-end and a 5′-end, wherein a terminal cap moiety (e.g., an inverted deoxy abasic moiety or inverted deoxy nucleotide moiety such as inverted thymidine) is present at the 5′-end, the 3′-end, or both of the 5′ and 3′ ends of the sense strand. In another embodiment, the antisense strand comprises a phosphorothioate internucleotide linkage at the 3′ end of the antisense strand. In another embodiment, the antisense strand comprises a glyceryl modification at the 3′ end. In another embodiment, the 5′-end of the antisense strand optionally includes a phosphate group.

In any of the above-described embodiments of a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a target gene, wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification, each of the two strands of the siNA molecule can comprise about 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides. In one embodiment, about 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides of each strand of the siNA molecule are base-paired to the complementary nucleotides of the other strand of the siNA molecule. In another embodiment, about 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides of each strand of the siNA molecule are base-paired to the complementary nucleotides of the other strand of the siNA molecule, wherein at least two 3′ terminal nucleotides of each strand of the siNA molecule are not base-paired to the nucleotides of the other strand of the siNA molecule. In another embodiment, each of the two 3′ terminal nucleotides of each fragment of the siNA molecule is a 2′-deoxy-pyrimidine, such as 2′-deoxy-thymidine. In one embodiment, each strand of the siNA molecule is base-paired to the complementary nucleotides of the other strand of the siNA molecule. In one embodiment, about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides of the antisense strand are base-paired to the nucleotide sequence of the target RNA or a portion thereof. In one embodiment, about 18 to about 25 (e.g., about 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides of the antisense strand are base-paired to the nucleotide sequence of the target RNA or a portion thereof.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a target gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of target RNA or a portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand. In one embodiment, each strand has at least two (e.g., 2, 3, 4, 5, or more) different chemical modifications, such as nucleotide sugar, base, or backbone modifications. In one embodiment, a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification. In one embodiment, a majority of the purine nucleotides present in the double-stranded siNA molecule comprises a sugar modification. In one embodiment, the 5′-end of the antisense strand optionally includes a phosphate group.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a target gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of target RNA or a portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification, and wherein the nucleotide sequence or a portion thereof of the antisense strand is complementary to a nucleotide sequence of the untranslated region or a portion thereof of the target RNA.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a target gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of target RNA or a portion thereof, wherein the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand, wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification, and wherein the nucleotide sequence of the antisense strand is complementary to a nucleotide sequence of the target RNA or a portion thereof that is present in the target RNA.

In one embodiment, the invention features a composition comprising a siNA molecule of the invention and a pharmaceutically acceptable carrier or diluent. In another embodiment, the invention features two or more differing siNA molecules of the invention (e.g. siNA molecules that target different regions of target RNA or siNA molecules that target HCV pathway RNA) and a pharmaceutically acceptable carrier or diluent.

In a non-limiting example, the introduction of chemically-modified nucleotides into nucleic acid molecules provides a powerful tool in overcoming potential limitations of in vivo stability and bioavailability inherent to native RNA molecules that are delivered exogenously. For example, the use of chemically-modified nucleic acid molecules can enable a lower dose of a particular nucleic acid molecule for a given therapeutic effect since chemically-modified nucleic acid molecules tend to have a longer half-life in serum. Furthermore, certain chemical modifications can improve the bioavailability of nucleic acid molecules by targeting particular cells or tissues and/or improving cellular uptake of the nucleic acid molecule. Therefore, even if the activity of a chemically-modified nucleic acid molecule is reduced as compared to a native nucleic acid molecule, for example, when compared to an all-RNA nucleic acid molecule, the overall activity of the modified nucleic acid molecule can be greater than that of the native molecule due to improved stability and/or delivery of the molecule. Unlike native unmodified siNA, chemically-modified siNA can also minimize the possibility of activating interferon activity or immunostimulation in humans. These properties therefore improve upon native siRNA or minimally modified siRNA's ability to mediate RNAi in various in vitro and in vivo settings, including use in both research and therapeutic applications. Applicant describes herein chemically modified siNA molecules with improved RNAi activity compared to corresponding unmodified or minimally modified siRNA molecules. The chemically modified siNA motifs disclosed herein provide the capacity to maintain RNAi activity that is substantially similar to unmodified or minimally modified active siRNA (see for example Elbashir et al., 2001, EMBO J., 20:6877-6888) while at the same time providing nuclease resistance and pharmacoketic properties suitable for use in therapeutic applications.

In any of the embodiments of siNA molecules described herein, the antisense region of a siNA molecule of the invention can comprise a phosphorothioate internucleotide linkage at the 3′-end of said antisense region. In any of the embodiments of siNA molecules described herein, the antisense region can comprise about one to about five phosphorothioate internucleotide linkages at the 5′-end of said antisense region. In any of the embodiments of siNA molecules described herein, the 3′-terminal nucleotide overhangs of a siNA molecule of the invention can comprise ribonucleotides or deoxyribonucleotides that are chemically-modified at a nucleic acid sugar, base, or backbone. In any of the embodiments of siNA molecules described herein, the 3′-terminal nucleotide overhangs can comprise one or more universal base ribonucleotides. In any of the embodiments of siNA molecules described herein, the 3′-terminal nucleotide overhangs can comprise one or more acyclic nucleotides.

One embodiment of the invention provides an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the invention in a manner that allows expression of the nucleic acid molecule. Another embodiment of the invention provides a mammalian cell comprising such an expression vector. The mammalian cell can be a human cell. The siNA molecule of the expression vector can comprise a sense region and an antisense region. The antisense region can comprise sequence complementary to a RNA or DNA sequence encoding a target and the sense region can comprise sequence complementary to the antisense region. The siNA molecule can comprise two distinct strands having complementary sense and antisense regions. The siNA molecule can comprise a single strand having complementary sense and antisense regions.

wherein each R1 and R2 is independently any nucleotide, non-nucleotide, or polynucleotide which can be naturally-occurring or chemically-modified and which can be included in the structure of the siNA molecule or serve as a point of attachment to the siNA molecule, each X and Y is independently O, S, N, alkyl, or substituted alkyl, each Z and W is independently O, S, N, alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, or acetyl and wherein W, X, Y, and Z are optionally not all O. In another embodiment, a backbone modification of the invention comprises a phosphonoacetate and/or thiophosphonoacetate internucleotide linkage (see for example Sheehan et al., 2003, Nucleic Acids Research, 31, 4109-4118).

The chemically-modified internucleotide linkages having Formula I, for example, wherein any Z, W, X, and/or Y independently comprises a sulphur atom, can be present in one or both oligonucleotide strands of the siNA duplex, for example, in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemically-modified internucleotide linkages having Formula I at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified internucleotide linkages having Formula I at the 5′-end of the sense strand, the antisense strand, or both strands. In another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidine nucleotides with chemically-modified internucleotide linkages having Formula I in the sense strand, the antisense strand, or both strands. In yet another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine nucleotides with chemically-modified internucleotide linkages having Formula I in the sense strand, the antisense strand, or both strands. In another embodiment, a siNA molecule of the invention having internucleotide linkage(s) of Formula I also comprises a chemically-modified nucleotide or non-nucleotide having any of Formulae I-VII.

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCH3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or a group having any of Formula I, II, III, IV, V, VI and/or VII, any of which can be included in the structure of the siNA molecule or serve as a point of attachment to the siNA molecule; R9 is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such as adenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other non-naturally occurring base that can be complementary or non-complementary to target RNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone, pyridinone, or any other non-naturally occurring universal base that can be complementary or non-complementary to target RNA. In one embodiment, R3 and/or R7 comprises a conjugate moiety and a linker (e.g., a nucleotide or non-nucleotide linker as described herein or otherwise known in the art). Non-limiting examples of conjugate moieties include ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; steroids, and polyamines, such as PEI, spermine or spermidine. In one embodiment, a nucleotide of the invention having Formula II is a 2′-deoxy-2′-fluoro nucleotide. In one embodiment, a nucleotide of the invention having Formula II is a 2′-O-methyl nucleotide. In one embodiment, a nucleotide of the invention having Formula II is a 2′-deoxy nucleotide.

The chemically-modified nucleotide or non-nucleotide of Formula II can be present in one or both oligonucleotide strands of the siNA duplex, for example in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more chemically-modified nucleotides or non-nucleotides of Formula II at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides or non-nucleotides of Formula II at the 5′-end of the sense strand, the antisense strand, or both strands. In another non-limiting example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides or non-nucleotides of Formula II at the 3′-end of the sense strand, the antisense strand, or both strands.

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCH3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or a group having any of Formula I, II, III, IV, V, VI and/or VII, any of which can be included in the structure of the siNA molecule or serve as a point of attachment to the siNA molecule; R9 is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such as adenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other non-naturally occurring base that can be employed to be complementary or non-complementary to target RNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone, pyridinone, or any other non-naturally occurring universal base that can be complementary or non-complementary to target RNA. In one embodiment, R3 and/or R7 comprises a conjugate moiety and a linker (e.g., a nucleotide or non-nucleotide linker as described herein or otherwise known in the art). Non-limiting examples of conjugate moieties include ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; steroids, and polyamines, such as PEI, spermine or spermidine.

The chemically-modified nucleotide or non-nucleotide of Formula III can be present in one or both oligonucleotide strands of the siNA duplex, for example, in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more chemically-modified nucleotides or non-nucleotides of Formula III at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide(s) or non-nucleotide(s) of Formula III at the 5′-end of the sense strand, the antisense strand, or both strands. In another non-limiting example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide or non-nucleotide of Formula III at the 3′-end of the sense strand, the antisense strand, or both strands.

In another embodiment, a siNA molecule of the invention comprises a nucleotide having Formula II or III, wherein the nucleotide having Formula II or III is in an inverted configuration. For example, the nucleotide having Formula II or III is connected to the siNA construct in a 3′-3′, 3′-2′, 2′-3′, or 5′-5′ configuration, such as at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

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

In one embodiment, the invention features a siNA molecule having a 5′-terminal phosphate group having Formula IV on the target-complementary strand, for example, a strand complementary to a target RNA, wherein the siNA molecule comprises an all RNA siNA molecule. In another embodiment, the invention features a siNA molecule having a 5′-terminal phosphate group having Formula IV on the target-complementary strand wherein the siNA molecule also comprises about 1 to about 3 (e.g., about 1, 2, or 3) nucleotide 3′-terminal nucleotide overhangs having about 1 to about 4 (e.g., about 1, 2, 3, or 4) deoxyribonucleotides on the 3′-end of one or both strands. In another embodiment, a 5′-terminal phosphate group having Formula IV is present on the target-complementary strand of a siNA molecule of the invention, for example a siNA molecule having chemical modifications having any of Formulae I-VII.

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

Each strand of the double stranded siNA molecule can have one or more chemical modifications such that each strand comprises a different pattern of chemical modifications. Several non-limiting examples of modification schemes that could give rise to different patterns of modifications are provided herein.

In one embodiment, the invention features a siNA molecule, wherein the sense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or 2′-deoxy-2′-fluoro nucleotides, with or without one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features a siNA molecule, wherein the sense strand comprises about 1 to about 5, specifically about 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or 2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about 5 or more, for example about 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.

In one embodiment, the invention features a siNA molecule, wherein the antisense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or 2′-deoxy-2′-fluoro nucleotides, with or without one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features a siNA molecule, wherein the antisense strand comprises about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or 2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about 5, for example about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule having about 1 to about 5 or more (specifically about 1, 2, 3, 4, 5 or more) phosphorothioate internucleotide linkages in each strand of the siNA molecule.

In another embodiment, the invention features a siNA molecule comprising 2′-5′ internucleotide linkages. The 2′-5′ internucleotide linkage(s) can be at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of one or both siNA sequence strands. In addition, the 2′-5′ internucleotide linkage(s) can be present at various other positions within one or both siNA sequence strands, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of a pyrimidine nucleotide in one or both strands of the siNA molecule can comprise a 2′-5′ internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of a purine nucleotide in one or both strands of the siNA molecule can comprise a 2′-5′ internucleotide linkage.

In another embodiment, a chemically-modified siNA molecule of the invention comprises a duplex having two strands, one or both of which can be chemically-modified, wherein each strand is independently about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length, wherein the duplex has about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the chemical modification comprises a structure having any of Formulae I-VII. For example, an exemplary chemically-modified siNA molecule of the invention comprises a duplex having two strands, one or both of which can be chemically-modified with a chemical modification having any of Formulae I-VII or any combination thereof, wherein each strand consists of about 21 nucleotides, each having a 2-nucleotide 3′-terminal nucleotide overhang, and wherein the duplex has about 19 base pairs. In another embodiment, a siNA molecule of the invention comprises a single stranded hairpin structure, wherein the siNA is about 36 to about 70 (e.g., about 36, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the siNA can include a chemical modification comprising a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises a linear oligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemically-modified with a chemical modification having any of Formulae I-VII or any combination thereof, wherein the linear oligonucleotide forms a hairpin structure having about 19 to about 21 (e.g., 19, 20, or 21) base pairs and a 2-nucleotide 3′-terminal nucleotide overhang. In another embodiment, a linear hairpin siNA molecule of the invention contains a stem loop motif, wherein the loop portion of the siNA molecule is biodegradable. For example, a linear hairpin siNA molecule of the invention is designed such that degradation of the loop portion of the siNA molecule in vivo can generate a double-stranded siNA molecule with 3′-terminal overhangs, such as 3′-terminal nucleotide overhangs comprising about 2 nucleotides.

In another embodiment, a siNA molecule of the invention comprises a hairpin structure, wherein the siNA is about 25 to about 50 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, and wherein the siNA can include one or more chemical modifications comprising a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises a linear oligonucleotide having about 25 to about 35 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that is chemically-modified with one or more chemical modifications having any of Formulae I-VII or any combination thereof, wherein the linear oligonucleotide forms a hairpin structure having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs and a 5′-terminal phosphate group that can be chemically modified as described herein (for example a 5′-terminal phosphate group having Formula IV). In another embodiment, a linear hairpin siNA molecule of the invention contains a stem loop motif, wherein the loop portion of the siNA molecule is biodegradable. In one embodiment, a linear hairpin siNA molecule of the invention comprises a loop portion comprising a non-nucleotide linker.

In another embodiment, a siNA molecule of the invention comprises an asymmetric hairpin structure, wherein the siNA is about 25 to about 50 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, and wherein the siNA can include one or more chemical modifications comprising a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises a linear oligonucleotide having about 25 to about 35 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that is chemically-modified with one or more chemical modifications having any of Formulae I-VII or any combination thereof, wherein the linear oligonucleotide forms an asymmetric hairpin structure having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs and a 5′-terminal phosphate group that can be chemically modified as described herein (for example a 5′-terminal phosphate group having Formula IV). In one embodiment, an asymmetric hairpin siNA molecule of the invention contains a stem loop motif, wherein the loop portion of the siNA molecule is biodegradable. In another embodiment, an asymmetric hairpin siNA molecule of the invention comprises a loop portion comprising a non-nucleotide linker.

In another embodiment, a siNA molecule of the invention comprises an asymmetric double stranded structure having separate polynucleotide strands comprising sense and antisense regions, wherein the antisense region is about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length, wherein the sense region is about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides in length, wherein the sense region and the antisense region have at least 3 complementary nucleotides, and wherein the siNA can include one or more chemical modifications comprising a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises an asymmetric double stranded structure having separate polynucleotide strands comprising sense and antisense regions, wherein the antisense region is about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) nucleotides in length and wherein the sense region is about 3 to about 15 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) nucleotides in length, wherein the sense region the antisense region have at least 3 complementary nucleotides, and wherein the siNA can include one or more chemical modifications comprising a structure having any of Formulae I-VII or any combination thereof. In another embodiment, the asymmetric double stranded siNA molecule can also have a 5′-terminal phosphate group that can be chemically modified as described herein (for example a 5′-terminal phosphate group having Formula IV).

In another embodiment, a siNA molecule of the invention comprises a circular nucleic acid molecule, wherein the siNA is about 38 to about 70 (e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the siNA can include a chemical modification, which comprises a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises a circular oligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemically-modified with a chemical modification having any of Formulae I-VII or any combination thereof, wherein the circular oligonucleotide forms a dumbbell shaped structure having about 19 base pairs and 2 loops.

In another embodiment, a circular siNA molecule of the invention contains two loop motifs, wherein one or both loop portions of the siNA molecule is biodegradable. For example, a circular siNA molecule of the invention is designed such that degradation of the loop portions of the siNA molecule in vivo can generate a double-stranded siNA molecule with 3′-terminal overhangs, such as 3′-terminal nucleotide overhangs comprising about 2 nucleotides.

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

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCH3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or a group having any of Formula I, II, III, IV, V, VI and/or VII, any of which can be included in the structure of the siNA molecule or serve as a point of attachment to the siNA molecule; R9 is O, S, CH2, S═O, CHF, or CF2. In one embodiment, R3 and/or R7 comprises a conjugate moiety and a linker (e.g., a nucleotide or non-nucleotide linker as described herein or otherwise known in the art). Non-limiting examples of conjugate moieties include ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; steroids, and polyamines, such as PEI, spermine or spermidine.

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

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCH3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or a group having any of Formula I, II, III, IV, V, VI and/or VII, any of which can be included in the structure of the siNA molecule or serve as a point of attachment to the siNA molecule; R9 is O, S, CH2, S═O, CHF, or CF2, and either R2, R3, R8 or R13 serve as points of attachment to the siNA molecule of the invention. In one embodiment, R3 and/or R7 comprises a conjugate moiety and a linker (e.g., a nucleotide or non-nucleotide linker as described herein or otherwise known in the art). Non-limiting examples of conjugate moieties include ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; steroids, and polyamines, such as PEI, spermine or spermidine.

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

wherein each n is independently an integer from 1 to 12, each R1, R2 and R3 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCH3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or a group having any of Formula I, II, III, IV, V, VI and/or VII, any of which can be included in the structure of the siNA molecule or serve as a point of attachment to the siNA molecule. In one embodiment, R3 and/or R1 comprises a conjugate moiety and a linker (e.g., a nucleotide or non-nucleotide linker as described herein or otherwise known in the art). Non-limiting examples of conjugate moieties include ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; steroids, and polyamines, such as PEI, spermine or spermidine.

By “ZIP code” sequences is meant, any peptide or protein sequence that is involved in cellular topogenic signaling mediated transport (see for example Ray et al., 2004, Science, 306(1501): 1505).

Each nucleotide within the double stranded siNA molecule can independently have a chemical modification comprising the structure of any of Formulae I-VIII. Thus, in one embodiment, one or more nucleotide positions of a siNA molecule of the invention comprises a chemical modification having structure of any of Formulae I-VII or any other modification herein. In one embodiment, each nucleotide position of a siNA molecule of the invention comprises a chemical modification having structure of any of Formulae I-VII or any other modification herein.

In one embodiment, one or more nucleotide positions of one or both strands of a double stranded siNA molecule of the invention comprises a chemical modification having structure of any of Formulae I-VII or any other modification herein. In one embodiment, each nucleotide position of one or both strands of a double stranded siNA molecule of the invention comprises a chemical modification having structure of any of Formulae I-VII or any other modification herein.

In another embodiment, the invention features a compound having Formula VII, wherein R1 and R2 are hydroxyl (OH) groups, n=1, and R3 comprises 0 and is the point of attachment to the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of one or both strands of a double-stranded siNA molecule of the invention or to a single-stranded siNA molecule of the invention. This modification is referred to herein as “glyceryl” (for example modification 6 in FIG. 10).

In another embodiment, a chemically modified nucleoside or non-nucleoside (e.g. a moiety having any of Formula V, VI or VII) of the invention is at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of a siNA molecule of the invention. For example, chemically modified nucleoside or non-nucleoside (e.g., a moiety having Formula V, VI or VII) can be present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the antisense strand, the sense strand, or both antisense and sense strands of the siNA molecule. In one embodiment, the chemically modified nucleoside or non-nucleoside (e.g., a moiety having Formula V, VI or VII) is present at the 5′-end and 3′-end of the sense strand and the 3′-end of the antisense strand of a double stranded siNA molecule of the invention. In one embodiment, the chemically modified nucleoside or non-nucleoside (e.g., a moiety having Formula V, VI or VII) is present at the terminal position of the 5′-end and 3′-end of the sense strand and the 3′-end of the antisense strand of a double stranded siNA molecule of the invention. In one embodiment, the chemically modified nucleoside or non-nucleoside (e.g., a moiety having Formula V, VI or VII) is present at the two terminal positions of the 5′-end and 3′-end of the sense strand and the 3′-end of the antisense strand of a double stranded siNA molecule of the invention. In one embodiment, the chemically modified nucleoside or non-nucleoside (e.g., a moiety having Formula V, VI or VII) is present at the penultimate position of the 5′-end and 3′-end of the sense strand and the 3′-end of the antisense strand of a double stranded siNA molecule of the invention. In addition, a moiety having Formula VII can be present at the 3′-end or the 5′-end of a hairpin siNA molecule as described herein.

In another embodiment, a siNA molecule of the invention comprises an abasic residue having Formula V or VI, wherein the abasic residue having Formula VI or VI is connected to the siNA construct in a 3′-3′, 3′-2′, 2′-3′, or 5′-5′ configuration, such as at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, a siNA molecule of the invention comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked nucleic acid (LNA) nucleotides, for example, at the 5′-end, the 3′-end, both of the 5′ and 3′-ends, or any combination thereof, of the siNA molecule.

In one embodiment, a siNA molecule of the invention comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 4′-thio nucleotides, for example, at the 5′-end, the 3′-end, both of the 5′ and 3′-ends, or any combination thereof, of the siNA molecule.

In another embodiment, a siNA molecule of the invention comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclic nucleotides, for example, at the 5′-end, the 3′-end, both of the 5′ and 3′-ends, or any combination thereof, of the siNA molecule.

In one embodiment, a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprises a sense strand or sense region having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) 2′-O-alkyl (e.g. 2′-O-methyl), 2′-deoxy-2′-fluoro, 2′-deoxy, FANA, or abasic chemical modifications or any combination thereof.

In one embodiment, a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprises an antisense strand or antisense region having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) 2′-O-alkyl (e.g. 2′-O-methyl), 2′-deoxy-2′-fluoro, 2′-deoxy, FANA, or abasic chemical modifications or any combination thereof.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are FANA pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are FANA pyrimidine nucleotides or alternately a plurality (ie. more than one) of pyrimidine nucleotides are FANA pyrimidine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region and an antisense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the sense region and the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality (ie. more than one) of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any (e.g., one or more or all) purine nucleotides present in the sense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality (ie. more than one) of purine nucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality (ie. more than one) of pyrimidine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality (ie. more than one) of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the sense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality (ie. more than one) of purine nucleotides are 2′-deoxy purine nucleotides), wherein any nucleotides comprising a 3′-terminal nucleotide overhang that are present in said sense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality (ie. more than one) of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the sense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality (ie. more than one) of purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality (ie. more than one) of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), wherein any (e.g., one or more or all) purine nucleotides present in the sense region are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality (ie. more than one) of purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides), and wherein any nucleotides comprising a 3′-terminal nucleotide overhang that are present in said sense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality (ie. more than one) of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality (ie. more than one) of purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality (ie. more than one) of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality (ie. more than one) of purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides), and wherein any nucleotides comprising a 3′-terminal nucleotide overhang that are present in said antisense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality (ie. more than one) of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality (ie. more than one) of purine nucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality (ie. more than one) of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality (ie. more than one) of purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) inside a cell or reconstituted in vitro system comprising a sense region, wherein one or more pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality (ie. more than one) of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and one or more purine nucleotides present in the sense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality (ie. more than one) of purine nucleotides are 2′-deoxy purine nucleotides), and an antisense region, wherein one or more pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality (ie. more than one) of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and one or more purine nucleotides present in the antisense region are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality (ie. more than one) of purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides). The sense region and/or the antisense region can have a terminal cap modification, such as any modification described herein or shown in FIG. 10, that is optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense and/or antisense sequence. The sense and/or antisense region can optionally further comprise a 3′-terminal nucleotide overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides. The overhang nucleotides can further comprise one or more (e.g., about 1, 2, 3, 4 or more) phosphorothioate, phosphonoacetate, and/or thiophosphonoacetate internucleotide linkages. Non-limiting examples of these chemically-modified siNAs are shown in FIGS. 4 and 5 and Table III herein. In any of these described embodiments, the purine nucleotides present in the sense region are alternatively 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides) and one or more purine nucleotides present in the antisense region are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality (ie. more than one) of purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides). Also, in any of these embodiments, one or more purine nucleotides present in the sense region are alternatively purine ribonucleotides (e.g., wherein all purine nucleotides are purine ribonucleotides or alternately a plurality (ie. more than one) of purine nucleotides are purine ribonucleotides) and any purine nucleotides present in the antisense region are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality (ie. more than one) of purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides). Additionally, in any of these embodiments, one or more purine nucleotides present in the sense region and/or present in the antisense region are alternatively selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides and 2′-O-methyl nucleotides (e.g., wherein all purine nucleotides are selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides and 2′-O-methyl nucleotides or alternately a plurality (ie. more than one) of purine nucleotides are selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides and 2′-O-methyl nucleotides).

In another embodiment, any modified nucleotides present in the siNA molecules of the invention, preferably in the antisense strand of the siNA molecules of the invention, but also optionally in the sense and/or both antisense and sense strands, comprise modified nucleotides having properties or characteristics similar to naturally occurring ribonucleotides. For example, the invention features siNA molecules including modified nucleotides having a Northern conformation (e.g., Northern pseudorotation cycle, see for example Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984) otherwise known as a “ribo-like” or “A-form helix” configuration. As such, chemically modified nucleotides present in the siNA molecules of the invention, preferably in the antisense strand of the siNA molecules of the invention, but also optionally in the sense and/or both antisense and sense strands, are resistant to nuclease degradation while at the same time maintaining the capacity to mediate RNAi. Non-limiting examples of nucleotides having a northern configuration include locked nucleic acid (LNA) nucleotides (e.g., 2′-O, 4′-C-methylene-(D-ribofuranosyl) nucleotides); 2′-methoxyethoxy (MOE) nucleotides; 2′-methyl-thio-ethyl, 2′-deoxy-2′-fluoro nucleotides, 2′-deoxy-2′-chloro nucleotides, 2′-azido nucleotides, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides, 4′-thio nucleotides and 2′-O-methyl nucleotides.

In one embodiment, the sense strand of a double stranded siNA molecule of the invention comprises a terminal cap moiety, (see for example FIG. 10) such as an inverted deoxyabasic moiety, at the 3′-end, 5′-end, or both 3′ and 5′-ends of the sense strand.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid molecule (siNA) capable of mediating RNA interference (RNAi) inside a cell or reconstituted in vitro system, wherein the chemical modification comprises a conjugate covalently attached to the chemically-modified siNA molecule. Non-limiting examples of conjugates contemplated by the invention include conjugates and ligands described in Vargeese et al., U.S. Ser. No. 10/427,160, filed Apr. 30, 2003, incorporated by reference herein in its entirety, including the drawings. In another embodiment, the conjugate is covalently attached to the chemically-modified siNA molecule via a biodegradable linker. In one embodiment, the conjugate molecule is attached at the 3′-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule. In another embodiment, the conjugate molecule is attached at the 5′-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule. In yet another embodiment, the conjugate molecule is attached both the 3′-end and 5′-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule, or any combination thereof. In one embodiment, a conjugate molecule of the invention comprises a molecule that facilitates delivery of a chemically-modified siNA molecule into a biological system, such as a cell. In another embodiment, the conjugate molecule attached to the chemically-modified siNA molecule is a ligand for a cellular receptor, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; steroids, and polyamines, such as PEI, spermine or spermidine. Examples of specific conjugate molecules contemplated by the instant invention that can be attached to chemically-modified siNA molecules are described in Vargeese et al., U.S. Ser. No. 10/201,394, filed Jul. 22, 2002 incorporated by reference herein. The type of conjugates used and the extent of conjugation of siNA molecules of the invention can be evaluated for improved pharmacokinetic profiles, bioavailability, and/or stability of siNA constructs while at the same time maintaining the ability of the siNA to mediate RNAi activity. As such, one skilled in the art can screen siNA constructs that are modified with various conjugates to determine whether the siNA conjugate complex possesses improved properties while maintaining the ability to mediate RNAi, for example in animal models as are generally known in the art.

In one embodiment, the invention features a short interfering nucleic acid (siNA) molecule of the invention, wherein the siNA further comprises a nucleotide, non-nucleotide, or mixed nucleotide/non-nucleotide linker that joins the sense region of the siNA to the antisense region of the siNA. In one embodiment, a nucleotide, non-nucleotide, or mixed nucleotide/non-nucleotide linker is used, for example, to attach a conjugate moiety to the siNA. In one embodiment, a nucleotide linker of the invention can be a linker of ≧2 nucleotides in length, for example about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In another embodiment, the nucleotide linker can be a nucleic acid aptamer. By “aptamer” or “nucleic acid aptamer” as used herein is meant a nucleic acid molecule that binds specifically to a target molecule wherein the nucleic acid molecule has sequence that comprises a sequence recognized by the target molecule in its natural setting. Alternately, an aptamer can be a nucleic acid molecule that binds to a target molecule where the target molecule does not naturally bind to a nucleic acid. The target molecule can be any molecule of interest. For example, the aptamer can be used to bind to a ligand-binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein. This is a non-limiting example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art. (See, for example, Gold et al., 1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J. Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser, 2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287, 820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.)

In yet another embodiment, a non-nucleotide linker of the invention comprises abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g. polyethylene glycols such as those having between 2 and 100 ethylene glycol units). Specific examples include those described by Seela and Kaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic Acids Res. 1987, 15:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 113:6324; Richardson and Schepartz, J. Am. Chem. Soc. 1991, 113:5109; Ma et al., Nucleic Acids Res. 1993, 21:2585 and Biochemistry 1993, 32:1751; Durand et al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides & Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301; Ono et al., Biochemistry 1991, 30:9914; Arnold et al., International Publication No. WO 89/02439; Usman et al., International Publication No. WO 95/06731; Dudycz et al., International Publication No. WO 95/11910 and Ferentz and Verdine, J. Am. Chem. Soc. 1991, 113:4000, all hereby incorporated by reference herein. A “non-nucleotide” further means any group or compound that can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the siNA molecule to retain RNAi activity or RNAi inhibitory to retain its inhibition activity. The group or compound can be abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine, for example at the C1 position of the sugar.

In one embodiment, the invention features a short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) inside a cell or reconstituted in vitro system, wherein one or both strands of the siNA molecule that are assembled from two separate oligonucleotides do not comprise any ribonucleotides (e.g., one or both strands of the siNA molecule are 100% chemically modified). For example, a siNA molecule can be assembled from a single oligonucleotide where the sense and antisense regions of the siNA comprise separate oligonucleotides that do not have any ribonucleotides (e.g., nucleotides having a 2′-OH group) present in the oligonucleotides. In another example, a siNA molecule can be assembled from a single oligonucleotide where the sense and antisense regions of the siNA are linked or circularized by a nucleotide or non-nucleotide linker as described herein, wherein the oligonucleotide does not have any ribonucleotides (e.g., nucleotides having a 2′-OH group) present in the oligonucleotide. Applicant has surprisingly found that the presence of ribonucleotides (e.g., nucleotides having a 2′-hydroxyl group) within the siNA molecule is not required or essential to support RNAi activity. As such, in one embodiment, all positions within the siNA can include chemically modified nucleotides and/or non-nucleotides such as nucleotides and or non-nucleotides having Formula I, II, III, IV, V, VI, or VII or any combination thereof to the extent that the ability of the siNA molecule to support RNAi activity in a cell is maintained.

In one embodiment, a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity in a cell or reconstituted in vitro system comprising a single stranded polynucleotide having complementarity to a target nucleic acid sequence. In another embodiment, the single stranded siNA molecule of the invention comprises a 5′-terminal phosphate group. In another embodiment, the single stranded siNA molecule of the invention comprises a 5′-terminal phosphate group and a 3′-terminal phosphate group (e.g., a 2′,3′-cyclic phosphate). In another embodiment, the single stranded siNA molecule of the invention comprises about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In yet another embodiment, the single stranded siNA molecule of the invention comprises one or more chemically modified nucleotides or non-nucleotides described herein. For example, all the positions within the siNA molecule can include chemically-modified nucleotides such as nucleotides having any of Formulae I-VII, or any combination thereof to the extent that the ability of the siNA molecule to support RNAi activity in a cell is maintained.

In one embodiment, a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity or that alternately modulates RNAi activity in a cell or reconstituted in vitro system comprising a single stranded polynucleotide having complementarity to a target nucleic acid sequence, wherein one or more pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein any purine nucleotides present in the antisense region are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purine nucleotides), and a terminal cap modification, such as any modification described herein or shown in FIG. 10, that is optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the antisense sequence. The siNA optionally further comprises about 1 to about 4 or more (e.g., about 1, 2, 3, 4 or more) terminal 2′-deoxynucleotides at the 3′-end of the siNA molecule, wherein the terminal nucleotides can further comprise one or more (e.g., 1, 2, 3, 4 or more) phosphorothioate, phosphonoacetate, and/or thiophosphonoacetate internucleotide linkages, and wherein the siNA optionally further comprises a terminal phosphate group, such as a 5′-terminal phosphate group. In any of these embodiments, any purine nucleotides present in the antisense region are alternatively 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides). Also, in any of these embodiments, any purine nucleotides present in the siNA (i.e., purine nucleotides present in the sense and/or antisense region) can alternatively be locked nucleic acid (LNA) nucleotides (e.g., wherein all purine nucleotides are LNA nucleotides or alternately a plurality of purine nucleotides are LNA nucleotides). Also, in any of these embodiments, any purine nucleotides present in the siNA are alternatively 2′-methoxyethyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-methoxyethyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-methoxyethyl purine nucleotides). In another embodiment, any modified nucleotides present in the single stranded siNA molecules of the invention comprise modified nucleotides having properties or characteristics similar to naturally occurring ribonucleotides. For example, the invention features siNA molecules including modified nucleotides having a Northern conformation (e.g., Northern pseudorotation cycle, see for example Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemically modified nucleotides present in the single stranded siNA molecules of the invention are preferably resistant to nuclease degradation while at the same time maintaining the capacity to mediate RNAi.

In one embodiment, a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprises a sense strand or sense region having two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) 2′-O-alkyl (e.g. 2′-O-methyl) modifications or any combination thereof. In another embodiment, the 2′-O-alkyl modification is at alternating position in the sense strand or sense region of the siNA, such as position 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 etc. or position 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 etc.

In one embodiment, a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprises an antisense strand or antisense region having two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) 2′-O-alkyl (e.g. 2′-O-methyl) modifications or any combination thereof. In another embodiment, the 2′-O-alkyl modification is at alternating position in the antisense strand or antisense region of the siNA, such as position 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 etc. or position 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 etc.

In one embodiment, a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprises a sense strand or sense region and an antisense strand or antisense region, each having two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) 2′-O-alkyl (e.g. 2′-O-methyl), 2′-deoxy-2′-fluoro, 2′-deoxy, or abasic chemical modifications or any combination thereof. In another embodiment, the 2′-O-alkyl modification is at alternating position in the sense strand or sense region of the siNA, such as position 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 etc. or position 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 etc. In another embodiment, the 2′-O-alkyl modification is at alternating position in the antisense strand or antisense region of the siNA, such as position 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 etc. or position 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 etc.

In one embodiment, a siNA molecule of the invention comprises chemically modified nucleotides or non-nucleotides (e.g., having any of Formulae I-VII, such as 2′-deoxy, 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy or 2′-O-methyl nucleotides) at alternating positions within one or more strands or regions of the siNA molecule. For example, such chemical modifications can be introduced at every other position of a RNA based siNA molecule, starting at either the first or second nucleotide from the 3′-end or 5′-end of the siNA. In a non-limiting example, a double stranded siNA molecule of the invention in which each strand of the siNA is 21 nucleotides in length is featured wherein positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21 of each strand are chemically modified (e.g., with compounds having any of Formulae I-VII, such as such as 2′-deoxy, 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy or 2′-O-methyl nucleotides). In another non-limiting example, a double stranded siNA molecule of the invention in which each strand of the siNA is 21 nucleotides in length is featured wherein positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 of each strand are chemically modified (e.g., with compounds having any of Formulae I-VII, such as such as 2′-deoxy, 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy or 2′-O-methyl nucleotides). In one embodiment, one strand of the double stranded siNA molecule comprises chemical modifications at positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 and chemical modifications at positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21. Such siNA molecules can further comprise terminal cap moieties and/or backbone modifications as described herein.

In one embodiment, a siNA molecule of the invention comprises the following features: if purine nucleotides are present at the 5′-end (e.g., at any of terminal nucleotide positions 1, 2, 3, 4, 5, or 6 from the 5′-end) of the antisense strand or antisense region (otherwise referred to as the guide sequence or guide strand) of the siNA molecule then such purine nucleosides are ribonucleotides. In another embodiment, the purine ribonucleotides, when present, are base paired to nucleotides of the sense strand or sense region (otherwise referred to as the passenger strand) of the siNA molecule. Such purine ribonucleotides can be present in a siNA stabilization motif that otherwise comprises modified nucleotides.

In one embodiment, a siNA molecule of the invention comprises the following features: if pyrimidine nucleotides are present at the 5′-end (e.g., at any of terminal nucleotide positions 1, 2, 3, 4, 5, or 6 from the 5′-end) of the antisense strand or antisense region (otherwise referred to as the guide sequence or guide strand) of the siNA molecule then such pyrimidine nucleosides are ribonucleotides. In another embodiment, the pyrimidine ribonucleotides, when present, are base paired to nucleotides of the sense strand or sense region (otherwise referred to as the passenger strand) of the siNA molecule. Such pyrimidine ribonucleotides can be present in a siNA stabilization motif that otherwise comprises modified nucleotides.

In one embodiment, a siNA molecule of the invention comprises the following features: if pyrimidine nucleotides are present at the 5′-end (e.g., at any of terminal nucleotide positions 1, 2, 3, 4, 5, or 6 from the 5′-end) of the antisense strand or antisense region (otherwise referred to as the guide sequence or guide strand) of the siNA molecule then such pyrimidine nucleosides are modified nucleotides. In another embodiment, the modified pyrimidine nucleotides, when present, are base paired to nucleotides of the sense strand or sense region (otherwise referred to as the passenger strand) of the siNA molecule. Non-limiting examples of modified pyrimidine nucleotides include those having any of Formulae I-VII, such as such as 2′-deoxy, 2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy or 2′-O-methyl nucleotides.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, SVI, SVII, SVIII, SIX, SX, SXII, SXIII, or SXIV comprises a terminal phosphate group at the 5′-end of the antisense strand or antisense region of the nucleic acid molecule.

In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, SVI, SVII, SVIII, SIX, SX, SXII, SXIII, or SXIV comprises X5=1, 2, or 3; each X1 and X2=1 or 2; X3=12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and X4=15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.

In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, SVI, SVII, SVIII, SIX, SX, SXII, SXIII, or SXIV comprises X5=1; each X1 and X2=2; X3=19, and X4=18.

In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, SVI, SVII, SVIII, SIX, SX, SXII, SXIII, or SXIV comprises X5=2; each X1 and X2=2; X3=19, and X4=17

In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, SVI, SVII, SVIII, SIX, SX, SXII, SXIII, or SXIV comprises X5=3; each X1 and X2=2; X3=19, and X4=16.

In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, SVI, SVII, SVIII, SIX, SX, SXII, SXIII, or SXIV comprises B at the 3′ and 5′ ends of the sense strand or sense region.

In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, SVI, SVII, SVIII, SIX, SX, SXII, SXIII, or SXIV comprises B at the 3′-end of the antisense strand or antisense region.

In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, SVI, SVII, SVIII, SIX, SX, SXII, SXIII, or SXIV further comprises one or more phosphorothioate internucleotide linkages at the first terminal (N) on the 3′ end of the sense strand, antisense strand, or both sense strand and antisense strands of the nucleic acid molecule. For example, a double stranded nucleic acid molecule can comprise X1 and/or X2=2 having overhanging nucleotide positions with a phosphorothioate internucleotide linkage, e.g., (NsN) where “s” indicates phosphorothioate.

In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, SVI, SVII, SVIII, SIX, SX, SXII, SXIII, or SXIV comprises (N) nucleotides in the antisense strand (lower strand) that are complementary to nucleotides in a target polynucleotide sequence (e.g., HCV target and/or HCV pathway/host target sequence) having complementary to the N and [N] nucleotides of the antisense (lower) strand.

In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, SVI, SVII, SVIII, SIX, SX, SXII, SXIII, or SXIV comprises (N) nucleotides in the sense strand (upper strand) that comprise nucleotide sequence corresponding a target polynucleotide sequence (e.g., HCV target and/or HCV pathway/host target sequence) having complementary to the antisense (lower) strand such that the contiguous (N) and N nucleotide sequence of the sense strand comprises nucleotide sequence of the target nucleic acid sequence (e.g., HCV target and/or HCV pathway/host target sequence).

In one embodiment, a double stranded nucleic acid molecule having any of structure SVIII or SXIV comprises B only at the 5′-end of the sense (upper) strand of the double stranded nucleic acid molecule.

In one embodiment, a double stranded nucleic acid molecule having any of structure SI, SII, SIII, SIV, SV, SVI, SVII, SVIII, SIX, SX, SXII, SXIII, or SXIV further comprises an unpaired terminal nucleotide at the 5′-end of the antisense (lower) strand. The unpaired nucleotide is not complementary to the sense (upper) strand. In one embodiment, the unpaired terminal nucleotide is complementary to a target polynucleotide sequence having complementary to the N and [N] nucleotides of the antisense (lower) strand. In another embodiment, the unpaired terminal nucleotide is not complementary to a target polynucleotide sequence having complementary to the N and [N] nucleotides of the antisense (lower) strand.

In one embodiment, a double stranded nucleic acid molecule having any of structure SVIII or SXIV comprises X6=1 and X3=10.

In one embodiment, a double stranded nucleic acid molecule having any of structure SVIII or SXIV comprises X6=2 and X3=9.

In one embodiment, the invention features a composition comprising a siNA molecule or double stranded nucleic acid molecule or RNAi inhibitor formulated as any of formulation LNP-051; LNP-053; LNP-054; LNP-069; LNP-073; LNP-077; LNP-080; LNP-082; LNP-083; LNP-060; LNP-061; LNP-086; LNP-097; LNP-098; LNP-099; LNP-100; LNP-101; LNP-102; LNP-103; or LNP-104 (see Table VI).

In one embodiment, the invention features a composition comprising a first double stranded nucleic and a second double stranded nucleic acid molecule each having a first strand and a second strand that are complementary to each other, wherein the second strand of the first double stranded nucleic acid molecule comprises sequence complementary to HCV sequence having SEQ ID NO: 1444 and the second strand of the second double stranded nucleic acid molecule comprises sequence complementary to HCV sequence having SEQ ID NO: 1417. In another embodiment, the first strand and the second strand of the first double stranded nucleic acid molecule comprise SEQ ID NOs: 1796 and 2010 respectively, and the first strand and the second strand of the second double stranded nucleic acid molecule comprise SEQ ID NOs: 1677 and 2011 respectively. In another embodiment, the first strand and the second strand of the first double stranded nucleic acid molecule comprise SEQ ID NOs: 1796 and 2012 respectively, and the first strand and the second strand of the second double stranded nucleic acid molecule comprise SEQ ID NOs: 1677 and 2013 respectively.

In another embodiment, the first strand and the second strand of the first double stranded nucleic acid molecule comprise SEQ ID NOs: 1796 and 2102 respectively, and the first strand and the second strand of the second double stranded nucleic acid molecule comprise SEQ ID NOs: 1677 and 2103 respectively.

In any of the above-described composition embodiments, the composition can further comprise various lipid and polyethyleneglycol-conjugates. In one embodiment, the composition further comprises a cationic lipid, a neutral lipid, and a polyethyleneglycol-conjugate. In another embodiment, the composition further comprises a cationic lipid, a neutral lipid, a polyethyleneglycol-conjugate, and a cholesterol. In another embodiment, the composition further comprises a polyethyleneglycol-conjugate, a cholesterol, and a surfactant. In one embodiment, the cationic lipid is selected from the group consisting of CLinDMA, pCLinDMA, eCLinDMA, DMOBA, and DMLBA. In one embodiment, the neutral lipid is selected from the group consisting of DSPC, DOBA, and cholesterol. In one embodiment, the polyethyleneglycol-conjugate is selected from the group consisting of a PEG-dimyristoyl glycerol and PEG-cholesterol. In one embodiment, the PEG is 2KPEG. In one embodiment, the surfactant is selected from the group consisting of palmityl alcohol, stearyl alcohol, oleyl alcohol and linoleyl alcohol. In one embodiment, the cationic lipid is CLinDMA, the neutral lipid is DSPC, the polyethylene glycol conjugate is 2KPEG-DMG, the cholesterol is cholesterol, and the surfactant is linoleyl alcohol. In one embodiment, the CLinDMA, the DSPC, the 2KPEG-DMG, the cholesterol, and the linoleyl alcohol are present in molar ratio of 43:38:10:2:7 respectively.

In any of the embodiments herein, the siNA molecule of the invention modulates expression of one or more targets via RNA interference or the inhibition of RNA interference. In one embodiment, the RNA interference is RISC mediated cleavage of the target (e.g., siRNA mediated RNA interference). In one embodiment, the RNA interference is translational inhibition of the target (e.g., miRNA mediated RNA interference). In one embodiment, the RNA interference is transcriptional inhibition of the target (e.g., siRNA mediated transcriptional silencing). In one embodiment, the RNA interference takes place in the cytoplasm. In one embodiment, the RNA interference takes place in the nucleus.

In any of the embodiments herein, the siNA molecule of the invention modulates expression of one or more targets via inhibition of an endogenous target RNA, such as an endogenous mRNA, siRNA, miRNA, or alternately though inhibition of RISC.

In one embodiment, the invention features one or more RNAi inhibitors that modulate the expression of one or more gene targets by miRNA inhibition, siRNA inhibition, or RISC inhibition.

In one embodiment, a RNAi inhibitor of the invention is a siNA molecule as described herein that has one or more strands that are complementary to one or more target miRNA or siRNA molecules.

In one embodiment, the RNAi inhibitor of the invention is an antisense molecule that is complementary to a target miRNA or siRNA molecule or a portion thereof. An antisense RNAi inhibitor of the invention can be of length of about 10 to about 40 nucleotides in length (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length). An antisense RNAi inhibitor of the invention can comprise one or more modified nucleotides or non-nucleotides as described herein (see for example molecules having any of Formulae I-VII herein or any combination thereof). In one embodiment, an antisense RNAi inhibitor of the invention can comprise one or more or all 2′-O-methyl nucleotides. In one embodiment, an antisense RNAi inhibitor of the invention can comprise one or more or all 2′-deoxy-2′-fluoro nucleotides. In one embodiment, an antisense RNAi inhibitor of the invention can comprise one or more or all 2′-O-methoxy-ethyl (also known as 2′-methoxyethoxy or MOE) nucleotides. In one embodiment, an antisense RNAi inhibitor of the invention can comprise one or more or all phosphorothioate internucleotide linkages. In one embodiment, an antisense RNA inhibitor or the invention can comprise a terminal cap moiety at the 3′-end, the 5′-end, or both the 5′ and 3′ ends of the antisense RNA inhibitor.

In one embodiment, a RNAi inhibitor of the invention is a nucleic acid aptamer having binding affinity for RISC, such as a regulatable aptamer (see for example An et al., 2006, RNA, 12:710-716). An aptamer RNAi inhibitor of the invention can be of length of about 10 to about 50 nucleotides in length (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length). An aptamer RNAi inhibitor of the invention can comprise one or more modified nucleotides or non-nucleotides as described herein (see for example molecules having any of Formulae I-VII herein or any combination thereof). In one embodiment, an aptamer RNAi inhibitor of the invention can comprise one or more or all 2′-O-methyl nucleotides. In one embodiment, an aptamer RNAi inhibitor of the invention can comprise one or more or all 2′-deoxy-2′-fluoro nucleotides. In one embodiment, an aptamer RNAi inhibitor of the invention can comprise one or more or all 2′-O-methoxy-ethyl (also known as 2′-methoxyethoxy or MOE) nucleotides. In one embodiment, an aptamer RNAi inhibitor of the invention can comprise one or more or all phosphorothioate internucleotide linkages. In one embodiment, an aptamer RNA inhibitor or the invention can comprise a terminal cap moiety at the 3′-end, the 5;′-end, or both the 5′ and 3′ ends of the aptamer RNA inhibitor.

In one embodiment, the invention features a method for modulating the expression of a target gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified or unmodified, wherein one of the siNA strands comprises a sequence complementary to RNA of the target gene; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the target gene in the cell.

In one embodiment, the invention features a method for modulating the expression of a target gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified or unmodified, wherein one of the siNA strands comprises a sequence complementary to RNA of the target gene and wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequence of the target RNA; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the target gene in the cell.

In another embodiment, the invention features a method for modulating the expression of more than one target gene within a cell comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified or unmodified, wherein one of the siNA strands comprises a sequence complementary to RNA of the target genes; and (b) introducing the siNA molecules into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the target genes in the cell.

In another embodiment, the invention features a method for modulating the expression of two or more target genes within a cell comprising: (a) synthesizing one or more siNA molecules of the invention, which can be chemically-modified or unmodified, wherein the siNA strands comprise sequences complementary to RNA of the target genes and wherein the sense strand sequences of the siNAs comprise sequences identical or substantially similar to the sequences of the target RNAs; and (b) introducing the siNA molecules into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the target genes in the cell.

In another embodiment, the invention features a method for modulating the expression of more than one target gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified or unmodified, wherein one of the siNA strands comprises a sequence complementary to RNA of the target gene and wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequences of the target RNAs; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the target genes in the cell.

In another embodiment, the invention features a method for modulating the expression of a target gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified or unmodified, wherein one of the siNA strands comprises a sequence complementary to RNA of the target gene, wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequences of the target RNA; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the target gene in the cell.

In one embodiment, siNA molecules of the invention are used as reagents in ex vivo applications. For example, siNA reagents are introduced into tissue or cells that are transplanted into a subject for therapeutic effect. The cells and/or tissue can be derived from an organism or subject that later receives the explant, or can be derived from another organism or subject prior to transplantation. The siNA molecules can be used to modulate the expression of one or more genes in the cells or tissue, such that the cells or tissue obtain a desired phenotype or are able to perform a function when transplanted in vivo. In one embodiment, certain target cells (e.g., liver cells) from a patient are extracted. These extracted cells are contacted with siNAs targeting a specific nucleotide sequence within the cells under conditions suitable for uptake of the siNAs by these cells (e.g. using delivery reagents such as cationic lipids, liposomes and the like or using techniques such as electroporation to facilitate the delivery of siNAs into cells). The cells are then reintroduced back into the same patient or other patients.

In one embodiment, the invention features a method of modulating the expression of a target gene in a tissue explant (e.g., liver or any other organ, tissue or cell as can be transplanted from one organism to another or back to the same organism from which the organ, tissue or cell is derived) comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the target gene; and (b) introducing the siNA molecule into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate (e.g., inhibit) the expression of the target gene in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate (e.g., inhibit) the expression of the target gene in that organism.

In one embodiment, the invention features a method of modulating the expression of a target gene in a tissue explant (e.g., liver or any other organ, tissue or cell as can be transplanted from one organism to another or back to the same organism from which the organ, tissue or cell is derived) comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the target gene and wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequence of the target RNA; and (b) introducing the siNA molecule into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate (e.g., inhibit) the expression of the target gene in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate (e.g., inhibit) the expression of the target gene in that organism.

In another embodiment, the invention features a method of modulating the expression of more than one target gene in a tissue explant (e.g., liver or any other organ, tissue or cell as can be transplanted from one organism to another or back to the same organism from which the organ, tissue or cell is derived) comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the target genes; and (b) introducing the siNA molecules into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate (e.g., inhibit) the expression of the target genes in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate (e.g., inhibit) the expression of the target genes in that organism.

In one embodiment, the invention features a method of modulating the expression of a target gene in a subject or organism comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the target gene; and (b) introducing the siNA molecule into the subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the target gene in the subject or organism. The level of target protein or RNA can be determined using various methods well-known in the art.

In another embodiment, the invention features a method of modulating the expression of more than one target gene in a subject or organism comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the target genes; and (b) introducing the siNA molecules into the subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the target genes in the subject or organism. The level of target protein or RNA can be determined as is known in the art.

In one embodiment, the invention features a method for modulating the expression of a target gene within a cell, comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the target gene; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate (e.g., inhibit) the expression of the target gene in the cell.

In another embodiment, the invention features a method for modulating the expression of more than one target gene within a cell, comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the target gene; and (b) contacting the cell in vitro or in vivo with the siNA molecule under conditions suitable to modulate (e.g., inhibit) the expression of the target genes in the cell.

In one embodiment, the invention features a method of modulating the expression of a target gene in a tissue explant ((e.g., any organ, tissue or cell as can be transplanted from one organism to another or back to the same organism from which the organ, tissue or cell is derived) comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the target gene; and (b) contacting a cell of the tissue explant derived from a particular subject or organism with the siNA molecule under conditions suitable to modulate (e.g., inhibit) the expression of the target gene in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the subject or organism the tissue was derived from or into another subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the target gene in that subject or organism.

In another embodiment, the invention features a method of modulating the expression of more than one target gene in a tissue explant (e.g., any organ, tissue or cell as can be transplanted from one organism to another or back to the same organism from which the organ, tissue or cell is derived) comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the target gene; and (b) introducing the siNA molecules into a cell of the tissue explant derived from a particular subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the target genes in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the subject or organism the tissue was derived from or into another subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the target genes in that subject or organism.

In one embodiment, the invention features a method of modulating the expression of a target gene in a subject or organism comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the target gene; and (b) introducing the siNA molecule into the subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the target gene in the subject or organism.

In another embodiment, the invention features a method of modulating the expression of more than one target gene in a subject or organism comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the target gene; and (b) introducing the siNA molecules into the subject or organism under conditions suitable to modulate (e.g., inhibit) the expression of the target genes in the subject or organism.

In one embodiment, the invention features a method of modulating the expression of a target gene in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate (e.g., inhibit) the expression of the target gene in the subject or organism.

In one embodiment, the invention features a method for treating or preventing a disease, disorder, trait or condition related to gene expression or activity in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the target gene in the subject or organism. The reduction of gene expression and thus reduction in the level of the respective protein/RNA relieves, to some extent, the symptoms of the disease, disorder, trait or condition.

In one embodiment, the invention features a method for treating or preventing HCV infection in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the HCV target gene in the subject or organism whereby the treatment or prevention of HCV infection can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as liver cells and tissues. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells involved in the maintenance or development of HCV infection in a subject or organism. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tissues or cells in the subject or organism. The siNA molecule can be combined with other therapeutic treatments and modalities as are known in the art for the treatment of or prevention of HCV infection in a subject or organism.

In one embodiment, the invention features a method for treating or preventing a liver failure or condition in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the HCV target gene in the subject or organism whereby the treatment or prevention of the liver failure or condition can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as liver cells and tissues involved in liver failure. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells involved in the maintenance or development of the liver failure or condition in a subject or organism. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tissues or cells in the subject or organism. The siNA molecule can be combined with other therapeutic treatments and modalities as are known in the art for the treatment of or prevention of liver failures, traits, disorders, or conditions in a subject or organism.

In one embodiment, the invention features a method for treating or preventing hepatocellular carcinoma in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the HCV target gene in the subject or organism whereby the treatment or prevention of hepatocellular carcinoma can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as liver cells and tissues involved in hepatocellular carcinoma. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells involved in the maintenance or development of hepatocellular carcinoma in a subject or organism. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tissues or cells in the subject or organism. The siNA molecule can be combined with other therapeutic treatments and modalities as are known in the art for the treatment of or prevention of hepatocellular carcinoma in a subject or organism.

In one embodiment, the invention features a method for treating or preventing an cirrhosis, disorder, trait or condition in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the HCV target gene in the subject or organism whereby the treatment or prevention of the cirrhosis, disorder, trait or condition can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as cells and tissues involved in the cirrhosis, disorder, trait or condition. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells involved in the maintenance or development of the cirrhosis, disorder, trait or condition in a subject or organism. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tissues or cells in the subject or organism. The siNA molecule can be combined with other therapeutic treatments and modalities as are known in the art for the treatment of or prevention of cirrhosis, traits, disorders, or conditions in a subject or organism.

In one embodiment, the invention features a method for treating or preventing HCV infection in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate (e.g., inhibit) the expression of an inhibitor of HCV gene expression in the subject or organism.

In one embodiment, the invention features a method for treating or preventing liver failure in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate (e.g., inhibit) the expression of an inhibitor of HCV gene expression in the subject or organism.

In one embodiment, the invention features a method for treating or preventing cirrhosis in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate (e.g., inhibit) the expression of an inhibitor of HCV gene expression in the subject or organism.

In one embodiment, the invention features a method for treating or preventing Hepatitis C Virus (HCV) infection in a subject, comprising administering to the subject PEG Interferon in combination with a siNA molecule of the invention; wherein the PEG Interferon and the siNA molecule are administered under conditions suitable for reducing or inhibiting the level of Hepatitis C Virus (HCV) in the subject compared to a subject not treated with the PEG Interferon and the siNA molecule. In one embodiment, a siNA molecule of the invention is formulated as a composition described in U.S. Provisional patent application No. 60/678,531 and in related U.S. Provisional patent application No. 60/703,946, filed Jul. 29, 2005, and U.S. Provisional patent application No. 60/737,024, filed Nov. 15, 2005 (Vargeese et al.), all of which are incorporated by reference herein in their entirety. Such siNA formulations are generally referred to as “lipid nucleic acid particles” (LNP).

In one embodiment, the invention features a method for treating or preventing Hepatitis C Virus (HCV) infection in a subject, comprising administering to the subject ribavirin in combination with a siNA molecule of the invention; wherein the ribavirin and the siNA are administered under conditions suitable for reducing or inhibiting the level of Hepatitis C Virus (HCV) in the subject compared to a subject not treated with the ribavirin and the siNA molecule. In one embodiment, the siNA molecule or double stranded nucleic acid molecule of the invention is formulated as a composition described in U.S. Provisional patent application No. 60/678,531 and in related U.S. Provisional patent application No. 60/703,946, filed Jul. 29, 2005, and U.S. Provisional patent application No. 60/737,024, filed Nov. 15, 2005 (Vargeese et al.).

In one embodiment, the invention features a method for treating or preventing Hepatitis C Virus (HCV) infection in a subject, comprising administering to the subject PEG Interferon and ribavirin in combination with a siNA molecule of the invention; wherein the PEG Interferon and ribavirin and the siNA molecule are administered under conditions suitable for reducing or inhibiting the level of Hepatitis C Virus (HCV) in the subject compared to a subject not treated with the PEG Interferon and ribavirin and the siNA molecule. In one embodiment, the siNA molecule or double stranded nucleic acid molecule of the invention is formulated as a composition described in U.S. Provisional patent application No. 60/678,531 and in related U.S. Provisional patent application No. 60/703,946, filed Jul. 29, 2005, and U.S. Provisional patent application No. 60/737,024, filed Nov. 15, 2005 (Vargeese et al.).

In one embodiment, the invention features a method for treating or preventing Hepatitis C Virus (HCV) infection in a subject, comprising administering to the subject PEG Interferon in combination with a chemically synthesized double stranded nucleic acid molecule; wherein (a) the double stranded nucleic acid molecule comprises a sense strand and an antisense strand; (b) each strand of the double stranded nucleic acid molecule is 15 to 28 nucleotides in length; (c) at least 15 nucleotides of the sense strand are complementary to the antisense strand(d) the antisense strand of the double stranded nucleic acid molecule has complementarity to a Hepatitis C Virus (HCV) HCV target RNA; and wherein the PEG Interferon and the double stranded nucleic acid molecule are administered under conditions suitable for reducing or inhibiting the level of Hepatitis C Virus (HCV) in the subject compared to a subject not treated with the PEG Interferon and the double stranded nucleic acid molecule. In one embodiment, the siNA molecule or double stranded nucleic acid molecule of the invention is formulated as a composition described in U.S. Provisional patent application No. 60/678,531 and in related U.S. Provisional patent application No. 60/703,946, filed Jul. 29, 2005, and U.S. Provisional patent application No. 60/737,024, filed Nov. 15, 2005 (Vargeese et al.).

In one embodiment, the invention features a method for treating or preventing Hepatitis C Virus (HCV) infection in a subject, comprising administering to the subject ribavirin in combination with a chemically synthesized double stranded nucleic acid molecule; wherein (a) the double stranded nucleic acid molecule comprises a sense strand and an antisense strand; (b) each strand of the double stranded nucleic acid molecule is 15 to 28 nucleotides in length; (c) at least 15 nucleotides of the sense strand are complementary to the antisense strand(d) the antisense strand of the double stranded nucleic acid molecule has complementarity to a Hepatitis C Virus (HCV) HCV target RNA; and wherein the ribavirin and the double stranded nucleic acid molecule are administered under conditions suitable for reducing or inhibiting the level of Hepatitis C Virus (HCV) in the subject compared to a subject not treated with the ribavirin and the double stranded nucleic acid molecule. In one embodiment, the siNA molecule or double stranded nucleic acid molecule of the invention is formulated as a composition described in U.S. Provisional patent application No. 60/678,531 and in related U.S. Provisional patent application No. 60/703,946, filed Jul. 29, 2005, and U.S. Provisional patent application No. 60/737,024, filed Nov. 15, 2005 (Vargeese et al.).

In one embodiment, the invention features a method for treating or preventing Hepatitis C Virus (HCV) infection in a subject, comprising administering to the subject PEG Interferon and ribavirin in combination with a chemically synthesized double stranded nucleic acid molecule; wherein (a) the double stranded nucleic acid molecule comprises a sense strand and an antisense strand; (b) each strand of the double stranded nucleic acid molecule is 15 to 28 nucleotides in length; (c) at least 15 nucleotides of the sense strand are complementary to the antisense strand(d) the antisense strand of the double stranded nucleic acid molecule has complementarity to a Hepatitis C Virus (HCV) HCV target RNA; and wherein the PEG Interferon and ribavirin and the double stranded nucleic acid molecule are administered under conditions suitable for reducing or inhibiting the level of Hepatitis C Virus (HCV) in the subject compared to a subject not treated with the PEG Interferon and ribavirin and the double stranded nucleic acid molecule. In one embodiment, the siNA molecule or double stranded nucleic acid molecule of the invention is formulated as a composition described in U.S. Provisional patent application No. 60/678,531 and in related U.S. Provisional patent application No. 60/703,946, filed Jul. 29, 2005, and U.S. Provisional patent application No. 60/737,024, filed Nov. 15, 2005 (Vargeese et al.).

In one embodiment, the invention features a method for treating or preventing Hepatitis C Virus (HCV) infection in a subject, comprising administering to the subject PEG Interferon in combination with a chemically synthesized double stranded nucleic acid molecule; wherein (a) the double stranded nucleic acid molecule comprises a sense strand and an antisense strand; (b) each strand of the double stranded nucleic acid molecule is 15 to 28 nucleotides in length; (c) at least 15 nucleotides of the sense strand are complementary to the antisense strand(d) the antisense strand of the double stranded nucleic acid molecule has complementarity to a Hepatitis C Virus (HCV) HCV target RNA; (e) at least 20% of the internal nucleotides of each strand of the double stranded nucleic acid molecule are modified nucleosides having a chemical modification; and (f) at least two of the chemical modifications are different from each other, and wherein the PEG Interferon and the double stranded nucleic acid molecule are administered under conditions suitable for reducing or inhibiting the level of Hepatitis C Virus (HCV) in the subject compared to a subject not treated with the PEG Interferon and the double stranded nucleic acid molecule. In one embodiment, the siNA molecule or double stranded nucleic acid molecule of the invention is formulated as a composition described in U.S. Provisional patent application No. 60/678,531 and in related U.S. Provisional patent application No. 60/703,946, filed Jul. 29, 2005, and U.S. Provisional patent application No. 60/737,024, filed Nov. 15, 2005 (Vargeese et al.).

In one embodiment, the invention features a method for treating or preventing Hepatitis C Virus (HCV) infection in a subject, comprising administering to the subject ribavirin in combination with a chemically synthesized double stranded nucleic acid molecule; wherein (a) the double stranded nucleic acid molecule comprises a sense strand and an antisense strand; (b) each strand of the double stranded nucleic acid molecule is 15 to 28 nucleotides in length; (c) at least 15 nucleotides of the sense strand are complementary to the antisense strand(d) the antisense strand of the double stranded nucleic acid molecule has complementarity to a Hepatitis C Virus (HCV) HCV target RNA; (e) at least 20% of the internal nucleotides of each strand of the double stranded nucleic acid molecule are modified nucleosides having a chemical modification; and (f) at least two of the chemical modifications are different from each other, and wherein the ribavirin and the double stranded nucleic acid molecule are administered under conditions suitable for reducing or inhibiting the level of Hepatitis C Virus (HCV) in the subject compared to a subject not treated with the ribavirin and the double stranded nucleic acid molecule. In one embodiment, the siNA molecule or double stranded nucleic acid molecule of the invention is formulated as a composition described in U.S. Provisional patent application No. 60/678,531 and in related U.S. Provisional patent application No. 60/703,946, filed Jul. 29, 2005, and U.S. Provisional patent application No. 60/737,024, filed Nov. 15, 2005 (Vargeese et al.).

In one embodiment, the invention features a method for treating or preventing Hepatitis C Virus (HCV) infection in a subject, comprising administering to the subject PEG Interferon and ribavirin in combination with a chemically synthesized double stranded nucleic acid molecule; wherein (a) the double stranded nucleic acid molecule comprises a sense strand and an antisense strand; (b) each strand of the double stranded nucleic acid molecule is 15 to 28 nucleotides in length; (c) at least 15 nucleotides of the sense strand are complementary to the antisense strand(d) the antisense strand of the double stranded nucleic acid molecule has complementarity to a Hepatitis C Virus (HCV) HCV target RNA; (e) at least 20% of the internal nucleotides of each strand of the double stranded nucleic acid molecule are modified nucleosides having a chemical modification; and (f) at least two of the chemical modifications are different from each other, and wherein the PEG Interferon and ribavirin and the double stranded nucleic acid molecule are administered under conditions suitable for reducing or inhibiting the level of Hepatitis C Virus (HCV) in the subject compared to a subject not treated with the PEG Interferon and ribavirin and the double stranded nucleic acid molecule. In one embodiment, the siNA molecule or double stranded nucleic acid molecule of the invention is formulated as a composition described in U.S. Provisional patent application No. 60/678,531 and in related U.S. Provisional patent application No. 60/703,946, filed Jul. 29, 2005, and U.S. Provisional patent application No. 60/737,024, filed Nov. 15, 2005 (Vargeese et al.).

In one embodiment, the invention features a method for treating or preventing Hepatitis C Virus (HCV) infection in a subject, comprising administering to the subject PEG Interferon in combination with a chemically synthesized double stranded nucleic acid molecule; wherein (a) the double stranded nucleic acid molecule comprises a sense strand and an antisense strand; (b) each strand of the double stranded nucleic acid molecule is 15 to 28 nucleotides in length; (c) at least 15 nucleotides of the sense strand are complementary to the antisense strand(d) the antisense strand of the double stranded nucleic acid molecule has complementarity to a Hepatitis C Virus (HCV) HCV target RNA; (e) at least 20% of the internal nucleotides of each strand of the double stranded nucleic acid molecule are modified nucleosides having a sugar modification; and (f) at least two of the sugar modifications are different from each other, and wherein the PEG Interferon and the double stranded nucleic acid molecule are administered under conditions suitable for reducing or inhibiting the level of Hepatitis C Virus (HCV) in the subject compared to a subject not treated with the PEG Interferon and the double stranded nucleic acid molecule. In one embodiment, the siNA molecule or double stranded nucleic acid molecule of the invention is formulated as a composition described in U.S. Provisional patent application No. 60/678,531 and in related U.S. Provisional patent application No. 60/703,946, filed Jul. 29, 2005, and U.S. Provisional patent application No. 60/737,024, filed Nov. 15, 2005 (Vargeese et al.).

In one embodiment, the invention features a method for treating or preventing Hepatitis C Virus (HCV) infection in a subject, comprising administering to the subject ribavirin in combination with a chemically synthesized double stranded nucleic acid molecule; wherein (a) the double stranded nucleic acid molecule comprises a sense strand and an antisense strand; (b) each strand of the double stranded nucleic acid molecule is 15 to 28 nucleotides in length; (c) at least 15 nucleotides of the sense strand are complementary to the antisense strand(d) the antisense strand of the double stranded nucleic acid molecule has complementarity to a Hepatitis C Virus (HCV) HCV target RNA; (e) at least 20% of the internal nucleotides of each strand of the double stranded nucleic acid molecule are modified nucleosides having a sugar modification; and (f) at least two of the sugar modifications are different from each other, and wherein the ribavirin and the double stranded nucleic acid molecule are administered under conditions suitable for reducing or inhibiting the level of Hepatitis C Virus (HCV) in the subject compared to a subject not treated with the ribavirin and the double stranded nucleic acid molecule. In one embodiment, the siNA molecule or double stranded nucleic acid molecule of the invention is formulated as a composition described in U.S. Provisional patent application No. 60/678,531 and in related U.S. Provisional patent application No. 60/703,946, filed Jul. 29, 2005, and U.S. Provisional patent application No. 60/737,024, filed Nov. 15, 2005 (Vargeese et al.).

In one embodiment, the invention features a method for treating or preventing Hepatitis C Virus (HCV) infection in a subject, comprising administering to the subject PEG Interferon and ribavirin in combination with a chemically synthesized double stranded nucleic acid molecule; wherein (a) the double stranded nucleic acid molecule comprises a sense strand and an antisense strand; (b) each strand of the double stranded nucleic acid molecule is 15 to 28 nucleotides in length; (c) at least 15 nucleotides of the sense strand are complementary to the antisense strand(d) the antisense strand of the double stranded nucleic acid molecule has complementarity to a Hepatitis C Virus (HCV) HCV target RNA; (e) at least 20% of the internal nucleotides of each strand of the double stranded nucleic acid molecule are modified nucleosides having a sugar modification; and (f) at least two of the sugar modifications are different from each other, and wherein the PEG Interferon and ribavirin and the double stranded nucleic acid molecule are administered under conditions suitable for reducing or inhibiting the level of Hepatitis C Virus (HCV) in the subject compared to a subject not treated with the PEG Interferon and ribavirin and the double stranded nucleic acid molecule. In one embodiment, the siNA molecule or double stranded nucleic acid molecule of the invention is formulated as a composition described in U.S. Provisional patent application No. 60/678,531 and in related U.S. Provisional patent application No. 60/703,946, filed Jul. 29, 2005, and U.S. Provisional patent application No. 60/737,024, filed Nov. 15, 2005 (Vargeese et al.).

In one embodiment, the invention features a method for treating or preventing a neurologic or neurodegenerative disease, disorder, trait or condition in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the HCV target gene in the subject or organism whereby the treatment or prevention of the neurologic or neurodegenerative disease, disorder, trait or condition can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as cells and tissues involved in the neurologic or neurodegenerative disease, disorder, trait or condition. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells involved in the maintenance or development of the neurologic or neurodegenerative disease, disorder, trait or condition in a subject or organism. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tissues or cells in the subject or organism. The siNA molecule can be combined with other therapeutic treatments and modalities as are known in the art for the treatment of or prevention of neurologic or neurodegenerative diseases, traits, disorders, or conditions in a subject or organism.

In one embodiment, the invention features a method for treating or preventing a metabolic disease, disorder, trait or condition in a subject or organism comprising contacting the subject or organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the HCV target gene in the subject or organism whereby the treatment or prevention of the metabolic disease, disorder, trait or condition can be achieved. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via local administration to relevant tissues or cells, such as cells and tissues involved in the metabolic disease, disorder, trait or condition. In one embodiment, the invention features contacting the subject or organism with a siNA molecule of the invention via systemic administration (such as via intravenous or subcutaneous administration of siNA) to relevant tissues or cells, such as tissues or cells involved in the maintenance or development of the metabolic disease, disorder, trait or condition in a subject or organism. The siNA molecule of the invention can be formulated or conjugated as described herein or otherwise known in the art to target appropriate tissues or cells in the subject or organism. The siNA molecule can be combined with other therapeutic treatments and modalities as are known in the art for the treatment of or prevention of metabolic diseases, traits, disorders, or conditions in a subject or organism.

In one embodiment, the invention features a composition comprising PEG Interferon and one or more double stranded nucleic acid molecules or siNA molecules of the invention in a pharmaceutically acceptable carrier or diluent. In another embodiment, the invention features a composition comprising PEG Interferon, ribavirin, Vertex VX-950, Actilon (CPG 10101), and/or Isatoribine (TLR-7 agonist) and one or more double stranded nucleic acid molecules or siNA molecules of the invention in a pharmaceutically acceptable carrier or diluent.

In one embodiment, a method of treatment of the invention features administration of a double stranded nucleic acid molecule of the invention in combination with one or more other therapeutic modalities, including Interferon (e.g., Interferon-alpha, or PEG interferon such as PEG-Intron, Rebetol, Rebetron, or Pegasys), ribavirin, Vertex VX-950, Actilon (CPG 10101), or Isatoribine (TLR-7 agonist). In another embodiment, such combination therapies can be utilized in any of the embodiments herein.

In one embodiment, the siNA molecule or double stranded nucleic acid molecule of the invention is formulated as a composition described in U.S. Provisional patent application No. 60/678,531 and in related U.S. Provisional patent application No. 60/703,946, filed Jul. 29, 2005, U.S. Provisional patent application No. 60/737,024, filed Nov. 15, 2005, and U.S. Ser. No. 11/353,630, filed Feb. 14, 2006 (Vargeese et al.).

In any of the methods herein for modulating the expression of one or more targets or for treating or preventing diseases, traits, conditions, or phenotypes in a cell, subject, or organism, the siNA molecule of the invention modulates expression of one or more targets via RNA interference. In one embodiment, the RNA interference is RISC mediated cleavage of the target (e.g., siRNA mediated RNA interference). In one embodiment, the RNA interference is translational inhibition of the target (e.g., miRNA mediated RNA interference). In one embodiment, the RNA interference is transcriptional inhibition of the target (e.g., siRNA mediated transcriptional silencing). In one embodiment, the RNA interference takes place in the cytoplasm. In one embodiment, the RNA interference takes place in the nucleus.

In any of the methods of treatment of the invention, the siNA can be administered to the subject as a course of treatment, for example administration at various time intervals, such as once per day over the course of treatment, once every two days over the course of treatment, once every three days over the course of treatment, once every four days over the course of treatment, once every five days over the course of treatment, once every six days over the course of treatment, once per week over the course of treatment, once every other week over the course of treatment, once per month over the course of treatment, etc. In one embodiment, the course of treatment is once every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. In one embodiment, the course of treatment is from about one to about 52 weeks or longer (e.g., indefinitely). In one embodiment, the course of treatment is from about one to about 48 months or longer (e.g., indefinitely).

In one embodiment, a course of treatment involves an initial course of treatment, such as once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks for a fixed interval (e.g., 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10× or more) followed by a maintenance course of treatment, such as once every 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, or more weeks for an additional fixed interval (e.g., 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10× or more).

In any of the methods of treatment of the invention, the siNA can be administered to the subject systemically as described herein or otherwise known in the art, either alone as a monotherapy or in combination with additional therapies described herein or as are known in the art. Systemic administration can include, for example, pulmonary (inhalation, nebulization etc.) intravenous, subcutaneous, intramuscular, catheterization, nasopharangeal, transdermal, or oral/gastrointestinal administration as is generally known in the art.

In one embodiment, in any of the methods of treatment or prevention of the invention, the siNA can be administered to the subject locally or to local tissues as described herein or otherwise known in the art, either alone as a monotherapy or in combination with additional therapies as are known in the art. Local administration can include, for example, inhalation, nebulization, catheterization, implantation, direct injection, dermal/transdermal application, stenting, ear/eye drops, or portal vein administration to relevant tissues, or any other local administration technique, method or procedure, as is generally known in the art.

In one embodiment, the invention features a method for administering siNA molecules and compositions of the invention to the inner ear, comprising, contacting the siNA with inner ear cells, tissues, or structures, under conditions suitable for the administration. In one embodiment, the administration comprises methods and devices as described in U.S. Pat. Nos. 5,421,818, 5,476,446, 5,474,529, 6,045,528, 6,440,102, 6,685,697, 6,120,484; and 5,572,594; all incorporated by reference herein and the teachings of Silverstein, 1999, Ear Nose Throat J., 78, 595-8, 600; and Jackson and Silverstein, 2002, Otolaryngol Clin North Am., 35, 639-53, and adapted for use the siNA molecules of the invention.

In another embodiment, the invention features a method of modulating the expression of more than one target gene in a subject or organism comprising contacting the subject or organism with one or more siNA molecules of the invention under conditions suitable to modulate (e.g., inhibit) the expression of the target genes in the subject or organism.

The siNA molecules of the invention can be designed to down regulate or inhibit target gene expression through RNAi targeting of a variety of nucleic acid molecules. In one embodiment, the siNA molecules of the invention are used to target various DNA corresponding to a target gene, for example via heterochromatic silencing or transcriptional inhibition. In one embodiment, the siNA molecules of the invention are used to target various RNAs corresponding to a target gene, for example via RNA target cleavage or translational inhibition. Non-limiting examples of such RNAs include messenger RNA (mRNA), non-coding RNA (ncRNA) or regulatory elements (see for example Mattick, 2005, Science, 309, 1527-1528 and Claverie, 2005, Science, 309, 1529-1530) which includes miRNA and other small RNAs, alternate RNA splice variants of target gene(s), post-transcriptionally modified RNA of target gene(s), pre-mRNA of target gene(s), and/or RNA templates. If alternate splicing produces a family of transcripts that are distinguished by usage of appropriate exons, the instant invention can be used to inhibit gene expression through the appropriate exons to specifically inhibit or to distinguish among the functions of gene family members. For example, a protein that contains an alternatively spliced transmembrane domain can be expressed in both membrane bound and secreted forms. Use of the invention to target the exon containing the transmembrane domain can be used to determine the functional consequences of pharmaceutical targeting of the membrane bound as opposed to the secreted form of the protein. Non-limiting examples of applications of the invention relating to targeting these RNA molecules include therapeutic pharmaceutical applications, cosmetic applications, veterinary applications, pharmaceutical discovery applications, molecular diagnostic and gene function applications, and gene mapping, for example using single nucleotide polymorphism mapping with siNA molecules of the invention. Such applications can be implemented using known gene sequences or from partial sequences available from an expressed sequence tag (EST).

In another embodiment, the siNA molecules of the invention are used to target conserved sequences corresponding to a gene family or gene families such as HCV family genes (e.g., all known HCV strains, groups of related HCV strains, or groups of divergent HCV strains). As such, siNA molecules targeting multiple HCV targets can provide increased therapeutic effect. In addition, siNA can be used to characterize pathways of gene function in a variety of applications. For example, the present invention can be used to inhibit the activity of target gene(s) in a pathway to determine the function of uncharacterized gene(s) in gene function analysis, mRNA function analysis, or translational analysis. The invention can be used to determine potential target gene pathways involved in various diseases and conditions toward pharmaceutical development. The invention can be used to understand pathways of gene expression involved in, for example proliferative diseases, disorders and conditions.

In one embodiment, siNA molecules can be used to characterize pathways of gene function in a variety of applications. For example, the present invention can be used to inhibit the activity of target gene(s) in a pathway to determine the function of uncharacterized gene(s) in gene function analysis, mRNA function analysis, or translational analysis. The invention can be used to determine potential target gene pathways involved in various diseases and conditions toward pharmaceutical development. The invention can be used to understand pathways of gene expression involved in, for example diseases, disorders, traits and conditions herein or otherwise known in the art.

In one embodiment, siNA molecule(s) and/or methods of the invention are used to down regulate the expression of gene(s) that encode RNA referred to by Genbank Accession, for example, target genes encoding RNA sequence(s) referred to herein by Genbank Accession number, for example, Genbank Accession Nos. shown herein (e.g. in Table I) and U.S. Provisional Patent Application No. 60/363,124, U.S. Ser. No. 10/923,536 and PCT/US03/05028, all incorporated by reference herein.

In one embodiment, the invention features a method comprising: (a) generating a library of siNA constructs having a predetermined complexity; and (b) assaying the siNA constructs of (a) above, under conditions suitable to determine RNAi target sites within the target RNA sequence. In one embodiment, the siNA molecules of (a) have strands of a fixed length, for example, about 23 nucleotides in length. In another embodiment, the siNA molecules of (a) are of differing length, for example having strands of about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length. In one embodiment, the assay can comprise a reconstituted in vitro siNA assay as described herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. In another embodiment, fragments of target RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target RNA sequence. The target RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by cellular expression in in vivo systems.

In one embodiment, the invention features a method comprising: (a) generating a randomized library of siNA constructs having a predetermined complexity, such as of 4^N, where N represents the number of base paired nucleotides in each of the siNA construct strands (eg. for a siNA construct having 21 nucleotide sense and antisense strands with 19 base pairs, the complexity would be 4¹⁹); and (b) assaying the siNA constructs of (a) above, under conditions suitable to determine RNAi target sites within the target target RNA sequence. In another embodiment, the siNA molecules of (a) have strands of a fixed length, for example about 23 nucleotides in length. In yet another embodiment, the siNA molecules of (a) are of differing length, for example having strands of about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length. In one embodiment, the assay can comprise a reconstituted in vitro siNA assay as described in Example 6 herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. In another embodiment, fragments of target RNA are analyzed for detectable levels of cleavage, for example, by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target target RNA sequence. The target target RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by cellular expression in in vivo systems.

In another embodiment, the invention features a method comprising: (a) analyzing the sequence of a RNA target encoded by a target gene; (b) synthesizing one or more sets of siNA molecules having sequence complementary to one or more regions of the RNA of (a); and (c) assaying the siNA molecules of (b) under conditions suitable to determine RNAi targets within the target RNA sequence. In one embodiment, the siNA molecules of (b) have strands of a fixed length, for example about 23 nucleotides in length. In another embodiment, the siNA molecules of (b) are of differing length, for example having strands of about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length. In one embodiment, the assay can comprise a reconstituted in vitro siNA assay as described herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. Fragments of target RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target RNA sequence. The target RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by expression in in vivo systems.

By “target site” is meant a sequence within a target RNA that is “targeted” for cleavage mediated by a siNA construct which contains sequences within its antisense region that are complementary to the target sequence.

By “detectable level of cleavage” is meant cleavage of target RNA (and formation of cleaved product RNAs) to an extent sufficient to discern cleavage products above the background of RNAs produced by random degradation of the target RNA. Production of cleavage products from 1-5% of the target RNA is sufficient to detect above the background for most methods of detection.

In one embodiment, the invention features a composition comprising a siNA molecule of the invention, which can be chemically-modified, in a pharmaceutically acceptable carrier or diluent. In another embodiment, the invention features a pharmaceutical composition comprising siNA molecules of the invention, which can be chemically-modified, targeting one or more genes in a pharmaceutically acceptable carrier or diluent. In another embodiment, the invention features a method for diagnosing a disease, trait, or condition in a subject comprising administering to the subject a composition of the invention under conditions suitable for the diagnosis of the disease, trait, or condition in the subject. In another embodiment, the invention features a method for treating or preventing a disease, trait, or condition, such as metabolic and/or cardiovascular diseases, traits, conditions, or disorders in a subject, comprising administering to the subject a composition of the invention under conditions suitable for the treatment or prevention of the disease, trait, or condition in the subject, alone or in conjunction with one or more other therapeutic compounds.

In another embodiment, the invention features a method for validating a target gene target, comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands includes a sequence complementary to RNA of a target gene; (b) introducing the siNA molecule into a cell, tissue, subject, or organism under conditions suitable for modulating expression of the target gene in the cell, tissue, subject, or organism; and (c) determining the function of the gene by assaying for any phenotypic change in the cell, tissue, subject, or organism.

In another embodiment, the invention features a method for validating a target comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands includes a sequence complementary to RNA of a target gene; (b) introducing the siNA molecule into a biological system under conditions suitable for modulating expression of the target gene in the biological system; and (c) determining the function of the gene by assaying for any phenotypic change in the biological system.

By “biological system” is meant, material, in a purified or unpurified form, from biological sources, including but not limited to human or animal, wherein the system comprises the components required for RNAi activity. The term “biological system” includes, for example, a cell, tissue, subject, or organism, or extract thereof. The term biological system also includes reconstituted RNAi systems that can be used in an in vitro setting.

By “phenotypic change” is meant any detectable change to a cell that occurs in response to contact or treatment with a nucleic acid molecule of the invention (e.g., siNA). Such detectable changes include, but are not limited to, changes in shape, size, proliferation, motility, protein expression or RNA expression or other physical or chemical changes as can be assayed by methods known in the art. The detectable change can also include expression of reporter genes/molecules such as Green Florescent Protein (GFP) or various tags that are used to identify an expressed protein or any other cellular component that can be assayed.

In one embodiment, the invention features a kit containing a siNA molecule of the invention, which can be chemically-modified, that can be used to modulate the expression of a target gene in a biological system, including, for example, in a cell, tissue, subject, or organism. In another embodiment, the invention features a kit containing more than one siNA molecule of the invention, which can be chemically-modified, that can be used to modulate the expression of more than one target gene in a biological system, including, for example, in a cell, tissue, subject, or organism.

In one embodiment, the invention features a cell containing one or more siNA molecules of the invention, which can be chemically-modified. In another embodiment, the cell containing a siNA molecule of the invention is a mammalian cell. In yet another embodiment, the cell containing a siNA molecule of the invention is a human cell.

In one embodiment, the synthesis of a siNA molecule of the invention, which can be chemically-modified, comprises: (a) synthesis of two complementary strands of the siNA molecule; (b) annealing the two complementary strands together under conditions suitable to obtain a double-stranded siNA molecule. In another embodiment, synthesis of the two complementary strands of the siNA molecule is by solid phase oligonucleotide synthesis. In yet another embodiment, synthesis of the two complementary strands of the siNA molecule is by solid phase tandem oligonucleotide synthesis.

In one embodiment, the invention features a method for synthesizing a siNA duplex molecule comprising: (a) synthesizing a first oligonucleotide sequence strand of the siNA molecule, wherein the first oligonucleotide sequence strand comprises a cleavable linker molecule that can be used as a scaffold for the synthesis of the second oligonucleotide sequence strand of the siNA; (b) synthesizing the second oligonucleotide sequence strand of siNA on the scaffold of the first oligonucleotide sequence strand, wherein the second oligonucleotide sequence strand further comprises a chemical moiety than can be used to purify the siNA duplex; (c) cleaving the linker molecule of (a) under conditions suitable for the two siNA oligonucleotide strands to hybridize and form a stable duplex; and (d) purifying the siNA duplex utilizing the chemical moiety of the second oligonucleotide sequence strand. In one embodiment, cleavage of the linker molecule in (c) above takes place during deprotection of the oligonucleotide, for example, under hydrolysis conditions using an alkylamine base such as methylamine. In one embodiment, the method of synthesis comprises solid phase synthesis on a solid support such as controlled pore glass (CPG) or polystyrene, wherein the first sequence of (a) is synthesized on a cleavable linker, such as a succinyl linker, using the solid support as a scaffold. The cleavable linker in (a) used as a scaffold for synthesizing the second strand can comprise similar reactivity as the solid support derivatized linker, such that cleavage of the solid support derivatized linker and the cleavable linker of (a) takes place concomitantly. In another embodiment, the chemical moiety of (b) that can be used to isolate the attached oligonucleotide sequence comprises a trityl group, for example a dimethoxytrityl group, which can be employed in a trityl-on synthesis strategy as described herein. In yet another embodiment, the chemical moiety, such as a dimethoxytrityl group, is removed during purification, for example, using acidic conditions.

In a further embodiment, the method for siNA synthesis is a solution phase synthesis or hybrid phase synthesis wherein both strands of the siNA duplex are synthesized in tandem using a cleavable linker attached to the first sequence which acts a scaffold for synthesis of the second sequence. Cleavage of the linker under conditions suitable for hybridization of the separate siNA sequence strands results in formation of the double-stranded siNA molecule.

In another embodiment, the invention features a method for synthesizing a siNA duplex molecule comprising: (a) synthesizing one oligonucleotide sequence strand of the siNA molecule, wherein the sequence comprises a cleavable linker molecule that can be used as a scaffold for the synthesis of another oligonucleotide sequence; (b) synthesizing a second oligonucleotide sequence having complementarity to the first sequence strand on the scaffold of (a), wherein the second sequence comprises the other strand of the double-stranded siNA molecule and wherein the second sequence further comprises a chemical moiety than can be used to isolate the attached oligonucleotide sequence; (c) purifying the product of (b) utilizing the chemical moiety of the second oligonucleotide sequence strand under conditions suitable for isolating the full-length sequence comprising both siNA oligonucleotide strands connected by the cleavable linker and under conditions suitable for the two siNA oligonucleotide strands to hybridize and form a stable duplex. In one embodiment, cleavage of the linker molecule in (c) above takes place during deprotection of the oligonucleotide, for example, under hydrolysis conditions. In another embodiment, cleavage of the linker molecule in (c) above takes place after deprotection of the oligonucleotide. In another embodiment, the method of synthesis comprises solid phase synthesis on a solid support such as controlled pore glass (CPG) or polystyrene, wherein the first sequence of (a) is synthesized on a cleavable linker, such as a succinyl linker, using the solid support as a scaffold. The cleavable linker in (a) used as a scaffold for synthesizing the second strand can comprise similar reactivity or differing reactivity as the solid support derivatized linker, such that cleavage of the solid support derivatized linker and the cleavable linker of (a) takes place either concomitantly or sequentially. In one embodiment, the chemical moiety of (b) that can be used to isolate the attached oligonucleotide sequence comprises a trityl group, for example a dimethoxytrityl group.

In another embodiment, the invention features a method for making a double-stranded siNA molecule in a single synthetic process comprising: (a) synthesizing an oligonucleotide having a first and a second sequence, wherein the first sequence is complementary to the second sequence, and the first oligonucleotide sequence is linked to the second sequence via a cleavable linker, and wherein a terminal 5′-protecting group, for example, a 5′-O-dimethoxytrityl group (5′-O-DMT) remains on the oligonucleotide having the second sequence; (b) deprotecting the oligonucleotide whereby the deprotection results in the cleavage of the linker joining the two oligonucleotide sequences; and (c) purifying the product of (b) under conditions suitable for isolating the double-stranded siNA molecule, for example using a trityl-on synthesis strategy as described herein.

In another embodiment, the method of synthesis of siNA molecules of the invention comprises the teachings of Scaringe et al., U.S. Pat. Nos. 5,889,136; 6,008,400; and 6,111,086, incorporated by reference herein in their entirety.

In one embodiment, the invention features siNA constructs that mediate RNAi against a target polynucleotide (e.g., RNA or DNA target), wherein the siNA construct comprises one or more chemical modifications, for example, one or more chemical modifications having any of Formulae I-VII or any combination thereof that increases the nuclease resistance of the siNA construct.

In another embodiment, the invention features a method for generating siNA molecules with increased nuclease resistance comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased nuclease resistance.

In another embodiment, the invention features a method for generating siNA molecules with improved toxicologic profiles (e.g., having attenuated or no immunostimulatory properties) comprising (a) introducing nucleotides having any of Formula I-VII (e.g., siNA motifs referred to in Table IV) or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved toxicologic profiles.

In another embodiment, the invention features a method for generating siNA formulations with improved toxicologic profiles (e.g., having attenuated or no immunostimulatory properties) comprising (a) generating a siNA formulation comprising a siNA molecule of the invention and a delivery vehicle or delivery particle as described herein or as otherwise known in the art, and (b) assaying the siNA formulation of step (a) under conditions suitable for isolating siNA formulations having improved toxicologic profiles.

In another embodiment, the invention features a method for generating siNA molecules that do not stimulate an interferon response (e.g., no interferon response or attenuated interferon response) in a cell, subject, or organism, comprising (a) introducing nucleotides having any of Formula I-VII (e.g., siNA motifs referred to in Table IV) or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules that do not stimulate an interferon response.

In another embodiment, the invention features a method for generating siNA formulations that do not stimulate an interferon response (e.g., no interferon response or attenuated interferon response) in a cell, subject, or organism, comprising (a) generating a siNA formulation comprising a siNA molecule of the invention and a delivery vehicle or delivery particle as described herein or as otherwise known in the art, and (b) assaying the siNA formulation of step (a) under conditions suitable for isolating siNA formulations that do not stimulate an interferon response. In one embodiment, the interferon comprises interferon alpha.

In another embodiment, the invention features a method for generating siNA molecules that do not stimulate an inflammatory or proinflammatory cytokine response (e.g., no cytokine response or attenuated cytokine response) in a cell, subject, or organism, comprising (a) introducing nucleotides having any of Formula I-VII (e.g., siNA motifs referred to in Table IV) or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules that do not stimulate a cytokine response. In one embodiment, the cytokine comprises an interleukin such as interleukin-6 (IL-6) and/or tumor necrosis alpha (TNF-α).

In another embodiment, the invention features a method for generating siNA formulations that do not stimulate an inflammatory or proinflammatory cytokine response (e.g., no cytokine response or attenuated cytokine response) in a cell, subject, or organism, comprising (a) generating a siNA formulation comprising a siNA molecule of the invention and a delivery vehicle or delivery particle as described herein or as otherwise known in the art, and (b) assaying the siNA formulation of step (a) under conditions suitable for isolating siNA formulations that do not stimulate a cytokine response. In one embodiment, the cytokine comprises an interleukin such as interleukin-6 (IL-6) and/or tumor necrosis alpha (TNF-α).

In another embodiment, the invention features a method for generating siNA molecules that do not stimulate Toll-like Receptor (TLR) response (e.g., no TLR response or attenuated TLR response) in a cell, subject, or organism, comprising (a) introducing nucleotides having any of Formula I-VII (e.g., siNA motifs referred to in Table IV) or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules that do not stimulate a TLR response. In one embodiment, the TLR comprises TLR3, TLR7, TLR8 and/or TLR9.

In another embodiment, the invention features a method for generating siNA formulations that do not stimulate a Toll-like Receptor (TLR) response (e.g., no TLR response or attenuated TLR response) in a cell, subject, or organism, comprising (a) generating a siNA formulation comprising a siNA molecule of the invention and a delivery vehicle or delivery particle as described herein or as otherwise known in the art, and (b) assaying the siNA formulation of step (a) under conditions suitable for isolating siNA formulations that do not stimulate a TLR response. In one embodiment, the TLR comprises TLR3, TLR7, TLR8 and/or TLR9.

In one embodiment, a chemically modified siNA molecule of the invention has an improved toxicologic profile compared to a corresponding siRNA molecule having no chemical modifications or fewer chemical modifications.

In one embodiment, the invention features a chemically synthesized double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a target RNA via RNA interference (RNAi), wherein: (a) each strand of said siNA molecule is about 18 to about 38 nucleotides in length; (b) one strand of said siNA molecule comprises nucleotide sequence having sufficient complementarity to said target RNA for the siNA molecule to direct cleavage of the target RNA via RNA interference; and (c) wherein the nucleotide positions within said siNA molecule are chemically modified to reduce the immunostimulatory properties of the siNA molecule to a level below that of a corresponding unmodified siRNA molecule. Such siNA molecules are said to have an improved toxicologic profile compared to an unmodified or minimally modified siNA.

By “improved toxicologic profile”, is meant that the chemically modified or formulated siNA construct exhibits decreased toxicity in a cell, subject, or organism compared to an unmodified or unformulated siNA, or siNA molecule having fewer modifications or modifications that are less effective in imparting improved toxicology. Such siNA molecules are also considered to have “improved RNAi activity”. In a non-limiting example, siNA molecules and formulations with improved toxicologic profiles are associated with reduced immunostimulatory properties, such as a reduced, decreased or attenuated immunostimulatory response in a cell, subject, or organism compared to an unmodified or unformulated siNA, or siNA molecule having fewer modifications or modifications that are less effective in imparting improved toxicology. Such an improved toxicologic profile is characterized by abrogated or reduced immunostimulation, such as reduction or abrogation of induction of interferons (e.g., interferon alpha), inflammatory cytokines (e.g., interleukins such as IL-6, and/or TNF-alpha), and/or toll like receptors (e.g., TLR-3, TLR-7, TLR-8, and/or TLR-9). In one embodiment, a siNA molecule or formulation with an improved toxicological profile comprises no ribonucleotides. In one embodiment, a siNA molecule or formulation with an improved toxicological profile comprises less than 5 ribonucleotides (e.g., 1, 2, 3, or 4 ribonucleotides). In one embodiment, a siNA molecule or formulation with an improved toxicological profile comprises Stab 7, Stab 8, Stab 11, Stab 12, Stab 13, Stab 16, Stab 17, Stab 18, Stab 19, Stab 20, Stab 23, Stab 24, Stab 25, Stab 26, Stab 27, Stab 28, Stab 29, Stab 30, Stab 31, Stab 32, Stab 33, Stab 34, Stab 35, Stab 36 or any combination thereof (see Table IV). Herein, numeric Stab chemistries include both 2′-fluoro and 2′-OCF3 versions of the chemistries shown in Table IV. For example, “Stab 7/8” refers to both Stab 7/8 and Stab 7F/8F etc. In one embodiment, a siNA molecule or formulation with an improved toxicological profile comprises a siNA molecule of the invention and a formulation as described in United States Patent Application Publication No. 20030077829, incorporated by reference herein in its entirety including the drawings.

In one embodiment, the level of immunostimulatory response associated with a given siNA molecule can be measured as is described herein or as is otherwise known in the art, for example by determining the level of PKR/interferon response, proliferation, B-cell activation, and/or cytokine production in assays to quantitate the immunostimulatory response of particular siNA molecules (see, for example, Leifer et al., 2003, J. Immunother. 26, 313-9; and U.S. Pat. No. 5,968,909, incorporated in its entirety by reference). In one embodiment, the reduced immunostimulatory response is between about 10% and about 100% compared to an unmodified or minimally modified siRNA molecule, e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% reduced immunostimulatory response. In one embodiment, the immunostimulatory response associated with a siNA molecule can be modulated by the degree of chemical modification. For example, a siNA molecule having between about 10% and about 100%, e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the nucleotide positions in the siNA molecule modified can be selected to have a corresponding degree of immunostimulatory properties as described herein.

In one embodiment, the degree of reduced immunostimulatory response is selected for optimized RNAi activity. For example, retaining a certain degree of immunostimulation can be preferred to treat viral infection, where less than 100% reduction in immunostimulation may be preferred for maximal antiviral activity (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduction in immunostimulation) whereas the inhibition of expression of an endogenous gene target may be preferred with siNA molecules that possess minimal immunostimulatory properties to prevent non-specific toxicity or off target effects (e.g., about 90% to about 100% reduction in immunostimulation).

In one embodiment, the invention features a chemically synthesized double stranded siNA molecule that directs cleavage of a target RNA via RNA interference (RNAi), wherein (a) each strand of said siNA molecule is about 18 to about 38 nucleotides in length; (b) one strand of said siNA molecule comprises nucleotide sequence having sufficient complementarity to said target RNA for the siNA molecule to direct cleavage of the target RNA via RNA interference; and (c) wherein one or more nucleotides of said siNA molecule are chemically modified to reduce the immunostimulatory properties of the siNA molecule to a level below that of a corresponding unmodified siNA molecule. In one embodiment, each stand comprises at least about 18 nucleotides that are complementary to the nucleotides of the other strand.

In another embodiment, the siNA molecule comprising modified nucleotides to reduce the immunostimulatory properties of the siNA molecule comprises an antisense region having nucleotide sequence that is complementary to a nucleotide sequence of a target gene or a portion thereof and further comprises a sense region, wherein said sense region comprises a nucleotide sequence substantially similar to the nucleotide sequence of said target gene or portion thereof. In one embodiment thereof, the antisense region and the sense region comprise about 18 to about 38 nucleotides, wherein said antisense region comprises at least about 18 nucleotides that are complementary to nucleotides of the sense region. In one embodiment thereof, the pyrimidine nucleotides in the sense region are 2′-O-methyl pyrimidine nucleotides. In another embodiment thereof, the purine nucleotides in the sense region are 2′-deoxy purine nucleotides. In yet another embodiment thereof, the pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides. In another embodiment thereof, the pyrimidine nucleotides of said antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides. In yet another embodiment thereof, the purine nucleotides of said antisense region are 2′-O-methyl purine nucleotides. In still another embodiment thereof, the purine nucleotides present in said antisense region comprise 2′-deoxypurine nucleotides. In another embodiment, the antisense region comprises a phosphorothioate internucleotide linkage at the 3′ end of said antisense region. In another embodiment, the antisense region comprises a glyceryl modification at a 3′ end of said antisense region.

In other embodiments, the siNA molecule comprising modified nucleotides to reduce the immunostimulatory properties of the siNA molecule can comprise any of the structural features of siNA molecules described herein. In other embodiments, the siNA molecule comprising modified nucleotides to reduce the immunostimulatory properties of the siNA molecule can comprise any of the chemical modifications of siNA molecules described herein.

In one embodiment, the invention features a method for generating a chemically synthesized double stranded siNA molecule having chemically modified nucleotides to reduce the immunostimulatory properties of the siNA molecule, comprising (a) introducing one or more modified nucleotides in the siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating an siNA molecule having reduced immunostimulatory properties compared to a corresponding siNA molecule having unmodified nucleotides. Each strand of the siNA molecule is about 18 to about 38 nucleotides in length. One strand of the siNA molecule comprises nucleotide sequence having sufficient complementarity to the target RNA for the siNA molecule to direct cleavage of the target RNA via RNA interference. In one embodiment, the reduced immunostimulatory properties comprise an abrogated or reduced induction of inflammatory or proinflammatory cytokines, such as interleukin-6 (IL-6) or tumor necrosis alpha (TNF-α), in response to the siNA being introduced in a cell, tissue, or organism. In another embodiment, the reduced immunostimulatory properties comprise an abrogated or reduced induction of Toll Like Receptors (TLRs), such as TLR3, TLR7, TLR8 or TLR9, in response to the siNA being introduced in a cell, tissue, or organism. In another embodiment, the reduced immunostimulatory properties comprise an abrogated or reduced induction of interferons, such as interferon alpha, in response to the siNA being introduced in a cell, tissue, or organism.

In another embodiment, the invention features a method for generating siNA molecules with increased binding affinity between the sense and antisense strands of the siNA molecule comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased binding affinity between the sense and antisense strands of the siNA molecule.

In one embodiment, the invention features siNA constructs that mediate RNAi against a target polynucleotide, wherein the siNA construct comprises one or more chemical modifications described herein that modulates the binding affinity between the antisense strand of the siNA construct and a complementary target RNA sequence within a cell.

In one embodiment, the invention features siNA constructs that mediate RNAi against a target polynucleotide, wherein the siNA construct comprises one or more chemical modifications described herein that modulates the binding affinity between the antisense strand of the siNA construct and a complementary target DNA sequence within a cell.

In another embodiment, the invention features a method for generating siNA molecules with increased binding affinity between the antisense strand of the siNA molecule and a complementary target RNA sequence comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased binding affinity between the antisense strand of the siNA molecule and a complementary target RNA sequence.

In another embodiment, the invention features a method for generating siNA molecules with increased binding affinity between the antisense strand of the siNA molecule and a complementary target DNA sequence comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased binding affinity between the antisense strand of the siNA molecule and a complementary target DNA sequence.

In one embodiment, the invention features siNA constructs that mediate RNAi against a target polynucleotide, wherein the siNA construct comprises one or more chemical modifications described herein that modulate the polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to the chemically-modified siNA construct.

In another embodiment, the invention features a method for generating siNA molecules capable of mediating increased polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to a chemically-modified siNA molecule comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules capable of mediating increased polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to the chemically-modified siNA molecule.

In one embodiment, the invention features chemically-modified siNA constructs that mediate RNAi against a target polynucleotide in a cell, wherein the chemical modifications do not significantly effect the interaction of siNA with a target RNA molecule, DNA molecule and/or proteins or other factors that are essential for RNAi in a manner that would decrease the efficacy of RNAi mediated by such siNA constructs.

In another embodiment, the invention features a method for generating siNA molecules with improved RNAi specificity against polynucleotide targets comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved RNAi specificity. In one embodiment, improved specificity comprises having reduced off target effects compared to an unmodified siNA molecule. For example, introduction of terminal cap moieties at the 3′-end, 5′-end, or both 3′ and 5′-ends of the sense strand or region of a siNA molecule of the invention can direct the siNA to have improved specificity by preventing the sense strand or sense region from acting as a template for RNAi activity against a corresponding target having complementarity to the sense strand or sense region.

In another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against a target polynucleotide comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved RNAi activity.

In yet another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against a target RNA comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved RNAi activity against the target RNA.

In yet another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against a target DNA comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved RNAi activity against the target DNA.

In another embodiment, the invention features a method for generating siNA molecules against a target polynucleotide with improved cellular uptake comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved cellular uptake.

In one embodiment, the invention features siNA constructs that mediate RNAi against a target polynucleotide, wherein the siNA construct comprises one or more chemical modifications described herein that increases the bioavailability of the siNA construct, for example, by attaching polymeric conjugates such as polyethyleneglycol or equivalent conjugates that improve the pharmacokinetics of the siNA construct, or by attaching conjugates that target specific tissue types or cell types in vivo. Non-limiting examples of such conjugates are described in Vargeese et al., U.S. Ser. No. 10/201,394 incorporated by reference herein.

In one embodiment, the invention features a method for generating siNA molecules of the invention with improved bioavailability comprising (a) introducing a conjugate into the structure of a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability. Such conjugates can include ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; cholesterol derivatives, polyamines, such as spermine or spermidine; and others.

In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein said second sequence is chemically modified in a manner that it can no longer act as a guide sequence for efficiently mediating RNA interference and/or be recognized by cellular proteins that facilitate RNAi. In one embodiment, the first nucleotide sequence of the siNA is chemically modified as described herein. In one embodiment, the first nucleotide sequence of the siNA is not modified (e.g., is all RNA).

In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein the second sequence is designed or modified in a manner that prevents its entry into the RNAi pathway as a guide sequence or as a sequence that is complementary to a target nucleic acid (e.g., RNA) sequence. In one embodiment, the first nucleotide sequence of the siNA is chemically modified as described herein. In one embodiment, the first nucleotide sequence of the siNA is not modified (e.g., is all RNA). Such design or modifications are expected to enhance the activity of siNA and/or improve the specificity of siNA molecules of the invention. These modifications are also expected to minimize any off-target effects and/or associated toxicity.

In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein said second sequence comprises a terminal cap moiety at the 5′-end of said second sequence. In one embodiment, the terminal cap moiety comprises an inverted abasic, inverted deoxy abasic, inverted nucleotide moiety, a group shown in FIG. 10, an alkyl or cycloalkyl group, a heterocycle, or any other group that prevents RNAi activity in which the second sequence serves as a guide sequence or template for RNAi.

In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein said second sequence comprises a terminal cap moiety at the 5′-end and 3′-end of said second sequence. In one embodiment, each terminal cap moiety individually comprises an inverted abasic, inverted deoxy abasic, inverted nucleotide moiety, a group shown in FIG. 10, an alkyl or cycloalkyl group, a heterocycle, or any other group that prevents RNAi activity in which the second sequence serves as a guide sequence or template for RNAi.

In one embodiment, the invention features a method for generating siNA molecules of the invention with improved specificity for down regulating or inhibiting the expression of a target nucleic acid (e.g., a DNA or RNA such as a gene or its corresponding RNA), comprising (a) introducing one or more chemical modifications into the structure of a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved specificity. In another embodiment, the chemical modification used to improve specificity comprises terminal cap modifications at the 5′-end, 3′-end, or both 5′ and 3′-ends of the siNA molecule. The terminal cap modifications can comprise, for example, structures shown in FIG. 10 (e.g. inverted deoxyabasic moieties) or any other chemical modification that renders a portion of the siNA molecule (e.g. the sense strand) incapable of mediating RNA interference against an off target nucleic acid sequence. In a non-limiting example, a siNA molecule is designed such that only the antisense sequence of the siNA molecule can serve as a guide sequence for RISC mediated degradation of a corresponding target RNA sequence. This can be accomplished by rendering the sense sequence of the siNA inactive by introducing chemical modifications to the sense strand that preclude recognition of the sense strand as a guide sequence by RNAi machinery. In one embodiment, such chemical modifications comprise any chemical group at the 5′-end of the sense strand of the siNA, or any other group that serves to render the sense strand inactive as a guide sequence for mediating RNA interference. These modifications, for example, can result in a molecule where the 5′-end of the sense strand no longer has a free 5′-hydroxyl (5′-OH) or a free 5′-phosphate group (e.g., phosphate, diphosphate, triphosphate, cyclic phosphate etc.). Non-limiting examples of such siNA constructs are described herein, such as “Stab 9/10”, “Stab 7/8”, “Stab 7/19”, “Stab 17/22”, “Stab 23/24”, “Stab 24/25”, and “Stab 24/26” (e.g., any siNA having Stab 7, 9, 17, 23, or 24 sense strands) chemistries and variants thereof (see Table IV) wherein the 5′-end and 3′-end of the sense strand of the siNA do not comprise a hydroxyl group or phosphate group. Herein, numeric Stab chemistries include both 2′-fluoro and 2′-OCF3 versions of the chemistries shown in Table IV. For example, “Stab 7/8” refers to both Stab 7/8 and Stab 7F/8F etc.

In one embodiment, the invention features a method for screening siNA molecules that are active in mediating RNA interference against a target nucleic acid sequence comprising (a) generating a plurality of unmodified siNA molecules, (b) screening the siNA molecules of step (a) under conditions suitable for isolating siNA molecules that are active in mediating RNA interference against the target nucleic acid sequence, and (c) introducing chemical modifications (e.g. chemical modifications as described herein or as otherwise known in the art) into the active siNA molecules of (b). In one embodiment, the method further comprises re-screening the chemically modified siNA molecules of step (c) under conditions suitable for isolating chemically modified siNA molecules that are active in mediating RNA interference against the target nucleic acid sequence.

In one embodiment, the invention features a method for screening chemically modified siNA molecules that are active in mediating RNA interference against a target nucleic acid sequence comprising (a) generating a plurality of chemically modified siNA molecules (e.g. siNA molecules as described herein or as otherwise known in the art), and (b) screening the siNA molecules of step (a) under conditions suitable for isolating chemically modified siNA molecules that are active in mediating RNA interference against the target nucleic acid sequence.

The term “ligand” refers to any compound or molecule, such as a drug, peptide, hormone, or neurotransmitter, that is capable of interacting with another compound, such as a receptor, either directly or indirectly. The receptor that interacts with a ligand can be present on the surface of a cell or can alternately be an intercellular receptor. Interaction of the ligand with the receptor can result in a biochemical reaction, or can simply be a physical interaction or association.

In another embodiment, the invention features a method for generating siNA molecules of the invention with improved bioavailability comprising (a) introducing an excipient formulation to a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability. Such excipients include polymers such as cyclodextrins, lipids, cationic lipids, polyamines, phospholipids, nanoparticles, receptors, ligands, and others.

In another embodiment, the invention features a method for generating siNA molecules of the invention with improved bioavailability comprising (a) introducing nucleotides having any of Formulae I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability.

In another embodiment, polyethylene glycol (PEG) can be covalently attached to siNA compounds of the present invention. The attached PEG can be any molecular weight, preferably from about 100 to about 50,000 daltons (Da).

The present invention can be used alone or as a component of a kit having at least one of the reagents necessary to carry out the in vitro or in vivo introduction of RNA to test samples and/or subjects. For example, preferred components of the kit include a siNA molecule of the invention and a vehicle that promotes introduction of the siNA into cells of interest as described herein (e.g., using lipids and other methods of transfection known in the art, see for example Beigelman et al, U.S. Pat. No. 6,395,713). The kit can be used for target validation, such as in determining gene function and/or activity, or in drug optimization, and in drug discovery (see for example Usman et al., U.S. Ser. No. 60/402,996). Such a kit can also include instructions to allow a user of the kit to practice the invention.

The term “short interfering nucleic acid”, “siNA”, “short interfering RNA”, “siRNA”, “short interfering nucleic acid molecule”, “short interfering oligonucleotide molecule”, or “chemically-modified short interfering nucleic acid molecule” as used herein refers to any nucleic acid molecule capable of inhibiting or down regulating gene expression or viral replication by mediating RNA interference “RNAi” or gene silencing in a sequence-specific manner. These terms can refer to both individual nucleic acid molecules, a plurality of such nucleic acid molecules, or pools of such nucleic acid molecules. The siNA can be a double-stranded nucleic acid molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e., each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure, for example wherein the double stranded region is about 15 to about 30, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs; the antisense strand comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof (e.g., about 15 to about 25 or more nucleotides of the siNA molecule are complementary to the target nucleic acid or a portion thereof). Alternatively, the siNA is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siNA are linked by means of a nucleic acid based or non-nucleic acid-based linker(s). The siNA can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siNA can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siNA molecule capable of mediating RNAi. The siNA can also comprise a single stranded polynucleotide having nucleotide sequence complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof (for example, where such siNA molecule does not require the presence within the siNA molecule of nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), wherein the single stranded polynucleotide can further comprise a terminal phosphate group, such as a 5′-phosphate (see for example Martinez et al., 2002, Cell., 110, 563-574 and Schwarz et al., 2002, Molecular Cell, 10, 537-568), or 5′,3′-diphosphate. In certain embodiments, the siNA molecule of the invention comprises separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic interactions, and/or stacking interactions. In certain embodiments, the siNA molecules of the invention comprise nucleotide sequence that is complementary to nucleotide sequence of a target gene. In another embodiment, the siNA molecule of the invention interacts with nucleotide sequence of a target gene in a manner that causes inhibition of expression of the target gene. As used herein, siNA molecules need not be limited to those molecules containing only RNA, but further encompasses chemically-modified nucleotides and non-nucleotides. In certain embodiments, the short interfering nucleic acid molecules of the invention lack 2′-hydroxy (2′-OH) containing nucleotides. Applicant describes in certain embodiments short interfering nucleic acids that do not require the presence of nucleotides having a 2′-hydroxy group for mediating RNAi and as such, short interfering nucleic acid molecules of the invention optionally do not include any ribonucleotides (e.g., nucleotides having a 2′-OH group). Such siNA molecules that do not require the presence of ribonucleotides within the siNA molecule to support RNAi can however have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2′-OH groups. Optionally, siNA molecules can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions. The modified short interfering nucleic acid molecules of the invention can also be referred to as short interfering modified oligonucleotides “siMON.” As used herein, the term siNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically-modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. Non limiting examples of siNA molecules of the invention are shown in FIGS. 4-6, and Tables II and III herein. Such siNA molecules are distinct from other nucleic acid technologies known in the art that mediate inhibition of gene expression, such as ribozymes, antisense, triplex forming, aptamer, 2,5-A chimera, or decoy oligonucleotides.

By “RNA interference” or “RNAi” is meant a biological process of inhibiting or down regulating gene expression in a cell as is generally known in the art and which is mediated by short interfering nucleic acid molecules, see for example Zamore and Haley, 2005, Science, 309, 1519-1524; Vaughn and Martienssen, 2005, Science, 309, 1525-1526; Zamore et al., 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al., International PCT Publication No. WO 00/44895; Zernicka-Goetz et al., International PCT Publication No. WO 01/36646; Fire, International PCT Publication No. WO 99/32619; Plaetinck et al., International PCT Publication No. WO 00/01846; Mello and Fire, International PCT Publication No. WO 01/29058; Deschamps-Depaillette, International PCT Publication No. WO 99/07409; and Li et al., International PCT Publication No. WO 00/44914; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237; Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002, RNA, 8, 842-850; Reinhart et al., 2002, gene & Dev., 16, 1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831). In addition, as used herein, the term RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, transcriptional inhibition, or epigenetics. For example, siNA molecules of the invention can be used to epigenetically silence genes at both the post-transcriptional level or the pre-transcriptional level. In a non-limiting example, epigenetic modulation of gene expression by siNA molecules of the invention can result from siNA mediated modification of chromatin structure or methylation patterns to alter gene expression (see, for example, Verdel et al., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237). In another non-limiting example, modulation of gene expression by siNA molecules of the invention can result from siNA mediated cleavage of RNA (either coding or non-coding RNA) via RISC, or alternately, translational inhibition as is known in the art. In another embodiment, modulation of gene expression by siNA molecules of the invention can result from transcriptional inhibition (see for example Janowski et al., 2005, Nature Chemical Biology, 1, 216-222).

In one embodiment, a siNA molecule of the invention is a duplex forming oligonucleotide “DFO”, (see for example FIGS. 14-15 and Vaish et al., U.S. Ser. No. 10/727,780 filed Dec. 3, 2003 and International PCT Application No. US04/16390, filed May 24, 2004).

In one embodiment, a siNA molecule of the invention is a multifunctional siNA, (see for example FIGS. 16-28 and Jadhav et al., U.S. Ser. No. 60/543,480 filed Feb. 10, 2004 and International PCT Application No. US04/16390, filed May 24, 2004). In one embodiment, the multifunctional siNA of the invention can comprise sequence targeting, for example, two or more regions of target RNA (see for example target sequences in Tables II and III). In one embodiment, the multifunctional siNA of the invention can comprise sequence targeting one or more different targets, including coding regions and non-coding regions of HCV or HCV pathway/host RNA.

By “asymmetric hairpin” as used herein is meant a linear siNA molecule comprising an antisense region, a loop portion that can comprise nucleotides or non-nucleotides, and a sense region that comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complementary nucleotides to base pair with the antisense region and form a duplex with loop. For example, an asymmetric hairpin siNA molecule of the invention can comprise an antisense region having length sufficient to mediate RNAi in a cell or in vitro system (e.g. about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and a loop region comprising about 4 to about 12 (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, or 12) nucleotides, and a sense region having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides that are complementary to the antisense region. The asymmetric hairpin siNA molecule can also comprise a 5′-terminal phosphate group that can be chemically modified. The loop portion of the asymmetric hairpin siNA molecule can comprise nucleotides, non-nucleotides, linker molecules, or conjugate molecules as described herein.

By “asymmetric duplex” as used herein is meant a siNA molecule having two separate strands comprising a sense region and an antisense region, wherein the sense region comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complementary nucleotides to base pair with the antisense region and form a duplex. For example, an asymmetric duplex siNA molecule of the invention can comprise an antisense region having length sufficient to mediate RNAi in a cell or in vitro system (e.g., about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and a sense region having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides that are complementary to the antisense region.

By “RNAi inhibitor” is meant any molecule that can down regulate, reduce or inhibit RNA interference function or activity in a cell or organism. An RNAi inhibitor can down regulate, reduce or inhibit RNAi (e.g., RNAi mediated cleavage of a target polynucleotide, translational inhibition, or transcriptional silencing) by interaction with or interfering the function of any component of the RNAi pathway, including protein components such as RISC, or nucleic acid components such as miRNAs or siRNAs. A RNAi inhibitor can be a siNA molecule, an antisense molecule, an aptamer, or a small molecule that interacts with or interferes with the function of RISC, a miRNA, or a siRNA or any other component of the RNAi pathway in a cell or organism. By inhibiting RNAi (e.g., RNAi mediated cleavage of a target polynucleotide, translational inhibition, or transcriptional silencing), a RNAi inhibitor of the invention can be used to modulate (e.g, up-regulate or down regulate) the expression of a target gene. In one embodiment, a RNA inhibitor of the invention is used to up-regulate gene expression by interfering with (e.g., reducing or preventing) endogenous down-regulation or inhibition of gene expression through translational inhibition, transcriptional silencing, or RISC mediated cleavage of a polynucleotide (e.g., mRNA). By interfering with mechanisms of endogenous repression, silencing, or inhibition of gene expression, RNAi inhibitors of the invention can therefore be used to up-regulate gene expression for the treatment of diseases, traits, or conditions resulting from a loss of function. In one embodiment, the term “RNAi inhibitor” is used in place of the term “siNA” in the various embodiments herein, for example, with the effect of increasing gene expression for the treatment of loss of function diseases, traits, and/or conditions.

By “aptamer” or “nucleic acid aptamer” as used herein is meant a polynucleotide that binds specifically to a target molecule wherein the nucleic acid molecule has sequence that is distinct from sequence recognized by the target molecule in its natural setting. Alternately, an aptamer can be a nucleic acid molecule that binds to a target molecule where the target molecule does not naturally bind to a nucleic acid. The target molecule can be any molecule of interest. For example, the aptamer can be used to bind to a ligand-binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein. This is a non-limiting example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art, see for example Gold et al., 1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J. Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser, 2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287, 820; and Jayasena, 1999, Clinical Chemistry, 45, 1628. Aptamer molecules of the invention can be chemically modified as is generally known in the art or as described herein.

The term “antisense nucleic acid”, as used herein, refers to a nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review, see Stein and Cheng, 1993 Science 261, 1004 and Woolf et al., U.S. Pat. No. 5,849,902) by steric interaction or by RNase H mediated target recognition. Typically, antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule. However, in certain embodiments, an antisense molecule can bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule can bind such that the antisense molecule forms a loop. Thus, the antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence or both. For a review of current antisense strategies, see Schmajuk et al., 1999, J. Biol. Chem., 274, 21783-21789, Delihas et al., 1997, Nature, 15, 751-753, Stein et al., 1997, Antisense N. A. Drug Dev., 7, 151, Crooke, 2000, Methods Enzymol., 313, 3-45; Crooke, 1998, Biotech. genet. Eng. Rev., 15, 121-157, Crooke, 1997, Ad. Pharmacol., 40, 1-49. In addition, antisense DNA or antisense modified with 2′-MOE and other modifications as are known in the art can be used to target RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex. The antisense oligonucleotides can comprise one or more RNAse H activating region, which is capable of activating RNAse H cleavage of a target RNA. Antisense DNA can be synthesized chemically or expressed via the use of a single stranded DNA expression vector or equivalent thereof. Antisense molecules of the invention can be chemically modified as is generally known in the art or as described herein.

By “modulate” is meant that the expression of the gene, or level of a RNA molecule or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the modulator. For example, the term “modulate” can mean “inhibit,” but the use of the word “modulate” is not limited to this definition.

By “inhibit”, “down-regulate”, or “reduce”, it is meant that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, is reduced below that observed in the absence of the nucleic acid molecules (e.g., siNA) of the invention. In one embodiment, inhibition, down-regulation or reduction with an siNA molecule is below that level observed in the presence of an inactive or attenuated molecule. In another embodiment, inhibition, down-regulation, or reduction with siNA molecules is below that level observed in the presence of, for example, an siNA molecule with scrambled sequence or with mismatches. In another embodiment, inhibition, down-regulation, or reduction of gene expression with a nucleic acid molecule of the instant invention is greater in the presence of the nucleic acid molecule than in its absence. In one embodiment, inhibition, down regulation, or reduction of gene expression is associated with post transcriptional silencing, such as RNAi mediated cleavage of a target nucleic acid molecule (e.g. RNA) or inhibition of translation. In one embodiment, inhibition, down regulation, or reduction of gene expression is associated with pretranscriptional silencing, such as by alterations in DNA methylation patterns and DNA chromatin structure.

By “up-regulate”, or “promote”, it is meant that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, is increased above that observed in the absence of the nucleic acid molecules (e.g., siNA) of the invention. In one embodiment, up-regulation or promotion of gene expression with an siNA molecule is above that level observed in the presence of an inactive or attenuated molecule. In another embodiment, up-regulation or promotion of gene expression with siNA molecules is above that level observed in the presence of, for example, an siNA molecule with scrambled sequence or with mismatches. In another embodiment, up-regulation or promotion of gene expression with a nucleic acid molecule of the instant invention is greater in the presence of the nucleic acid molecule than in its absence. In one embodiment, up-regulation or promotion of gene expression is associated with inhibition of RNA mediated gene silencing, such as RNAi mediated cleavage or silencing of a coding or non-coding RNA target that down regulates, inhibits, or silences the expression of the gene of interest to be up-regulated. The down regulation of gene expression can, for example, be induced by a coding RNA or its encoded protein, such as through negative feedback or antagonistic effects. The down regulation of gene expression can, for example, be induced by a non-coding RNA having regulatory control over a gene of interest, for example by silencing expression of the gene via translational inhibition, chromatin structure, methylation, RISC mediated RNA cleavage, or translational inhibition. As such, inhibition or down regulation of targets that down regulate, suppress, or silence a gene of interest can be used to up-regulate or promote expression of the gene of interest toward therapeutic use.

In one embodiment, a RNAi inhibitor of the invention is used to up regulate gene expression by inhibiting RNAi or gene silencing. For example, a RNAi inhibitor of the invention can be used to treat loss of function diseases and conditions by up-regulating gene expression, such as in instances of haploinsufficiency where one allele of a particular gene harbors a mutation (e.g., a frameshift, missense, or nonsense mutation) resulting in a loss of function of the protein encoded by the mutant allele. In such instances, the RNAi inhibitor can be used to up regulate expression of the protein encoded by the wild type or functional allele, thus correcting the haploinsufficiency by compensating for the mutant or null allele. In another embodiment, a siNA molecule of the invention is used to down regulate expression of a toxic gain of function allele while a RNAi inhibitor of the invention is used concomitantly to up regulate expression of the wild type or functional allele, such as in the treatment of diseases, traits, or conditions herein or otherwise known in the art (see for example Rhodes et al., 2004, PNAS USA, 101:11147-11152 and Meisler et al. 2005, The Journal of Clinical Investigation, 115:2010-2017).

By “gene”, or “target gene” or “target DNA”, is meant a nucleic acid that encodes an RNA, for example, nucleic acid sequences including, but not limited to, structural genes encoding a polypeptide. A gene or target gene can also encode a functional RNA (FRNA) or non-coding RNA (ncRNA), such as small temporal RNA (stRNA), micro RNA (miRNA), small nuclear RNA (snRNA), short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomal RNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof. Such non-coding RNAs can serve as target nucleic acid molecules for siNA mediated RNA interference in modulating the activity of fRNA or ncRNA involved in functional or regulatory cellular processes. Abberant fRNA or ncRNA activity leading to disease can therefore be modulated by siNA molecules of the invention. siNA molecules targeting fRNA and ncRNA can also be used to manipulate or alter the genotype or phenotype of a subject, organism or cell, by intervening in cellular processes such as genetic imprinting, transcription, translation, or nucleic acid processing (e.g., transamination, methylation etc.). The target gene can be a gene derived from a cell, an endogenous gene, a transgene, or exogenous genes such as genes of a pathogen, for example a virus, which is present in the cell after infection thereof. The cell containing the target gene can be derived from or contained in any organism, for example a plant, animal, protozoan, virus, bacterium, or fungus. Non-limiting examples of plants include monocots, dicots, or gymnosperms. Non-limiting examples of animals include vertebrates or invertebrates. Non-limiting examples of fungi include molds or yeasts. For a review, see for example Snyder and Gerstein, 2003, Science, 300, 258-260.

By “non-canonical base pair” is meant any non-Watson Crick base pair, such as mismatches and/or wobble base pairs, including flipped mismatches, single hydrogen bond mismatches, trans-type mismatches, triple base interactions, and quadruple base interactions. Non-limiting examples of such non-canonical base pairs include, but are not limited to, AC reverse Hoogsteen, AC wobble, AU reverse Hoogsteen, GU wobble, AA N7 amino, CC 2-carbonyl-amino(H1)-N3-amino(H2), GA sheared, UC 4-carbonyl-amino, UU imino-carbonyl, AC reverse wobble, AU Hoogsteen, AU reverse Watson Crick, CG reverse Watson Crick, GC N3-amino-amino N3, AA N1-amino symmetric, AA N7-amino symmetric, GA N7-N1 amino-carbonyl, GA+carbonyl-amino N7-N1, GG N1-carbonyl symmetric, GG N3-amino symmetric, CC carbonyl-amino symmetric, CC N3-amino symmetric, UU 2-carbonyl-imino symmetric, UU 4-carbonyl-imino symmetric, AA amino-N3, AA N1-amino, AC amino 2-carbonyl, AC N3-amino, AC N7-amino, AU amino-4-carbonyl, AU N1-imino, AU N3-imino, AU N7-imino, CC carbonyl-amino, GA amino-N1, GA amino-N7, GA carbonyl-amino, GA N3-amino, GC amino-N3, GC carbonyl-amino, GC N3-amino, GC N7-amino, GG amino-N7, GG carbonyl-imino, GG N7-amino, GU amino-2-carbonyl, GU carbonyl-imino, GU imino-2-carbonyl, GU N7-imino, psiU imino-2-carbonyl, UC 4-carbonyl-amino, UC imino-carbonyl, UU imino-4-carbonyl, AC C2-H—N3, GA carbonyl-C2-H, UU imino-4-carbonyl 2 carbonyl-C5-H, AC amino(A) N3(C)-carbonyl, GC imino amino-carbonyl, Gpsi imino-2-carbonyl amino-2-carbonyl, and GU imino amino-2-carbonyl base pairs.

By “HCV” as used herein is meant, any hepatitis C virus or HCV protein, peptide, or polypeptide having HCV activity, such as encoded by HCV Genbank Accession Nos. shown in Table I. The term HCV also refers to nucleic acid sequences encoding any HCV protein, peptide, or polypeptide having HCV activity. The term “HCV” is also meant to include other HCV encoding sequence, such as other HCV isoforms, mutant HCV genes, splice variants of HCV genes, and HCV gene polymorphisms. In one embodiment, the term HCV as used herein refers to cellular or host proteins or polynucleotides encoding such proteins or that are otherwise involved in HCV infection and/or replication.

By “target” as used herein is meant, any target protein, peptide, or polypeptide, such as encoded by Genbank Accession Nos. herein and in U.S. Ser. No. 10/923,536 and U.S. Ser. No. 10/923,536, both incorporated by reference herein. The term “target” also refers to nucleic acid sequences or target polynucleotide sequence encoding any target protein, peptide, or polypeptide, such as proteins, peptides, or polypeptides encoded by sequences having Genbank Accession Nos. shown herein or in U.S. Provisional Patent Application No. 60/363,124, U.S. Ser. No. 10/923,536 and U.S. Ser. No. 10/923,536. The target of interest can include target polynucleotide sequences, such as target DNA or target RNA. The term “target” is also meant to include other sequences, such as differing isoforms, mutant target genes, splice variants of target polynucleotides, target polymorphisms, and non-coding (e.g., ncRNA, miRNA, sRNA) or other regulatory polynucleotide sequences as described herein. Therefore, in various embodiments of the invention, a double stranded nucleic acid molecule of the invention (e.g., siNA) having complementarity to a target RNA can be used to inhibit or down regulate miRNA or other ncRNA activity. In one embodiment, inhibition of miRNA or ncRNA activity can be used to down regulate or inhibit gene expression (e.g., gene targets described herein or otherwise known in the art) or viral replication (e.g., viral targets described herein or otherwise known in the art) that is dependent on miRNA or ncRNA activity. In another embodiment, inhibition of miRNA or ncRNA activity by double stranded nucleic acid molecules of the invention (e.g. siNA) having complementarity to the miRNA or ncRNA can be used to up regulate or promote target gene expression (e.g., gene targets described herein or otherwise known in the art) where the expression of such genes is down regulated, suppressed, or silenced by the miRNA or ncRNA. Such up-regulation of gene expression can be used to treat diseases and conditions associated with a loss of function or haploinsufficiency as are generally known in the art.

By “pathway target” or “host target” is meant any target involved in pathways of gene expression or activity or cellular or host proteins or polynucleotides encoding such proteins or that are otherwise involved in HCV infection and/or replication. For example, any given target can have related pathway or host targets that can include upstream, downstream, or modifier genes in a biologic pathway. These pathway and host target genes can provide additive or synergistic effects in the treatment of diseases, conditions, and traits herein.

In one embodiment, the target is any target RNA or a portion thereof.

In one embodiment, the target is any target DNA or a portion thereof.

In one embodiment, the target is any target mRNA or a portion thereof.

In one embodiment, the target is any target miRNA or a portion thereof.

In one embodiment, the target is any target siRNA or a portion thereof.

In one embodiment, the target is any target stRNA or a portion thereof.

In one embodiment, the target is a target and or pathway target or a portion thereof.

In one embodiment, the target is any (e.g., one or more) of target sequences described herein and/or in U.S. Provisional Patent Application No. 60/363,124, U.S. Ser. No. 10/923,536 and/or PCT/US03/05028, or a portion thereof. In one embodiment, the target is any (e.g., one or more) of target sequences shown in Tables I, II, or III or a portion thereof. In another embodiment, the target is a siRNA, miRNA, or stRNA corresponding to any (e.g., one or more) target, upper strand, or lower strand sequence shown in Table II or Table III or a portion thereof. In another embodiment, the target is any siRNA, miRNA, or stRNA corresponding any (e.g., one or more) sequence corresponding to a sequence herein or described in U.S. Provisional Patent Application No. 60/363,124, U.S. Ser. No. 10/923,536 and/or PCT/US03/05028.

By “homologous sequence” is meant, a nucleotide sequence that is shared by one or more polynucleotide sequences, such as genes, gene transcripts and/or non-coding polynucleotides. For example, a homologous sequence can be a nucleotide sequence that is shared by two or more genes encoding related but different proteins, such as different members of a gene family, different protein epitopes, different protein isoforms or completely divergent genes, such as a cytokine and its corresponding receptors. A homologous sequence can be a nucleotide sequence that is shared by two or more non-coding polynucleotides, such as noncoding DNA or RNA, regulatory sequences, introns, and sites of transcriptional control or regulation. Homologous sequences can also include conserved sequence regions shared by more than one polynucleotide sequence. Homology does not need to be perfect homology (e.g., 100%), as partially homologous sequences are also contemplated by the instant invention (e.g., 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% etc.).

By “conserved sequence region” is meant, a nucleotide sequence of one or more regions in a polynucleotide does not vary significantly between generations or from one biological system, subject, or organism to another biological system, subject, or organism. The polynucleotide can include both coding and non-coding DNA and RNA.

By “sense region” is meant a nucleotide sequence of a siNA molecule having complementarity to an antisense region of the siNA molecule. In addition, the sense region of a siNA molecule can comprise a nucleic acid sequence having homology with a target nucleic acid sequence. In one embodiment, the sense region of the siNA molecule is referred to as the sense strand or passenger strand.

By “antisense region” is meant a nucleotide sequence of a siNA molecule having complementarity to a target nucleic acid sequence. In addition, the antisense region of a siNA molecule can optionally comprise a nucleic acid sequence having complementarity to a sense region of the siNA molecule. In one embodiment, the antisense region of the siNA molecule is referred to as the antisense strand or guide strand.

By “target nucleic acid” or “target polynucleotide” is meant any nucleic acid sequence (e.g, any target and/or pathway target sequence) whose expression or activity is to be modulated. The target nucleic acid can be DNA or RNA. In one embodiment, a target nucleic acid of the invention is target RNA or DNA.

By “complementarity” is meant that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types as described herein. In one embodiment, a double stranded nucleic acid molecule of the invention, such as an siNA molecule, wherein each strand is between 15 and 30 nucleotides in length, comprises between about 10% and about 100% (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) complementarity between the two strands of the double stranded nucleic acid molecule. In another embodiment, a double stranded nucleic acid molecule of the invention, such as an siNA molecule, where one strand is the sense strand and the other stand is the antisense strand, wherein each strand is between 15 and 30 nucleotides in length, comprises between at least about 10% and about 100% (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) complementarity between the nucleotide sequence in the antisense strand of the double stranded nucleic acid molecule and the nucleotide sequence of its corresponding target nucleic acid molecule, such as a target RNA or target mRNA or viral RNA. In one embodiment, a double stranded nucleic acid molecule of the invention, such as an siNA molecule, where one strand comprises nucleotide sequence that is referred to as the sense region and the other strand comprises a nucleotide sequence that is referred to as the antisense region, wherein each strand is between 15 and 30 nucleotides in length, comprises between about 10% and about 100% (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) complementarity between the sense region and the antisense region of the double stranded nucleic acid molecule. In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785). A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10 nucleotides in the first oligonucleotide being based paired to a second nucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%, 80%, 90%, and 100% complementary respectively). In one embodiment, a siNA molecule of the invention has perfect complementarity between the sense strand or sense region and the antisense strand or antisense region of the siNA molecule. In one embodiment, a siNA molecule of the invention is perfectly complementary to a corresponding target nucleic acid molecule. “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. In one embodiment, a siNA molecule of the invention comprises about 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides that are complementary to one or more target nucleic acid molecules or a portion thereof. In one embodiment, a siNA molecule of the invention has partial complementarity (i.e., less than 100% complementarity) between the sense strand or sense region and the antisense strand or antisense region of the siNA molecule or between the antisense strand or antisense region of the siNA molecule and a corresponding target nucleic acid molecule. For example, partial complementarity can include various mismatches or non-based paired nucleotides (e.g., 1, 2, 3, 4, 5 or more mismatches or non-based paired nucleotides) within the siNA structure which can result in bulges, loops, or overhangs that result between the between the sense strand or sense region and the antisense strand or antisense region of the siNA molecule or between the antisense strand or antisense region of the siNA molecule and a corresponding target nucleic acid molecule.

In one embodiment, a double stranded nucleic acid molecule of the invention, such as siNA molecule, has perfect complementarity between the sense strand or sense region and the antisense strand or antisense region of the nucleic acid molecule. In one embodiment, double stranded nucleic acid molecule of the invention, such as siNA molecule, is perfectly complementary to a corresponding target nucleic acid molecule.

In one embodiment, double stranded nucleic acid molecule of the invention, such as siNA molecule, has partial complementarity (i.e., less than 100% complementarity) between the sense strand or sense region and the antisense strand or antisense region of the double stranded nucleic acid molecule or between the antisense strand or antisense region of the nucleic acid molecule and a corresponding target nucleic acid molecule. For example, partial complementarity can include various mismatches or non-base paired nucleotides (e.g., 1, 2, 3, 4, 5 or more mismatches or non-based paired nucleotides, such as nucleotide bulges) within the double stranded nucleic acid molecule, structure which can result in bulges, loops, or overhangs that result between the sense strand or sense region and the antisense strand or antisense region of the double stranded nucleic acid molecule or between the antisense strand or antisense region of the double stranded nucleic acid molecule and a corresponding target nucleic acid molecule.

In one embodiment, double stranded nucleic acid molecule of the invention is a microRNA (miRNA). By “microRNA” or “miRNA” is meant, a small double stranded RNA that regulates the expression of target messenger RNAs either by mRNA cleavage, translational repression/inhibition or heterochromatic silencing (see for example Ambros, 2004, Nature, 431, 350-355; Bartel, 2004, Cell, 116, 281-297; Cullen, 2004, Virus Research., 102, 3-9; He et al., 2004, Nat. Rev. genet., 5, 522-531; Ying et al., 2004, gene, 342, 25-28; and Sethupathy et al., 2006, RNA, 12:192-197). In one embodiment, the microRNA of the invention, has partial complementarity (i.e., less than 100% complementarity) between the sense strand or sense region and the antisense strand or antisense region of the miRNA molecule or between the antisense strand or antisense region of the miRNA and a corresponding target nucleic acid molecule. For example, partial complementarity can include various mismatches or non-base paired nucleotides (e.g., 1, 2, 3, 4, 5 or more mismatches or non-based paired nucleotides, such as nucleotide bulges) within the double stranded nucleic acid molecule, structure which can result in bulges, loops, or overhangs that result between the sense strand or sense region and the antisense strand or antisense region of the miRNA or between the antisense strand or antisense region of the miRNA and a corresponding target nucleic acid molecule.

In one embodiment, siNA molecules of the invention that down regulate or reduce target gene expression are used for treating, preventing or reducing HCV infection, liver failure, hepatocellular carcinoma, or cirrhosis in a subject or organism as described herein or otherwise known in the art.

In one embodiment of the present invention, each sequence of a siNA molecule of the invention is independently about 15 to about 30 nucleotides in length, in specific embodiments about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In another embodiment, the siNA duplexes of the invention independently comprise about 15 to about 30 base pairs (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30). In another embodiment, one or more strands of the siNA molecule of the invention independently comprises about 15 to about 30 nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) that are complementary to a target nucleic acid molecule. In yet another embodiment, siNA molecules of the invention comprising hairpin or circular structures are about 35 to about 55 (e.g., about 35, 40, 45, 50 or 55) nucleotides in length, or about 38 to about 44 (e.g., about 38, 39, 40, 41, 42, 43, or 44) nucleotides in length and comprising about 15 to about 25 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs. Exemplary siNA molecules of the invention are shown in Tables II and III and/or FIGS. 4-5.

As used herein “cell” is used in its usual biological sense, and does not refer to an entire multicellular organism, e.g., specifically does not refer to a human. The cell can be present in an organism, e.g., birds, plants and mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell). The cell can be of somatic or germ line origin, totipotent or pluripotent, dividing or non-dividing. The cell can also be derived from or can comprise a gamete or embryo, a stem cell, or a fully differentiated cell. The cell can be an isolated cell, purified cell, or substantially purified cell as is generally recognized in the art.

The siNA molecules of the invention are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through local delivery to the lung, with or without their incorporation in biopolymers. In particular embodiments, the nucleic acid molecules of the invention comprise sequences shown in Tables II and III and/or FIGS. 4-5. Examples of such nucleic acid molecules consist essentially of sequences defined in these tables and figures. Furthermore, the chemically modified constructs described in Table IV and the lipid nanoparticle (LNP) formulations shown in Table VI can be applied to any siNA sequence or group of siNA sequences of the invention.

In another aspect, the invention provides mammalian cells containing one or more siNA molecules of this invention. The one or more siNA molecules can independently be targeted to the same or different sites within a target polynucleotide of the invention.

By “RNA” is meant a molecule comprising at least one ribonucleotide residue. By “ribonucleotide” is meant a nucleotide with a hydroxyl group at the 2′ position of a β-D-ribofuranose moiety. The terms include double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the instant invention can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.

By “subject” is meant an organism, which is a donor or recipient of explanted cells or the cells themselves. “Subject” also refers to an organism to which the nucleic acid molecules of the invention can be administered. A subject can be a mammal or mammalian cells, including a human or human cells. In one embodiment, the subject is an infant (e.g., subjects that are less than 1 month old, or 1, 2, 3, 4, 5, 6, 7, 8, 9 10, 11, or 12 months old). In one embodiment, the subject is a toddler (e.g., 1, 2, 3, 4, 5 or 6 years old). In one embodiment, the subject is a senior (e.g., anyone over the age of about 65 years of age).

By “chemical modification” as used herein is meant any modification of chemical structure of the nucleotides that differs from nucleotides of native siRNA or RNA. The term “chemical modification” encompasses the addition, substitution, or modification of native siRNA or RNA nucleosides and nucleotides with modified nucleosides and modified nucleotides as described herein or as is otherwise known in the art. Non-limiting examples of such chemical modifications include without limitation compositions having any of Formulae I, II, III, IV, V, VI, or VII herein, phosphorothioate internucleotide linkages, 2′-deoxyribonucleotides, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, 4′-thio ribonucleotides, 2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides (see for example U.S. Ser. No. 10/981,966 filed Nov. 5, 2004, incorporated by reference herein), FANA, “universal base” nucleotides, “acyclic” nucleotides, 5-C-methyl nucleotides, terminal glyceryl and/or inverted deoxy abasic residue incorporation, or a modification having any of Formulae I-VII herein. In one embodiment, the nucleic acid molecules of the invention (e.g, dsRNA, siNA etc.) are partially modified (e.g., about 5%, 10,%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% modified) with chemical modifications. In another embodiment, the nucleic acid molecules of the invention (e.g, dsRNA, siNA etc.) are completely modified (e.g., about 100% modified) with chemical modifications.

The term “phosphorothioate” as used herein refers to an internucleotide linkage having Formula I, wherein Z and/or W comprise a sulfur atom. Hence, the term phosphorothioate refers to both phosphorothioate and phosphorodithioate internucleotide linkages.

The term “phosphonoacetate” as used herein refers to an internucleotide linkage having Formula I, wherein Z and/or W comprise an acetyl or protected acetyl group.

The term “thiophosphonoacetate” as used herein refers to an internucleotide linkage having Formula I, wherein Z comprises an acetyl or protected acetyl group and W comprises a sulfur atom or alternately W comprises an acetyl or protected acetyl group and Z comprises a sulfur atom.

The term “universal base” as used herein refers to nucleotide base analogs that form base pairs with each of the natural DNA/RNA bases with little discrimination between them. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art (see for example Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

The term “acyclic nucleotide” as used herein refers to any nucleotide having an acyclic ribose sugar, for example where any of the ribose carbons (C1, C2, C3, C4, or C5), are independently or in combination absent from the nucleotide.

The nucleic acid molecules of the instant invention, individually, or in combination or in conjunction with other drugs, can be used to for preventing or treating diseases, disorders, conditions, and traits described herein or otherwise known in the art, in a subject or organism.

In one embodiment, the siNA molecules of the invention can be administered to a subject or can be administered to other appropriate cells evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.

In a further embodiment, the siNA molecules can be used in combination with other known treatments to prevent or treat diseases, disorders, or conditions in a subject or organism. For example, the described molecules could be used in combination with one or more known compounds, treatments, or procedures to prevent or treat diseases, disorders, conditions, and traits described herein in a subject or organism as are known in the art.

In one embodiment, the invention features an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the invention, in a manner which allows expression of the siNA molecule. For example, the vector can contain sequence(s) encoding both strands of a siNA molecule comprising a duplex. The vector can also contain sequence(s) encoding a single nucleic acid molecule that is self-complementary and thus forms a siNA molecule. Non-limiting examples of such expression vectors are described in Paul et al., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500; and Novina et al., 2002, Nature Medicine, advance online publication doi:10.1038/nm725.

In another embodiment, the invention features a mammalian cell, for example, a human cell, including an expression vector of the invention.

In yet another embodiment, the expression vector of the invention comprises a sequence for a siNA molecule having complementarity to a RNA molecule referred to by a Genbank Accession numbers, for example Genbank Accession Nos. described herein or in U.S. Provisional Patent Application No. 60/363,124, U.S. Ser. No. 10/923,536 and/or PCT/US03/05028.

In one embodiment, an expression vector of the invention comprises a nucleic acid sequence encoding two or more siNA molecules, which can be the same or different.

In another aspect of the invention, siNA molecules that interact with target RNA molecules and down-regulate gene encoding target RNA molecules (for example target RNA molecules referred to by Genbank Accession numbers herein) are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. The recombinant vectors capable of expressing the siNA molecules can be delivered as described herein, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of siNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the siNA molecules bind and down-regulate gene function or expression via RNA interference (RNAi). Delivery of siNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell.

By “vectors” is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a non-limiting example of a scheme for the synthesis of siNA molecules. The complementary siNA sequence strands, strand 1 and strand 2, are synthesized in tandem and are connected by a cleavable linkage, such as a nucleotide succinate or abasic succinate, which can be the same or different from the cleavable linker used for solid phase synthesis on a solid support. The synthesis can be either solid phase or solution phase, in the example shown, the synthesis is a solid phase synthesis. The synthesis is performed such that a protecting group, such as a dimethoxytrityl group, remains intact on the terminal nucleotide of the tandem oligonucleotide. Upon cleavage and deprotection of the oligonucleotide, the two siNA strands spontaneously hybridize to form a siNA duplex, which allows the purification of the duplex by utilizing the properties of the terminal protecting group, for example by applying a trityl on purification method wherein only duplexes/oligonucleotides with the terminal protecting group are isolated.

FIG. 2 shows a MALDI-TOF mass spectrum of a purified siNA duplex synthesized by a method of the invention. The two peaks shown correspond to the predicted mass of the separate siNA sequence strands. This result demonstrates that the siNA duplex generated from tandem synthesis can be purified as a single entity using a simple trityl-on purification methodology.

FIG. 3 shows a non-limiting proposed mechanistic representation of target RNA degradation involved in RNAi. Double-stranded RNA (dsRNA), which is generated by RNA-dependent RNA polymerase (RdRP) from foreign single-stranded RNA, for example viral, transposon, or other exogenous RNA, activates the DICER enzyme that in turn generates siNA duplexes. Alternately, synthetic or expressed siNA can be introduced directly into a cell by appropriate means. An active siNA complex forms which recognizes a target RNA, resulting in degradation of the target RNA by the RISC endonuclease complex or in the synthesis of additional RNA by RNA-dependent RNA polymerase (RdRP), which can activate DICER and result in additional siNA molecules, thereby amplifying the RNAi response.

FIG. 4A-F shows non-limiting examples of chemically-modified siNA constructs of the present invention. In the figure, N stands for any nucleotide (adenosine, guanosine, cytosine, uridine, or optionally thymidine, for example thymidine can be substituted in the overhanging regions designated by parenthesis (N N). Various modifications are shown for the sense and antisense strands of the siNA constructs. The (N N) nucleotide positions can be chemically modified as described herein (e.g., 2′-O-methyl, 2′-deoxy-2′-fluoro etc.) and can be either derived from a corresponding target nucleic acid sequence or not (see for example FIG. 6C). Furthermore, the sequences shown in FIG. 4 can optionally include a ribonucleotide at the 9^thposition from the 5′-end of the sense strand or the 11^thposition based on the 5′-end of the guide strand by counting 11 nucleotide positions in from the 5′-terminus of the guide strand (see FIG. 6C).

FIG. 4A: The sense strand comprises 21 nucleotides wherein the two terminal 3′-nucleotides are optionally base paired and wherein all nucleotides present are ribonucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all nucleotides present are ribonucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4B: The sense strand comprises 21 nucleotides wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the sense and antisense strand.

FIG. 4C: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-O-methyl or 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4D: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein and wherein and all purine nucleotides that may be present are 2′-deoxy nucleotides. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4E: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4F: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein and wherein and all purine nucleotides that may be present are 2′-deoxy nucleotides. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and having one 3′-terminal phosphorothioate internucleotide linkage and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-deoxy nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the antisense strand. The antisense strand of constructs A-F comprise sequence complementary to any target nucleic acid sequence of the invention. Furthermore, when a glyceryl moiety (L) is present at the 3′-end of the antisense strand for any construct shown in FIG. 4 A-F, the modified internucleotide linkage is optional.

FIG. 5A-F shows non-limiting examples of specific chemically-modified siNA sequences of the invention. A-F applies the chemical modifications described in FIG. 4A-F to an exemplary HCV siNA sequence. Such chemical modifications can be applied to any siNA sequence for any target. Furthermore, the sequences shown in FIG. 5 can optionally include a ribonucleotide at the 9^thposition from the 5′-end of the sense strand or the 11^thposition based on the 5′-end of the guide strand by counting 11 nucleotide positions in from the 5′-terminus of the guide strand (see FIG. 6C). In addition, the sequences shown in FIG. 5 can optionally include terminal ribonucleotides at up to about 4 positions at the 5′-end of the antisense strand (e.g., about 1, 2, 3, or 4 terminal ribonucleotides at the 5′-end of the antisense strand).

FIG. 6A-C shows non-limiting examples of different siNA constructs of the invention.

The examples shown in FIG. 6A (constructs 1, 2, and 3) have 19 representative base pairs; however, different embodiments of the invention include any number of base pairs described herein. Bracketed regions represent nucleotide overhangs, for example, comprising about 1, 2, 3, or 4 nucleotides in length, preferably about 2 nucleotides. Constructs 1 and 2 can be used independently for RNAi activity. Construct 2 can comprise a polynucleotide or non-nucleotide linker, which can optionally be designed as a biodegradable linker. In one embodiment, the loop structure shown in construct 2 can comprise a biodegradable linker that results in the formation of construct 1 in vivo and/or in vitro. In another example, construct 3 can be used to generate construct 2 under the same principle wherein a linker is used to generate the active siNA construct 2 in vivo and/or in vitro, which can optionally utilize another biodegradable linker to generate the active siNA construct 1 in vivo and/or in vitro. As such, the stability and/or activity of the siNA constructs can be modulated based on the design of the siNA construct for use in vivo or in vitro and/or in vitro.

The examples shown in FIG. 6B represent different variations of double stranded nucleic acid molecule of the invention, such as microRNA, that can include overhangs, bulges, loops, and stem-loops resulting from partial complementarity. Such motifs having bulges, loops, and stem-loops are generally characteristics of miRNA. The bulges, loops, and stem-loops can result from any degree of partial complementarity, such as mismatches or bulges of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides in one or both strands of the double stranded nucleic acid molecule of the invention.

The example shown in FIG. 6C represents a model double stranded nucleic acid molecule of the invention comprising a 19 base pair duplex of two 21 nucleotide sequences having dinucleotide 3′-overhangs. The top strand (1) represents the sense strand (passenger strand), the middle strand (2) represents the antisense (guide strand), and the lower strand (3) represents a target polynucleotide sequence. The dinucleotide overhangs (NN) can comprise sequence derived from the target polynucleotide. For example, the 3′-(NN) sequence in the guide strand can be complementary to the 5′-[NN] sequence of the target polynucleotide. In addition, the 5′-(NN) sequence of the passenger strand can comprise the same sequence as the 5′-[NN] sequence of the target polynucleotide sequence. In other embodiments, the overhangs (NN) are not derived from the target polynucleotide sequence, for example where the 3′-(NN) sequence in the guide strand are not complementary to the 5′-[NN] sequence of the target polynucleotide and the 5′-(NN) sequence of the passenger strand can comprise different sequence from the 5′-[NN] sequence of the target polynucleotide sequence. In additional embodiments, any (NN) nucleotides are chemically modified, e.g., as 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or other modifications herein. Furthermore, the passenger strand can comprise a ribonucleotide position N of the passenger strand. For the representative 19 base pair 21 mer duplex shown, position N can be 9 nucleotides in from the 3′ end of the passenger strand. However, in duplexes of differing length, the position N is determined based on the 5′-end of the guide strand by counting 11 nucleotide positions in from the 5′-terminus of the guide strand and picking the corresponding base paired nucleotide in the passenger strand. Cleavage by Ago2 takes place between positions 10 and 11 as indicated by the arrow. In additional embodiments, there are two ribonucleotides, NN, at positions 10 and 11 based on the 5′-end of the guide strand by counting 10 and 11 nucleotide positions in from the 5′-terminus of the guide strand and picking the corresponding base paired nucleotides in the passenger strand.

FIG. 7A-C is a diagrammatic representation of a scheme utilized in generating an expression cassette to generate siNA hairpin constructs.

FIG. 7A: A DNA oligomer is synthesized with a 5′-restriction site (R1) sequence followed by a region having sequence identical (sense region of siNA) to a predetermined target sequence, wherein the sense region comprises, for example, about 19, 20, 21, or 22 nucleotides (N) in length, which is followed by a loop sequence of defined sequence (X), comprising, for example, about 3 to about 10 nucleotides.

FIG. 7B: The synthetic construct is then extended by DNA polymerase to generate a hairpin structure having self-complementary sequence that will result in a siNA transcript having specificity for a target sequence and having self-complementary sense and antisense regions.

FIG. 7C: The construct is heated (for example to about 95° C.) to linearize the sequence, thus allowing extension of a complementary second DNA strand using a primer to the 3′-restriction sequence of the first strand. The double-stranded DNA is then inserted into an appropriate vector for expression in cells. The construct can be designed such that a 3′-terminal nucleotide overhang results from the transcription, for example, by engineering restriction sites and/or utilizing a poly-U termination region as described in Paul et al., 2002, Nature Biotechnology, 29, 505-508.

FIG. 8A-C is a diagrammatic representation of a scheme utilized in generating an expression cassette to generate double-stranded siNA constructs.

FIG. 8A: A DNA oligomer is synthesized with a 5′-restriction (R1) site sequence followed by a region having sequence identical (sense region of siNA) to a predetermined target sequence, wherein the sense region comprises, for example, about 19, 20, 21, or 22 nucleotides (N) in length, and which is followed by a 3′-restriction site (R2) which is adjacent to a loop sequence of defined sequence (X).

FIG. 8B: The synthetic construct is then extended by DNA polymerase to generate a hairpin structure having self-complementary sequence.

FIG. 8C: The construct is processed by restriction enzymes specific to R1 and R2 to generate a double-stranded DNA which is then inserted into an appropriate vector for expression in cells. The transcription cassette is designed such that a U6 promoter region flanks each side of the dsDNA which generates the separate sense and antisense strands of the siNA. Poly T termination sequences can be added to the constructs to generate U overhangs in the resulting transcript.

FIG. 9A-E is a diagrammatic representation of a method used to determine target sites for siNA mediated RNAi within a particular target nucleic acid sequence, such as messenger RNA.

FIG. 9A: A pool of siNA oligonucleotides are synthesized wherein the antisense region of the siNA constructs has complementarity to target sites across the target nucleic acid sequence, and wherein the sense region comprises sequence complementary to the antisense region of the siNA.

FIGS. 9B&C: (FIG. 9B) The sequences are pooled and are inserted into vectors such that (FIG. 9C) transfection of a vector into cells results in the expression of the siNA.

FIG. 9D: Cells are sorted based on phenotypic change that is associated with modulation of the target nucleic acid sequence.

FIG. 9E: The siNA is isolated from the sorted cells and is sequenced to identify efficacious target sites within the target nucleic acid sequence.

FIG. 10 shows non-limiting examples of different stabilization chemistries (1-10) that can be used, for example, to stabilize the 3′-end of siNA sequences of the invention, including (1) [3-3′]-inverted deoxyribose; (2) deoxyribonucleotide; (3) [5′-3′]-3′-deoxyribonucleotide; (4) [5′-3′]-ribonucleotide; (5) [5′-3′]-3′-O-methyl ribonucleotide; (6) 3′-glyceryl; (7) [3′-5′]-3′-deoxyribonucleotide; (8) [3′-3′]-deoxyribonucleotide; (9) [5′-2′]-deoxyribonucleotide; and (10) [5-3′]-dideoxyribonucleotide. In addition to modified and unmodified backbone chemistries indicated in the figure, these chemistries can be combined with different backbone modifications as described herein, for example, backbone modifications having Formula I. In addition, the 2′-deoxy nucleotide shown 5′ to the terminal modifications shown can be another modified or unmodified nucleotide or non-nucleotide described herein, for example modifications having any of Formulae I-VII or any combination thereof.

FIG. 11 shows a non-limiting example of a strategy used to identify chemically modified siNA constructs of the invention that are nuclease resistant while preserving the ability to mediate RNAi activity. Chemical modifications are introduced into the siNA construct based on educated design parameters (e.g. introducing 2′-modifications, base modifications, backbone modifications, terminal cap modifications etc). The modified construct in tested in an appropriate system (e.g. human serum for nuclease resistance, shown, or an animal model for PK/delivery parameters). In parallel, the siNA construct is tested for RNAi activity, for example in a cell culture system such as a luciferase reporter assay). Lead siNA constructs are then identified which possess a particular characteristic while maintaining RNAi activity, and can be further modified and assayed once again. This same approach can be used to identify siNA-conjugate molecules with improved pharmacokinetic profiles, delivery, and RNAi activity.

FIG. 12 shows non-limiting examples of phosphorylated siNA molecules of the invention, including linear and duplex constructs and asymmetric derivatives thereof.

FIG. 13 shows non-limiting examples of chemically modified terminal phosphate groups of the invention.

FIG. 14A shows a non-limiting example of methodology used to design self complementary DFO constructs utilizing palindrome and/or repeat nucleic acid sequences that are identified in a target nucleic acid sequence. (i) A palindrome or repeat sequence is identified in a nucleic acid target sequence. (ii) A sequence is designed that is complementary to the target nucleic acid sequence and the palindrome sequence. (iii) An inverse repeat sequence of the non-palindrome/repeat portion of the complementary sequence is appended to the 3′-end of the complementary sequence to generate a self complementary DFO molecule comprising sequence complementary to the nucleic acid target. (iv) The DFO molecule can self-assemble to form a double stranded oligonucleotide. FIG. 14B shows a non-limiting representative example of a duplex forming oligonucleotide sequence. FIG. 14C shows a non-limiting example of the self assembly schematic of a representative duplex forming oligonucleotide sequence. FIG. 14D shows a non-limiting example of the self assembly schematic of a representative duplex forming oligonucleotide sequence followed by interaction with a target nucleic acid sequence resulting in modulation of gene expression.

FIG. 15 shows a non-limiting example of the design of self complementary DFO constructs utilizing palindrome and/or repeat nucleic acid sequences that are incorporated into the DFO constructs that have sequence complementary to any target nucleic acid sequence of interest. Incorporation of these palindrome/repeat sequences allow the design of DFO constructs that form duplexes in which each strand is capable of mediating modulation of target gene expression, for example by RNAi. First, the target sequence is identified. A complementary sequence is then generated in which nucleotide or non-nucleotide modifications (shown as X or Y) are introduced into the complementary sequence that generate an artificial palindrome (shown as XYXYXY in the Figure). An inverse repeat of the non-palindrome/repeat complementary sequence is appended to the 3′-end of the complementary sequence to generate a self complementary DFO comprising sequence complementary to the nucleic acid target. The DFO can self-assemble to form a double stranded oligonucleotide.

FIG. 16 shows non-limiting examples of multifunctional siNA molecules of the invention comprising two separate polynucleotide sequences that are each capable of mediating RNAi directed cleavage of differing target nucleic acid sequences. FIG. 16A shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first and second complementary regions are situated at the 3′-ends of each polynucleotide sequence in the multifunctional siNA. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. FIG. 16B shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first and second complementary regions are situated at the 5′-ends of each polynucleotide sequence in the multifunctional siNA. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences.

FIG. 17 shows non-limiting examples of multifunctional siNA molecules of the invention comprising a single polynucleotide sequence comprising distinct regions that are each capable of mediating RNAi directed cleavage of differing target nucleic acid sequences. FIG. 17A shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the second complementary region is situated at the 3′-end of the polynucleotide sequence in the multifunctional siNA. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. FIG. 17B shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first complementary region is situated at the 5′-end of the polynucleotide sequence in the multifunctional siNA. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. In one embodiment, these multifunctional siNA constructs are processed in vivo or in vitro to generate multifunctional siNA constructs as shown in FIG. 16.

FIG. 18 shows non-limiting examples of multifunctional siNA molecules of the invention comprising two separate polynucleotide sequences that are each capable of mediating RNAi directed cleavage of differing target nucleic acid sequences and wherein the multifunctional siNA construct further comprises a self complementary, palindrome, or repeat region, thus enabling shorter bifunctional siNA constructs that can mediate RNA interference against differing target nucleic acid sequences. FIG. 18A shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first and second complementary regions are situated at the 3′-ends of each polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. FIG. 18B shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first and second complementary regions are situated at the 5′-ends of each polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences.

FIG. 19 shows non-limiting examples of multifunctional siNA molecules of the invention comprising a single polynucleotide sequence comprising distinct regions that are each capable of mediating RNAi directed cleavage of differing target nucleic acid sequences and wherein the multifunctional siNA construct further comprises a self complementary, palindrome, or repeat region, thus enabling shorter bifunctional siNA constructs that can mediate RNA interference against differing target nucleic acid sequences. FIG. 19A shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the second complementary region is situated at the 3′-end of the polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. FIG. 19B shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first complementary region is situated at the 5′-end of the polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. In one embodiment, these multifunctional siNA constructs are processed in vivo or in vitro to generate multifunctional siNA constructs as shown in FIG. 18.

FIG. 20 shows a non-limiting example of how multifunctional siNA molecules of the invention can target two separate target nucleic acid molecules, such as separate RNA molecules encoding differing proteins (e.g., any of targets herein), for example, a cytokine and its corresponding receptor, differing viral strains, a virus and a cellular protein involved in viral infection or replication, or differing proteins involved in a common or divergent biologic pathway that is implicated in the maintenance of progression of disease. Each strand of the multifunctional siNA construct comprises a region having complementarity to separate target nucleic acid molecules. The multifunctional siNA molecule is designed such that each strand of the siNA can be utilized by the RISC complex to initiate RNA interference mediated cleavage of its corresponding target. These design parameters can include destabilization of each end of the siNA construct (see for example Schwarz et al., 2003, Cell, 115, 199-208). Such destabilization can be accomplished for example by using guanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), or destabilizing chemically modified nucleotides at terminal nucleotide positions as is known in the art.

FIG. 21 shows a non-limiting example of how multifunctional siNA molecules of the invention can target two separate target nucleic acid sequences within the same target nucleic acid molecule, such as alternate coding regions of a RNA, coding and non-coding regions of a RNA, or alternate splice variant regions of a RNA. Each strand of the multifunctional siNA construct comprises a region having complementarity to the separate regions of the target nucleic acid molecule. The multifunctional siNA molecule is designed such that each strand of the siNA can be utilized by the RISC complex to initiate RNA interference mediated cleavage of its corresponding target region. These design parameters can include destabilization of each end of the siNA construct (see for example Schwarz et al., 2003, Cell, 115, 199-208). Such destabilization can be accomplished for example by using guanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), or destabilizing chemically modified nucleotides at terminal nucleotide positions as is known in the art.

FIG. 22(A-H) shows non-limiting examples of tethered multifunctional siNA constructs of the invention. In the examples shown, a linker (e.g., nucleotide or non-nucleotide linker) connects two siNA regions (e.g., two sense, two antisense, or alternately a sense and an antisense region together. Separate sense (or sense and antisense) sequences corresponding to a first target sequence and second target sequence are hybridized to their corresponding sense and/or antisense sequences in the multifunctional siNA. In addition, various conjugates, ligands, aptamers, polymers or reporter molecules can be attached to the linker region for selective or improved delivery and/or pharmacokinetic properties.

FIG. 23 shows a non-limiting example of various dendrimer based multifunctional siNA designs.

FIG. 24 shows a non-limiting example of various supramolecular multifunctional siNA designs.

FIG. 25 shows a non-limiting example of a dicer enabled multifunctional siNA design using a 30 nucleotide precursor siNA construct. A 30 base pair duplex is cleaved by Dicer into 22 and 8 base pair products from either end (8 b.p. fragments not shown). For ease of presentation the overhangs generated by dicer are not shown—but can be compensated for. Three targeting sequences are shown. The required sequence identity overlapped is indicated by grey boxes. The N's of the parent 30 b.p. siNA are suggested sites of 2′-OH positions to enable Dicer cleavage if this is tested in stabilized chemistries. Note that processing of a 30mer duplex by Dicer RNase III does not give a precise 22+8 cleavage, but rather produces a series of closely related products (with 22+8 being the primary site). Therefore, processing by Dicer will yield a series of active siNAs.

FIG. 26 shows a non-limiting example of a dicer enabled multifunctional siNA design using a 40 nucleotide precursor siNA construct. A 40 base pair duplex is cleaved by Dicer into 20 base pair products from either end. For ease of presentation the overhangs generated by dicer are not shown—but can be compensated for. Four targeting sequences are shown. The target sequences having homology are enclosed by boxes. This design format can be extended to larger RNAs. If chemically stabilized siNAs are bound by Dicer, then strategically located ribonucleotide linkages can enable designer cleavage products that permit our more extensive repertoire of multiifunctional designs. For example cleavage products not limited to the Dicer standard of approximately 22-nucleotides can allow multifunctional siNA constructs with a target sequence identity overlap ranging from, for example, about 3 to about 15 nucleotides.

FIG. 27 shows a non-limiting example of additional multifunctional siNA construct designs of the invention. In one example, a conjugate, ligand, aptamer, label, or other moiety is attached to a region of the multifunctional siNA to enable improved delivery or pharmacokinetic profiling.

FIG. 28 shows a non-limiting example of additional multifunctional siNA construct designs of the invention. In one example, a conjugate, ligand, aptamer, label, or other moiety is attached to a region of the multifunctional siNA to enable improved delivery or pharmacokinetic profiling.

FIG. 29 shows a non-limiting example of a cholesterol linked phosphoramidite that can be used to synthesize cholesterol conjugated siNA molecules of the invention. An example is shown with the cholesterol moiety linked to the 5′-end of the sense strand of a siNA molecule.

FIG. 30 shows a non-limiting example of a double stranded nucleic acid molecule cocktail formulation targeting GBV-B in a marmoset model of HCV infection. GBV-B provides a small animal model for testing antiviral compounds and vaccines for HCV infection. Two animals were inoculated with GBV-B and IV treatment with the active formulated siNA (Sirna Compound Nos. 33149/47677 and 31703/38756, Formulation LNP-086; see Tables III and VI) at 3 mg/kg was initiated one day post infection. Another 2 animals were inoculated with GBV-B and were untreated to serve as negative controls. The animals were monitored to determine the effect of the therapy of GBV-B infection. Blood draws were performed over the course of the study to determine viral titers. Dosing of formulated siNA in the treated animals was repeated at days 1, 3, and 7 after inoculation at day 0. As shown in the figure, these animals show a profound inhibition of GBV-B over a three week time course compared to the untreated control animals.

FIG. 31 shows a non-limiting example of inhibition of GBV infection in an animal with established GBV infection that was treated with active formulated siNA (Sirna Compound Nos. 33149/47677 and 31703/38756, Formulation LNP-086; see Tables III and VI) at days 28, 31, and 35 post infection. This animal showed a decrease in viral titer down to the limit of detection following the dosing of active compound compared to historic untreated controls.

DETAILED DESCRIPTION OF THE INVENTION
Mechanism of Action of Nucleic Acid Molecules of the Invention

The discussion that follows discusses the proposed mechanism of RNA interference mediated by short interfering RNA as is presently known, and is not meant to be limiting and is not an admission of prior art. Applicant demonstrates herein that chemically-modified short interfering nucleic acids possess similar or improved capacity to mediate RNAi as do siRNA molecules and are expected to possess improved stability and activity in vivo; therefore, this discussion is not meant to be limiting only to siRNA and can be applied to siNA as a whole. By “improved capacity to mediate RNAi” or “improved RNAi activity” is meant to include RNAi activity measured in vitro and/or in vivo where the RNAi activity is a reflection of both the ability of the siNA to mediate RNAi and the stability of the siNAs of the invention. In this invention, the product of these activities can be increased in vitro and/or in vivo compared to an all RNA siRNA or a siNA containing a plurality of ribonucleotides. In some cases, the activity or stability of the siNA molecule can be decreased (i.e., less than ten-fold), but the overall activity of the siNA molecule is enhanced in vitro and/or in vivo.

RNA interference refers to the process of sequence specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Fire et al., 1998, Nature, 391, 806). The corresponding process in plants is commonly referred to as post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes which is commonly shared by diverse flora and phyla (Fire et al., 1999, Trends genet., 15, 358). Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA or viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response though a mechanism that has yet to be fully characterized. This mechanism appears to be different from the interferon response that results from dsRNA-mediated activation of protein kinase PKR and 2′,5′-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as Dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Berstein et al., 2001, Nature, 409, 363). Short interfering RNAs derived from Dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes. Dicer has also been implicated in the excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001, Science, 293, 834). The RNAi response also features an endonuclease complex containing a siRNA, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence homologous to the siRNA. Cleavage of the target RNA takes place in the middle of the region complementary to the guide sequence of the siRNA duplex (Elbashir et al., 2001, genes Dev., 15, 188). In addition, RNA interference can also involve small RNA (e.g., micro-RNA or miRNA) mediated gene silencing, presumably though cellular mechanisms that regulate chromatin structure and thereby prevent transcription of target gene sequences (see for example Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237). As such, siNA molecules of the invention can be used to mediate gene silencing via interaction with RNA transcripts or alternately by interaction with particular gene sequences, wherein such interaction results in gene silencing either at the transcriptional level or post-transcriptional level.

RNAi has been studied in a variety of systems. Fire et al., 1998, Nature, 391, 806, were the first to observe RNAI in C. elegans. Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated by dsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001, Nature, 411, 494, describe RNAi induced by introduction of duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells. Recent work in Drosophila embryonic lysates has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies have shown that 21 nucleotide siRNA duplexes are most active when containing two 2-nucleotide 3′-terminal nucleotide overhangs. Furthermore, substitution of one or both siRNA strands with 2′-deoxy or 2′-O-methyl nucleotides abolishes RNAi activity, whereas substitution of 3′-terminal siRNA nucleotides with deoxy nucleotides was shown to be tolerated. Mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity. In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5′-end of the siRNA guide sequence rather than the 3′-end (Elbashir et al., 2001, EMBO J., 20, 6877). Other studies have indicated that a 5′-phosphate on the target-complementary strand of a siRNA duplex is required for siRNA activity and that ATP is utilized to maintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309); however, siRNA molecules lacking a 5′-phosphate are active when introduced exogenously, suggesting that 5′-phosphorylation of siRNA constructs may occur in vivo.

Duplex Forming Oligonucleotides (DFO) of the Invention

In one embodiment, the invention features siNA molecules comprising duplex forming oligonucleotides (DFO) that can self-assemble into double stranded oligonucleotides. The duplex forming oligonucleotides of the invention can be chemically synthesized or expressed from transcription units and/or vectors. The DFO molecules of the instant invention provide useful reagents and methods for a variety of therapeutic, diagnostic, agricultural, veterinary, target validation, genomic discovery, genetic engineering and pharmacogenomic applications.

Applicant demonstrates herein that certain oligonucleotides, referred to herein for convenience but not limitation as duplex forming oligonucleotides or DFO molecules, are potent mediators of sequence specific regulation of gene expression. The oligonucleotides of the invention are distinct from other nucleic acid sequences known in the art (e.g., siRNA, miRNA, stRNA, shRNA, antisense oligonucleotides etc.) in that they represent a class of linear polynucleotide sequences that are designed to self-assemble into double stranded oligonucleotides, where each strand in the double stranded oligonucleotides comprises a nucleotide sequence that is complementary to a target nucleic acid molecule. Nucleic acid molecules of the invention can thus self assemble into functional duplexes in which each strand of the duplex comprises the same polynucleotide sequence and each strand comprises a nucleotide sequence that is complementary to a target nucleic acid molecule.

Generally, double stranded oligonucleotides are formed by the assembly of two distinct oligonucleotide sequences where the oligonucleotide sequence of one strand is complementary to the oligonucleotide sequence of the second strand; such double stranded oligonucleotides are assembled from two separate oligonucleotides, or from a single molecule that folds on itself to form a double stranded structure, often referred to in the field as hairpin stem-loop structure (e.g., shRNA or short hairpin RNA). These double stranded oligonucleotides known in the art all have a common feature in that each strand of the duplex has a distinct nucleotide sequence.

Distinct from the double stranded nucleic acid molecules known in the art, the applicants have developed a novel, potentially cost effective and simplified method of forming a double stranded nucleic acid molecule starting from a single stranded or linear oligonucleotide. The two strands of the double stranded oligonucleotide formed according to the instant invention have the same nucleotide sequence and are not covalently linked to each other. Such double-stranded oligonucleotides molecules can be readily linked post-synthetically by methods and reagents known in the art and are within the scope of the invention. In one embodiment, the single stranded oligonucleotide of the invention (the duplex forming oligonucleotide) that forms a double stranded oligonucleotide comprises a first region and a second region, where the second region includes a nucleotide sequence that is an inverted repeat of the nucleotide sequence in the first region, or a portion thereof, such that the single stranded oligonucleotide self assembles to form a duplex oligonucleotide in which the nucleotide sequence of one strand of the duplex is the same as the nucleotide sequence of the second strand. Non-limiting examples of such duplex forming oligonucleotides are illustrated in FIGS. 14 and 15. These duplex forming oligonucleotides (DFOs) can optionally include certain palindrome or repeat sequences where such palindrome or repeat sequences are present in between the first region and the second region of the DFO.

In one embodiment, the invention features a duplex forming oligonucleotide (DFO) molecule, wherein the DFO comprises a duplex forming self complementary nucleic acid sequence that has nucleotide sequence complementary to a target nucleic acid sequence. The DFO molecule can comprise a single self complementary sequence or a duplex resulting from assembly of such self complementary sequences.

In one embodiment, a duplex forming oligonucleotide (DFO) of the invention comprises a first region and a second region, wherein the second region comprises a nucleotide sequence comprising an inverted repeat of nucleotide sequence of the first region such that the DFO molecule can assemble into a double stranded oligonucleotide. Such double stranded oligonucleotides can act as a short interfering nucleic acid (siNA) to modulate gene expression. Each strand of the double stranded oligonucleotide duplex formed by DFO molecules of the invention can comprise a nucleotide sequence region that is complementary to the same nucleotide sequence in a target nucleic acid molecule (e.g., target RNA).

In one embodiment, the invention features a single stranded DFO that can assemble into a double stranded oligonucleotide. The applicant has surprisingly found that a single stranded oligonucleotide with nucleotide regions of self complementarity can readily assemble into duplex oligonucleotide constructs. Such DFOs can assemble into duplexes that can inhibit gene expression in a sequence specific manner. The DFO molecules of the invention comprise a first region with nucleotide sequence that is complementary to the nucleotide sequence of a second region and where the sequence of the first region is complementary to a target nucleic acid (e.g., RNA). The DFO can form a double stranded oligonucleotide wherein a portion of each strand of the double stranded oligonucleotide comprises a sequence complementary to a target nucleic acid sequence.

In one embodiment, the invention features a double stranded oligonucleotide, wherein the two strands of the double stranded oligonucleotide are not covalently linked to each other, and wherein each strand of the double stranded oligonucleotide comprises a nucleotide sequence that is complementary to the same nucleotide sequence in a target nucleic acid molecule or a portion thereof (e.g., target RNA target). In another embodiment, the two strands of the double stranded oligonucleotide share an identical nucleotide sequence of at least about 15, preferably at least about 16, 17, 18, 19, 20, or 21 nucleotides.

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

5′-p-XZX′-3′

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

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

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

5′-p-XZX′-3′

3′-X′ZX-p-5′

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

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

5′-p-XX′-3′

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

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

5′-p-XX′-3′

3′-X′X-p-5′

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

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

5′-p-Z-3′

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

In one embodiment, a DFO molecule having any of Formula DFO-I, DFO-I(a), DFO-I(b), DFO-II(a) or DFO-II can comprise chemical modifications as described herein without limitation, such as, for example, nucleotides having any of Formulae I-VII, stabilization chemistries as described in Table IV, or any other combination of modified nucleotides and non-nucleotides as described in the various embodiments herein.

In one embodiment, the palidrome or repeat sequence or modified nucleotide (e.g., nucleotide with a modified base, such as 2-amino purine or a universal base) in Z of DFO constructs having Formula DFO-I, DFO-I(a) and DFO-I(b), comprises chemically modified nucleotides that are able to interact with a portion of the target nucleic acid sequence (e.g., modified base analogs that can form Watson Crick base pairs or non-Watson Crick base pairs).

In one embodiment, a DFO molecule of the invention, for example a DFO having Formula DFO-I or DFO-II, comprises about 15 to about 40 nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides). In one embodiment, a DFO molecule of the invention comprises one or more chemical modifications. In a non-limiting example, the introduction of chemically modified nucleotides and/or non-nucleotides into nucleic acid molecules of the invention provides a powerful tool in overcoming potential limitations of in vivo stability and bioavailability inherent to unmodified RNA molecules that are delivered exogenously. For example, the use of chemically modified nucleic acid molecules can enable a lower dose of a particular nucleic acid molecule for a given therapeutic effect since chemically modified nucleic acid molecules tend to have a longer half-life in serum or in cells or tissues. Furthermore, certain chemical modifications can improve the bioavailability and/or potency of nucleic acid molecules by not only enhancing half-life but also facilitating the targeting of nucleic acid molecules to particular organs, cells or tissues and/or improving cellular uptake of the nucleic acid molecules. Therefore, even if the activity of a chemically modified nucleic acid molecule is reduced in vitro as compared to a native/unmodified nucleic acid molecule, for example when compared to an unmodified RNA molecule, the overall activity of the modified nucleic acid molecule can be greater than the native or unmodified nucleic acid molecule due to improved stability, potency, duration of effect, bioavailability and/or delivery of the molecule.

Multifunctional or Multi-Targeted siNA Molecules of the Invention

In one embodiment, the invention features siNA molecules comprising multifunctional short interfering nucleic acid (multifunctional siNA) molecules that modulate the expression of one or more target genes in a biologic system, such as a cell, tissue, or organism. The multifunctional short interfering nucleic acid (multifunctional siNA) molecules of the invention can target more than one region of the target nucleic acid sequence or can target sequences of more than one distinct target nucleic acid molecules (e.g., target and/or pathway target RNA and/or DNA sequences). The multifunctional siNA molecules of the invention can be chemically synthesized or expressed from transcription units and/or vectors. The multifunctional siNA molecules of the instant invention provide useful reagents and methods for a variety of human applications, therapeutic, diagnostic, agricultural, veterinary, target validation, genomic discovery, genetic engineering and pharmacogenomic applications.

Applicant demonstrates herein that certain oligonucleotides, referred to herein for convenience but not limitation as multifunctional short interfering nucleic acid or multifunctional siNA molecules, are potent mediators of sequence specific regulation of gene expression. The multifunctional siNA molecules of the invention are distinct from other nucleic acid sequences known in the art (e.g., siRNA, miRNA, stRNA, shRNA, antisense oligonucleotides, etc.) in that they represent a class of polynucleotide molecules that are designed such that each strand in the multifunctional siNA construct comprises a nucleotide sequence that is complementary to a distinct nucleic acid sequence in one or more target nucleic acid molecules. A single multifunctional siNA molecule (generally a double-stranded molecule) of the invention can thus target more than one (e.g., 2, 3, 4, 5, or more) differing target nucleic acid target molecules. Nucleic acid molecules of the invention can also target more than one (e.g., 2, 3, 4, 5, or more) region of the same target nucleic acid sequence. As such multifunctional siNA molecules of the invention are useful in down regulating or inhibiting the expression of one or more target nucleic acid molecules. For example, a multifunctional siNA molecule of the invention can target nucleic acid molecules encoding a virus or viral proteins and corresponding cellular proteins required for viral infection and/or replication, or differing strains of a particular virus (e.g., HCV). By reducing or inhibiting expression of more than one target nucleic acid molecule with one multifunctional siNA construct, multifunctional siNA molecules of the invention represent a class of potent therapeutic agents that can provide simultaneous inhibition of multiple targets within a disease or pathogen related pathway. Such simultaneous inhibition can provide synergistic therapeutic treatment strategies without the need for separate preclinical and clinical development efforts or complex regulatory approval process.

Use of multifunctional siNA molecules that target more then one region of a target nucleic acid molecule (e.g., target RNA or DNA) is expected to provide potent inhibition of gene expression. For example, a single multifunctional siNA construct of the invention can target both conserved and variable regions of a target nucleic acid molecule (e.g., target RNA or DNA), thereby allowing down regulation or inhibition of, for example, different target isoforms or variants to optimize therapeutic efficacy and minimize toxicity, or allowing for targeting of both coding and non-coding regions of the target nucleic acid molecule.

Generally, double stranded oligonucleotides are formed by the assembly of two distinct oligonucleotides where the oligonucleotide sequence of one strand is complementary to the oligonucleotide sequence of the second strand; such double stranded oligonucleotides are generally assembled from two separate oligonucleotides (e.g., siRNA). Alternately, a duplex can be formed from a single molecule that folds on itself (e.g., shRNA or short hairpin RNA). These double stranded oligonucleotides are known in the art to mediate RNA interference and all have a common feature wherein only one nucleotide sequence region (guide sequence or the antisense sequence) has complementarity to a target nucleic acid sequence, and the other strand (sense sequence) comprises nucleotide sequence that is homologous to the target nucleic acid sequence. generally, the antisense sequence is retained in the active RISC complex and guides the RISC to the target nucleotide sequence by means of complementary base-pairing of the antisense sequence with the target sequence for mediating sequence-specific RNA interference. It is known in the art that in some cell culture systems, certain types of unmodified siRNAs can exhibit “off target” effects. It is hypothesized that this off-target effect involves the participation of the sense sequence instead of the antisense sequence of the siRNA in the RISC complex (see for example Schwarz et al., 2003, Cell, 115, 199-208). In this instance the sense sequence is believed to direct the RISC complex to a sequence (off-target sequence) that is distinct from the intended target sequence, resulting in the inhibition of the off-target sequence. In these double stranded nucleic acid molecules, each strand is complementary to a distinct target nucleic acid sequence. However, the off-targets that are affected by these dsRNAs are not entirely predictable and are non-specific.

Distinct from the double stranded nucleic acid molecules known in the art, the applicants have developed a novel, potentially cost effective and simplified method of down regulating or inhibiting the expression of more than one target nucleic acid sequence using a single multifunctional siNA construct. The multifunctional siNA molecules of the invention are designed to be double-stranded or partially double stranded, such that a portion of each strand or region of the multifunctional siNA is complementary to a target nucleic acid sequence of choice. As such, the multifunctional siNA molecules of the invention are not limited to targeting sequences that are complementary to each other, but rather to any two differing target nucleic acid sequences. Multifunctional siNA molecules of the invention are designed such that each strand or region of the multifunctional siNA molecule, that is complementary to a given target nucleic acid sequence, is of suitable length (e.g., from about 16 to about 28 nucleotides in length, preferably from about 18 to about 28 nucleotides in length) for mediating RNA interference against the target nucleic acid sequence. The complementarity between the target nucleic acid sequence and a strand or region of the multifunctional siNA must be sufficient (at least about 8 base pairs) for cleavage of the target nucleic acid sequence by RNA interference. Multifunctional siNA of the invention is expected to minimize off-target effects seen with certain siRNA sequences, such as those described in Schwarz et al., supra.

It has been reported that dsRNAs of length between 29 base pairs and 36 base pairs (Tuschl et al., International PCT Publication No. WO 02/44321) do not mediate RNAi. One reason these dsRNAs are inactive may be the lack of turnover or dissociation of the strand that interacts with the target RNA sequence, such that the RISC complex is not able to efficiently interact with multiple copies of the target RNA resulting in a significant decrease in the potency and efficiency of the RNAi process. Applicant has surprisingly found that the multifunctional siNAs of the invention can overcome this hurdle and are capable of enhancing the efficiency and potency of RNAi process. As such, in certain embodiments of the invention, multifunctional siNAs of length of about 29 to about 36 base pairs can be designed such that, a portion of each strand of the multifunctional siNA molecule comprises a nucleotide sequence region that is complementary to a target nucleic acid of length sufficient to mediate RNAi efficiently (e.g., about 15 to about 23 base pairs) and a nucleotide sequence region that is not complementary to the target nucleic acid. By having both complementary and non-complementary portions in each strand of the multifunctional siNA, the multifunctional siNA can mediate RNA interference against a target nucleic acid sequence without being prohibitive to turnover or dissociation (e.g., where the length of each strand is too long to mediate RNAi against the respective target nucleic acid sequence). Furthermore, design of multifunctional siNA molecules of the invention with internal overlapping regions allows the multifunctional siNA molecules to be of favorable (decreased) size for mediating RNA interference and of size that is well suited for use as a therapeutic agent (e.g., wherein each strand is independently from about 18 to about 28 nucleotides in length). Non-limiting examples are illustrated in FIGS. 16-28.

In one embodiment, a multifunctional siNA molecule of the invention comprises a first region and a second region, where the first region of the multifunctional siNA comprises a nucleotide sequence complementary to a nucleic acid sequence of a first target nucleic acid molecule, and the second region of the multifunctional siNA comprises nucleic acid sequence complementary to a nucleic acid sequence of a second target nucleic acid molecule. In one embodiment, a multifunctional siNA molecule of the invention comprises a first region and a second region, where the first region of the multifunctional siNA comprises nucleotide sequence complementary to a nucleic acid sequence of the first region of a target nucleic acid molecule, and the second region of the multifunctional siNA comprises nucleotide sequence complementary to a nucleic acid sequence of a second region of a the target nucleic acid molecule. In another embodiment, the first region and second region of the multifunctional siNA can comprise separate nucleic acid sequences that share some degree of complementarity (e.g., from about 1 to about 10 complementary nucleotides). In certain embodiments, multifunctional siNA constructs comprising separate nucleic acid sequences can be readily linked post-synthetically by methods and reagents known in the art and such linked constructs are within the scope of the invention. Alternately, the first region and second region of the multifunctional siNA can comprise a single nucleic acid sequence having some degree of self complementarity, such as in a hairpin or stem-loop structure. Non-limiting examples of such double stranded and hairpin multifunctional short interfering nucleic acids are illustrated in FIGS. 16 and 17 respectively. These multifunctional short interfering nucleic acids (multifunctional siNAs) can optionally include certain overlapping nucleotide sequence where such overlapping nucleotide sequence is present in between the first region and the second region of the multifunctional siNA (see for example FIGS. 18 and 19).

In one embodiment, the invention features a multifunctional short interfering nucleic acid (multifunctional siNA) molecule, wherein each strand of the multifunctional siNA independently comprises a first region of nucleic acid sequence that is complementary to a distinct target nucleic acid sequence and the second region of nucleotide sequence that is not complementary to the target sequence. The target nucleic acid sequence of each strand is in the same target nucleic acid molecule or different target nucleic acid molecules.

In another embodiment, the multifunctional siNA comprises two strands, where: (a) the first strand comprises a region having sequence complementarity to a target nucleic acid sequence (complementary region 1) and a region having no sequence complementarity to the target nucleotide sequence (non-complementary region 1); (b) the second strand of the multifunction siNA comprises a region having sequence complementarity to a target nucleic acid sequence that is distinct from the target nucleotide sequence complementary to the first strand nucleotide sequence (complementary region 2), and a region having no sequence complementarity to the target nucleotide sequence of complementary region 2 (non-complementary region 2); (c) the complementary region 1 of the first strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the non-complementary region 2 of the second strand and the complementary region 2 of the second strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the non-complementary region 1 of the first strand. The target nucleic acid sequence of complementary region 1 and complementary region 2 is in the same target nucleic acid molecule or different target nucleic acid molecules.

In another embodiment, the multifunctional siNA comprises two strands, where: (a) the first strand comprises a region having sequence complementarity to a target nucleic acid sequence derived from a gene (e.g., a first gene) (complementary region 1) and a region having no sequence complementarity to the target nucleotide sequence of complementary region 1 (non-complementary region 1); (b) the second strand of the multifunction siNA comprises a region having sequence complementarity to a target nucleic acid sequence derived from a gene (e.g., a second gene) that is distinct from the gene of complementary region 1 (complementary region 2), and a region having no sequence complementarity to the target nucleotide sequence of complementary region 2 (non-complementary region 2); (c) the complementary region 1 of the first strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the non-complementary region 2 of the second strand and the complementary region 2 of the second strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the non-complementary region 1 of the first strand.

In another embodiment, the multifunctional siNA comprises two strands, where: (a) the first strand comprises a region having sequence complementarity to a target nucleic acid sequence derived from a gene (e.g., gene) (complementary region 1) and a region having no sequence complementarity to the target nucleotide sequence of complementary region 1 (non-complementary region 1); (b) the second strand of the multifunction siNA comprises a region having sequence complementarity to a target nucleic acid sequence distinct from the target nucleic acid sequence of complementary region 1 (complementary region 2), provided, however, that the target nucleic acid sequence for complementary region 1 and target nucleic acid sequence for complementary region 2 are both derived from the same gene, and a region having no sequence complementarity to the target nucleotide sequence of complementary region 2 (non-complementary region 2); (c) the complementary region 1 of the first strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the non-complementary region 2 of the second strand and the complementary region 2 of the second strand comprises a nucleotide sequence that is complementary to nucleotide sequence in the non-complementary region 1 of the first strand.

In one embodiment, the invention features a multifunctional short interfering nucleic acid (multifunctional siNA) molecule, wherein the multifunctional siNA comprises two complementary nucleic acid sequences in which the first sequence comprises a first region having nucleotide sequence complementary to nucleotide sequence within a first target nucleic acid molecule, and in which the second sequence comprises a first region having nucleotide sequence complementary to a distinct nucleotide sequence within the same target nucleic acid molecule. Preferably, the first region of the first sequence is also complementary to the nucleotide sequence of the second region of the second sequence, and where the first region of the second sequence is complementary to the nucleotide sequence of the second region of the first sequence.

In one embodiment, the invention features a multifunctional short interfering nucleic acid (multifunctional siNA) molecule, wherein the multifunctional siNA comprises two complementary nucleic acid sequences in which the first sequence comprises a first region having a nucleotide sequence complementary to a nucleotide sequence within a first target nucleic acid molecule, and in which the second sequence comprises a first region having a nucleotide sequence complementary to a distinct nucleotide sequence within a second target nucleic acid molecule. Preferably, the first region of the first sequence is also complementary to the nucleotide sequence of the second region of the second sequence, and where the first region of the second sequence is complementary to the nucleotide sequence of the second region of the first sequence.

In one embodiment, the invention features a multifunctional siNA molecule comprising a first region and a second region, where the first region comprises a nucleic acid sequence having about 18 to about 28 nucleotides complementary to a nucleic acid sequence within a first target nucleic acid molecule, and the second region comprises nucleotide sequence having about 18 to about 28 nucleotides complementary to a distinct nucleic acid sequence within a second target nucleic acid molecule.

In one embodiment, the invention features a multifunctional siNA molecule comprising a first region and a second region, where the first region comprises nucleic acid sequence having about 18 to about 28 nucleotides complementary to a nucleic acid sequence within a target nucleic acid molecule, and the second region comprises nucleotide sequence having about 18 to about 28 nucleotides complementary to a distinct nucleic acid sequence within the same target nucleic acid molecule.

In one embodiment, the invention features a double stranded multifunctional short interfering nucleic acid (multifunctional siNA) molecule, wherein one strand of the multifunctional siNA comprises a first region having nucleotide sequence complementary to a first target nucleic acid sequence, and the second strand comprises a first region having a nucleotide sequence complementary to a second target nucleic acid sequence. The first and second target nucleic acid sequences can be present in separate target nucleic acid molecules or can be different regions within the same target nucleic acid molecule. As such, multifunctional siNA molecules of the invention can be used to target the expression of different genes, splice variants of the same gene, both mutant and conserved regions of one or more gene transcripts, or both coding and non-coding sequences of the same or differing genes or gene transcripts.

In one embodiment, a target nucleic acid molecule of the invention encodes a single protein. In another embodiment, a target nucleic acid molecule encodes more than one protein (e.g., 1, 2, 3, 4, 5 or more proteins). As such, a multifunctional siNA construct of the invention can be used to down regulate or inhibit the expression of several proteins. For example, a multifunctional siNA molecule comprising a region in one strand having nucleotide sequence complementarity to a first target nucleic acid sequence derived from a viral genome (e.g., HCV) and the second strand comprising a region with nucleotide sequence complementarity to a second target nucleic acid sequence present in target nucleic acid molecules derived from genes encoding two proteins (e.g., two differing host proteins involved in the HCV life-cycle) can be used to down regulate, inhibit, or shut down a particular biologic pathway by targeting, for example, a viral RNA (e.g., HCV RNA) and one or more host RNAs that are involved in viral infection or the viral life-cycle (e.g., La antigen or interferon regulatory factors).

In one embodiment the invention takes advantage of conserved nucleotide sequences present in different gene variants. By designing multifunctional siNAs in a manner where one strand includes a sequence that is complementary to one or more target nucleic acid sequences that are conserved among various target gene family members and the other strand optionally includes sequence that is complementary to pathway target nucleic acid sequence, it is possible to selectively and effectively inhibit a target gene disease related biological pathway using a single multifunctional siNA.

In another nonlimiting example, a multifunctional siNA molecule comprising a region in one strand having a nucleotide sequence complementarity to a first target nucleic acid sequence (e.g., conserved sequence) present in a target nucleic acid molecule such as a viral genome (e.g., HCV RNA) and the second strand comprising a region having a nucleotide sequence complementarity to a second target nucleic acid sequence (e.g., conserved sequence) present in target nucleic acid molecule derived from a gene encoding a viral protein (e.g., HCV proteins) to down regulate, inhibit, or shut down the viral replication and infection by targeting the viral genome and viral encoded proteins necessary for viral infection or replication.

In one embodiment the invention takes advantage of conserved nucleotide sequences present in different strains, isotypes or forms of a virus and genes encoded by these different strains, isotypes and forms of the virus (e.g., HCV). By designing multifunctional siNAs in a manner where one strand includes a sequence that is complementary to target nucleic acid sequence conserved among various strains, isotypes or forms of a virus and the other strand includes sequence that is complementary to target nucleic acid sequence conserved in a protein encoded by the virus, it is possible to selectively and effectively inhibit viral replication or infection using a single multifunctional siNA.

In one embodiment, a multifunctional short interfering nucleic acid (multifunctional siNA) of the invention comprises a first region and a second region, wherein the first region comprises a nucleotide sequence complementary to a HCV viral RNA encoding one or more HCV viruses (e.g., one or more strains of HCV) and the second region comprises a nucleotide sequence complementary to a viral RNA encoding one or more interferon agonist proteins. In one embodiment, the first region can comprise a nucleotide sequence complementary to shared or conserved RNA sequences of differing HCV viral strains or classes of HCV viral strains. Non-limiting example of interferon agonist proteins include any protein that is capable of inhibition or suppressing RNA silencing (e.g., RNA binding proteins such as E3L or NS1 or equivalents thereof, see for example Li et al., 2004, PNAS, 101, 1350-1355).

In one embodiment, a multifunctional short interfering nucleic acid (multifunctional siNA) of the invention comprises a first region and a second region, wherein the first region comprises nucleotide sequence complementary to a HCV viral RNA and the second region comprises nucleotide sequence complementary to a cellular RNA that is involved in HCV viral infection and/or replication. Non-limiting examples of cellular RNAs involved in viral infection and/or replication include cellular receptors, cell surface molecules, cellular enzymes, cellular transcription factors, and/or cytokines, second messengers, and cellular accessory molecules including, but not limited to, La antigen, FAS, interferon agonist proteins (e.g., E3L or NS1 or equivalents thereof, see for example Li et al., 2004, PNAS, 101, 1350-1355), interferon regulatory factors (IRFs); cellular PKR protein kinase (PKR); human eukaryotic initiation factors 2B (elF2B gamma and/or elF2gamma); human DEAD Box protein (DDX3); and cellular proteins that bind to the poly(U) tract of the HCV 3′-UTR, such as polypyrimidine tract-binding protein.

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

5′-p-XZX′-3′

3′-Y′ZY-p-5′

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

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

5′-p-XX′-3′

3′-Y′Y-p-5′

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

In one embodiment, the lengths of oligonucleotides Y and Y′ are identical. In another embodiment, the lengths of oligonucleotides Y and Y′ are not identical. In one embodiment, the double stranded oligonucleotide construct of Formula I(a) includes one or more, specifically 1, 2, 3 or 4, mismatches, to the extent such mismatches do not significantly diminish the ability of the double stranded oligonucleotide to inhibit target gene expression.

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

XX′

Y′—W—Y

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

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

XX′

Y′—W—Y

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

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

XX′

Y′—W—Y

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

In one embodiment, regions X and Y of multifunctional siNA molecule of the invention (e.g., having any of Formula MF-I-MF-V), are complementary to different target nucleic acid sequences that are portions of the same target nucleic acid molecule. In one embodiment, such target nucleic acid sequences are at different locations within the coding region of a RNA transcript. In one embodiment, such target nucleic acid sequences comprise coding and non-coding regions of the same RNA transcript. In one embodiment, such target nucleic acid sequences comprise regions of alternately spliced transcripts or precursors of such alternately spliced transcripts.

In one embodiment, a multifunctional siNA molecule having any of Formula MF-I-MF-V can comprise chemical modifications as described herein without limitation, such as, for example, nucleotides having any of Formulae I-VII described herein, stabilization chemistries as described in Table IV, or any other combination of modified nucleotides and non-nucleotides as described in the various embodiments herein.

In one embodiment, the palidrome or repeat sequence or modified nucleotide (e.g., nucleotide with a modified base, such as 2-amino purine or a universal base) in Z of multifunctional siNA constructs having Formula MF-I or MF-II comprises chemically modified nucleotides that are able to interact with a portion of the target nucleic acid sequence (e.g., modified base analogs that can form Watson Crick base pairs or non-Watson Crick base pairs).

In one embodiment, a multifunctional siNA molecule of the invention, for example each strand of a multifunctional siNA having MF-I-MF-V, independently comprises about 15 to about 40 nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides). In one embodiment, a multifunctional siNA molecule of the invention comprises one or more chemical modifications. In a non-limiting example, the introduction of chemically modified nucleotides and/or non-nucleotides into nucleic acid molecules of the invention provides a powerful tool in overcoming potential limitations of in vivo stability and bioavailability inherent to unmodified RNA molecules that are delivered exogenously. For example, the use of chemically modified nucleic acid molecules can enable a lower dose of a particular nucleic acid molecule for a given therapeutic effect since chemically modified nucleic acid molecules tend to have a longer half-life in serum or in cells or tissues. Furthermore, certain chemical modifications can improve the bioavailability and/or potency of nucleic acid molecules by not only enhancing half-life but also facilitating the targeting of nucleic acid molecules to particular organs, cells or tissues and/or improving cellular uptake of the nucleic acid molecules. Therefore, even if the activity of a chemically modified nucleic acid molecule is reduced in vitro as compared to a native/unmodified nucleic acid molecule, for example when compared to an unmodified RNA molecule, the overall activity of the modified nucleic acid molecule can be greater than the native or unmodified nucleic acid molecule due to improved stability, potency, duration of effect, bioavailability and/or delivery of the molecule.

In another embodiment, the invention features multifunctional siNAs, wherein the multifunctional siNAs are assembled from two separate double-stranded siNAs, with one of the ends of each sense strand is tethered to the end of the sense strand of the other siNA molecule, such that the two antisense siNA strands are annealed to their corresponding sense strand that are tethered to each other at one end (see FIG. 22). The tethers or linkers can be nucleotide-based linkers or non-nucleotide based linkers as generally known in the art and as described herein.

In one embodiment, the invention features a multifunctional siNA, wherein the multifunctional siNA is assembled from two separate double-stranded siNAs, with the 5′-end of one antisense strand of the siNA is tethered to the 3′-end of the antisense strand of the other siNA molecule, such that the 5′-end of the one of the sense siNA strands annealed to their corresponding antisense sense strand that are tethered to each other at one end, faces the 3′-end of the other sense strand (see FIG. 22 (G-H)). In one embodiment, the linkage between the 5′-end of the first antisense strand and the 3′-end of the second antisense strand is designed in such a way as to be readily cleavable (e.g., biodegradable linker) such that the 5′ end of each antisense strand of the multifunctional siNA has a free 5′-end suitable to mediate RNA interference-based cleavage of the target RNA. The tethers or linkers can be nucleotide-based linkers or non-nucleotide based linkers as generally known in the art and as described herein.

In one embodiment, the invention features a multifunctional siNA, wherein the multifunctional siNA is assembled from two separate double-stranded siNAs, with the 5′-end of one antisense strand of the siNA is tethered to the 5′-end of the antisense strand of the other siNA molecule, such that the 3′-end of the one of the sense siNA strands annealed to their corresponding antisense sense strand that are tethered to each other at one end, faces the 3′-end of the other sense strand (see FIG. 22 (E)). In one embodiment, the linkage between the 5′-end of the first antisense strand and the 5′-end of the second antisense strand is designed in such a way as to be readily cleavable (e.g., biodegradable linker) such that the 5′ end of each antisense strand of the multifunctional siNA has a free 5′-end suitable to mediate RNA interference-based cleavage of the target RNA. The tethers or linkers can be nucleotide-based linkers or non-nucleotide based linkers as generally known in the art and as described herein.

In one embodiment, the invention features a multifunctional siNA, wherein the multifunctional siNA is assembled from two separate double-stranded siNAs, with the 3′-end of one antisense strand of the siNA is tethered to the 3′-end of the antisense strand of the other siNA molecule, such that the 5′-end of the one of the sense siNA strands annealed to their corresponding antisense sense strand that are tethered to each other at one end, faces the 3′-end of the other sense strand (see FIG. 22 (F)). In one embodiment, the linkage between the 5′-end of the first antisense strand and the 5′-end of the second antisense strand is designed in such a way as to be readily cleavable (e.g., biodegradable linker) such that the 5′ end of each antisense strand of the multifunctional siNA has a free 5′-end suitable to mediate RNA interference-based cleavage of the target RNA. The tethers or linkers can be nucleotide-based linkers or non-nucleotide based linkers as generally known in the art and as described herein.

In any of the above embodiments, a first target nucleic acid sequence or second target nucleic acid sequence can independently comprise HCV RNA or a portion thereof or a polynucleotide coding or non-coding sequence of cellular or host target that is involved in HCV infection or replication, or disease processes associated with HCV infection such as such as cellular receptors, cell surface molecules, cellular enzymes, cellular transcription factors, and/or cytokines, second messengers, and cellular accessory molecules including, but not limited to, La antigen (see for example Costa-Mattioli et al., 2004, Mol Cell Biol., 24, 6861-70, e.g., Genbank Accession No. NM_—003142); FAS (e.g., Genbank Accession No. NM_—000043) or FAS ligand (e.g., Genbank Accession No. NM_—000639); interferon regulatory factors (IRFs; e.g., Genbank Accession No. AF082503.1); cellular PKR protein kinase (e.g., Genbank Accession No. XM_—002661.7); human eukaryotic initiation factors 2B (elF2Bgamma; e.g., Genbank Accession No. AF256223, and/or elF2gamma; e.g., Genbank Accession No. NM_—006874.1); human DEAD Box protein (DDX3; e.g., Genbank Accession No. XM_—018021.2); and cellular proteins that bind to the poly(U) tract of the HCV 3′-UTR, such as polypyrimidine tract-binding protein (e.g., Genbank Accession Nos. NM_—031991.1 and XM_—042972.3). In one embodiment, the first HCV target nucleic acid sequence is a HCV RNA or a portion thereof and the second HCV target nucleic acid sequence is a HCV RNA of a portion thereof. In one embodiment, the first HCV target nucleic acid sequence is a HCV RNA or a portion thereof and the second HCV target nucleic acid sequence is a host RNA or a portion thereof. In one embodiment, the first HCV target nucleic acid sequence is a host RNA or a portion thereof and the second HCV target nucleic acid sequence is a host RNA or a portion thereof. In one embodiment, the first HCV target nucleic acid sequence is a host RNA or a portion thereof and the second HCV target nucleic acid sequence is a HCV RNA or a portion thereof.

Synthesis of Nucleic Acid Molecules

Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. In this invention, small nucleic acid motifs (“small” refers to nucleic acid motifs no more than 100 nucleotides in length, preferably no more than 80 nucleotides in length, and most preferably no more than 50 nucleotides in length; e.g., individual siNA oligonucleotide sequences or siNA sequences synthesized in tandem) are preferably used for exogenous delivery. The simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of protein and/or RNA structure. Exemplary molecules of the instant invention are chemically synthesized, and others can similarly be synthesized.

Oligonucleotides (e.g., certain modified oligonucleotides or portions of oligonucleotides lacking ribonucleotides) are synthesized using protocols known in the art, for example as described in Caruthers et al., 1992, Methods in Enzymology 211, 3-19, Thompson et al., International PCT Publication No. WO 99/54459, Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol. Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, and Brennan, U.S. Pat. No. 6,001,311. All of these references are incorporated herein by reference. The synthesis of oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 2.5 min coupling step for 2′-O-methylated nucleotides and a 45 second coupling step for 2′-deoxy nucleotides or 2′-deoxy-2′-fluoro nucleotides. Table V outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 μmol scale can be performed on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycle of 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 22-fold excess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite and a 70-fold excess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol) can be used in each coupling cycle of deoxy residues relative to polymer-bound 5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include the following: detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solution is 16.9 mM 12, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems, Inc.). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile) is used.

Deprotection of the DNA-based oligonucleotides is performed as follows: the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aqueous methylamine (1 mL) at 65° C. for 10 minutes. After cooling to −20° C., the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder. In one embodiment, the nucleic acid molecules of the invention are synthesized, deprotected, and analyzed according to methods described in U.S. Pat. No. 6,995,259, U.S. Pat. No. 6,686,463, U.S. Pat. No. 6,673,918, U.S. Pat. No. 6,649,751, U.S. Pat. No. 6,989,442, and U.S. Ser. No. 10/190,359, all incorporated by reference herein in their entirety.

The method of synthesis used for RNA including certain siNA molecules of the invention follows the procedure as described in Usman et al., 1987, J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684 Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5 min coupling step for alkylsilyl protected nucleotides and a 2.5 min coupling step for 2′-O-methylated nucleotides. Table V outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycle of 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol) of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess of S-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in each coupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include the following: detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM 12, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems, Inc.). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide 0.05 M in acetonitrile) is used.

Deprotection of the RNA is performed using either a two-pot or one-pot protocol. For the two-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10 min. After cooling to −20° C., the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder. The base deprotected oligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300 μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mL TEA-3HF to provide a 1.4 M HF concentration) and heated to 65° C. After 1.5 h, the oligomer is quenched with 1.5 M NH₄HCO₃. In one embodiment, the nucleic acid molecules of the invention are synthesized, deprotected, and analyzed according to methods described in U.S. Pat. No. 6,995,259, U.S. Pat. No. 6,686,463, U.S. Pat. No. 6,673,918, U.S. Pat. No. 6,649,751, U.S. Pat. No. 6,989,442, and U.S. Ser. No. 10/190,359, all incorporated by reference herein in their entirety.

Alternatively, for the one-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL) at 65° C. for 15 minutes. The vial is brought to room temperature TEA-3HF (0.1 mL) is added and the vial is heated at 65° C. for 15 minutes. The sample is cooled at −20° C. and then quenched with 1.5 M NH₄HCO₃.

For purification of the trityl-on oligomers, the quenched NH₄HCO₃solution is loaded onto a C-18 containing cartridge that had been prewashed with acetonitrile followed by 50 mM TEAA. After washing the loaded cartridge with water, the RNA is detritylated with 0.5% TFA for 13 minutes. The cartridge is then washed again with water, salt exchanged with 1 M NaCl and washed with water again. The oligonucleotide is then eluted with 30% acetonitrile.

The average stepwise coupling yields are typically >98% (Wincott et al., 1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in the art will recognize that the scale of synthesis can be adapted to be larger or smaller than the example described above including but not limited to 96-well format.

Alternatively, the nucleic acid molecules of the present invention can be synthesized separately and joined together post-synthetically, for example, by ligation (Moore et al., 1992, Science 256, 9923; Draper et al., International PCT publication No. WO 93/23569; Shabarova et al., 1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204), or by hybridization following synthesis and/or deprotection.

The siNA molecules of the invention can also be synthesized via a tandem synthesis methodology as described in Example 1 herein, wherein both siNA strands are synthesized as a single contiguous oligonucleotide fragment or strand separated by a cleavable linker which is subsequently cleaved to provide separate siNA fragments or strands that hybridize and permit purification of the siNA duplex. The linker can be a polynucleotide linker or a non-nucleotide linker. The tandem synthesis of siNA as described herein can be readily adapted to both multiwell/multiplate synthesis platforms such as 96 well or similarly larger multi-well platforms. The tandem synthesis of siNA as described herein can also be readily adapted to large scale synthesis platforms employing batch reactors, synthesis columns and the like.

A siNA molecule can also be assembled from two distinct nucleic acid strands or fragments wherein one fragment includes the sense region and the second fragment includes the antisense region of the RNA molecule.

The nucleic acid molecules of the present invention can be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H (for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163). siNA constructs can be purified by gel electrophoresis using general methods or can be purified by high pressure liquid chromatography (HPLC; see Wincott et al., supra, the totality of which is hereby incorporated herein by reference) and re-suspended in water.

In another aspect of the invention, siNA molecules of the invention are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. The recombinant vectors capable of expressing the siNA molecules can be delivered as described herein, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of siNA molecules.

Optimizing Activity of the Nucleic Acid Molecule of the Invention.

Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) can prevent their degradation by serum ribonucleases, which can increase their potency (see e.g., Eckstein et al., International Publication No. WO 92/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman and Cedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162; Sproat, U.S. Pat. No. 5,334,711; Gold et al., U.S. Pat. No. 6,300,074; and Burgin et al., supra; all of which are incorporated by reference herein). All of the above references describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of the nucleic acid molecules described herein. Modifications that enhance their efficacy in cells, and removal of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements are desired.

There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy. For example, oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35, 14090). Sugar modification of nucleic acid molecules have been extensively described in the art (see Eckstein et al., International Publication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344, 565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. International Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al., International PCT publication No. WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al., International PCT Publication No. WO 98/13526; Thompson et al., U.S. Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al., 1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010; all of the references are hereby incorporated in their totality by reference herein). Such publications describe general methods and strategies to determine the location of incorporation of sugar, base and/or phosphate modifications and the like into nucleic acid molecules without modulating catalysis, and are incorporated by reference herein. In view of such teachings, similar modifications can be used as described herein to modify the siNA nucleic acid molecules of the instant invention so long as the ability of siNA to promote RNAI is cells is not significantly inhibited.

In one embodiment, a nucleic acid molecule of the invention is chemically modified as described in US 20050020521, incorporated by reference herein in its entirety.

While chemical modification of oligonucleotide internucleotide linkages with phosphorothioate, phosphorodithioate, and/or 5′-methylphosphonate linkages improves stability, excessive modifications can cause some toxicity or decreased activity. Therefore, when designing nucleic acid molecules, the amount of these internucleotide linkages should be minimized. The reduction in the concentration of these linkages should lower toxicity, resulting in increased efficacy and higher specificity of these molecules.

Short interfering nucleic acid (siNA) molecules having chemical modifications that maintain or enhance activity are provided. Such a nucleic acid is also generally more resistant to nucleases than an unmodified nucleic acid. Accordingly, the in vitro and/or in vivo activity should not be significantly lowered. In cases in which modulation is the goal, therapeutic nucleic acid molecules delivered exogenously should optimally be stable within cells until translation of the target RNA has been modulated long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state. Improvements in the chemical synthesis of RNA and DNA (Wincott et al., 1995, Nucleic Acids Res. 23, 2677; Caruthers et al., 1992, Methods in Enzymology 211, 3-19 (incorporated by reference herein)) have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability, as described above.

In one embodiment, nucleic acid molecules of the invention include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clamp nucleotides. A G-clamp nucleotide is a modified cytosine analog wherein the modifications confer the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine within a duplex, see for example Lin and Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. A single G-clamp analog substitution within an oligonucleotide can result in substantially enhanced helical thermal stability and mismatch discrimination when hybridized to complementary oligonucleotides. The inclusion of such nucleotides in nucleic acid molecules of the invention results in both enhanced affinity and specificity to nucleic acid targets, complementary sequences, or template strands. In another embodiment, nucleic acid molecules of the invention include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA “locked nucleic acid” nucleotides such as a 2′,4′-C methylene bicyclo nucleotide (see for example Wengel et al., International PCT Publication No. WO 00/66604 and WO 99/14226).

In another embodiment, the invention features conjugates and/or complexes of siNA molecules of the invention. Such conjugates and/or complexes can be used to facilitate delivery of siNA molecules into a biological system, such as a cell. The conjugates and complexes provided by the instant invention can impart therapeutic activity by transferring therapeutic compounds across cellular membranes, altering the pharmacokinetics, and/or modulating the localization of nucleic acid molecules of the invention. The present invention encompasses the design and synthesis of novel conjugates and complexes for the delivery of molecules, including, but not limited to, small molecules, lipids, cholesterol, phospholipids, nucleosides, nucleotides, nucleic acids, antibodies, toxins, negatively charged polymers and other polymers, for example proteins, peptides, hormones, carbohydrates, polyethylene glycols, or polyamines, across cellular membranes. In general, the transporters described are designed to be used either individually or as part of a multi-component system, with or without degradable linkers. These compounds are expected to improve delivery and/or localization of nucleic acid molecules of the invention into a number of cell types originating from different tissues, in the presence or absence of serum (see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of the molecules described herein can be attached to biologically active molecules via linkers that are biodegradable, such as biodegradable nucleic acid linker molecules.

The term “biodegradable linker” as used herein, refers to a nucleic acid or non-nucleic acid linker molecule that is designed as a biodegradable linker to connect one molecule to another molecule, for example, a biologically active molecule to a siNA molecule of the invention or the sense and antisense strands of a siNA molecule of the invention. The biodegradable linker is designed such that its stability can be modulated for a particular purpose, such as delivery to a particular tissue or cell type. The stability of a nucleic acid-based biodegradable linker molecule can be modulated by using various chemistries, for example combinations of ribonucleotides, deoxyribonucleotides, and chemically-modified nucleotides, such as 2′-O-methyl, 2′-fluoro, 2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified or base modified nucleotides. The biodegradable nucleic acid linker molecule can be a dimer, trimer, tetramer or longer nucleic acid molecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprise a single nucleotide with a phosphorus-based linkage, for example, a phosphoramidate or phosphodiester linkage. The biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base modifications.

The term “biodegradable” as used herein, refers to degradation in a biological system, for example, enzymatic degradation or chemical degradation.

The term “biologically active molecule” as used herein refers to compounds or molecules that are capable of eliciting or modifying a biological response in a system. Non-limiting examples of biologically active siNA molecules either alone or in combination with other molecules contemplated by the instant invention include therapeutically active molecules such as antibodies, cholesterol, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers, decoys and analogs thereof. Biologically active molecules of the invention also include molecules capable of modulating the pharmacokinetics and/or pharmacodynamics of other biologically active molecules, for example, lipids and polymers such as polyamines, polyamides, polyethylene glycol and other polyethers.

The term “phospholipid” as used herein, refers to a hydrophobic molecule comprising at least one phosphorus group. For example, a phospholipid can comprise a phosphorus-containing group and saturated or unsaturated alkyl group, optionally substituted with OH, COOH, oxo, amine, or substituted or unsubstituted aryl groups.

Therapeutic nucleic acid molecules (e.g., siNA molecules) delivered exogenously optimally are stable within cells until reverse transcription of the RNA has been modulated long enough to reduce the levels of the RNA transcript. The nucleic acid molecules are resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of nucleic acid molecules described in the instant invention and in the art have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above.

In yet another embodiment, siNA molecules having chemical modifications that maintain or enhance enzymatic activity of proteins involved in RNAi are provided. Such nucleic acids are also generally more resistant to nucleases than unmodified nucleic acids. Thus, in vitro and/or in vivo the activity should not be significantly lowered.

Use of the nucleic acid-based molecules of the invention will lead to better treatments by affording the possibility of combination therapies (e.g., multiple siNA molecules targeted to different genes; nucleic acid molecules coupled with known small molecule modulators; or intermittent treatment with combinations of molecules, including different motifs and/or other chemical or biological molecules). The treatment of subjects with siNA molecules can also include combinations of different types of nucleic acid molecules, such as enzymatic nucleic acid molecules (ribozymes), allozymes, antisense, 2,5-A oligoadenylate, decoys, and aptamers.

In another aspect a siNA molecule of the invention comprises one or more 5′ and/or a 3′-cap structure, for example, on only the sense siNA strand, the antisense siNA strand, or both siNA strands.

By “cap structure” is meant chemical modifications, which have been incorporated at either terminus of the oligonucleotide (see, for example, Adamic et al., U.S. Pat. No. 5,998,203, incorporated by reference herein). These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and may help in delivery and/or localization within a cell. The cap may be present at the 5′-terminus (5′-cap) or at the 3′-terminal (3′-cap) or may be present on both termini. In non-limiting examples, the 5′-cap includes, but is not limited to, glyceryl, inverted deoxy abasic residue (moiety); 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety; 1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety. Non-limiting examples of cap moieties are shown in FIG. 10.

Non-limiting examples of the 3′-cap include, but are not limited to, glyceryl, inverted deoxy abasic residue (moiety), 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasic moiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediol phosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5′-mercapto moieties (for more details see Beaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by reference herein).

By the term “non-nucleotide” is meant any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine and therefore lacks a base at the 1′-position.

An “alkyl” group refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group can be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂or N(CH₃)₂, amino, or SH. The term also includes alkenyl groups that are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12 carbons. More preferably, it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkenyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂, halogen, N(CH₃)₂, amino, or SH. The term “alkyl” also includes alkynyl groups that have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has 1 to 12 carbons. More preferably, it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂or N(CH₃)₂, amino or SH.

Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An “aryl” group refers to an aromatic group that has at least one ring having a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted. The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An “alkylaryl” group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above). Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An “amide” refers to an —C(O)—NH—R, where R is either alkyl, aryl, alkylaryl or hydrogen. An “ester” refers to an —C(O)—OR′, where R is either alkyl, aryl, alkylaryl or hydrogen.

By “nucleotide” as used herein is as recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1′ position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see, for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No. WO 92/07065; Usman et al., International PCT Publication No. WO 93/15187; Uhlman & Peyman, supra, all are hereby incorporated by reference herein). There are several examples of modified nucleic acid bases known in the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some of the non-limiting examples of base modifications that can be introduced into nucleic acid molecules include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra). By “modified bases” in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1′ position or their equivalents.

In one embodiment, the invention features modified siNA molecules, with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions. For a review of oligonucleotide backbone modifications, see Hunziker and Leumann, 1995, Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417, and Mesmaeker et al., 1994, Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39.

By “abasic” is meant sugar moieties lacking a nucleobase or having a hydrogen atom (H) or other other non-nucleobase chemical groups in place of a nucleobase at the 1′ position of the sugar moiety, see for example Adamic et al., U.S. Pat. No. 5,998,203. In one embodiment, an abasic moiety of the invention is a ribose, deoxyribose, or dideoxyribose sugar.

By “unmodified nucleoside” is meant one of the bases adenine, cytosine, guanine, thymine, or uracil joined to the 1′ carbon of β-D-ribo-furanose.

By “modified nucleoside” is meant any nucleotide base which contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate. Non-limiting examples of modified nucleotides are shown by Formulae I-VII and/or other modifications described herein.

In connection with 2′-modified nucleotides as described for the present invention, by “amino” is meant 2′—NH₂or 2′-O—NH₂, which can be modified or unmodified. Such modified groups are described, for example, in Eckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S. Pat. No. 6,248,878, which are both incorporated by reference in their entireties.

Various modifications to nucleic acid siNA structure can be made to enhance the utility of these molecules. Such modifications will enhance shelf-life, half-life in vitro, stability, and ease of introduction of such oligonucleotides to the target site, e.g., to enhance penetration of cellular membranes, and confer the ability to recognize and bind to targeted cells.

Administration of Nucleic Acid Molecules

A siNA molecule of the invention can be adapted for use to treat, prevent, inhibit, or reduce HCV infection, liver failure, hepatocellular carcinoma, cirrhosis and/or any other trait, disease or condition that is related to or will respond to the levels of HCV in a cell or tissue, alone or in combination with other therapies. In one embodiment, the siNA molecules of the invention and formulations or compositions thereof are administered to the liver as is generally known in the art (see for example Wen et al., 2004, World J Gastroenterol., 10, 244-9; Murao et al., 2002, Pharm Res., 19, 1808-14; Liu et al., 2003, Gene Ther., 10, 180-7; Hong et al., 2003, J Pharm Pharmacol., 54, 51-8; Herrmann et al., 2004, Arch Virol., 149, 1611-7; and Matsuno et al., 2003, Gene Ther., 10, 1559-66).

In one embodiment, a siNA composition of the invention can comprise a delivery vehicle, including liposomes, for administration to a subject, carriers and diluents and their salts, and/or can be present in pharmaceutically acceptable formulations. Methods for the delivery of nucleic acid molecules are described in Akhtar et al., 1992, Trends Cell Bio., 2, 139; Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al., 1999, Mol. Membr. Biol., 16, 129-140; Hofland and Huang, 1999, Handb. Exp. Pharmacol., 137, 165-192; and Lee et al., 2000, ACS Symp. Ser., 752, 184-192, all of which are incorporated herein by reference. Beigelman et al., U.S. Pat. No. 6,395,713 and Sullivan et al., PCT WO 94/02595 further describe the general methods for delivery of nucleic acid molecules. These protocols can be utilized for the delivery of virtually any nucleic acid molecule. Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins (see for example Gonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074; Wang et al., International PCT publication Nos. WO 03/47518 and WO 03/46185), poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see for example U.S. Pat. No. 6,447,796 and US Patent Application Publication No. US 2002130430), biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (O'Hare and Normand, International PCT Publication No. WO 00/53722). In another embodiment, the nucleic acid molecules of the invention can also be formulated or complexed with polyethyleneimine and derivatives thereof, such as polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives. In one embodiment, the nucleic acid molecules of the invention are formulated as described in United States Patent Application Publication No. 20030077829, incorporated by reference herein in its entirety.

In one embodiment, a siNA molecule of the invention is formulated as a composition described in U.S. Provisional patent application No. 60/678,531 and in related U.S. Provisional patent application No. 60/703,946, filed Jul. 29, 2005, U.S. Provisional patent application No. 60/737,024, filed Nov. 15, 2005, and U.S. Ser. No. 11/353,630, filed Feb. 14, 2006 (Vargeese et al.), all of which are incorporated by reference herein in their entirety. Such siNA formulations are generally referred to as “lipid nucleic acid particles” (LNP). In one embodiment, a siNA molecule of the invention is formulated with one or more LNP compositions described herein in Table VI (see U.S. Ser. No. 11/353,630 supra).

In one embodiment, the siNA molecules of the invention and formulations or compositions thereof are administered to lung tissues and cells as is described in US 2006/0062758; US 2006/0014289; and US 2004/0077540.

In one embodiment, a siNA molecule of the invention is complexed with membrane disruptive agents such as those described in U.S. Patent Application Publication No. 20010007666, incorporated by reference herein in its entirety including the drawings. In another embodiment, the membrane disruptive agent or agents and the siNA molecule are also complexed with a cationic lipid or helper lipid molecule, such as those lipids described in U.S. Pat. No. 6,235,310, incorporated by reference herein in its entirety including the drawings.

In one embodiment, a siNA molecule of the invention is complexed with delivery systems as described in U.S. Patent Application Publication No. 2003077829 and International PCT Publication Nos. WO 00/03683 and WO 02/087541, all incorporated by reference herein in their entirety including the drawings.

In one embodiment, a siNA molecule of the invention is complexed with delivery systems as is generally described in U.S. Patent Application Publication Nos. US-20050287551; US-20050164220; US-20050191627; US-20050118594; US-20050153919; US-20050085486; and US-20030158133; all incorporated by reference herein in their entirety including the drawings.

In one embodiment, the nucleic acid molecules of the invention are administered to skeletal tissues (e.g., bone, cartilage, tendon, ligament) or bone metastatic tumors via atelocollagen complexation or conjugation (see for example Takeshita et al., 2005, PNAS, 102, 12177-12182). Therefore, in one embodiment, the instant invention features one or more dsiNA molecules as a composition complexed with atelocollagen. In another embodiment, the instant invention features one or more siNA molecules conjugated to atelocollagen via a linker as described herein or otherwise known in the art.

In one embodiment, the nucleic acid molecules of the invention and formulations thereof (e.g., LNP formulations of double stranded nucleic acid molecules of the invention) are administered via pulmonary delivery, such as by inhalation of an aerosol or spray dried formulation administered by an inhalation device or nebulizer, providing rapid local uptake of the nucleic acid molecules into relevant pulmonary tissues. Solid particulate compositions containing respirable dry particles of micronized nucleic acid compositions can be prepared by grinding dried or lyophilized nucleic acid compositions, and then passing the micronized composition through, for example, a 400 mesh screen to break up or separate out large agglomerates. A solid particulate composition comprising the nucleic acid compositions of the invention can optionally contain a dispersant which serves to facilitate the formation of an aerosol as well as other therapeutic compounds. A suitable dispersant is lactose, which can be blended with the nucleic acid compound in any suitable ratio, such as a 1 to 1 ratio by weight.

Aerosols of liquid particles comprising a nucleic acid composition of the invention can be produced by any suitable means, such as with a nebulizer (see for example U.S. Pat. No. 4,501,729). Nebulizers are commercially available devices which transform solutions or suspensions of an active ingredient into a therapeutic aerosol mist either by means of acceleration of a compressed gas, typically air or oxygen, through a narrow venturi orifice or by means of ultrasonic agitation. Suitable formulations for use in nebulizers comprise the active ingredient in a liquid carrier in an amount of up to 40% w/w preferably less than 20% w/w of the formulation. The carrier is typically water or a dilute aqueous alcoholic solution, preferably made isotonic with body fluids by the addition of, for example, sodium chloride or other suitable salts. Optional additives include preservatives if the formulation is not prepared sterile, for example, methyl hydroxybenzoate, anti-oxidants, flavorings, volatile oils, buffering agents and emulsifiers and other formulation surfactants. The aerosols of solid particles comprising the active composition and surfactant can likewise be produced with any solid particulate aerosol generator. Aerosol generators for administering solid particulate therapeutics to a subject produce particles which are respirable, as explained above, and generate a volume of aerosol containing a predetermined metered dose of a therapeutic composition at a rate suitable for human administration.

In one embodiment, a solid particulate aerosol generator of the invention is an insufflator. Suitable formulations for administration by insufflation include finely comminuted powders which can be delivered by means of an insufflator. In the insufflator, the powder, e.g., a metered dose thereof effective to carry out the treatments described herein, is contained in capsules or cartridges, typically made of gelatin or plastic, which are either pierced or opened in situ and the powder delivered by air drawn through the device upon inhalation or by means of a manually-operated pump. The powder employed in the insufflator consists either solely of the active ingredient or of a powder blend comprising the active ingredient, a suitable powder diluent, such as lactose, and an optional surfactant. The active ingredient typically comprises from 0.1 to 100 w/w of the formulation. A second type of illustrative aerosol generator comprises a metered dose inhaler. Metered dose inhalers are pressurized aerosol dispensers, typically containing a suspension or solution formulation of the active ingredient in a liquified propellant. During use these devices discharge the formulation through a valve adapted to deliver a metered volume to produce a fine particle spray containing the active ingredient. Suitable propellants include certain chlorofluorocarbon compounds, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof. The formulation can additionally contain one or more co-solvents, for example, ethanol, emulsifiers and other formulation surfactants, such as oleic acid or sorbitan trioleate, anti-oxidants and suitable flavoring agents. Other methods for pulmonary delivery are described in, for example US Patent Application No. 20040037780, and U.S. Pat. Nos. 6,592,904; 6,582,728; 6,565,885, all incorporated by reference herein.

In one embodiment, the siNA and LNP compositions and formulations provided herein for use in pulmonary delivery further comprise one or more surfactants. Suitable surfactants or surfactant components for enhancing the uptake of the compositions of the invention include synthetic and natural as well as full and truncated forms of surfactant protein A, surfactant protein B, surfactant protein C, surfactant protein D and surfactant Protein E, di-saturated phosphatidylcholine (other than dipalmitoyl), dipalmitoylphosphatidylchol-ine, phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine; phosphatidic acid, ubiquinones, lysophosphatidylethanolamine, lysophosphatidylcholine, palmitoyl-lysophosphatidylcholine, dehydroepiandrosterone, dolichols, sulfatidic acid, glycerol-3-phosphate, dihydroxyacetone phosphate, glycerol, glycero-3-phosphocholine, dihydroxyacetone, palmitate, cytidine diphosphate (CDP) diacylglycerol, CDP choline, choline, choline phosphate; as well as natural and artificial lamelar bodies which are the natural carrier vehicles for the components of surfactant, omega-3 fatty acids, polyenic acid, polyenoic acid, lecithin, palmitinic acid, non-ionic block copolymers of ethylene or propylene oxides, polyoxypropylene, monomeric and polymeric, polyoxyethylene, monomeric and polymeric, poly (vinyl amine) with dextran and/or alkanoyl side chains, Brij 35, Triton X-100 and synthetic surfactants ALEC, Exosurf, Survan and Atovaquone, among others. These surfactants may be used either as single or part of a multiple component surfactant in a formulation, or as covalently bound additions to the 5′ and/or 3′ ends of the nucleic acid component of a pharmaceutical composition herein.

The composition of the present invention may be administered into the respiratory system as a formulation including particles of respirable size, e.g. particles of a size sufficiently small to pass through the nose, mouth and larynx upon inhalation and through the bronchi and alveoli of the lungs. In general, respirable particles range from about 0.5 to 10 microns in size. Particles of non-respirable size which are included in the aerosol tend to deposit in the throat and be swallowed, and the quantity of non-respirable particles in the aerosol is thus minimized. For nasal administration, a particle size in the range of 10-500 um is preferred to ensure retention in the nasal cavity.

In one embodiment, the invention features the use of methods to deliver the nucleic acid molecules of the instant invention to the central nervous system and/or peripheral nervous system. Experiments have demonstrated the efficient in vivo uptake of nucleic acids by neurons. As an example of local administration of nucleic acids to nerve cells, Sommer et al., 1998, Antisense Nuc. Acid Drug Dev., 8, 75, describe a study in which a 15mer phosphorothioate antisense nucleic acid molecule to c-fos is administered to rats via microinjection into the brain. Antisense molecules labeled with tetramethylrhodamine-isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC) were taken up by exclusively by neurons thirty minutes post-injection. A diffuse cytoplasmic staining and nuclear staining was observed in these cells. As an example of systemic administration of nucleic acid to nerve cells, Epa et al., 2000, Antisense Nuc. Acid Drug Dev., 10, 469, describe an in vivo mouse study in which beta-cyclodextrin-adamantane-oligonucleotide conjugates were used to target the p75 neurotrophin receptor in neuronally differentiated PC12 cells. Following a two week course of IP administration, pronounced uptake of p75 neurotrophin receptor antisense was observed in dorsal root ganglion (DRG) cells. In addition, a marked and consistent down-regulation of p75 was observed in DRG neurons. Additional approaches to the targeting of nucleic acid to neurons are described in Broaddus et al., 1998, J. Neurosurg., 88(4), 734; Karle et al., 1997, Eur. J. Pharmocol., 340(2/3), 153; Bannai et al., 1998, Brain Research, 784(1,2), 304; Rajakumar et al., 1997, Synapse, 26(3), 199; Wu-pong et al., 1999, BioPharm, 12(1), 32; Bannai et al., 1998, Brain Res. Protoc., 3(1), 83; Simantov et al., 1996, Neuroscience, 74(1), 39. Nucleic acid molecules of the invention are therefore amenable to delivery to and uptake by cells that express repeat expansion allelic variants for modulation of RE gene expression. The delivery of nucleic acid molecules of the invention, targeting RE is provided by a variety of different strategies. Traditional approaches to CNS delivery that can be used include, but are not limited to, intrathecal and intracerebroventricular administration, implantation of catheters and pumps, direct injection or perfusion at the site of injury or lesion, injection into the brain arterial system, or by chemical or osmotic opening of the blood-brain barrier. Other approaches can include the use of various transport and carrier systems, for example though the use of conjugates and biodegradable polymers. Furthermore, gene therapy approaches, for example as described in Kaplitt et al., U.S. Pat. No. 6,180,613 and Davidson, WO 04/013280, can be used to express nucleic acid molecules in the CNS.

The delivery of nucleic acid molecules of the invention to the CNS is provided by a variety of different strategies. Traditional approaches to CNS delivery that can be used include, but are not limited to, intrathecal and intracerebroventricular administration, implantation of catheters and pumps, direct injection or perfusion at the site of injury or lesion, injection into the brain arterial system, or by chemical or osmotic opening of the blood-brain barrier. Other approaches can include the use of various transport and carrier systems, for example though the use of conjugates and biodegradable polymers. Furthermore, gene therapy approaches, for example as described in Kaplitt et al., U.S. Pat. No. 6,180,613 and Davidson, WO 04/013280, can be used to express nucleic acid molecules in the CNS.

In one embodiment, the invention features the use of methods to deliver the nucleic acid molecules of the instant invention to hematopoietic cells, including monocytes and lymphocytes. These methods are described in detail by Hartmann et al., 1998, J. Phamacol. Exp. Ther., 285(2), 920-928; Kronenwett et al., 1998, Blood, 91(3), 852-862; Filion and Phillips, 1997, Biochim. Biophys. Acta., 1329(2), 345-356; Ma and Wei, 1996, Leuk. Res., 20(11/12), 925-930; and Bongartz et al., 1994, Nucleic Acids Research, 22(22), 4681-8. Such methods, as described above, include the use of free oligonucleotide, cationic lipid formulations, liposome formulations including pH sensitive liposomes and immunoliposomes, and bioconjugates including oligonucleotides conjugated to fusogenic peptides, for the transfection of hematopoietic cells with oligonucleotides.

In one embodiment, siNA compounds and compositions of the invention are administered either systemically or locally about every 1-50 weeks (e.g., about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 weeks), alone or in combination with other compounds and/or therapies herein. In one embodiment, siNA compounds and compositions of the invention are administered systemically (e.g., via intravenous, subcutaneous, intramuscular, infusion, pump, implant etc.) about every 1-50 weeks (e.g., about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 weeks), alone or in combination with other compounds and/or therapies described herein and/or otherwise known in the art.

In one embodiment, delivery systems of the invention include, for example, aqueous and nonaqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone). In one embodiment, the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer. Examples of liposomes which can be used in this invention include the following: (1) CellFectin, 1:1.5 (M/M) liposome formulation of the cationic lipid N,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmit-y-spermine and dioleoyl phosphatidylethanolamine (DOPE) (GIBCO BRL); (2) Cytofectin GSV, 2:1 (M/M) liposome formulation of a cationic lipid and DOPE (Glen Research); (3) DOTAP (N-[1-(2,3-dioleoyloxy)-N,N,N-tri-methyl-ammoniummethylsulfate) (Boehringer Manheim); and (4) Lipofectamine, 3:1 (M/M) liposome formulation of the polycationic lipid DOSPA and the neutral lipid DOPE (GIBCO BRL).

In one embodiment, delivery systems of the invention include patches, tablets, suppositories, pessaries, gels and creams, and can contain excipients such as solubilizers and enhancers (e.g., propylene glycol, bile salts and amino acids), and other vehicles (e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid).

In one embodiment, siNA molecules of the invention are formulated or complexed with polyethylenimine (e.g., linear or branched PEI) and/or polyethylenimine derivatives, including for example grafted PEIs such as galactose PEI, cholesterol PEI, antibody derivatized PEI, and polyethylene glycol PEI (PEG-PEI) derivatives thereof (see for example Ogris et al., 2001, AAPA PharmSci, 3, 1-11; Furgeson et al., 2003, Bioconjugate Chem., 14, 840-847; Kunath et al., 2002, Pharmaceutical Research, 19, 810-817; Choi et al., 2001, Bull. Korean Chem. Soc., 22, 46-52; Bettinger et al., 1999, Bioconjugate Chem., 10, 558-561; Peterson et al., 2002, Bioconjugate Chem., 13, 845-854; Erbacher et al., 1999, Journal of gene Medicine Preprint, 1, 1-18; Godbey et al., 1999., PNAS USA, 96, 5177-5181; Godbey et al., 1999, Journal of Controlled Release, 60, 149-160; Diebold et al., 1999, Journal of Biological Chemistry, 274, 19087-19094; Thomas and Klibanov, 2002, PNAS USA, 99, 14640-14645; and Sagara, U.S. Pat. No. 6,586,524, incorporated by reference herein.

In one embodiment, a siNA molecule of the invention comprises a bioconjugate, for example a nucleic acid conjugate as described in Vargeese et al., U.S. Ser. No. 10/427,160, filed Apr. 30, 2003; U.S. Pat. No. 6,528,631; U.S. Pat. No. 6,335,434; U.S. Pat. No. 6,235,886; U.S. Pat. No. 6,153,737; U.S. Pat. No. 5,214,136; U.S. Pat. No. 5,138,045, all incorporated by reference herein.

Thus, the invention features a pharmaceutical composition comprising one or more nucleic acid(s) of the invention in an acceptable carrier, such as a stabilizer, buffer, and the like. The polynucleotides of the invention can be administered (e.g., RNA, DNA or protein) and introduced to a subject by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition. When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed. The compositions of the present invention can also be formulated and used as creams, gels, sprays, oils and other suitable compositions for topical, dermal, or transdermal administration as is known in the art.

The present invention also includes pharmaceutically acceptable formulations of the compounds described. These formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.

A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic or local administration, into a cell or subject, including for example a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged nucleic acid is desirable for delivery). For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms that prevent the composition or formulation from exerting its effect.

In one embodiment, siNA molecules of the invention are administered to a subject by systemic administration in a pharmaceutically acceptable composition or formulation. By “systemic administration” is meant in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body. Administration routes that lead to systemic absorption include, without limitation: intravenous, subcutaneous, portal vein, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these administration routes exposes the siNA molecules of the invention to an accessible diseased tissue (e.g., lung). The rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other drug carrier comprising the compounds of the instant invention can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES). A liposome formulation that can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach can provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells.

By “pharmaceutically acceptable formulation” or “pharmaceutically acceptable composition” is meant, a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity. Non-limiting examples of agents suitable for formulation with the nucleic acid molecules of the instant invention include: P-glycoprotein inhibitors (such as Pluronic P85); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery (Emerich, D F et al, 1999, Cell Transplant, 8, 47-58); and loaded nanoparticles, such as those made of polybutylcyanoacrylate. Other non-limiting examples of delivery strategies for the nucleic acid molecules of the instant invention include material described in Boado et al., 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge et al., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058.

The invention also features the use of a composition comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes) and nucleic acid molecules of the invention. These formulations offer a method for increasing the accumulation of drugs (e.g., siNA) in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011). Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995, Biochim. Biophys. Acta, 1238, 86-90). The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995, 42, 24864-24870; Choi et al., International PCT Publication No. WO 96/10391; Ansell et al., International PCT Publication No. WO 96/10390; Holland et al., International PCT Publication No. WO 96/10392). Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.

In one embodiment, a liposomal formulation of the invention comprises a double stranded nucleic acid molecule of the invention (e.g, siNA) formulated or complexed with compounds and compositions described in U.S. Pat. Nos. 6,858,224; 6,534,484; 6,287,591; 6,835,395; 6,586,410; 6,858,225; 6,815,432; U.S. Pat. Nos. 6,586,001; 6,120,798; U.S. Pat. No. 6,977,223; U.S. Pat. Nos. 6,998,115; 5,981,501; 5,976,567; 5,705,385; US 2006/0019912; US 2006/0019258; US 2006/0008909; US 2005/0255153; US 2005/0079212; US 2005/0008689; US 2003/0077829, US 2005/0064595, US 2005/0175682, US 2005/0118253; US 2004/0071654; US 2005/0244504; US 2005/0265961 and US 2003/0077829, all of which are incorporated by reference herein in their entirety.

The present invention also includes compositions prepared for storage or administration that include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985), hereby incorporated by reference herein. For example, preservatives, stabilizers, dyes and flavoring agents can be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents can be used.

A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize. generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer.

The nucleic acid molecules of the invention and formulations thereof can be administered orally, topically, parenterally, by inhalation or spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and/or vehicles. The term parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like. In addition, there is provided a pharmaceutical formulation comprising a nucleic acid molecule of the invention and a pharmaceutically acceptable carrier. One or more nucleic acid molecules of the invention can be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients. The pharmaceutical compositions containing nucleic acid molecules of the invention can be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.

Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients can be, for example, inert diluents; such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets can be uncoated or they can be coated by known techniques. In some cases such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in a mixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents can be added to provide palatable oral preparations. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents or suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, can also be present.

Pharmaceutical compositions of the invention can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions can also contain sweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations can also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The nucleic acid molecules of the invention can also be administered in the form of suppositories, e.g., for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols.

Nucleic acid molecules of the invention can be administered parenterally in a sterile medium. The drug, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle.

Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per subject per day). The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration. Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient.

It is understood that the specific dose level for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

For administration to non-human animals, the composition can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water.

The nucleic acid molecules of the present invention can also be administered to a subject in combination with other therapeutic compounds to increase the overall therapeutic effect. The use of multiple compounds to treat an indication can increase the beneficial effects while reducing the presence of side effects.

In one embodiment, the invention comprises compositions suitable for administering nucleic acid molecules of the invention to specific cell types. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu, 1987, J. Biol. Chem. 262, 4429-4432) is unique to hepatocytes and binds branched galactose-terminal glycoproteins, such as asialoorosomucoid (ASOR). In another example, the folate receptor is overexpressed in many cancer cells. Binding of such glycoproteins, synthetic glycoconjugates, or folates to the receptor takes place with an affinity that strongly depends on the degree of branching of the oligosaccharide chain, for example, triatennary structures are bound with greater affinity than biatenarry or monoatennary chains (Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al., 1982, J. Biol. Chem., 257, 939-945). Lee and Lee, 1987, Glycoconjugate J., 4, 317-328, obtained this high specificity through the use of N-acetyl-D-galactosamine as the carbohydrate moiety, which has higher affinity for the receptor, compared to galactose. This “clustering effect” has also been described for the binding and uptake of mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom et al., 1981, J. Med. Chem., 24, 1388-1395). The use of galactose, galactosamine, or folate based conjugates to transport exogenous compounds across cell membranes can provide a targeted delivery approach to, for example, the treatment of liver disease, cancers of the liver, or other cancers. The use of bioconjugates can also provide a reduction in the required dose of therapeutic compounds required for treatment. Furthermore, therapeutic bioavailability, pharmacodynamics, and pharmacokinetic parameters can be modulated through the use of nucleic acid bioconjugates of the invention. Non-limiting examples of such bioconjugates are described in Vargeese et al., U.S. Ser. No. 10/201,394, filed Aug. 13, 2001; and Matulic-Adamic et al., U.S. Ser. No. 60/362,016, filed Mar. 6, 2002.

Alternatively, certain siNA molecules of the instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci., USA 83, 399; Scanlon et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Dropulic et al., 1992, J. Virol., 66, 1432-41; Weerasinghe et al., 1991, J. Virol., 65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Sarver et al., 1990 Science, 247, 1222-1225; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Good et al., 1997, gene Therapy, 4, 45. Those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector. The activity of such nucleic acids can be augmented by their release from the primary transcript by a enzymatic nucleic acid (Draper et al., PCT WO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992, Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al., 1993, Nucleic Acids Res., 21, 3249-55; Chowrira et al., 1994, J. Biol. Chem., 269, 25856.

In another aspect of the invention, RNA molecules of the present invention can be expressed from transcription units (see for example Couture et al., 1996, TIG., 12, 510) inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. In another embodiment, pol III based constructs are used to express nucleic acid molecules of the invention (see for example Thompson, U.S. Pats. Nos. 5,902,880 and 6,146,886). The recombinant vectors capable of expressing the siNA molecules can be delivered as described above, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of nucleic acid molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the siNA molecule interacts with the target mRNA and generates an RNAi response. Delivery of siNA molecule expressing vectors can be systemic, such as by intravenous or intra-muscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell (for a review see Couture et al, 1996, TIG., 12, 510).

In one aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the instant invention. The expression vector can encode one or both strands of a siNA duplex, or a single self-complementary strand that self hybridizes into a siNA duplex. The nucleic acid sequences encoding the siNA molecules of the instant invention can be operably linked in a manner that allows expression of the siNA molecule (see for example Paul et al., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500; and Novina et al., 2002, Nature Medicine, advance online publication doi: 10.1038/nm725).

In another aspect, the invention features an expression vector comprising: a) a transcription initiation region (e.g., eukaryotic pol I, II or III initiation region); b) a transcription termination region (e.g., eukaryotic pol I, II or III termination region); and c) a nucleic acid sequence encoding at least one of the siNA molecules of the instant invention, wherein said sequence is operably linked to said initiation region and said termination region in a manner that allows expression and/or delivery of the siNA molecule. The vector can optionally include an open reading frame (ORF) for a protein operably linked on the 5′ side or the 3′-side of the sequence encoding the siNA of the invention; and/or an intron (intervening sequences).

Transcription of the siNA molecule sequences can be driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters are expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type depends on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. US A, 87, 6743-7; Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993, Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol. Cell. Biol., 10, 4529-37). Several investigators have demonstrated that nucleic acid molecules expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 6340-4; L'Huillier et al., 1992, EMBO J., 11, 4411-8; Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. U.S.A., 90, 8000-4; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech, 1993, Science, 262, 1566). More specifically, transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful in generating high concentrations of desired RNA molecules such as siNA in cells (Thompson et al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al., 1994, Nucleic Acid Res., 22, 2830; Noonberg et al., U.S. Pat. No. 5,624,803; Good et al., 1997, gene Ther., 4, 45; Beigelman et al., International PCT Publication No. WO 96/18736. The above siNA transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors) (for a review see Couture and Stinchcomb, 1996, supra).

In another aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the siNA molecules of the invention in a manner that allows expression of that siNA molecule. The expression vector comprises in one embodiment; a) a transcription initiation region; b) a transcription termination region; and c) a nucleic acid sequence encoding at least one strand of the siNA molecule, wherein the sequence is operably linked to the initiation region and the termination region in a manner that allows expression and/or delivery of the siNA molecule.

In another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; and d) a nucleic acid sequence encoding at least one strand of a siNA molecule, wherein the sequence is operably linked to the 3′-end of the open reading frame and wherein the sequence is operably linked to the initiation region, the open reading frame and the termination region in a manner that allows expression and/or delivery of the siNA molecule. In yet another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; and d) a nucleic acid sequence encoding at least one siNA molecule, wherein the sequence is operably linked to the initiation region, the intron and the termination region in a manner which allows expression and/or delivery of the nucleic acid molecule.

In another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; and e) a nucleic acid sequence encoding at least one strand of a siNA molecule, wherein the sequence is operably linked to the 3′-end of the open reading frame and wherein the sequence is operably linked to the initiation region, the intron, the open reading frame and the termination region in a manner which allows expression and/or delivery of the siNA molecule.

HCV Biology and Biochemistry

In 1989, the Hepatitis C Virus (HCV) was determined to be an RNA virus and was identified as the causative agent of most non-A non-B viral Hepatitis (Choo et al., 1989, Science, 244, 359-362). Unlike retroviruses such as HIV, HCV does not go though a DNA replication phase and no integrated forms of the viral genome into the host chromosome have been detected (Houghton et al., 1991, Hepatology, 14, 381-388). Rather, replication of the coding (plus) strand is mediated by the production of a replicative (minus) strand leading to the generation of several copies of plus strand HCV RNA. The genome consists of a single, large, open-reading frame that is translated into a polyprotein (Kato et al., 1991, FEBS Letters, 280: 325-328). This polyprotein subsequently undergoes post-translational cleavage, producing several viral proteins (Leinbach et al., 1994, Virology, 204:163-169).

Examination of the 9.5-kilobase genome of HCV has demonstrated that the viral nucleic acid can mutate at a high rate (Smith et al., 1997 Mol. Evol. 45, 238-246). This rate of mutation has led to the evolution of several distinct genotypes of HCV that share approximately 70% sequence identity (Simmonds et al., 1994, J. Gen. Virol. 75, 1053-1061). It is important to note that these sequences are evolutionarily quite distant. For example, the genetic identity between humans and primates such as the chimpanzee is approximately 98%. In addition, it has been demonstrated that an HCV infection in an individual patient is composed of several distinct and evolving quasispecies that have 98% identity at the RNA level. Thus, the HCV genome is hypervariable and continuously changing. Although the HCV genome is hypervariable, there are 3 regions of the genome that are highly conserved. These conserved sequences occur in the 5′ and 3′ non-coding regions as well as the 5′-end of the core protein coding region and are thought to be vital for HCV RNA replication as well as translation of the HCV polyprotein. Thus, therapeutic agents that target these conserved HCV genomic regions may have a significant impact over a wide range of HCV genotypes. Moreover, it is unlikely that drug resistance will occur with enzymatic nucleic acids specific to conserved regions of the HCV genome. In contrast, therapeutic modalities that target inhibition of enzymes such as the viral proteases or helicase are likely to result in the selection for drug resistant strains since the RNA for these viral encoded enzymes is located in the hypervariable portion of the HCV genome.

After initial exposure to HCV, a patient experiences a transient rise in liver enzymes, which indicates that inflammatory processes are occurring (Alter et al., IN: Seeff L B, Lewis J H, eds. Current Perspectives in Hepatology. New York: Plenum Medical Book Co; 1989:83-89). This elevation in liver enzymes occurs at least 4 weeks after the initial exposure and may last for up to two months (Farci et al., 1991, New England Journal of Medicine. 325, 98-104). Prior to the rise in liver enzymes, it is possible to detect HCV RNA in the patient's serum using RT-PCR analysis (Takahashi et al., 1993, American Journal of Gastroenterology. 88, 240-243). This stage of the disease is called the acute stage and usually goes undetected since 75% of patients with acute viral hepatitis from HCV infection are asymptomatic. The remaining 25% of these patients develop jaundice or other symptoms of hepatitis.

Although acute HCV infection is a benign disease, as many as 80% of acute HCV patients progress to chronic liver disease as evidenced by persistent elevation of serum alanine aminotransferase (ALT) levels and by continual presence of circulating HCV RNA (Sherlock, 1992, Lancet, 339, 802). The natural progression of chronic HCV infection over a 10 to 20 year period leads to cirrhosis in 20 to 50% of patients (Davis et al., 1993, Infectious Agents and Disease, 2, 150, 154) and progression of HCV infection to hepatocellular carcinoma has been well documented (Liang et al., 1993, Hepatology. 18, 1326-1333; Tong et al., 1994, Western Journal of Medicine, 160, 133-138). There have been no studies that have determined sub-populations that are most likely to progress to cirrhosis and/or hepatocellular carcinoma, thus all patients have equal risk of progression.

It is important to note that the survival for patients diagnosed with hepatocellular carcinoma is only 0.9 to 12.8 months from initial diagnosis (Takahashi et al., 1993, American Journal of Gastroenterology. 88, 240-243). Treatment of hepatocellular carcinoma with chemotherapeutic agents has not proven effective and only 10% of patients will benefit from surgery due to extensive tumor invasion of the liver (Trinchet et al., 1994, Presse Medicine. 23, 831-833). Given the aggressive nature of primary hepatocellular carcinoma, the only viable treatment alternative to surgery is liver transplantation (Pichlmayr et al., 1994, Hepatology. 20, 33S-40S).

Upon progression to cirrhosis, patients with chronic HCV infection present with clinical features, which are common to clinical cirrhosis regardless of the initial cause (D'Amico et al., 1986, Digestive Diseases and Sciences. 31, 468-475). These clinical features may include: bleeding esophageal varices, ascites, jaundice, and encephalopathy (Zakim D, Boyer T D. Hepatology a textbook of liver disease. Second Edition Volume 1. 1990 W.B. Saunders Company. Philadelphia). In the early stages of cirrhosis, patients are classified as compensated, the stage at which the patient's liver is still able to detoxify metabolites in the blood-stream although liver tissue damage has occurred. In addition, most patients with compensated liver disease are asymptomatic and the minority with symptoms report only minor symptoms, such as dyspepsia and weakness. In the later stages of cirrhosis, patients are classified as decompensated, the stage at which the ability of the liver to detoxify metabolites in the bloodstream is diminished. It is at the decompensated stage that the clinical features described above present.

In 1986, D'Amico et al. described the clinical manifestations and survival rates in 1155 patients with both alcoholic and viral associated cirrhosis (D'Amico supra). Of the 1155 patients, 435 (37%) had compensated disease although 70% were asymptomatic at the beginning of the study. The remaining 720 patients (63%) had decompensated liver disease with 78% presenting with a history of ascites, 31% with jaundice, 17% had bleeding and 16% had encephalopathy. Hepatocellular carcinoma was observed in six (0.5%) patients with compensated disease and in 30 (2.6%) patients with decompensated disease.

Over the course of six years, the patients with compensated cirrhosis developed clinical features of decompensated disease at a rate of 10% per year. In most cases, ascites was the first presentation of decompensation. In addition, hepatocellular carcinoma developed in 59 patients who initially presented with compensated disease by the end of the six-year study.

With respect to survival, the D'Amico study indicated that the five-year survival rate for all patients in the study was only 40%. The six-year survival rate for the patients who initially had compensated cirrhosis was 54% while the six-year survival rate for patients who initially presented with decompensated disease was only 21%. There were no significant differences in the survival rates between the patients who had alcoholic cirrhosis and the patients with viral related cirrhosis. The major causes of death for the patients in the D'Amico study were liver failure in 49%; hepatocellular carcinoma in 22%; and bleeding in 13% (D'Amico supra).

Chronic Hepatitis C is a slowly progressing inflammatory disease of the liver, mediated by a virus (HCV) that can lead to cirrhosis, liver failure and/or hepatocellular carcinoma over a period of 10 to 20 years. In the US, it is estimated that infection with HCV accounts for 50,000 new cases of acute hepatitis in the United States each year (NIH Consensus Development Conference Statement on Management of Hepatitis C March 1997). The prevalence of HCV in the United States is estimated at 1.8% and the CDC places the number of chronically infected Americans at approximately 4.5 million people. The CDC also estimates that up to 10,000 deaths per year are caused by chronic HCV infection.

Numerous well controlled clinical trials using interferon (IFN-alpha) in the treatment of chronic HCV infection have demonstrated that treatment three times a week results in lowering of serum ALT values in approximately 50% (40%-70%) of patients by the end of 6 months of therapy (Davis et al., 1989, New England Journal of Medicine, 321, 1501-1506; Marcellin et al., 1991, Hepatology, 13, 393-397; Tong et al., 1997, Hepatology, 26, 747-754; Tong et al., 1997, Hepatology, 26, 1640-1645). However, following cessation of interferon treatment, approximately 50% of the responding patients relapsed, resulting in a “durable” response rate as assessed by normalization of serum ALT concentrations of approximately 20-25%.

Direct measurement of HCV RNA is possible through use of either the branched-DNA or Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) analysis. In general, RT-PCR methodology is more sensitive and leads to a more accurate assessment of the clinical course (Tong et al., supra). Studies that have examined six months of type 1 interferon therapy using changes in HCV RNA values as a clinical endpoint have demonstrated that up to 35% of patients have a loss of HCV RNA by the end of therapy (Marcellin et al., supra). However, as with the ALT endpoint, about 50% of the patients relapse within six months following cessation of therapy, resulting in a durable virologic response of only 12% (Marcellin et al., supra). Studies that have examined 48 weeks of therapy have demonstrated that the sustained virological response is up to 25% (NIH consensus statement: 1997). Thus, standard of care for treatment of chronic HCV infection with type 1 interferon is now 48 weeks of therapy using changes in HCV RNA concentrations as the primary assessment of efficacy (Hoofnagle et al., 1997, New England Journal of Medicine, 336, 347-356).

Side effects resulting from treatment with type 1 interferons can be divided into four general categories, which include: (1) Influenza-like symptoms; (2) Neuropsychiatric; (3) Laboratory abnormalities; and (4) Miscellaneous (Dusheiko et al., 1994, Journal of Viral Hepatitis, 1, 3-5). Examples of influenza-like symptoms include fatigue, fever, myalgia, malaise, appetite loss, tachycardia, rigors, headache, and arthralgias. The influenza-like symptoms are usually short-lived and tend to abate after the first four weeks of dosing (Dushieko et al., supra). Neuropsychiatric side effects include irritability, apathy, mood changes, insomnia, cognitive changes, and depression. The most important of these neuropsychiatric side effects is depression and patients who have a history of depression should not be given type 1 interferon. Laboratory abnormalities include reduction in myeloid cells, including granulocytes, platelets and to a lesser extent red blood cells. These changes in blood cell counts rarely lead to any significant clinical sequellae (Dushieko et al., supra). In addition, increases in triglyceride concentrations and elevations in serum alanine and aspartate aminotransferase concentration have been observed. Finally, thyroid abnormalities have been reported. These thyroid abnormalities are usually reversible after cessation of interferon therapy and can be controlled with appropriate medication while on therapy. Miscellaneous side effects include nausea, diarrhea, abdominal and back pain, pruritus, alopecia, and rhinorrhea. In general, most side effects will abate after 4 to 8 weeks of therapy (Dushieko et al., supra).

The use of small interfering nucleic acid molecules targeting HCV genes and cellular/host gene targets associated with the HIV life cycle therefore provides a class of novel therapeutic agents that can be used in the treatment and diagnosis of HCV infection, liver failure, hepatocellular carcinoma, cirrhosis or any other disease or condition that responds to modulation (e.g., inhibition) of HCV genes in a subject or organism.

EXAMPLES

The following are non-limiting examples showing the selection, isolation, synthesis and activity of nucleic acids of the instant invention.

Example 1
Tandem Synthesis of siNA Constructs

Exemplary siNA molecules of the invention are synthesized in tandem using a cleavable linker, for example, a succinyl-based linker. Tandem synthesis as described herein is followed by a one-step purification process that provides RNAi molecules in high yield. This approach is highly amenable to siNA synthesis in support of high throughput RNAi screening, and can be readily adapted to multi-column or multi-well synthesis platforms.

After completing a tandem synthesis of a siNA oligo and its complement in which the 5′-terminal dimethoxytrityl (5′-O-DMT) group remains intact (trityl on synthesis), the oligonucleotides are deprotected as described above. Following deprotection, the siNA sequence strands are allowed to spontaneously hybridize. This hybridization yields a duplex in which one strand has retained the 5′-O-DMT group while the complementary strand comprises a terminal 5′-hydroxyl. The newly formed duplex behaves as a single molecule during routine solid-phase extraction purification (Trityl-On purification) even though only one molecule has a dimethoxytrityl group. Because the strands form a stable duplex, this dimethoxytrityl group (or an equivalent group, such as other trityl groups or other hydrophobic moieties) is all that is required to purify the pair of oligos, for example, by using a C18 cartridge.

Standard phosphoramidite synthesis chemistry is used up to the point of introducing a tandem linker, such as an inverted deoxy abasic succinate or glyceryl succinate linker (see FIG. 1) or an equivalent cleavable linker. A non-limiting example of linker coupling conditions that can be used includes a hindered base such as diisopropylethylamine (DIPA) and/or DMAP in the presence of an activator reagent such as Bromotripyrrolidinophosphoniumhexafluororophosphate (PyBrOP). After the linker is coupled, standard synthesis chemistry is utilized to complete synthesis of the second sequence leaving the terminal the 5′-O-DMT intact. Following synthesis, the resulting oligonucleotide is deprotected according to the procedures described herein and quenched with a suitable buffer, for example with 50 mM NaOAc or 1.5M NH₄H₂CO₃.

Purification of the siNA duplex can be readily accomplished using solid phase extraction, for example, using a Waters C18 SepPak 1 g cartridge conditioned with 1 column volume (CV) of acetonitrile, 2 CV H2O, and 2 CV 50 mM NaOAc. The sample is loaded and then washed with 1 CV H2O or 50 mM NaOAc. Failure sequences are eluted with 1 CV 14% ACN (Aqueous with 50 mM NaOAc and 50 mM NaCl). The column is then washed, for example with 1 CV H2O followed by on-column detritylation, for example by passing 1 CV of 1% aqueous trifluoroacetic acid (TFA) over the column, then adding a second CV of 1% aqueous TFA to the column and allowing to stand for approximately 10 minutes. The remaining TFA solution is removed and the column washed with H2O followed by 1 CV 1M NaCl and additional H2O. The siNA duplex product is then eluted, for example, using 1 CV 20% aqueous CAN.

FIG. 2 provides an example of MALDI-TOF mass spectrometry analysis of a purified siNA construct in which each peak corresponds to the calculated mass of an individual siNA strand of the siNA duplex. The same purified siNA provides three peaks when analyzed by capillary gel electrophoresis (CGE), one peak presumably corresponding to the duplex siNA, and two peaks presumably corresponding to the separate siNA sequence strands. Ion exchange HPLC analysis of the same siNA contract only shows a single peak. Testing of the purified siNA construct using a luciferase reporter assay described below demonstrated the same RNAi activity compared to siNA constructs generated from separately synthesized oligonucleotide sequence strands.

Example 2
Identification of Potential siNA Target Sites in any RNA Sequence

The sequence of an RNA target of interest, such as a human mRNA transcript (e.g., any of sequences referred to herein by GenBank Accession Number), is screened for target sites, for example by using a computer folding algorithm. In a non-limiting example, the sequence of a gene or RNA gene transcript derived from a database, such as Genbank, is used to generate siNA targets having complementarity to the target. Such sequences can be obtained from a database, or can be determined experimentally as known in the art. Target sites that are known, for example, those target sites determined to be effective target sites based on studies with other nucleic acid molecules, for example ribozymes or antisense, or those targets known to be associated with a disease, trait, or condition such as those sites containing mutations or deletions, can be used to design siNA molecules targeting those sites. Various parameters can be used to determine which sites are the most suitable target sites within the target RNA sequence. These parameters include but are not limited to secondary or tertiary RNA structure, the nucleotide base composition of the target sequence, the degree of homology between various regions of the target sequence, or the relative position of the target sequence within the RNA transcript. Based on these determinations, any number of target sites within the RNA transcript can be chosen to screen siNA molecules for efficacy, for example by using in vitro RNA cleavage assays, cell culture, or animal models. In a non-limiting example, anywhere from 1 to 1000 target sites are chosen within the transcript based on the size of the siNA construct to be used. High throughput screening assays can be developed for screening siNA molecules using methods known in the art, such as with multi-well or multi-plate assays to determine efficient reduction in target gene expression.

Example 3
Selection of siNA Molecule Target Sites in a RNA

The following non-limiting steps can be used to carry out the selection of siNAs targeting a given gene sequence or transcript.

1. The target sequence is parsed in silico into a list of all fragments or subsequences of a particular length, for example 23 nucleotide fragments, contained within the target sequence. This step is typically carried out using a custom Perl script, but commercial sequence analysis programs such as Oligo, MacVector, or the GCG Wisconsin Package can be employed as well.

2. In some instances the siNAs correspond to more than one target sequence; such would be the case for example in targeting different transcripts of the same gene, targeting different transcripts of more than one gene, or for targeting both the human gene and an animal homolog. In this case, a subsequence list of a particular length is generated for each of the targets, and then the lists are compared to find matching sequences in each list. The subsequences are then ranked according to the number of target sequences that contain the given subsequence; the goal is to find subsequences that are present in most or all of the target sequences. Alternately, the ranking can identify subsequences that are unique to a target sequence, such as a mutant target sequence. Such an approach would enable the use of siNA to target specifically the mutant sequence and not effect the expression of the normal sequence.

3. In some instances the siNA subsequences are absent in one or more sequences while present in the desired target sequence; such would be the case if the siNA targets a gene with a paralogous family member that is to remain untargeted. As in case 2 above, a subsequence list of a particular length is generated for each of the targets, and then the lists are compared to find sequences that are present in the target gene but are absent in the untargeted paralog.

4. The ranked siNA subsequences can be further analyzed and ranked according to GC content. A preference can be given to sites containing 30-70% GC, with a further preference to sites containing 40-60% GC.

5. The ranked siNA subsequences can be further analyzed and ranked according to self-folding and internal hairpins. Weaker internal folds are preferred; strong hairpin structures are to be avoided.

6. The ranked siNA subsequences can be further analyzed and ranked according to whether they have runs of GGG or CCC in the sequence. GGG (or even more Gs) in either strand can make oligonucleotide synthesis problematic and can potentially interfere with RNAi activity, so it is avoided whenever better sequences are available. CCC is searched in the target strand because that will place GGG in the antisense strand.

7. The ranked siNA subsequences can be further analyzed and ranked according to whether they have the dinucleotide UU (uridine dinucleotide) on the 3′-end of the sequence, and/or AA on the 5′-end of the sequence (to yield 3′ UU on the antisense sequence). These sequences allow one to design siNA molecules with terminal TT thymidine dinucleotides.

8. Four or five target sites are chosen from the ranked list of subsequences as described above. For example, in subsequences having 23 nucleotides, the right 21 nucleotides of each chosen 23-mer subsequence are then designed and synthesized for the upper (sense) strand of the siNA duplex, while the reverse complement of the left 21 nucleotides of each chosen 23-mer subsequence are then designed and synthesized for the lower (antisense) strand of the siNA duplex (see Table II). If terminal TT residues are desired for the sequence (as described in paragraph 7), then the two 3′ terminal nucleotides of both the sense and antisense strands are replaced by TT prior to synthesizing the oligos.

9. The siNA molecules are screened in an in vitro, cell culture or animal model system to identify the most active siNA molecule or the most preferred target site within the target RNA sequence.

10. Other design considerations can be used when selecting target nucleic acid sequences, see, for example, Reynolds et al., 2004, Nature Biotechnology Advanced Online Publication, 1 Feb. 2004, doi:10.1038/nbt936 and Ui-Tei et al., 2004, Nucleic Acids Research, 32, doi:10.1093/nar/gkh247.

In an alternate approach, a pool of siNA constructs specific to a target sequence is used to screen for target sites in cells expressing target RNA, such as cultured Jurkat, HeLa, A549 or 293T cells. The general strategy used in this approach is shown in FIG. 9. Cells expressing the target RNA are transfected with the pool of siNA constructs and cells that demonstrate a phenotype associated with target inhibition are sorted. The pool of siNA constructs can be expressed from transcription cassettes inserted into appropriate vectors (see for example FIG. 7 and FIG. 8). The siNA from cells demonstrating a positive phenotypic change (e.g., decreased proliferation, decreased target mRNA levels or decreased target protein expression), are sequenced to determine the most suitable target site(s) within the target target RNA sequence.

In one embodiment, siNA molecules of the invention are selected using the following methodology. The following guidelines were compiled to predict hyper-active siNAs that contain chemical modifications described herein. These rules emerged from a comparative analysis of hyper-active (>75% knockdown of target mRNA levels) and inactive (<75% knockdown of target mRNA levels) siNAs against several different targets. A total of 242 siNA sequences were analyzed. Thirty-five siNAs out of 242 siNAs were grouped into hyper-active and the remaining siNAs were grouped into inactive groups. The hyper-active siNAs clearly showed a preference for certain bases at particular nucleotide positions within the siNA sequence. For example, A or U nucleobase was overwhelmingly present at position 19 of the sense strand in hyper-active siNAs and opposite was true for inactive siNAs. There was also a pattern of a A/U rich (3 out of 5 bases as A or U) region between positions 15-19 and G/C rich region between positions 1-5 (3 out of 5 bases as G or C) of the sense strand in hyperactive siNAs. As shown in Table VII, 12 such patterns were identified that were characteristics of hyper-active siNAs. It is to be noted that not every pattern was present in each hyper-active siNA. Thus, to design an algorithm for predicting hyper-active siNAs, a different score was assigned for each pattern. Depending on how frequently such patterns occur in hyper-active siNAs versus inactive siNAs, the design parameters were assigned a score with the highest being 10. If a certain nucleobase is not preferred at a position, then a negative score was assigned. For example, at positions 9 and 13 of the sense strand, a G nucleotide was not preferred in hyper-active siNAs and therefore they were given score of −3(minus 3). The differential score for each pattern is given in Table VII. The pattern #4 was given a maximum score of −100. This is mainly to weed out any sequence that contains string of 4Gs or 4Cs as they can be highly incompatible for synthesis and can allow sequences to self-aggregate, thus rendering the siNA inactive. Using this algorithm, the highest score possible for any siNA is 66. As there are numerous siNA sequences possible against any given target of reasonable size (1000 nucleotides), this algorithm is useful to generate hyper-active siNAs.

In one embodiment, rules 1-11 shown in Table VII are used to generate active siNA molecules of the invention. In another embodiment, rules 1-12 shown in Table VII are used to generate active siNA molecules of the invention.

Example 4
siNA Design

siNA target sites were chosen by analyzing sequences of the target and optionally prioritizing the target sites on the basis of the rules presented in Example 3 above, and alternately on the basis of folding (structure of any given sequence analyzed to determine siNA accessibility to the target), or by using a library of siNA molecules as described in Example 3, or alternately by using an in vitro siNA system as described in Example 6 herein. siNA molecules were designed that could bind each target and are selected using the algorithm above and are optionally individually analyzed by computer folding to assess whether the siNA molecule can interact with the target sequence. Varying the length of the siNA molecules can be chosen to optimize activity. generally, a sufficient number of complementary nucleotide bases are chosen to bind to, or otherwise interact with, the target RNA, but the degree of complementarity can be modulated to accommodate siNA duplexes or varying length or base composition. By using such methodologies, siNA molecules can be designed to target sites within any known RNA sequence, for example those RNA sequences corresponding to the any gene transcript.

Target sequences are analysed to generate targets from which double stranded siNA are designed (Table II). To generate synthetic siNA constructs, the algorithm described in Example 3 is utilized to pick active double stranded constructs and chemically modified versions thereof. For example, in Table II, the target sequence is shown, along with the upper (sense strand) and lower (antisense strand) of the siNA duplex. Multifunctional siNAs are designed by searching for homologous sites between different target sequences (e.g., from about 5 to about 15 nucleotide regions of shared homology) and allowing for non-canonical base pairs (e.g. G:U wobble base pairing) or mismatched base pairs.

Chemically modified siNA constructs were designed as described herein (see for example Table III) to provide nuclease stability for systemic administration in vivo and/or improved pharmacokinetic, localization, and delivery properties while preserving the ability to mediate RNAi activity. Chemical modifications as described herein are introduced synthetically using synthetic methods described herein and those generally known in the art. The synthetic siNA constructs are then assayed for nuclease stability in serum and/or cellular/tissue extracts (e.g. liver extracts). The synthetic siNA constructs are also tested in parallel for RNAi activity using an appropriate assay, such as a luciferase reporter assay as described herein or another suitable assay that can quantity RNAi activity. Synthetic siNA constructs that possess both nuclease stability and RNAi activity can be further modified and re-evaluated in stability and activity assays. The chemical modifications of the stabilized active siNA constructs can then be applied to any siNA sequence targeting any chosen RNA and used, for example, in target screening assays to pick lead siNA compounds for therapeutic development (see for example FIG. 11).

Example 5
Chemical Synthesis and Purification of siNA

siNA molecules can be designed to interact with various sites in the RNA message, for example, target sequences within the RNA sequences described herein. The sequence of one strand of the siNA molecule(s) is complementary to the target site sequences described above. The siNA molecules can be chemically synthesized using methods described herein. Inactive siNA molecules that are used as control sequences can be synthesized by scrambling the sequence of the siNA molecules such that it is not complementary to the target sequence. generally, siNA constructs can by synthesized using solid phase oligonucleotide synthesis methods as described herein (see for example Usman et al., U.S. Pat. Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098; 6,362,323; 6,437,117; 6,469,158; Scaringe et al., U.S. Pat. Nos. 6,111,086; 6,008,400; 6,111,086 all incorporated by reference herein in their entirety).

In a non-limiting example, RNA oligonucleotides are synthesized in a stepwise fashion using the phosphoramidite chemistry as is known in the art. Standard phosphoramidite chemistry involves the use of nucleosides comprising any of 5′-O-dimethoxytrityl, 2′-O-tert-butyldimethylsilyl, 3′-O-2-Cyanoethyl N,N-diisopropylphosphoroamidite groups, and exocyclic amine protecting groups (e.g. N6-benzoyl adenosine, N4 acetyl cytidine, and N2-isobutyryl guanosine). Alternately, 2′-O—Silyl Ethers can be used in conjunction with acid-labile 2′-O-orthoester protecting groups in the synthesis of RNA as described by Scaringe supra. Differing 2′ chemistries can require different protecting groups, for example 2′-deoxy-2′-amino nucleosides can utilize N-phthaloyl protection as described by Usman et al., U.S. Pat. No. 5,631,360, incorporated by reference herein in its entirety).

During solid phase synthesis, each nucleotide is added sequentially (3′- to 5′-direction) to the solid support-bound oligonucleotide. The first nucleoside at the 3′-end of the chain is covalently attached to a solid support (e.g., controlled pore glass or polystyrene) using various linkers. The nucleotide precursor, a ribonucleoside phosphoramidite, and activator are combined resulting in the coupling of the second nucleoside phosphoramidite onto the 5′-end of the first nucleoside. The support is then washed and any unreacted 5′-hydroxyl groups are capped with a capping reagent such as acetic anhydride to yield inactive 5′-acetyl moieties. The trivalent phosphorus linkage is then oxidized to a more stable phosphate linkage. At the end of the nucleotide addition cycle, the 5′-O-protecting group is cleaved under suitable conditions (e.g., acidic conditions for trityl-based groups and fluoride for silyl-based groups). The cycle is repeated for each subsequent nucleotide.

Modification of synthesis conditions can be used to optimize coupling efficiency, for example by using differing coupling times, differing reagent/phosphoramidite concentrations, differing contact times, differing solid supports and solid support linker chemistries depending on the particular chemical composition of the siNA to be synthesized. Deprotection and purification of the siNA can be performed as is generally described in Usman et al., U.S. Pat. No. 5,831,071, U.S. Pat. No. 6,353,098, U.S. Pat. No. 6,437,117, and Bellon et al., U.S. Pat. No. 6,054,576, U.S. Pat. No. 6,162,909, U.S. Pat. No. 6,303,773, or Scaringe supra, incorporated by reference herein in their entireties. Additionally, deprotection conditions can be modified to provide the best possible yield and purity of siNA constructs. For example, applicant has observed that oligonucleotides comprising 2′-deoxy-2′-fluoro nucleotides can degrade under inappropriate deprotection conditions. Such oligonucleotides are deprotected using aqueous methylamine at about 35° C. for 30 minutes. If the 2′-deoxy-2′-fluoro containing oligonucleotide also comprises ribonucleotides, after deprotection with aqueous methylamine at about 35° C. for 30 minutes, TEA-HF is added and the reaction maintained at about 65° C. for an additional 15 minutes. The deprotected single strands of siNA are purified by anion exchange to achieve a high purity while maintaining high yields. To form the siNA duplex molecule the single strands are combined in equal molar ratios in a saline solution to form the duplex. The duplex siNA is concentrated and desalted by tangential filtration prior to lyophilization

Example 6
RNAi In Vitro Assay to Assess siNA Activity

An in vitro assay that recapitulates RNAi in a cell-free system is used to evaluate siNA constructs targeting target RNA targets. The assay comprises the system described by Tuschl et al., 1999, genes and Development, 13, 3191-3197 and Zamore et al., 2000, Cell, 101, 25-33 adapted for use with a target RNA. A Drosophila extract derived from syncytial blastoderm is used to reconstitute RNAi activity in vitro. Target RNA is generated via in vitro transcription from an appropriate target expressing plasmid using T7 RNA polymerase or via chemical synthesis as described herein. Sense and antisense siNA strands (for example 20 uM each) are annealed by incubation in buffer (such as 100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for 1 minute at 90° C. followed by 1 hour at 37° C., then diluted in lysis buffer (for example 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate). Annealing can be monitored by gel electrophoresis on an agarose gel in TBE buffer and stained with ethidium bromide. The Drosophila lysate is prepared using zero to two-hour-old embryos from Oregon R flies collected on yeasted molasses agar that are dechorionated and lysed. The lysate is centrifuged and the supernatant isolated. The assay comprises a reaction mixture containing 50% lysate [vol/vol], RNA (10-50 μM final concentration), and 10% [vol/vol] lysis buffer containing siNA (10 nM final concentration). The reaction mixture also contains 10 mM creatine phosphate, 10 ug/ml creatine phosphokinase, 100 um GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP, 5 mM DTT, 0.1 U/uL RNasin (Promega), and 100 uM of each amino acid. The final concentration of potassium acetate is adjusted to 100 mM. The reactions are pre-assembled on ice and preincubated at 25° C. for 10 minutes before adding RNA, then incubated at 25° C. for an additional 60 minutes. Reactions are quenched with 4 volumes of 1.25× Passive Lysis Buffer (Promega). Target RNA cleavage is assayed by RT-PCR analysis or other methods known in the art and are compared to control reactions in which siNA is omitted from the reaction.

Alternately, internally-labeled target RNA for the assay is prepared by in vitro transcription in the presence of [alpha-³²P] CTP, passed over a G50 Sephadex column by spin chromatography and used as target RNA without further purification. Optionally, target RNA is 5′-³²P-end labeled using T4 polynucleotide kinase enzyme. Assays are performed as described above and target RNA and the specific RNA cleavage products generated by RNAi are visualized on an autoradiograph of a gel. The percentage of cleavage is determined by PHOSPHOR IMAGER® (autoradiography) quantitation of bands representing intact control RNA or RNA from control reactions without siNA and the cleavage products generated by the assay.

In one embodiment, this assay is used to determine target sites in the target RNA target for siNA mediated RNAi cleavage, wherein a plurality of siNA constructs are screened for RNAi mediated cleavage of the target RNA target, for example, by analyzing the assay reaction by electrophoresis of labeled target RNA, or by northern blotting, as well as by other methodology well known in the art.

Example 7
Nucleic Acid Inhibition of HCV Target RNA In Vivo

siNA molecules targeted to the human HCV RNA are designed and synthesized as described above. These nucleic acid molecules can be tested for cleavage activity in vivo, for example, using the following procedure. The target sequences and the nucleotide location within the HCV RNA are given in Tables II and III.

Two formats are used to test the efficacy of siNAs targeting HCV. First, the reagents are tested in cell culture using, for example, human hepatoma (Huh7) cells, to determine the extent of RNA and protein inhibition. siNA reagents (e.g.; see Tables II and III) are selected against the HCV target as described herein. RNA inhibition is measured after delivery of these reagents by a suitable transfection agent to, for example, cultured epidermal keratinocytes. Relative amounts of target RNA are measured versus actin using real-time PCR monitoring of amplification (eg., ABI 7700 TAQMAN®). A comparison is made to a mixture of oligonucleotide sequences made to unrelated targets or to a randomized siNA control with the same overall length and chemistry, but randomly substituted at each position. Primary and secondary lead reagents are chosen for the target and optimization performed. After an optimal transfection agent concentration is chosen, a RNA time-course of inhibition is performed with the lead siNA molecule. In addition, a cell-plating format can be used to determine RNA inhibition.

In addition, a cell-plating format can be used to determine RNA inhibition. This system is described in Rice et al., U.S. Pat. No. 5,874,565 and U.S. Pat. No. 6,127,116, both incorporated by reference herein.

Delivery of siNA to Cells

Huh7b cells stably transfected with the HCV subgenomic replicon Clone A or Ava.5 are seeded, for example, at 8.5×10³cells per well of a 96-well platein DMEM (Gibco) the day before transfection. siNA (final concentration, for example 25 nM) and cationic lipid Lipofectamine2000 (e.g., final concentration 0.5 ul/well) are complexed in Optimem (Gibco) at 37° C. for 20 minutes in polypropylene microtubes. Following vortexing, the complexed siNA is added to each well and incubated for 24-72 hours.

TAQMAN® (Real-Time PCR Monitoring of Amplification) and Lightcycler Quantification of mRNA

Total RNA is prepared from cells following siNA delivery, for example, using Qiagen RNA purification kits for 6-well or Rneasy extraction kits for 96-well assays. For TAQMAN® analysis (real-time PCR monitoring of amplification), dual-labeled probes are synthesized with the reporter dye, FAM or JOE, covalently linked at the 5′-end and the quencher dye TAMRA conjugated to the 3′-end. One-step RT-PCR amplifications are performed on, for example, an ABI PRISM 7700 Sequence Detector using 50 μl reactions consisting of 10 μl total RNA, 100 nM forward primer, 900 nM reverse primer, 100 nM probe, 1× TaqMan PCR reaction buffer (PE-Applied Biosystems), 5.5 mM MgCl₂, 300 μM each dATP, dCTP, dGTP, and dTTP, 10 U RNase Inhibitor (Promega), 1.25 U AMPLITAQ GOLD° (DNA polymerase) (PE-Applied Biosystems) and 10 U M-MLV Reverse Transcriptase (Promega). The thermal cycling conditions can consist of 30 minutes at 48° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95° C. and 1 minute at 60° C. Quantitation of mRNA levels is determined relative to standards generated from serially diluted total cellular RNA (300, 100, 33, 11 ng/reaction) and normalizing to β-actin or GAPDH mRNA in parallel TAQMAN® reactions (real-time PCR monitoring of amplification). For each gene of interest an upper and lower primer and a fluorescently labeled probe are designed. Real time incorporation of SYBR Green I dye into a specific PCR product can be measured in glass capillary tubes using a lightcyler. A standard curve is generated for each primer pair using control cRNA. Values are represented as relative expression to GAPDH in each sample.

Western Blotting

Total RNA is prepared from cells following siNA delivery, for example, using Ambion Rnaqueous 4-PCR purification kit for large scale extractions, or Ambion Rnaqueous-96 purification kit for 96-well assays. For Taqman analysis, dual-labeled probes are synthesized with, for example, the reporter dyes FAM or VIC covalently linked at the 5′-end and the quencher dye TAMARA conjugated to the 3′-end. One-step RT-PCR amplifications are performed on, for example, an ABI PRISM 7700 Sequence detector using 50 uL reactions consisting of 10 uL total RNA, 100 nM forward primer, 100 nM reverse primer, 100 nM probe, 1× TaqMan PCR reaction buffer (PE-Applied Biosystems), 5.5 mM MgCl2, 100 uM each dATP, dCTP, dGTP and dTTP, 0.2 U RNase Inhibitor (Promega), 0.025 U AmpliTaq Gold (PE-Applied Biosystems) and 0.2 U M-MLV Reverse Transcriptase (Promega). The thermal cycling conditions can consist of 30 minutes at 48° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95° C. and 1 minute at 60° C. Quantitation of target mRNA level is determined relative to standards generated from serially diluted total cellular RNA (300, 100, 30, 10 ng/r×n) and normalizing to, for example, 36B4 mRNA in either parallel or same tube TaqMan reactions. For HCV Replicon mRNA quantitation, PCR primers and probe specific for the neomycin gene were used:

neo-forward primer,

(SEQ ID NO: 2150)

5′-CCGGCTACCTGCCCATTC-3′;

neo-reverse primer,

(SEQ ID NO: 2151)

5′-CCAGATCATCCTGATCGACAAG-3′;

neo-probe,

(SEQ ID NO: 2152)

5′FAM-ACATCGCATCGAGCGAGCACGTAC-TAMARA3′;

For normalization, 36B4 PCR primers and probe were used:

36B4-forward primer,

(SEQ ID NO: 2153)

5′-TCTATCATCAACGGGTACAAACGA-3′;

36B4 reverse primer,

(SEQ ID NO: 2154)

5′-CTTTTCAGCAAGTGGGAAGGTG-3′;

36B4 probe,

(SEQ ID NO: 2155)

5′VIC-CCTGGCCTTGTCTGTGGAGACGGATTA-TAMARA3′;

Example 8
Models Useful to Evaluate the Down-Regulation of HCV Gene Expression
Cell Culture

Although there have been reports of replication of HCV in cell culture (see below), these systems are difficult to reproduce and have proven unreliable. Therefore, as was the case for development of other anti-HCV therapeutics, such as interferon and ribavirin, after demonstration of safety in animal studies applicant can proceed directly into a clinical feasibility study.

Several recent reports have documented in vitro growth of HCV in human cell lines (Mizutani et al., Biochem Biophys Res Commun 1996 227(3):822-826; Tagawa et al., Journal of Gasteroenterology and Hepatology 1995 10(5):523-527; Cribier et al., Journal of General Virology 76(10):2485-2491; Seipp et al., Journal of General Virology 1997 78(10)2467-2478; Iacovacci et al., Research Virology 1997 148(2):147-151; Iocavacci et al., Hepatology 1997 26(5) 1328-1337; Ito et al., Journal of General Virology 1996 77(5):1043-1054; Nakajima et al., Journal of Virology 1996 70(5):3325-3329; Mizutani et al., Journal of Virology 1996 70(10):7219-7223; Valli et al., Res Virol 1995 146(4): 285-288; Kato et al., Biochem Biophys Res Comm 1995 206(3):863-869). Replication of HCV has been reported in both T and B cell lines, as well as cell lines derived from human hepatocytes. Detection of low level replication was documented using either RT-PCR based assays or the b-DNA assay. It is important to note that the most recent publications regarding HCV cell cultures document replication for up to 6-months. However, the level of HCV replication observed in these cell lines has not been robust enough for screening of antiviral compounds.

In addition to cell lines that can be infected with HCV, several groups have reported the successful transformation of cell lines with cDNA clones of full-length or partial HCV genomes (Harada et al., Journal of General Virology, 1995, 76(5)1215-1221; Haramatsu et al., Journal of Viral Hepatitis 1997 4S(1):61-67; Dash et al., American Journal of Pathology 1997 151(2):363-373; Mizuno et al., Gasteroenterology 1995 109(6):1933-40; Yoo et al., Journal Of Virology 1995 69(1):32-38).

The recent development of subgenomic HCV RNA replicons capable of successfully replicating in the human hepatoma cell line, Huh7, represents a significant advance toward a dependable cell culture model. These replicons contain the neomycin gene upstream of the HCV nonstructural genes allowing for the selection of replicative RNAs in Huh7 cells. Initially, RNA replication was detected at a low frequency (Lohmann et al. Science 1999 285: 110-113) but the identification of replicons with cell-adaptive mutations in the NS5A region has improved the efficiency of replication 10,000-fold (Blight et al. Science 2000 290:1972-1975). Steps in the HCV life cycle, such as translation, protein processing, and RNA replication are recapitulated in the subgenomic replicon systems, but early events (viral attachment and uncoating) and viral assembly is absent. Inclusion of the structural genes of HCV within the replicons results in the production of HCV core and envelope proteins, but virus assembly does not occur (Pietschmann et al. Journal of Virology 2002 76: 4008-4021). Such replicon systems have been used to study siRNA mediated inhibition of HCV RNA, see for example, Randall et al., 2003, PNAS USA, 100, 235-240.

In several cell culture systems, cationic lipids have been shown to enhance the bioavailability of oligonucleotides to cells in culture (Bennet, et al., 1992, Mol. Pharmacology, 41, 1023-1033). In one embodiment, siNA molecules of the invention are complexed with cationic lipids for cell culture experiments. siNA and cationic lipid mixtures are prepared in serum-free DMEM immediately prior to addition to the cells. DMEM plus additives are warmed to room temperature (about 20-25° C.) and cationic lipid is added to the final desired concentration and the solution is vortexed briefly. siNA molecules are added to the final desired concentration and the solution is again vortexed briefly and incubated for 10 minutes at room temperature. In dose response experiments, the RNA/lipid complex is serially diluted into DMEM following the 10 minute incubation.

Animal Models

Evaluating the efficacy of anti-HCV agents in animal models is an important prerequisite to human clinical trials. The best characterized animal system for HCV infection is the chimpanzee. Moreover, the chronic hepatitis that results from HCV infection in chimpanzees and humans is very similar. Although clinically relevant, the chimpanzee model suffers from several practical impediments that make use of this model difficult. These include high cost, long incubation requirements and lack of sufficient quantities of animals. Due to these factors, a number of groups have attempted to develop rodent models of chronic hepatitis C infection. While direct infection has not been possible, several groups have reported on the stable transfection of either portions or entire HCV genomes into rodents (Yamamoto et al., Hepatology 1995 22(3): 847-855; Galun et al., Journal of Infectious Disease 1995 172(1):25-30; Koike et al., Journal of general Virology 1995 76(12)3031-3038; Pasquinelli et al., Hepatology 1997 25(3): 719-727; Hayashi et al., Princess Takamatsu Symp 1995 25:1430149; Mariya et al., Journal of General Virology 1997 78(7) 1527-1531; Takehara et al., Hepatology 1995 21(3):746-751; Kawamura et al., Hepatology 1997 25(4): 1014-1021). In addition, transplantation of HCV infected human liver into immunocompromised mice results in prolonged detection of HCV RNA in the animal's blood.

A method for expressing hepatitis C virus in an in vivo animal model has been developed (Vierling, International PCT Publication No. WO 99/16307). Viable, HCV infected human hepatocytes are transplanted into a liver parenchyma of a scid/scid mouse host. The scid/scid mouse host is then maintained in a viable state, whereby viable, morphologically intact human hepatocytes persist in the donor tissue and hepatitis C virus is replicated in the persisting human hepatocytes. This model provides an effective means for the study of HCV inhibition by enzymatic nucleic acids in vivo.

As such, these models can be used in evaluating the efficacy of siNA molecules of the invention in inhibiting HCV expression. These models and others can similarly be used to evaluate the safety and efficacy of siNA molecules of the invention in a pre-clinical setting.

Example 9
RNAi Mediated Inhibition of Target Gene Expression

In Vitro siNA Mediated Inhibition of Target RNA

siNA constructs (Table III) are tested for efficacy in reducing HCV RNA expression in, for example, Huh7 cells. Cells are plated approximately 24 hours before transfection in 96-well plates at 5,000-7,500 cells/well, 100 μl/well, such that at the time of transfection cells are 70-90% confluent. For transfection, annealed siNAs are mixed with the transfection reagent (Lipofectamine 2000, Invitrogen) in a volume of 50 μl/well and incubated for 20 minutes at room temperature. The siNA transfection mixtures are added to cells to give a final siNA concentration of 25 nM in a volume of 150 μl. Each siNA transfection mixture is added to 3 wells for triplicate siNA treatments. Cells are incubated at 37° for 24 hours in the continued presence of the siNA transfection mixture. At 24 hours, RNA is prepared from each well of treated cells. The supernatants with the transfection mixtures are first removed and discarded, then the cells are lysed and RNA prepared from each well. Target gene expression following treatment is evaluated by RT-PCR for the target gene and for a control gene (36B4, an RNA polymerase subunit) for normalization. The triplicate data is averaged and the standard deviations determined for each treatment. Normalized data are graphed and the percent reduction of target mRNA by active siNAs in comparison to their respective inverted control siNAs is determined.

Example 10
Evaluation of siNA Molecules in Marmosets

GBV-B is very closely related to human hepatitis C virus and causes hepatitis in tamarins and marmosets. Thus, GBV-B provides a small animal model for testing antiviral compounds and vaccines for HCV infection. This study investigated the efficacy of LNP formulated double stranded siNA molecules targeting HCV RNA sites 293 and 316. The GBV-B model is an excellent system to test whether this therapy is likely to work on humans chronically infected with HCV.

In the study, 2 animals were inoculated with GBV-B and IV treatment with the active formulated siNA (Sirna Compound Nos. 33149/47677 and 31703/38756, Formulation LNP-086; see Tables III and VI) at 3 mg/kg was initiated one day post infection. Active compositions were formulated as is described in U.S. Provisional patent application No. 60/737,024, filed Nov. 15, 2005. Another 2 animals were inoculated with GBV-B and were untreated to serve as negative controls. The animals were monitored to determine the effect of the therapy of GBV-B infection. Blood draws were performed over the course of the study to determine viral titers. Dosing of formulated siNA in the treated animals was repeated at days 1, 3, and 7 after inoculation at day 0. These animals show a profound inhibition of GBV-B over a three week time course compared to the untreated control animals (see FIG. 30). In addition, an animal with established GBV infection was treated with active formulated siNA (Sirna Compound Nos. 33149/47677 and 31703/38756, Formulation LNP-086; see Tables III and VI) at days 28, 31, and 35 post infection. This animal showed a decrease in viral titer down to the limit of detection following the dosing of active compound (see FIG. 31) compared to historic untreated controls.

Example 11
Evaluation of siNA Molecules in Chimpanzees

This study is used to evaluate double stranded nucleic acid antiviral formulations for the ability to suppress hepatitis C virus (HCV) replication in HCV-infected chimpanzees. The compound formulation contains siNA directed at the HCV viral genome and that mediates degradation of the viral RNA via RNA interference. The compound is administered by IV. The chimpanzees to be used are selected from a group of HCV chronic animals. The study is conducted in two phases: pharmacokinetics and efficacy.

The pharmacokinetics portion of the study is conducted in two non-HCV-infected animals. Blood samples are obtained at time 0, 15 min, 30 min, 24 hr, and days 3, 7 and 14. Liver biopsies are obtained at 24 hr and 14 days. The efficacy involves testing of the antiviral compound in 2 or more HCV infected chimpanzees.

Animals receive 4 weekly IV injections with the antiviral siNA formulation. Blood samples are obtained at −4, −2, and 0 weeks, then weekly for 6 weeks, and then every other week for 4 additional weeks. Liver biopsies are obtained at −4 weeks and +4 weeks (one week after last injection). A blood and tissue sampling schedule is shown below. The animals are monitored for blood chemistries and CBC at all bleeds. At the sign of any serious adverse effects, treatment is stopped. For each animal, a total of 11 blood samples are requested to monitor the viral RNA levels in the serum. Liver needle biopsies are requested at two time points for analysis of viral RNA load in the liver, level of siNA compound targeted to liver, the presence of siNA induced viral RNA cleavage products, and changes in liver gene expression. Viral RNA is monitored by real time TaqMan quantitative RT-PCR. Serum samples are extracted in duplicate and run in quadruplicate. Liver RNA levels are run in duplicate.

Schedule for SINA PK Study in 2 Uninfected Chimpanzees

Days Weeks

Biopsy
Dosing IV
Serum
CBC
Chemistries
Liver

Day 0,
X
10 ml
2 ml
3 ml

Day 0, 15 min

10 ml
2 ml
3 ml

Day 0, 30 min

10 ml
2 ml
3 ml

Day 1, 24 hr

10 ml
2 ml
3 ml
X

Day 3

10 ml
2 ml
3 ml

Day 7

10 ml
2 ml
3 ml

Day 14

10 ml
2 ml
3 ml
X

Blood, 1 × SST tube to Lanford Lab. Processed in 1 ml frozen aliquots.

Biopsies frozen.

Schedule for SINA Efficacy Study in 2 HCV Chronic Chimpanzees

Weeks

Biopsy
Dosing IV
Serum
CBC
Chemistries
Liver

Pre −4

20 ml
2 ml
3 ml
X

Pre −2

20 ml
2 ml
3 ml

Day 0, Wk 0
X
20 ml
2 ml
3 ml

Day 7, Wk 1
X
20 ml
2 ml
3 ml

Wk 2
X
20 ml
2 ml
3 ml

Wk 3
X
20 ml
2 ml
3 ml

Wk 4

20 ml
2 ml
3 ml
X

Wk 5

20 ml
2 ml
3 ml

Wk 6

20 ml
2 ml
3 ml

Wk 8

20 ml
2 ml
3 ml

Wk 10

20 ml
2 ml
3 ml

Blood, 2 × SST tube to Lanford Lab. Processed in 1 ml frozen aliquots.

Biopsies frozen. Divided in half, process half for RNA with RNAzol for viral RNA.

Example 12
Safety, Tolerability, PK, PD and Anti-Viral Effects of Single and Multiple Dose Administration of SIRNA-AV34 in Interferon-Naïve and Experienced Patients with Chronic HCV Infection

The primary objective of this study are to establish the MTD of single and multiple doses of Sirna-034 in HCV-positive patients. A secondary objective is to evaluate the single dose and steady-state pharmacokinetics of Sirna-034; to evaluate the pharmacodynamics of single and multiple doses of Sirna-034; and to assess the effect of single and multiple doses of Sirna-034 on indices of viral replication and infectivity.

This is a Phase I/II, randomized, double-blind, placebo-controlled, single- and multiple-dose escalation study of Sirna-034 in patients with chronic hepatitis C infection and compensated hepatic function. Patients meeting eligibility criteria are be enrolled into 4-6 sequential groups for the SAD portion of the study. Each patient is admitted to a clinical research unit for treatment administration and discharged 36 hours after dosing unless further intensive monitoring is considered necessary by the investigator. Patients have daily visits to the clinic for five additional days, and receive a follow-up phone call for SAEs 30 days after dosing. Dose escalation is dependent on safety parameters (physical examination findings, vital signs, adverse events, and laboratory values) from the preceding group.

At screening for the SAD phase, patients undergo phlebotomy for assessment of serum chemistry, hematology, coagulation parameters, serum beta-HCG (women only), HIV antibody status, HBsAg, alpha feto-protein, quantitative HCV viral RNA and HCV genotyping, and provide urine for urinalysis. During the treatment phase, clinical laboratories (chemistry, hematology, coagulation parameters and urinalysis) are assessed pre-dose, and 24 hours, 3 d and 6 d after dosing. ECG are assessed pre-dose and 4-6 hours and 6 days after dosing. Physical exam is conducted pre-dose and 6 days after dosing. Serum samples for PK analysis are collected pre-dose, and 30 minutes, 1, 2, 3, 4, 6, 8, 12, 24 and 36 hours, and 2, 3, 4, 5, and 6 days after dosing. Assessments of viral RNA and potentially another marker of viral infectivity is performed pre-dose and 1, 2, 3, 4, 5, and 6 days after dosing.

Once the dose of Sirna-034 that is well-tolerated results in approximately 90% reduction in viral load has been determined, patients are enrolled into the MAD portion of the study. Patients from the SAD portion of the study who choose to participate in the MAD phase undergo limited re-screening consisting of interval history, physical exam, clinical laboratory assessment, and quantitative HCV viral RNA. New enrollees undergo full re-screening as outlined for the SAD phase, above. Patients enrolled in once weekly dosing regimen visit the study site weekly×4, 7 days after the last dose, and receive a telephone call for SAEs 30 d after the last dose. Patients randomized to an every other week dosing regimen visit the study site 2 times and have similar post-treatment follow-up. Dose escalation is dependent on safety parameters (physical examination findings, vital signs, adverse events, and laboratory values) from the preceding group.

During the MAD treatment phase, clinical laboratories (chemistry, hematology, coagulation parameters and urinalysis) are assessed prior to administration of each dose, and 7 days after the last dose. ECG is assessed before the first dose, and 7 days after the last dose. Physical exam is conducted pre-dose, after the last dose and 7 days after the last dose. Serum samples for PK analysis are collected before and 2, 4, 8, 12 and 24 hours after the first dose, before each subsequent dose, and 2, 4, 8, 12 and 24 hours and 7 days after the last dose. Assessments of viral RNA and potentially another marker of viral infectivity is performed prior to the first dose and then weekly up to 7 days after the last dose.

Diagnosis and Main Criteria for Inclusion/Exclusion:

Men and women who are not of child-bearing potential; 18 to 60 years of age; HCV-positive; elevated ALT at screening and on one other occasion within the prior 6 months; HIV negative; HBsAg negative; normal PT, PTT, hemoglobin, bilirubin, albumin and alpha feto-protein; platelet count >100K; no other known cause of liver disease; either interferon naïve, relapsed after interferon, or non-responder to interferon.

Dosage and Mode of Administration:

Liquid solution for IV injection, 0.1 to 10 mg/ml of Sirna-034. For the SAD portion of the study, the starting dose is TBD, but estimate 1/50 of NOAEL from 4 week monkey toxicology study. Dose to be escalated to MTD. For the MAD portion of the study, the single dose that produces 90% reduction of viral load, if well-tolerated as single doses, will be administered to sequential cohorts dosed either weekly or every other week×4 weeks.

Duration of Patient Participation/Duration of Study/Duration of Treatment:

For SAD period, duration of treatment will be 42 days (range, 38 to 49) consisting of a 1-14 day screening period, 1-day treatment with 36 hours in inpatient observation, five additional days of outpatient observation and 30 day SAE follow-up (via telephone). During the MAD phase, the same patients, as well as newly-recruited patients as necessary, will be treated for 72 days (range, 65 to 79) consisting of a 0-14 day screening period, 4 week treatment period, 7 day follow-up period and 30 day SAE follow-up (via telephone). As a result, the duration of patient participation for those patients enrolled in both phases of the study may be up to 128 days, not including any intervening periods between the SAD and MAD portions of the study.

Reference Therapy, Dosage and Mode of Administration:

Placebo will be formulated as a liquid solution for IV injection and be identical in appearance to Sirna-034.

Criteria for Evaluation

For Efficacy and Pharmacodynamics, HCV viral RNA will be evaluated possibly along with a novel biologic marker of HCV capsid protein processing or viral infectivity. For Safety, vital signs, adverse events, standard laboratory tests, and physical examinations will be monitored.

Statistical Methods

Incidence of adverse events, signs, symptoms, ECG parameters and laboratory findings; descriptive statistics of changes in ECG parameters and laboratory values, PK and PD parameters; and exploratory analyses of measures of viral load, replication and infectivity.

The main objective of the Clinical Plan is to assess the safety and efficacy of Sirna-034 as a potential treatment for Chronic Hepatitis C in such a way as to support the Target Profile (TP) and meet requirements for registration. The total number of subjects to be studied in the entire plan is about 1200.

This CP for Sirna-034 is designed to validate the TP and to meet regulatory requirements for registration as a treatment for Chronic Hepatitis C. The initial claim will be that Sirna-034 is indicated for the treatment of Chronic Hepatitis C in combination with PEG-Interferon and ribavirin in patient non responding to PEG-Interferon and ribavirin, who have compensate liver disease and 18 year of age and older.

A Phase I/II Dose Escalation study in patients with compensated and previously treated or untreated chronic hepatitis C will establish the safety of multiple dose administration (weekly ascending doses for 4 week or every two weeks for four weeks), the extent of systemic exposure to the compound, and establish a “proof of mechanism” as effective monotherapy through analysis of serum HCV RNA (reduction of 1-2 logs).

This will be followed by a formal Phase II dose finding study in combination PEG-Interferon and ribavirin in clinically active hepatitis C patients non responding to PEG-Interferon and ribavirin. The 48-week study will provide data on the safety, effectiveness and PK of the triple combination, with Sirna-034 administered weekly.

The Phase III studies will be randomized, placebo-controlled studies which will confirm evidence of efficacy and provide further safety data of the compound. Two pivotal randomized, placebo-controlled multi-national Phase III studies will be designed to confirm efficacy and provide safety data to support registration. Primary efficacy endpoints will be at 24 weeks following the end of 48-week treatment, represented by undetectable HCV RNA and normalization of ALT. At the time of registration, over 1200 patients will have been exposed to Sirna-034.

Sirna-034 is a modified anti-HCV siNA consisting of two Sirna duplexes (Sirna Compound Nos. 33149/47677 and 31703/38756, (SEQ ID NOs: 1796/2102 and 1677/2103) formulated as LNP-086, which consists of CLinDMA/DSPC/Cholesterol/PEG-DMG/Linoleyl alcohol in a molar ration of 43/38/10/2/7; see Tables III and VI) that has the potential to inhibit HCV replication. Sirna-034 targets HCV mRNA sites 293 and 316. Sirna-034 is expected to improve the patient's overall symptomatic response to conventional therapy (PEG-Interferon and ribavirin), and will provide the best opportunity to safely minimize a subject's susceptibility to the risk of the disability, morbidity and mortality caused by HCV infection.

Example 13
Indications

The present body of knowledge in HCV research indicates the need for methods to assay HCV activity and for compounds that can regulate HCV expression for research, diagnostic, and therapeutic use. As described herein, the nucleic acid molecules of the present invention can be used in assays to diagnose disease state related of HCV levels. In addition, the nucleic acid molecules can be used to treat disease state related to HCV levels.

Particular degenerative and disease states that can be associated with HCV expression modulation include, but are not limited to, HCV infection, liver failure, hepatocellular carcinoma, cirrhosis, and/or other disease states associated with HCV infection.

Example 14
Interferons

Interferons represent a non-limiting example of a class of compounds that can be used in conduction with the siNA molecules of the invention for treating the diseases and/or conditions described herein. Type I interferons (IFN) are a class of natural cytokines that includes a family of greater than 25 IFN-α (Pesta, 1986, Methods Enzymol. 119, 3-14) as well as IFN-β, and IFN-ω. Although evolutionarily derived from the same gene (Diaz et al., 1994, Genomics 22, 540-552), there are many differences in the primary sequence of these molecules, implying an evolutionary divergence in biologic activity. All type I IFN share a common pattern of biologic effects that begin with binding of the IFN to the cell surface receptor (Pfeffer & Strulovici, 1992, Transmembrane secondary messengers for IFN-α/β. In: Interferon. Principles and Medical Applications, S. Baron, D. H. Coopenhaver, F. Dianzani, W. R. Fleischmann Jr., T. K. Hughes Jr., G. R. Kimpel, D. W. Niesel, G. J. Stanton, and S. K. Tyring, eds. 151-160). Binding is followed by activation of tyrosine kinases, including the Janus tyrosine kinases and the STAT proteins, which leads to the production of several IFN-stimulated gene products (Johnson et al., 1994, Sci. Am. 270, 68-75). The IFN-stimulated gene products are responsible for the pleotropic biologic effects of type I IFN, including antiviral, antiproliferative, and immunomodulatory effects, cytokine induction, and HLA class I and class II regulation (Pestka et al., 1987, Annu. Rev. Biochem 56, 727). Examples of IFN-stimulated gene products include 2-5-oligoadenylate synthetase (2-5 OAS), β2-microglobulin, neopterin, p68 kinases, and the Mx protein (Chebath & Revel, 1992, The 2-5 A system: 2-5 A synthetase, isospecies and functions. In: Interferon. Principles and Medical Applications, S. Baron, D. H. Coopenhaver, F. Dianzani, W. R. Jr. Fleischmann, T. K. Jr Hughes, G. R. Kimpel, D. W. Niesel, G. J. Stanton, and S. K. Tyring, eds., pp. 225-236; Samuel, 1992, The RNA-dependent P1/eIF-2α protein kinase. In: Interferon. Principles and Medical Applications. S. Baron, D. H. Coopenhaver, F. Dianzani, W. R. Fleischmann Jr., T. K. Hughes Jr., G. R. Kimpel, D. W. Niesel, G. H. Stanton, and S. K. Tyring, eds. 237-250; Horisberger, 1992, Mx protein: function and Mechanism of Action. In: Interferon. Principles and Medical Applications. S. Baron, D. H. Coopenhaver, F. Dianzani, W. R. Fleischmann Jr., T. K. Hughes Jr., G. R. Kimpel, D. W. Niesel, G. H. Stanton, and S. K. Tyring, eds. 215-224). Although all type I IFN have similar biologic effects, not all the activities are shared by each type I IFN, and in many cases, the extent of activity varies quite substantially for each IFN subtype (Fish et al, 1989, J. Interferon Res. 9, 97-114; Ozes et al., 1992, J. Interferon Res. 12, 55-59). More specifically, investigations into the properties of different subtypes of IFN-α and molecular hybrids of IFN-α have shown differences in pharmacologic properties (Rubinstein, 1987, J. Interferon Res. 7, 545-551). These pharmacologic differences can arise from as few as three amino acid residue changes (Lee et al., 1982, Cancer Res. 42, 1312-1316).

Eighty-five to 166 amino acids are conserved in the known IFN-α subtypes. Excluding the IFN-α pseudogenes, there are approximately 25 known distinct IFN-α subtypes. Pairwise comparisons of these nonallelic subtypes show primary sequence differences ranging from 2% to 23%. In addition to the naturally occurring IFNs, a non-natural recombinant type I interferon known as consensus interferon (CIFN) has been synthesized as a therapeutic compound (Tong et al., 1997, Hepatology 26, 747-754).

Interferon is currently in use for at least 12 different indications, including infectious and autoimmune diseases and cancer (Borden, 1992, N. Engl. J. Med. 326, 1491-1492). For autoimmune diseases, IFN has been utilized for treatment of rheumatoid arthritis, multiple sclerosis, and Crohn's disease. For treatment of cancer, IFN has been used alone or in combination with a number of different compounds. Specific types of cancers for which IFN has been used include squamous cell carcinomas, melanomas, hypernephromas, hemangiomas, hairy cell leukemia, and Kaposi's sarcoma. In the treatment of infectious diseases, IFNs increase the phagocytic activity of macrophages and cytotoxicity of lymphocytes and inhibits the propagation of cellular pathogens. Specific indications for which IFN has been used as treatment include hepatitis B, human papillomavirus types 6 and 11 (i.e. genital warts) (Leventhal et al., 1991, N Engl J Med 325, 613-617), chronic granulomatous disease, and hepatitis C virus.

Numerous well controlled clinical trials using IFN-alpha in the treatment of chronic HCV infection have demonstrated that treatment three times a week results in lowering of serum ALT values in approximately 50% (range 40% to 70%) of patients by the end of 6 months of therapy (Davis et al., 1989, N. Engl. J. Med. 321, 1501-1506; Marcellin et al., 1991, Hepatology 13, 393-397; Tong et al., 1997, Hepatology 26, 747-754; Tong et al., Hepatology 26, 1640-1645). However, following cessation of interferon treatment, approximately 50% of the responding patients relapsed, resulting in a “durable” response rate as assessed by normalization of serum ALT concentrations of approximately 20 to 25%. In addition, studies that have examined six months of type 1 interferon therapy using changes in HCV RNA values as a clinical endpoint have demonstrated that up to 35% of patients will have a loss of HCV RNA by the end of therapy (Tong et al., 1997, supra). However, as with the ALT endpoint, about 50% of the patients relapse six months following cessation of therapy resulting in a durable virologic response of only 12% (23). Studies that have examined 48 weeks of therapy have demonstrated that the sustained virological response is up to 25%.

Pegylated interferons, i.e., interferons conjugated with polyethylene glycol (PEG), have demonstrated improved characteristics over interferon. Advantages incurred by PEG conjugation can include an improved pharmacokinetic profile compared to interferons lacking PEG, thus imparting more convenient dosing regimes, improved tolerance, and improved antiviral efficacy. Such improvements have been demonstrated in clinical studies of both polyethylene glycol interferon alfa-2a (PEGASYS, Roche) and polyethylene glycol interferon alfa-2b (VIRAFERON PEG, PEG-INTRON, Enzon/Schering Plough).

siNA molecules in combination with interferons and polyethylene glycol interferons have the potential to improve the effectiveness of treatment of HCV or any of the other indications discussed above. siNA molecules targeting RNAs associated with HCV infection can be used individually or in combination with other therapies such as interferons and polyethylene glycol interferons and to achieve enhanced efficacy.

Example 15
Multifunctional siNA Inhibition of Target RNA Expression

Multifunctional siNA Design

Once target sites have been identified for multifunctional siNA constructs, each strand of the siNA is designed with a complementary region of length, for example, of about 18 to about 28 nucleotides, that is complementary to a different target nucleic acid sequence. Each complementary region is designed with an adjacent flanking region of about 4 to about 22 nucleotides that is not complementary to the target sequence, but which comprises complementarity to the complementary region of the other sequence (see for example FIG. 16). Hairpin constructs can likewise be designed (see for example FIG. 17). Identification of complementary, palindrome or repeat sequences that are shared between the different target nucleic acid sequences can be used to shorten the overall length of the multifunctional siNA constructs (see for example FIGS. 18 and 19).

In a non-limiting example, three additional categories of additional multifunctional siNA designs are presented that allow a single siNA molecule to silence multiple targets. The first method utilizes linkers to join siNAs (or multifunctional siNAs) in a direct manner. This can allow the most potent siNAs to be joined without creating a long, continuous stretch of RNA that has potential to trigger an interferon response. The second method is a dendrimeric extension of the overlapping or the linked multifunctional design; or alternatively the organization of siNA in a supramolecular format. The third method uses helix lengths greater than 30 base pairs. Processing of these siNAs by Dicer will reveal new, active 5′ antisense ends. Therefore, the long siNAs can target the sites defined by the original 5′ ends and those defined by the new ends that are created by Dicer processing. When used in combination with traditional multifunctional siNAs (where the sense and antisense strands each define a target) the approach can be used for example to target 4 or more sites.

I. Tethered Bifunctional siNAs

The basic idea is a novel approach to the design of multifunctional siNAs in which two antisense siNA strands are annealed to a single sense strand. The sense strand oligonucleotide contains a linker (e.g., non-nucleotide linker as described herein) and two segments that anneal to the antisense siNA strands (see FIG. 22). The linkers can also optionally comprise nucleotide-based linkers. Several potential advantages and variations to this approach include, but are not limited to:

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

Dendrimer and Supramolecular siNAs

In the dendrimer siNA approach, the synthesis of siNA is initiated by first synthesizing the dendrimer template followed by attaching various functional siNAs. Various constructs are depicted in FIG. 23. The number of functional siNAs that can be attached is only limited by the dimensions of the dendrimer used.

Supramolecular Approach to Multifunctional siNA

The supramolecular format simplifies the challenges of dendrimer synthesis. In this format, the siNA strands are synthesized by standard RNA chemistry, followed by annealing of various complementary strands. The individual strand synthesis contains an antisense sense sequence of one siNA at the 5′-end followed by a nucleic acid or synthetic linker, such as hexaethyleneglyol, which in turn is followed by sense strand of another siNA in 5′ to 3′ direction. Thus, the synthesis of siNA strands can be carried out in a standard 3′ to 5′ direction. Representative examples of trifunctional and tetrafunctional siNAs are depicted in FIG. 24. Based on a similar principle, higher functionality siNA constructs can be designed as long as efficient annealing of various strands is achieved.

Dicer Enabled Multifunctional siNA

Using bioinformatic analysis of multiple targets, stretches of identical sequences shared between differing target sequences can be identified ranging from about two to about fourteen nucleotides in length. These identical regions can be designed into extended siNA helixes (e.g., >30 base pairs) such that the processing by Dicer reveals a secondary functional 5′-antisense site (see for example FIG. 25). For example, when the first 17 nucleotides of a siNA antisense strand (e.g., 21 nucleotide strands in a duplex with 3′-TT overhangs) are complementary to a target RNA, robust silencing was observed at 25 nM. 80% silencing was observed with only 16 nucleotide complementarity in the same format.

Incorporation of this property into the designs of siNAs of about 30 to 40 or more base pairs results in additional multifunctional siNA constructs. The example in FIG. 25 illustrates how a 30 base-pair duplex can target three distinct sequences after processing by Dicer-RNaseIII; these sequences can be on the same mRNA or separate RNAs, such as viral and host factor messages, or multiple points along a given pathway (e.g., inflammatory cascades). Furthermore, a 40 base-pair duplex can combine a bifunctional design in tandem, to provide a single duplex targeting four target sequences. An even more extensive approach can include use of homologous sequences to enable five or six targets silenced for one multifunctional duplex. The example in FIG. 25 demonstrates how this can be achieved. A 30 base pair duplex is cleaved by Dicer into 22 and 8 base pair products from either end (8 b.p. fragments not shown). For ease of presentation the overhangs generated by dicer are not shown—but can be compensated for. Three targeting sequences are shown. The required sequence identity overlapped is indicated by grey boxes. The N's of the parent 30 b.p. siNA are suggested sites of 2′-OH positions to enable Dicer cleavage if this is tested in stabilized chemistries. Note that processing of a 30mer duplex by Dicer RNase III does not give a precise 22+8 cleavage, but rather produces a series of closely related products (with 22+8 being the primary site). Therefore, processing by Dicer will yield a series of active siNAs. Another non-limiting example is shown in FIG. 26. A 40 base pair duplex is cleaved by Dicer into 20 base pair products from either end. For ease of presentation the overhangs generated by dicer are not shown—but can be compensated for. Four targeting sequences are shown in four colors, blue, light-blue and red and orange. The required sequence identity overlapped is indicated by grey boxes. This design format can be extended to larger RNAs. If chemically stabilized siNAs are bound by Dicer, then strategically located ribonucleotide linkages can enable designer cleavage products that permit our more extensive repertoire of multifunctional designs. For example cleavage products not limited to the Dicer standard of approximately 22-nucleotides can allow multifunctional siNA constructs with a target sequence identity overlap ranging from, for example, about 3 to about 15 nucleotides.

Example 16
Diagnostic Uses

The siNA molecules of the invention can be used in a variety of diagnostic applications, such as in the identification of molecular targets (e.g., RNA) in a variety of applications, for example, in clinical, industrial, environmental, agricultural and/or research settings. Such diagnostic use of siNA molecules involves utilizing reconstituted RNAi systems, for example, using cellular lysates or partially purified cellular lysates. siNA molecules of this invention can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of endogenous or exogenous, for example viral, RNA in a cell. The close relationship between siNA activity and the structure of the target RNA allows the detection of mutations in any region of the molecule, which alters the base-pairing and three-dimensional structure of the target RNA. By using multiple siNA molecules described in this invention, one can map nucleotide changes, which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with siNA molecules can be used to inhibit gene expression and define the role of specified gene products in the progression of disease or infection. In this manner, other genetic targets can be defined as important mediators of the disease. These experiments will lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple siNA molecules targeted to different genes, siNA molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations siNA molecules and/or other chemical or biological molecules). Other in vitro uses of siNA molecules of this invention are well known in the art, and include detection of the presence of mRNAs associated with a disease, infection, or related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a siNA using standard methodologies, for example, fluorescence resonance emission transfer (FRET).

In a specific example, siNA molecules that cleave only wild-type or mutant forms of the target RNA are used for the assay. The first siNA molecules (i.e., those that cleave only wild-type forms of target RNA) are used to identify wild-type RNA present in the sample and the second siNA molecules (i.e., those that cleave only mutant forms of target RNA) are used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA are cleaved by both siNA molecules to demonstrate the relative siNA efficiencies in the reactions and the absence of cleavage of the “non-targeted” RNA species. The cleavage products from the synthetic substrates also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population. Thus, each analysis requires two siNA molecules, two substrates and one unknown sample, which is combined into six reactions. The presence of cleavage products is determined using an RNase protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells. The expression of mRNA whose protein product is implicated in the development of the phenotype (i.e., disease related or infection related) is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels is adequate and decreases the cost of the initial diagnosis. Higher mutant form to wild-type ratios are correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.

All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.

One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varying substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, such additional embodiments are within the scope of the present invention and the following claims. The present invention teaches one skilled in the art to test various combinations and/or substitutions of chemical modifications described herein toward generating nucleic acid constructs with improved activity for mediating RNAi activity. Such improved activity can comprise improved stability, improved bioavailability, and/or improved activation of cellular responses mediating RNAi. Therefore, the specific embodiments described herein are not limiting and one skilled in the art can readily appreciate that specific combinations of the modifications described herein can be tested without undue experimentation toward identifying siNA molecules with improved RNAi activity.

The invention illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of”, and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims.

In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

TABLE I

HCV Accession Numbers

Seq Name
Acc#
LOCUS

gi|329763|gb|M84754.1|HPCGENANTI
M84754.1
HPCGENANTI

gi|567059|gb|U16362.1|HCU16362
U16362.1
HCU16362

gi|5918956|gb|AF165059.1|AF165059
AF165059.1
AF165059

gi|385583|gb|S62220.1|S62220
S62220.1
S62220

gi|6010587|gb|AF177040.1|AF177040
AF177040.1
AF177040

gi|5748510|emb|AJ238800.1|HCJ238800
AJ238800.1
HCJ238800

gi|7650221|gb|AF207752.1|AF207752
AF207752.1
AF207752

gi|11559454|dbj|AB049094.1|AB049094
AB049094.1
AB049094

gi|3550760|dbj|D84263.1|D84263
D84263.1
D84263

gi|221610|dbj|D90208.1|HPCJCG
D90208.1
HPCJCG

gi|558520|dbj|D28917.1|HPCK3A
D28917.1
HPCK3A

gi|2176577|dbj|E08461.1|E08461
E08461.1
E08461

gi|6707285|gb|AF169005.1|AF169005
AF169005.1
AF169005

gi|12309923|emb|AX057094.1|AX057094
AX057094.1
AX057094

gi|6010585|gb|AF177039.1|AF177039
AF177039.1
AF177039

gi|7329202|gb|AF238482.1|AF238482
AF238482.1
AF238482

gi|11559464|dbj|AB049099.1|AB049099
AB049099.1
AB049099

gi|5918932|gb|AF165047.1|AF165047
AF165047.1
AF165047

gi|5918946|gb|AF165054.1|AF165054
AF165054.1
AF165054

gi|7650233|gb|AF207758.1|AF207758
AF207758.1
AF207758

gi|19568932|gb|AF483269.1|
AF483269.1

gi|7650247|gb|AF207765.1|AF207765
AF207765.1
AF207765

gi|12309919|emb|AX057086.1|AX057086
AX057086.1
AX057086

gi|5708597|dbj|E10839.1|E10839
E10839.1
E10839

gi|2327074|gb|AF011753.1|AF011753
AF011753.1
AF011753

gi|12310062|emb|AX057317.1|AX057317
AX057317.1
AX057317

gi|221606|dbj|D10750.1|HPCJ491
D10750.1
HPCJ491

gi|2174448|dbj|E06261.1|E06261
E06261.1
E06261

gi|3098640|gb|AF054251.1|AF054251
AF054251.1
AF054251

gi|18027684|gb|AF313916.1|AF313916
AF313916.1
AF313916

gi|329873|gb|M62321.1|HPCPLYPRE
M62321.1
HPCPLYPRE

gi|464177|dbj|D14853.1|HPCCGS
D14853.1
HPCCGS

gi|15422182|gb|AY051292.1|
AY051292.1

gi|676877|dbj|D49374.1|HPCFG
D49374.1
HPCFG

gi|1030706|dbj|D50480.1|HPCK1R1
D50480.1
HPCK1R1

gi|7650223|gb|AF207753.1|AF207753
AF207753.1
AF207753

gi|7650237|gb|AF207760.1|AF207760
AF207760.1
AF207760

gi|11559444|dbj|AB049089.1|AB049089
AB049089.1
AB049089

gi|3550762|dbj|D84264.1|D84264
D84264.1
D84264

gi|12831192|gb|AF333324.1|AF333324
AF333324.1
AF333324

gi|13122265|dbj|AB047641.1|AB047641
AB047641.1
AB047641

gi|7329204|gb|AF238483.1|AF238483
AF238483.1
AF238483

gi|11559468|dbj|AB049101.1|AB049101
AB049101.1
AB049101

gi|5918934|gb|AF165048.1|AF165048
AF165048.1
AF165048

gi|5918948|gb|AF165055.1|AF165055
AF165055.1
AF165055

gi|7650235|gb|AF207759.1|AF207759
AF207759.1
AF207759

gi|7650249|gb|AF207766.1|AF207766
AF207766.1
AF207766

gi|9843676|emb|AJ278830.1|HEC278830
AJ278830.1
HEC278830

gi|11559450|dbj|AB049092.1|AB049092
AB049092.1
AB049092

gi|2943783|dbj|D89815.1|D89815
D89815.1
D89815

gi|9626438|ref|NC_001433.1|
NC_001433.1

gi|12310134|emb|AX057395.1|AX057395
AX057395.1
AX057395

gi|11559460|dbj|AB049097.1|AB049097
AB049097.1
AB049097

gi|12309922|emb|AX057092.1|AX057092
AX057092.1
AX057092

gi|2174644|dbj|E06457.1|E06457
E06457.1
E06457

gi|2176559|dbj|E08443.1|E08443
E08443.1
E08443

gi|5918960|gb|AF165061.1|AF165061
AF165061.1
AF165061

gi|2326454|emb|Y12083.1|HCV12083
Y12083.1
HCV12083

gi|5918938|gb|AF165050.1|AF165050
AF165050.1
AF165050

gi|7650225|gb|AF207754.1|AF207754
AF207754.1
AF207754

gi|7650261|gb|AF207772.1|AF207772
AF207772.1
AF207772

gi|1030704|dbj|D50485.1|HPCK1S2
D50485.1
HPCK1S2

gi|3550758|dbj|D84262.1|D84262
D84262.1
D84262

gi|7650239|gb|AF207761.1|AF207761
AF207761.1
AF207761

gi|3550764|dbj|D84265.1|D84265
D84265.1
D84265

gi|7329206|gb|AF238484.1|AF238484
AF238484.1
AF238484

gi|2176516|dbj|E08399.1|E08399
E08399.1
E08399

gi|5918936|gb|AF165049.1|AF165049
AF165049.1
AF165049

gi|11559446|dbj|AB049090.1|AB049090
AB049090.1
AB049090

gi|5441837|emb|AJ242653.1|SSE242653
AJ242653.1
SSE242653

gi|3098641|gb|AF054252.1|AF054252
AF054252.1
AF054252

gi|4753720|emb|AJ132997.1|HCV132997
AJ132997.1
HCV132997

gi|5420376|emb|AJ238799.1|HCJ238799
AJ238799.1
HCJ238799

gi|11559440|dbj|AB049087.1|AB049087
AB049087.1
AB049087

gi|15529110|gb|AY045702.1|
AY045702.1

gi|560788|dbj|D30613.1|HPCPP
D30613.1
HPCPP

gi|11225869|emb|AX036253.1|AX036253
AX036253.1
AX036253

gi|11559456|dbj|AB049095.1|AB049095
AB049095.1
AB049095

gi|329770|gb|M58335.1|HPCHUMR
M58335.1
HPCHUMR

gi|6707279|gb|AF169002.1|AF169002
AF169002.1
AF169002

gi|221586|dbj|D10749.1|HPCHCJ1
D10749.1
HPCHCJ1

gi|2171981|dbj|E03766.1|E03766
E03766.1
E03766

gi|6010579|gb|AF177036.1|AF177036
AF177036.1
AF177036

gi|1030703|dbj|D50484.1|HPCK1S3
D50484.1
HPCK1S3

gi|3098650|gb|AF054257.1|AF054257
AF054257.1
AF054257

gi|5821154|dbj|AB016785.1|AB016785
AB016785.1
AB016785

gi|5918962|gb|AF165062.1|AF165062
AF165062.1
AF165062

gi|7650227|gb|AF207755.1|AF207755
AF207755.1
AF207755

gi|7650263|gb|AF207773.1|AF207773
AF207773.1
AF207773

gi|1183030|dbj|D63822.1|HPCJK046E2
D63822.1
HPCJK046E2

gi|13122271|dbj|AB047644.1|AB047644
AB047644.1
AB047644

gi|2443428|gb|U89019.1|HCU89019
U89019.1
HCU89019

gi|2462303|emb|Y13184.1|HCV1480
Y13184.1
HCV1480

gi|7329208|gb|AF238485.1|AF238485
AF238485.1
AF238485

gi|1160327|dbj|D14484.1|HPCJRNA
D14484.1
HPCJRNA

gi|12309921|emb|AX057090.1|AX057090
AX057090.1
AX057090

gi|3098643|gb|AF054253.1|AF054253
AF054253.1
AF054253

gi|21397075|gb|AF511948.1|
AF511948.1

gi|1030701|dbj|D50482.1|HPCK1R3
D50482.1
HPCK1R3

gi|1030702|dbj|D50483.1|HPCK1S1
D50483.1
HPCK1S1

gi|3098632|gb|AF054247.1|AF054247
AF054247.1
AF054247

gi|59478|emb|X61596.1|HCVJK1G
X61596.1
HCVJK1G

gi|3098652|gb|AF054258.1|AF054258
AF054258.1
AF054258

gi|5918950|gb|AF165056.1|AF165056
AF165056.1
AF165056

gi|7650251|gb|AF207767.1|AF207767
AF207767.1
AF207767

gi|5918964|gb|AF165063.1|AF165063
AF165063.1
AF165063

gi|5918928|gb|AF165045.1|AF165045
AF165045.1
AF165045

gi|5532421|gb|AF139594.1|AF139594
AF139594.1
AF139594

gi|13122267|dbj|AB047642.1|AB047642
AB047642.1
AB047642

gi|5441831|emb|AJ242651.1|SSE242651
AJ242651.1
SSE242651

gi|7650265|gb|AF207774.1|AF207774
AF207774.1
AF207774

gi|7650229|gb|AF207756.1|AF207756
AF207756.1
AF207756

gi|1183032|dbj|D63821.1|HPCJK049E1
D63821.1
HPCJK049E1

gi|2175714|dbj|E07579.1|E07579
E07579.1
E07579

gi|1212741|dbj|D45172.1|HPCHCPO
D45172.1
HPCHCPO

gi|5708511|dbj|E05027.1|E05027
E05027.1
E05027

gi|1483141|dbj|D50409.1|D50409
D50409.1
D50409

gi|13122261|dbj|AB047639.1|AB047639
AB047639.1
AB047639

gi|6521008|dbj|AB031663.1|AB031663
AB031663.1
AB031663

gi|633201|emb|X76918.1|HCVCENS1
X76918.1
HCVCENS1

gi|329737|gb|M67463.1|HPCCGAA
M67463.1
HPCCGAA

gi|11559452|dbj|AB049093.1|AB049093
AB049093.1
AB049093

gi|13619567|emb|AX100563.1|AX100563
AX100563.1
AX100563

gi|221604|dbj|D13558.1|HPCJ483
D13558.1
HPCJ483

gi|11225872|emb|AX036256.1|AX036256
AX036256.1
AX036256

gi|1749761|dbj|D89872.1|D89872
D89872.1
D89872

gi|5918940|gb|AF165051.1|AF165051
AF165051.1
AF165051

gi|4753718|emb|AJ132996.1|HCV132996
AJ132996.1
HCV132996

gi|7650241|gb|AF207762.1|AF207762
AF207762.1
AF207762

gi|3098645|gb|AF054254.1|AF054254
AF054254.1
AF054254

gi|9930556|gb|AF290978.1|AF290978
AF290978.1
AF290978

gi|11559462|dbj|AB049098.1|AB049098
AB049098.1
AB049098

gi|2764397|emb|AJ000009.1|HCVPOLYP
AJ000009.1
HCVPOLYP

gi|221608|dbj|D10988.1|HPCJ8G
D10988.1
HPCJ8G

gi|3098634|gb|AF054248.1|AF054248
AF054248.1
AF054248

gi|221650|dbj|D00944.1|HPCPOLP
D00944.1
HPCPOLP

gi|306286|gb|M96362.1|HPCUNKCDS
M96362.1
HPCUNKCDS

gi|3098654|gb|AF054259.1|AF054259
AF054259.1
AF054259

gi|5918952|gb|AF165057.1|AF165057
AF165057.1
AF165057

gi|7650253|gb|AF207768.1|AF207768
AF207768.1
AF207768

gi|5918966|gb|AF165064.1|AF165064
AF165064.1
AF165064

gi|15487693|gb|AF356827.1|AF356827
AF356827.1
AF356827

gi|5738246|gb|AF176573.1|AF176573
AF176573.1
AF176573

gi|11559448|dbj|AB049091.1|AB049091
AB049091.1
AB049091

gi|21397077|gb|AF511950.1|
AF511950.1

gi|3098638|gb|AF054250.1|AF054250
AF054250.1
AF054250

gi|6707281|gb|AF169003.1|AF169003
AF169003.1
AF169003

gi|329739|gb|L02836.1|HPCCGENOM
L02836.1
HPCCGENOM

gi|6010581|gb|AF177037.1|AF177037
AF177037.1
AF177037

gi|11559442|dbj|AB049088.1|AB049088
AB049088.1
AB049088

gi|21397076|gb|AF511949.1|
AF511949.1

gi|1030705|dbj|D50481.1|HPCK1R2
D50481.1
HPCK1R2

gi|2176384|dbj|E08264.1|E08264
E08264.1
E08264

gi|3660725|gb|AF064490.1|AF064490
AF064490.1
AF064490

gi|2252489|emb|Y11604.1|HCV4APOLY
Y11604.1
HCV4APOLY

gi|5918942|gb|AF165052.1|AF165052
AF165052.1
AF165052

gi|2895898|gb|AF046866.1|AF046866
AF046866.1
AF046866

gi|7650243|gb|AF207763.1|AF207763
AF207763.1
AF207763

gi|11559458|dbj|AB049096.1|AB049096
AB049096.1
AB049096

gi|13122263|dbj|AB047640.1|AB047640
AB047640.1
AB047640

gi|5708574|dbj|E08263.1|E08263
E08263.1
E08263

gi|7650257|gb|AF207770.1|AF207770
AF207770.1
AF207770

gi|3098647|gb|AF054255.1|AF054255
AF054255.1
AF054255

gi|11559466|dbj|AB049100.1|AB049100
AB049100.1
AB049100

gi|1181831|gb|U45476.1|HCU45476
U45476.1
HCU45476

gi|2327070|gb|AF011751.1|AF011751
AF011751.1
AF011751

gi|3098636|gb|AF054249.1|AF054249
AF054249.1
AF054249

gi|7329210|gb|AF238486.1|AF238486
AF238486.1
AF238486

gi|221612|dbj|D11168.1|HPCJTA
D11168.1
HPCJTA

gi|960359|dbj|D63857.1|HPVHCVN
D63857.1
HPVHCVN

gi|13122273|dbj|AB047645.1|AB047645
AB047645.1
AB047645

gi|5918954|gb|AF165058.1|AF165058
AF165058.1
AF165058

gi|7650255|gb|AF207769.1|AF207769
AF207769.1
AF207769

gi|437107|gb|U01214.1|HCU01214
U01214.1
HCU01214

gi|471116|dbj|D10934.1|HPCRNA
D10934.1
HPCRNA

gi|13026028|dbj|E66593.1|E66593
E66593.1
E66593

gi|2316097|gb|AF009606.1|AF009606
AF009606.1
AF009606

gi|6707283|gb|AF169004.1|AF169004
AF169004.1
AF169004

gi|514395|dbj|D17763.1|HPCEGS
D17763.1
HPCEGS

gi|9757541|dbj|AB030907.1|AB030907
AB030907.1
AB030907

gi|7329200|gb|AF238481.1|AF238481
AF238481.1
AF238481

gi|6010583|gb|AF177038.1|AF177038
AF177038.1
AF177038

gi|2172621|dbj|E04420.1|E04420
E04420.1
E04420

gi|8926244|gb|AF271632.1|AF271632
AF271632.1
AF271632

gi|5918930|gb|AF165046.1|AF165046
AF165046.1
AF165046

gi|7650231|gb|AF207757.1|AF207757
AF207757.1
AF207757

gi|5918944|gb|AF165053.1|AF165053
AF165053.1
AF165053

gi|7650245|gb|AF207764.1|AF207764
AF207764.1
AF207764

gi|12309920|emb|AX057088.1|AX057088
AX057088.1
AX057088

gi|5918958|gb|AF165060.1|AF165060
AF165060.1
AF165060

gi|7650259|gb|AF207771.1|AF207771
AF207771.1
AF207771

gi|7341102|gb|AF208024.1|AF208024
AF208024.1
AF208024

gi|3098649|gb|AF054256.1|AF054256
AF054256.1
AF054256

gi|1944375|dbj|D85516.1|D85516
D85516.1
D85516

gi|2327072|gb|AF011752.1|AF011752
AF011752.1
AF011752

gi|221614|dbj|D11355.1|HPCJTB
D11355.1
HPCJTB

gi|13122269|dbj|AB047643.1|AB047643
AB047643.1
AB047643

TABLE II

HCV siNA AND TARGET SEQUENCES

Seq

Seq

Seq

Sequence
ID
Upper seq
ID
Lower seq
ID

GCCCCGGGAGGUCUCGUAG
1
GCCCCGGGAGGUCUCGUAG
1
CUACGAGACCUCCCGGGGC
697

UGUGGUACUGCCUGAUAGG
2
UGUGGUACUGCCUGAUAGG
2
CCUAUCAGGCAGUACCACA
698

UUGUGGUACUGCCUGAUAG
3
UUGUGGUACUGCCUGAUAG
3
CUAUCAGGCAGUACCACAA
699

CCCCGGGAGGUCUCGUAGA
4
CCCCGGGAGGUCUCGUAGA
4
UCUACGAGACCUCCCGGGG
700

GUGGUACUGCCUGAUAGGG
5
GUGGUACUGCCUGAUAGGG
5
CCCUAUCAGGCAGUACCAC
701

CUGCCUGAUAGGGUGCUUG
6
CUGCCUGAUAGGGUGCUUG
6
CAAGCACCCUAUCAGGCAG
702

CCUUGUGGUACUGCCUGAU
7
CCUUGUGGUACUGCCUGAU
7
AUCAGGCAGUACCACAAGG
703

GCGAAAGGCCUUGUGGUAC
8
GCGAAAGGCCUUGUGGUAC
8
GUACCACAAGGCCUUUCGC
704

UACUGCCUGAUAGGGUGCU
9
UACUGCCUGAUAGGGUGCU
9
AGCACCCUAUCAGGCAGUA
705

GGUACUGCCUGAUAGGGUG
10
GGUACUGCCUGAUAGGGUG
10
CACCCUAUCAGGCAGUACC
706

AAAGGCCUUGUGGUACUGC
11
AAAGGCCUUGUGGUACUGC
11
GCAGUACCACAAGGCCUUU
707

AAGGCCUUGUGGUACUGCC
12
kAGGCCUUGUGGUACUGCC
12
GGCAGUACCACAAGGCCUU
708

CUUGUGGUACUGCCUGAUA
13
CUUGUGGUACUGCCUGAUA
13
UAUCAGGCAGUACCACAAG
709

AGGCCUUGUGGUACUGCCU
14
AGGCCUUGUGGUACUGCCU
14
AGGCAGUACCACAAGGCCU
710

GUACUGCCUGAUAGGGUGC
15
GUACUGCCUGAUAGGGUGC
15
GCACCCUAUCAGGCAGUAC
711

ACUGCCUGAUAGGGUGCUU
16
ACUGCCUGAUAGGGUGCUU
16
AAGCACCCUAUCAGGCAGU
712

CUUGCGAGUGCCCCGGGAG
17
CUUGCGAGUGCCCCGGGAG
17
CUCCCGGGGCACUCGCAAG
713

CUGAUAGGGUGCUUGCGAG
18
CUGAUAGGGUGCUUGCGAG
18
CUCGCAAGCACCCUAUCAG
714

UUGCGAGUGCCCCGGGAGG
19
UUGCGAGUGCCCCGGGAGG
19
CCUCCCGGGGCACUCGCAA
715

CCUGAUAGGGUGCUUGCGA
20
CCUGAUAGGGUGCUUGCGA
20
UCGCAAGCACCCUAUCAGG
716

GGCCUUGUGGUACUGCCUG
21
GGCCUUGUGGUACUGCCUG
21
CAGGCAGUACCACAAGGCC
717

GCUUGCGAGUGCCCCGGGA
22
GCUUGCGAGUGCCCCGGGA
22
UCCCGGGGCACUCGCAAGC
718

UGCCUGAUAGGGUGCUUGC
23
UGCCUGAUAGGGUGCUUGC
23
GCAAGCACCCUAUCAGGCA
719

GAAAGGCCUUGUGGUACUG
24
GAAAGGCCUUGUGGUACUG
24
CAGUACCACAAGGCCUUUC
720

GCCUGAUAGGGUGCUUGCG
25
GCCUGAUAGGGUGCUUGCG
25
CGCAAGCACCCUAUCAGGC
721

CGAAAGGCCUUGUGGUACU
26
CGAAAGGCCUUGUGGUACU
26
AGUACCACAAGGCCUUUCG
722

GCCUUGUGGUACUGCCUGA
27
GCCUUGUGGUACUGCCUGA
27
UCAGGCAGUACCACAAGGC
723

GAGUGCCCCGGGAGGUCUC
28
GAGUGCCCCGGGAGGUCUC
28
GAGACCUCCCGGGGCACUC
724

CCCGGGAGGUCUCGUAGAC
29
CCCGGGAGGUCUCGUAGAC
29
GUCUACGAGACCUCCCGGG
725

UGCGAGUGCCCCGGGAGGU
30
UGCGAGUGCCCCGGGAGGU
30
ACCUCCCGGGGCACUCGCA
726

UGGUACUGCCUGAUAGGGU
31
UGGUACUGCCUGAUAGGGU
31
ACCCUAUCAGGCAGUACCA
727

CCGGUGAGUACACCGGAAU
32
CCGGUGAGUACACCGGAAU
32
AUUCCGGUGUACUCACCGG
728

GCGAGUGCCCCGGGAGGUC
33
GCGAGUGCCCCGGGAGGUC
33
GACCUCCCGGGGCACUCGC
729

CGAGUGCCCCGGGAGGUCU
34
CGAGUGCCCCGGGAGGUCU
34
AGACCUCCCGGGGCACUCG
730

UGCCCCGGGAGGUCUCGUA
35
UGCCCCGGGAGGUCUCGUA
35
UACGAGACCUCCCGGGGCA
731

GUGCCCCGGGAGGUCUCGU
36
GUGCCCCGGGAGGUCUCGU
36
ACGAGACCUCCCGGGGCAC
732

AGUGCCCCGGGAGGUCUCG
37
AGUGCCCCGGGAGGUCUCG
37
CGAGACCUCCCGGGGCACU
733

CCGGGAGGUCUCGUAGACC
38
CCGGGAGGUCUCGUAGACC
38
GGUCUACGAGACCUCCCGG
734

UGAUAGGGUGCUUGCGAGU
39
UGAUAGGGUGCUUGCGAGU
39
ACUCGCAAGCACCCUAUCA
735

GUGCUUGCGAGUGCCCCGG
40
GUGCUUGCGAGUGCCCCGG
40
CCGGGGCACUCGCAAGCAC
736

AUAGGGUGCUUGCGAGUGC
41
AUAGGGUGCUUGCGAGUGC
41
GCACUCGCAAGCACCCUAU
737

GGGUGCUUGCGAGUGCCCC
42
GGGUGCUUGCGAGUGCCCC
42
GGGGCACUCGCAAGCACCC
738

CGGGAGGUCUCGUAGACCG
43
CGGGAGGUCUCGUAGACCG
43
CGGUCUACGAGACCUCCCG
739

GGGAGGUCUCGUAGACCGU
44
GGGAGGUCUCGUAGACCGU
44
ACGGUCUACGAGACCUCCC
740

GAUAGGGUGCUUGCGAGUG
45
GAUAGGGUGCUUGCGAGUG
45
CACUCGCAAGCACCCUAUC
741

GGAGGUCUCGUAGACCGUG
46
GGAGGUCUCGUAGACCGUG
46
CACGGUCUACGAGACCUCC
742

AGGGUGCUUGCGAGUGCCC
47
AGGGUGCUUGCGAGUGCCC
47
GGGCACUCGCAAGCACCCU
743

UGCUUGCGAGUGCCCCGGG
48
UGCUUGCGAGUGCCCCGGG
48
CCCGGGGCACUCGCAAGCA
744

GGUGCUUGCGAGUGCCCCG
49
GGUGCUUGCGAGUGCCCCG
49
CGGGGCACUCGCAAGCACC
745

UAGGGUGCUUGCGAGUGCC
50
UAGGGUGCUUGCGAGUGCC
50
GGCACUCGCAAGCACCCUA
746

AGGUCUCGUAGACCGUGCA
51
AGGUCUCGUAGACCGUGCA
51
UGCACGGUCUACGAGACCU
747

GAGGUCUCGUAGACCGUGC
52
GAGGUCUCGUAGACCGUGC
52
GCACGGUCUACGAGACCUC
748

GGAACCGGUGAGUACACCG
53
GGAACCGGUGAGUACACCG
53
CGGUGUACUCACCGGUUCC
749

CGGAACCGGUGAGUACACC
54
CGGAACCGGUGAGUACACC
54
GGUGUACUCACCGGUUCCG
750

CGGUGAGUACACCGGAAUU
55
CGGUGAGUACACCGGAAUU
55
AAUUCCGGUGUACUCACCG
751

GCGGAACCGGUGAGUACAC
56
GCGGAACCGGUGAGUACAC
56
GUGUACUCACCGGUUCCGC
752

AACCGGUGAGUACACCGGA
57
AACCGGUGAGUACACCGGA
57
UCCGGUGUACUCACCGGUU
753

ACCGGUGAGUACACCGGAA
58
ACCGGUGAGUACACCGGAA
58
UUCCGGUGUACUCACCGGU
754

CUGCGGAACCGGUGAGUAC
59
CUGCGGAACCGGUGAGUAC
59
GUACUCACCGGUUCCGCAG
755

GUCUGCGGAACCGGUGAGU
60
GUCUGCGGAACCGGUGAGU
60
ACUCACCGGUUCCGCAGAC
756

GAACCGGUGAGUACACCGG
61
GAACCGGUGAGUACACCGG
61
CCGGUGUACUCACCGGUUC
757

UGCGGAACCGGUGAGUACA
62
UGCGGAACCGGUGAGUACA
62
UGUACUCACCGGUUCCGCA
758

UCUGCGGAACCGGUGAGUA
63
UCUGCGGAACCGGUGAGUA
63
UACUCACCGGUUCCGCAGA
759

GGGAGAGCCAUAGUGGUCU
64
GGGAGAGCCAUAGUGGUCU
64
AGACCACUAUGGCUCUCCC
760

GUGGUCUGCGGAACCGGUG
65
GUGGUCUGCGGAACCGGUG
65
CACCGGUUCCGCAGACCAC
761

GGUCUGCGGAACCGGUGAG
66
GGUCUGCGGAACCGGUGAG
66
CUCACCGGUUCCGCAGACC
762

CGGGAGAGCCAUAGUGGUC
67
CGGGAGAGCCAUAGUGGUC
67
GACCACUAUGGCUCUCCCG
763

CCGGGAGAGCCAUAGUGGU
68
CCGGGAGAGCCAUAGUGGU
68
ACCACUAUGGCUCUCCCGG
764

UGGUCUGCGGAACCGGUGA
69
UGGUCUGCGGAACCGGUGA
69
UCACCGGUUCCGCAGACCA
765

GUGAGUACACCGGAAUUGC
70
GUGAGUACACCGGAAUUGC
70
GCAAUUCCGGUGUACUCAC
766

UGAGUACACCGGAAUUGCC
71
UGAGUACACCGGAAUUGCC
71
GGCAAUUCCGGUGUACUCA
767

GGUGAGUACACCGGAAUUG
72
GGUGAGUACACCGGAAUUG
72
CAAUUCCGGUGUACUCACC
768

GAGCCAUAGUGGUCUGCGG
73
GAGCCAUAGUGGUCUGCGG
73
CCGCAGACCACUAUGGCUC
769

AGAGCCAUAGUGGUCUGCG
74
AGAGCCAUAGUGGUCUGCG
74
CGCAGACCACUAUGGCUCU
770

UAGUGGUCUGCGGAACCGG
75
UAGUGGUCUGCGGAACCGG
75
CCGGUUCCGCAGACCACUA
771

AUAGUGGUCUGCGGAACCG
76
AUAGUGGUCUGCGGAACCG
76
CGGUUCCGCAGACCACUAU
772

GAGAGCCAUAGUGGUCUGC
77
GAGAGCCAUAGUGGUCUGC
77
GCAGACCACUAUGGCUCUC
773

GCCAUAGUGGUCUGCGGAA
78
GCCAUAGUGGUCUGCGGAA
78
UUCCGCAGACCACUAUGGC
774

AGUGGUCUGCGGAACCGGU
79
AGUGGUCUGCGGAACCGGU
79
ACCGGUUCCGCAGACCACU
775

CAUAGUGGUCUGCGGAACC
80
CAUAGUGGUCUGCGGAACC
80
GGUUCCGCAGACCACUAUG
776

AGCCAUAGUGGUCUGCGGA
81
AGCCAUAGUGGUCUGCGGA
81
UCCGCAGACCACUAUGGCU
777

CCAUAGUGGUCUGCGGAAC
82
CCAUAGUGGUCUGCGGAAC
82
GUUCCGCAGACCACUAUGG
778

CCCCUCCCGGGAGAGCCAU
83
CCCCUCCCGGGAGAGCCAU
83
AUGGCUCUCCCGGGAGGGG
779

GGAGAGCCAUAGUGGUCUG
84
GGAGAGCCAUAGUGGUCUG
84
CAGACCACUAUGGCUCUCC
780

CCCGGGAGAGCCAUAGUGG
85
CCCGGGAGAGCCAUAGUGG
85
CCACUAUGGCUCUCCCGGG
781

CCCCCUCCCGGGAGAGCCA
86
CCCCCUCCCGGGAGAGCCA
86
UGGCUCUCCCGGGAGGGGG
782

UCCCGGGAGAGCCAUAGUG
87
UCCCGGGAGAGCCAUAGUG
87
CACUAUGGCUCUCCCGGGA
783

CCCCCCUCCCGGGAGAGCC
88
CCCCCCUCCCGGGAGAGCC
88
GGCUCUCCCGGGAGGGGGG
784

CCCUCCCGGGAGAGCCAUA
89
CCCUCCCGGGAGAGCCAUA
89
UAUGGCUCUCCCGGGAGGG
785

CCUCCCGGGAGAGCCAUAG
90
CCUCCCGGGAGAGCCAUAG
90
CUAUGGCUCUCCCGGGAGG
786

CUCCCGGGAGAGCCAUAGU
91
CUCCCGGGAGAGCCAUAGU
91
ACUAUGGCUCUCCCGGGAG
787

UGUUGCCGCGCAGGGGCCC
92
UGUUGCCGCGCAGGGGCCC
92
GGGCCCCUGCGCGGCAACA
788

CCCCCCCUCCCGGGAGAGC
93
CCCCCCCUCCCGGGAGAGC
93
GCUCUCCCGGGAGGGGGGG
789

CAUGGCGUUAGUAUGAGUG
94
CAUGGCGUUAGUAUGAGUG
94
CACUCAUACUAACGCCAUG
790

UAGCCAUGGCGUUAGUAUG
95
UAGCCAUGGCGUUAGUAUG
95
CAUACUAACGCCAUGGCUA
791

AGCCAUGGCGUUAGUAUGA
96
AGCCAUGGCGUUAGUAUGA
96
UCAUACUAACGCCAUGGCU
792

CCAUGGCGUUAGUAUGAGU
97
CCAUGGCGUUAGUAUGAGU
97
ACUCAUACUAACGCCAUGG
793

AUGGCGUUAGUAUGAGUGU
98
AUGGCGUUAGUAUGAGUGU
98
ACACUCAUACUAACGCCAU
794

AAGCGUCUAGCCAUGGCGU
99
AAGCGUCUAGCCAUGGCGU
99
ACGCCAUGGCUAGACGCUU
795

GUCUAGCCAUGGCGUUAGU
100
GUCUAGCCAUGGCGUUAGU
100
ACUAACGCCAUGGCUAGAC
796

AAAGCGUCUAGCCAUGGCG
101
AAAGCGUCUAGCCAUGGCG
101
CGCCAUGGCUAGACGCUUU
797

GCGUCUAGCCAUGGCGUUA
102
GCGUCUAGCCAUGGCGUUA
102
UAACGCCAUGGCUAGACGC
798

GCCAUGGCGUUAGUAUGAG
103
GCCAUGGCGUUAGUAUGAG
103
CUCAUACUAACGCCAUGGC
799

AGCGUCUAGCCAUGGCGUU
104
AGCGUCUAGCCAUGGCGUU
104
AACGCCAUGGCUAGACGCU
800

CGUCUAGCCAUGGCGUUAG
105
CGUCUAGCCAUGGCGUUAG
105
CUAACGCCAUGGCUAGACG
801

UCUAGCCAUGGCGUUAGUA
106
UCUAGCCAUGGCGUUAGUA
106
UACUAACGCCAUGGCUAGA
802

GAAAGCGUCUAGCCAUGGC
107
GAAAGCGUCUAGCCAUGGC
107
GCCAUGGCUAGACGCUUUC
803

CUAGCCAUGGCGUUAGUAU
108
CUAGCCAUGGCGUUAGUAU
108
AUACUAACGCCAUGGCUAG
804

CACUCCCCUGUGAGGAACU
109
CACUCCCCUGUGAGGAACU
109
AGUUCCUCACAGGGGAGUG
805

ACCUCAAAGAAAAACCAAA
110
ACCUCAAAGAAAAACCAAA
110
UUUGGUUUUUCUUUGAGGU
806

CGCAGAAAGCGUCUAGCCA
111
CGCAGAAAGCGUCUAGCCA
111
UGGCUAGACGCUUUCUGCG
807

GGGUAAGGUCAUCGAUACC
112
GGGUAAGGUCAUCGAUACC
112
GGUAUCGAUGACCUUACCC
808

CAGAAAGCGUCUAGCCAUG
113
CAGAAAGCGUCUAGCCAUG
113
CAUGGCUAGACGCUUUCUG
809

AAACCUCAAAGAAAAACCA
114
AAACCUCAAAGAAAAACCA
114
UGGUUUUUCUUUGAGGUUU
810

GCAGAAAGCGUCUAGCCAU
115
GCAGAAAGCGUCUAGCCAU
115
AUGGCUAGACGCUUUCUGC
811

AGAAAGCGUCUAGCCAUGG
116
AGAAAGCGUCUAGCCAUGG
116
CCAUGGCUAGACGCUUUCU
812

ACGCAGAAAGCGUCUAGCC
117
ACGCAGAAAGCGUCUAGCC
117
GGCUAGACGCUUUCUGCGU
813

AACCUCAAAGAAAAACCAA
118
AACCUCAAAGAAAAACCAA
118
UUGGUUUUUCUUUGAGGUU
814

UGGGUAAGGUCAUCGAUAC
119
UGGGUAAGGUCAUCGAUAC
119
GUAUCGAUGACCUUACCCA
815

GUAAGGUCAUCGAUACCCU
120
GUAAGGUCAUCGAUACCCU
120
AGGGUAUCGAUGACCUUAC
816

UUCACGCAGAAAGCGUCUA
121
UUCACGCAGAAAGCGUCUA
121
UAGACGCUUUCUGCGUGAA
817

GGUAAGGUCAUCGAUACCC
122
GGUAAGGUCAUCGAUACCC
122
GGGUAUCGAUGACCUUACC
818

AUCACUCCCCUGUGAGGAA
123
AUCACUCCCCUGUGAGGAA
123
UUCCUCACAGGGGAGUGAU
819

UCACUCCCCUGUGAGGAAC
124
UCACUCCCCUGUGAGGAAC
124
GUUCCUCACAGGGGAGUGA
820

UGUCUUCACGCAGAAAGCG
125
UGUCUUCACGCAGAAAGCG
125
CGCUUUCUGCGUGAAGACA
821

UCACGCAGAAAGCGUCUAG
126
UCACGCAGAAAGCGUCUAG
126
CUAGACGCUUUCUGCGUGA
822

CACGCAGAAAGCGUCUAGC
127
CACGCAGAAAGCGUCUAGC
127
GCUAGACGCUUUCUGCGUG
823

GACCGGGUCCUUUCUUGGA
128
GACCGGGUCCUUUCUUGGA
128
UCCAAGAAAGGACCCGGUC
824

GAGGAACUACUGUCUUCAC
129
GAGGAACUACUGUCUUCAC
129
GUGAAGACAGUAGUUCCUC
825

CUGUGAGGAACUACUGUCU
130
CUGUGAGGAACUACUGUCU
130
AGACAGUAGUUCCUCACAG
826

GGAACUACUGUCUUCACGC
131
GGAACUACUGUCUUCACGC
131
GCGUGAAGACAGUAGUUCC
827

ACUCCCCUGUGAGGAACUA
132
ACUCCCCUGUGAGGAACUA
132
UAGUUCCUCACAGGGGAGU
828

GUCUUCACGCAGAAAGCGU
133
GUCUUCACGCAGAAAGCGU
133
ACGCUUUCUGCGUGAAGAC
829

AGGAACUACUGUCUUCACG
134
AGGAACUACUGUCUUCACG
134
CGUGAAGACAGUAGUUCCU
830

CCUGUGAGGAACUACUGUC
135
CCUGUGAGGAACUACUGUC
135
GACAGUAGUUCCUCACAGG
831

UGUGAGGAACUACUGUCUU
136
UGUGAGGAACUACUGUCUU
136
AAGACAGUAGUUCCUCACA
832

UCUUCACGCAGAAAGCGUC
137
UCUUCACGCAGAAAGCGUC
137
GACGCUUUCUGCGUGAAGA
833

GAACUACUGUCUUCACGCA
138
GAACUACUGUCUUCACGCA
138
UGCGUGAAGACAGUAGUUC
834

CCCUGUGAGGAACUACUGU
139
CCCUGUGAGGAACUACUGU
139
ACAGUAGUUCCUCACAGGG
835

CUUCACGCAGAAAGCGUCU
140
CUUCACGCAGAAAGCGUCU
140
AGACGCUUUCUGCGUGAAG
836

UGAGGAACUACUGUCUUCA
141
UGAGGAACUACUGUCUUCA
141
UGAAGACAGUAGUUCCUCA
837

UGGCGUUAGUAUGAGUGUC
142
UGGCGUUAGUAUGAGUGUC
142
GACACUCAUACUAACGCCA
838

CCCCUGUGAGGAACUACUG
143
CCCCUGUGAGGAACUACUG
143
CAGUAGUUCCUCACAGGGG
839

GUGAGGAACUACUGUCUUC
144
GUGAGGAACUACUGUCUUC
144
GAAGACAGUAGUUCCUCAC
840

GGCGUUAGUAUGAGUGUCG
145
GGCGUUAGUAUGAGUGUCG
145
CGACACUCAUACUAACGCC
841

GCCGAGUAGUGUUGGGUCG
146
GCCGAGUAGUGUUGGGUCG
146
CGACCCAACACUACUCGGC
842

ACUGUCUUCACGCAGAAAG
147
ACUGUCUUCACGCAGAAAG
147
CUUUCUGCGUGAAGACAGU
843

UGGGUCGCGAAAGGCCUUG
148
UGGGUCGCGAAAGGCCUUG
148
CAAGGCCUUUCGCGACCCA
844

CUACUGUCUUCACGCAGAA
149
CUACUGUCUUCACGCAGAA
149
UUCUGCGUGAAGACAGUAG
845

CGAGUAGUGUUGGGUCGCG
150
CGAGUAGUGUUGGGUCGCG
150
CGCGACCCAACACUACUCG
846

GUAGUGUUGGGUCGCGAAA
151
GUAGUGUUGGGUCGCGAAA
151
UUUCGCGACCCAACACUAC
847

UAAACCUCAAAGAAAAACC
152
UAAACCUCAAAGAAAAACC
152
GGUUUUUCUUUGAGGUUUA
848

CCGAGUAGUGUUGGGUCGC
153
CCGAGUAGUGUUGGGUCGC
153
GCGACCCAACACUACUCGG
849

AGCCGAGUAGUGUUGGGUC
154
AGCCGAGUAGUGUUGGGUC
154
GACCCAACACUACUCGGCU
850

GUCGCGAAAGGCCUUGUGG
155
GUCGCGAAAGGCCUUGUGG
155
CCACAAGGCCUUUCGCGAC
851

UAGUGUUGGGUCGCGAAAG
156
UAGUGUUGGGUCGCGAAAG
156
CUUUCGCGACCCAACACUA
852

CUAGCCGAGUAGUGUUGGG
157
CUAGCCGAGUAGUGUUGGG
157
CCCAACACUACUCGGCUAG
853

GAGUAGUGUUGGGUCGCGA
158
GAGUAGUGUUGGGUCGCGA
158
UCGCGACCCAACACUACUC
854

UCGCGAAAGGCCUUGUGGU
159
UCGCGAAAGGCCUUGUGGU
159
ACCACAAGGCCUUUCGCGA
855

GCGUUAGUAUGAGUGUCGU
160
GCGUUAGUAUGAGUGUCGU
160
ACGACACUCAUACUAACGC
856

UAGCCGAGUAGUGUUGGGU
161
UAGCCGAGUAGUGUUGGGU
161
ACCCAACACUACUCGGCUA
857

AACUACUGUCUUCACGCAG
162
AACUACUGUCUUCACGCAG
162
CUGCGUGAAGACAGUAGUU
858

CGCGAAAGGCCUUGUGGUA
163
CGCGAAAGGCCUUGUGGUA
163
UACCACAAGGCCUUUCGCG
859

AGUGUUGGGUCGCGAAAGG
164
AGUGUUGGGUCGCGAAAGG
164
CCUUUCGCGACCCAACACU
860

GUUGGGUCGCGAAAGGCCU
165
GUUGGGUCGCGAAAGGCCU
165
AGGCCUUUCGCGACCCAAC
861

AGUAGUGUUGGGUCGCGAA
166
AGUAGUGUUGGGUCGCGAA
166
UUCGCGACCCAACACUACU
862

UUGGGUCGCGAAAGGCCUU
167
UUGGGUCGCGAAAGGCCUU
167
kAGGCCUUUCGCGACCCAA
863

UCCCCUGUGAGGAACUACU
168
UCCCCUGUGAGGAACUACU
168
AGUAGUUCCUCACAGGGGA
864

UACUGUCUUCACGCAGAAA
169
UACUGUCUUCACGCAGAAA
169
UUUCUGCGUGAAGACAGUA
865

GUGUUGGGUCGCGAAAGGC
170
GUGUUGGGUCGCGAAAGGC
170
GCCUUUCGCGACCCAACAC
866

ACUACUGUCUUCACGCAGA
171
ACUACUGUCUUCACGCAGA
171
UCUGCGUGAAGACAGUAGU
867

CUGUCUUCACGCAGAAAGC
172
CUGUCUUCACGCAGAAAGC
172
GCUUUCUGCGUGAAGACAG
868

GGGUCGCGAAAGGCCUUGU
173
GGGUCGCGAAAGGCCUUGU
173
ACAAGGCCUUUCGCGACCC
869

CCUAAACCUCAAAGAAAAA
174
CCUAAACCUCAAAGAAAAA
174
UUUUUCUUUGAGGUUUAGG
870

GGUCGCGAAAGGCCUUGUG
175
GGUCGCGAAAGGCCUUGUG
175
CACAAGGCCUUUCGCGACC
871

CUAAACCUCAAAGAAAAAC
176
CUAAACCUCAAAGAAAAAC
176
GUUUUUCUUUGAGGUUUAG
872

UGUUGGGUCGCGAAAGGCC
177
UGUUGGGUCGCGAAAGGCC
177
GGCCUUUCGCGACCCAACA
873

CUCCCCUGUGAGGAACUAC
178
CUCCCCUGUGAGGAACUAC
178
GUAGUUCCUCACAGGGGAG
874

UCCUAAACCUCAAAGAAAA
179
UCCUAAACCUCAAAGAAAA
179
UUUUCUUUGAGGUUUAGGA
875

ACCGGGUCCUUUCUUGGAU
180
ACCGGGUCCUUUCUUGGAU
180
AUCCAAGAAAGGACCCGGU
876

AAUCCUAAACCUCAAAGAA
181
AAUCCUAAACCUCAAAGAA
181
UUCUUUGAGGUUUAGGAUU
877

UCAAUGCCUGGAGAUUUGG
182
UCAAUGCCUGGAGAUUUGG
182
CCAAAUCUCCAGGCAUUGA
878

AUGCCUGGAGAUUUGGGCG
183
AUGCCUGGAGAUUUGGGCG
183
CGCCCAAAUCUCCAGGCAU
879

AAUGCCUGGAGAUUUGGGC
184
AAUGCCUGGAGAUUUGGGC
184
GCCCAAAUCUCCAGGCAUU
880

CCGACCUCAUGGGGUACAU
185
CCGACCUCAUGGGGUACAU
185
AUGUACCCCAUGAGGUCGG
881

GCUCAAUGCCUGGAGAUUU
186
GCUCAAUGCCUGGAGAUUU
186
AAAUCUCCAGGCAUUGAGC
882

CUCAAUGCCUGGAGAUUUG
187
CUCAAUGCCUGGAGAUUUG
187
CAAAUCUCCAGGCAUUGAG
883

GCUAGCCGAGUAGUGUUGG
188
GCUAGCCGAGUAGUGUUGG
188
CCAACACUACUCGGCUAGC
884

CGCUCAAUGCCUGGAGAUU
189
CGCUCAAUGCCUGGAGAUU
189
AAUCUCCAGGCAUUGAGCG
885

CAAUGCCUGGAGAUUUGGG
190
CAAUGCCUGGAGAUUUGGG
190
CCCAAAUCUCCAGGCAUUG
886

GCCGACCUCAUGGGGUACA
191
GCCGACCUCAUGGGGUACA
191
UGUACCCCAUGAGGUCGGC
887

AUCCUAAACCUCAAAGAAA
192
AUCCUAAACCUCAAAGAAA
192
UUUCUUUGAGGUUUAGGAU
888

AGAUUUGGGCGUGCCCCCG
193
AGAUUUGGGCGUGCCCCCG
193
CGGGGGCACGCCCAAAUCU
889

CCCGCUCAAUGCCUGGAGA
194
CCCGCUCAAUGCCUGGAGA
194
UCUCCAGGCAUUGAGCGGG
890

GAGAUUUGGGCGUGCCCCC
195
GAGAUUUGGGCGUGCCCCC
195
GGGGGCACGCCCAAAUCUC
891

GGAGAUUUGGGCGUGCCCC
196
GGAGAUUUGGGCGUGCCCC
196
GGGGCACGCCCAAAUCUCC
892

GAUUUGGGCGUGCCCCCGC
197
GAUUUGGGCGUGCCCCCGC
197
GCGGGGGCACGCCCAAAUC
893

CCGCUCAAUGCCUGGAGAU
198
CCGCUCAAUGCCUGGAGAU
198
AUCUCCAGGCAUUGAGCGG
894

AGUACACCGGAAUUGCCAG
199
AGUACACCGGAAUUGCCAG
199
CUGGCAAUUCCGGUGUACU
895

UACACCGGAAUUGCCAGGA
200
UACACCGGAAUUGCCAGGA
200
UCCUGGCAAUUCCGGUGUA
896

GAGUACACCGGAAUUGCCA
201
GAGUACACCGGAAUUGCCA
201
UGGCAAUUCCGGUGUACUC
897

GUACACCGGAAUUGCCAGG
202
GUACACCGGAAUUGCCAGG
202
CCUGGCAAUUCCGGUGUAC
898

UUGCCGCGCAGGGGCCCCA
203
UUGCCGCGCAGGGGCCCCA
203
UGGGGCCCCUGCGCGGCAA
899

CUGGAGAUUUGGGCGUGCC
204
CUGGAGAUUUGGGCGUGCC
204
GGCACGCCCAAAUCUCCAG
900

GUUGCCGCGCAGGGGCCCC
205
GUUGCCGCGCAGGGGCCCC
205
GGGGCCCCUGCGCGGCAAC
901

GCCUGGAGAUUUGGGCGUG
206
GCCUGGAGAUUUGGGCGUG
206
CACGCCCAAAUCUCCAGGC
902

UGGAGAUUUGGGCGUGCCC
207
UGGAGAUUUGGGCGUGCCC
207
GGGCACGCCCAAAUCUCCA
903

CCUGGAGAUUUGGGCGUGC
208
CCUGGAGAUUUGGGCGUGC
208
GCACGCCCAAAUCUCCAGG
904

UGCUAGCCGAGUAGUGUUG
209
UGCUAGCCGAGUAGUGUUG
209
CAACACUACUCGGCUAGCA
905

UGCCUGGAGAUUUGGGCGU
210
UGCCUGGAGAUUUGGGCGU
210
ACGCCCAAAUCUCCAGGCA
906

CUGCUAGCCGAGUAGUGUU
211
CUGCUAGCCGAGUAGUGUU
211
AACACUACUCGGCUAGCAG
907

ACUGCUAGCCGAGUAGUGU
212
ACUGCUAGCCGAGUAGUGU
212
ACACUACUCGGCUAGCAGU
908

GACUGCUAGCCGAGUAGUG
213
GACUGCUAGCCGAGUAGUG
213
CACUACUCGGCUAGCAGUC
909

AGACUGCUAGCCGAGUAGU
214
AGACUGCUAGCCGAGUAGU
214
ACUACUCGGCUAGCAGUCU
910

ACCCGCUCAAUGCCUGGAG
215
ACCCGCUCAAUGCCUGGAG
215
CUCCAGGCAUUGAGCGGGU
911

AACCCGCUCAAUGCCUGGA
216
AACCCGCUCAAUGCCUGGA
216
UCCAGGCAUUGAGCGGGUU
912

UGCCGCGCAGGGGCCCCAG
217
UGCCGCGCAGGGGCCCCAG
217
CUGGGGCCCCUGCGCGGCA
913

AGGGGCCCCAGGUUGGGUG
218
AGGGGCCCCAGGUUGGGUG
218
CACCCAACCUGGGGCCCCU
914

GGGCCCCAGGUUGGGUGUG
219
GGGCCCCAGGUUGGGUGUG
219
CACACCCAACCUGGGGCCC
915

CAGGGGCCCCAGGUUGGGU
220
CAGGGGCCCCAGGUUGGGU
220
ACCCAACCUGGGGCCCCUG
916

GGCCCCAGGUUGGGUGUGC
221
GGCCCCAGGUUGGGUGUGC
221
GCACACCCAACCUGGGGCC
917

CGCAGGGGCCCCAGGUUGG
222
CGCAGGGGCCCCAGGUUGG
222
CCAACCUGGGGCCCCUGCG
918

UGGGCAGGAUGGCUCCUGU
223
UGGGCAGGAUGGCUCCUGU
223
ACAGGAGCCAUCCUGCCCA
919

GCCCCAGGUUGGGUGUGCG
224
GCCCCAGGUUGGGUGUGCG
224
CGCACACCCAACCUGGGGC
920

GCAGGGGCCCCAGGUUGGG
225
GCAGGGGCCCCAGGUUGGG
225
CCCAACCUGGGGCCCCUGC
921

GGGCAGGAUGGCUCCUGUC
226
GGGCAGGAUGGCUCCUGUC
226
GACAGGAGCCAUCCUGCCC
922

GGGGCCCCAGGUUGGGUGU
227
GGGGCCCCAGGUUGGGUGU
227
ACACCCAACCUGGGGCCCC
923

GCCGCGCAGGGGCCCCAGG
228
GCCGCGCAGGGGCCCCAGG
228
CCUGGGGCCCCUGCGCGGC
924

GCGCAGGGGCCCCAGGUUG
229
GCGCAGGGGCCCCAGGUUG
229
CAACCUGGGGCCCCUGCGC
925

CGCGCAGGGGCCCCAGGUU
230
CGCGCAGGGGCCCCAGGUU
230
AACCUGGGGCCCCUGCGCG
926

CCGCGCAGGGGCCCCAGGU
231
CCGCGCAGGGGCCCCAGGU
231
ACCUGGGGCCCCUGCGCGG
927

AGGACGACCGGGUCCUUUC
232
AGGACGACCGGGUCCUUUC
232
GAAAGGACCCGGUCGUCCU
928

CAGGACGACCGGGUCCUUU
233
CAGGACGACCGGGUCCUUU
233
AAAGGACCCGGUCGUCCUG
929

UGCCAGGACGACCGGGUCC
234
UGCCAGGACGACCGGGUCC
234
GGACCCGGUCGUCCUGGCA
930

AUUGCCAGGACGACCGGGU
235
AUUGCCAGGACGACCGGGU
235
ACCCGGUCGUCCUGGCAAU
931

AAUUGCCAGGACGACCGGG
236
AAUUGCCAGGACGACCGGG
236
CCCGGUCGUCCUGGCAAUU
932

UUGCCAGGACGACCGGGUC
237
UUGCCAGGACGACCGGGUC
237
GACCCGGUCGUCCUGGCAA
933

CCAGGACGACCGGGUCCUU
238
CCAGGACGACCGGGUCCUU
238
AAGGACCCGGUCGUCCUGG
934

GCCAGGACGACCGGGUCCU
239
GCCAGGACGACCGGGUCCU
239
AGGACCCGGUCGUCCUGGC
935

GAAUUGCCAGGACGACCGG
240
GAAUUGCCAGGACGACCGG
240
CCGGUCGUCCUGGCAAUUC
936

ACGACCGGGUCCUUUCUUG
241
ACGACCGGGUCCUUUCUUG
241
CAAGAAAGGACCCGGUCGU
937

GACGACCGGGUCCUUUCUU
242
GACGACCGGGUCCUUUCUU
242
AAGAAAGGACCCGGUCGUC
938

CGACCGGGUCCUUUCUUGG
243
CGACCGGGUCCUUUCUUGG
243
CCAAGAAAGGACCCGGUCG
939

GGACGACCGGGUCCUUUCU
244
GGACGACCGGGUCCUUUCU
244
AGAAAGGACCCGGUCGUCC
940

CCGGAAUUGCCAGGACGAC
245
CCGGAAUUGCCAGGACGAC
245
GUCGUCCUGGCAAUUCCGG
941

ACACCGGAAUUGCCAGGAC
246
ACACCGGAAUUGCCAGGAC
246
GUCCUGGCAAUUCCGGUGU
942

ACCGGAAUUGCCAGGACGA
247
ACCGGAAUUGCCAGGACGA
247
UCGUCCUGGCAAUUCCGGU
943

CGGAAUUGCCAGGACGACC
248
CGGAAUUGCCAGGACGACC
248
GGUCGUCCUGGCAAUUCCG
944

GGAAUUGCCAGGACGACCG
249
GGAAUUGCCAGGACGACCG
249
CGGUCGUCCUGGCAAUUCC
945

CACCGGAAUUGCCAGGACG
250
CACCGGAAUUGCCAGGACG
250
CGUCCUGGCAAUUCCGGUG
946

CCCCAGGUUGGGUGUGCGC
251
CCCCAGGUUGGGUGUGCGC
251
GCGCACACCCAACCUGGGG
947

GAUCGUUGGUGGAGUUUAC
252
GAUCGUUGGUGGAGUUUAC
252
GUAAACUCCACCAACGAUC
948

CAGAUCGUUGGUGGAGUUU
253
CAGAUCGUUGGUGGAGUUU
253
AAACUCCACCAACGAUCUG
949

AGAUCGUUGGUGGAGUUUA
254
AGAUCGUUGGUGGAGUUUA
254
UAAACUCCACCAACGAUCU
950

CCCAGGUUGGGUGUGCGCG
255
CCCAGGUUGGGUGUGCGCG
255
CGCGCACACCCAACCUGGG
951

CCAGGUUGGGUGUGCGCGC
256
CCAGGUUGGGUGUGCGCGC
256
GCGCGCACACCCAACCUGG
952

AGGUUGGGUGUGCGCGCGA
257
AGGUUGGGUGUGCGCGCGA
257
UCGCGCGCACACCCAACCU
953

CAGGUUGGGUGUGCGCGCG
258
CAGGUUGGGUGUGCGCGCG
258
CGCGCGCACACCCAACCUG
954

GGUUGGGUGUGCGCGCGAC
259
GGUUGGGUGUGCGCGCGAC
259
GUCGCGCGCACACCCAACC
955

GAAAAACCAAACGUAACAC
260
GAAAAACCAAACGUAACAC
260
GUGUUACGUUUGGUUUUUC
956

AGAAAAACCAAACGUAACA
261
AGAAAAACCAAACGUAACA
261
UGUUACGUUUGGUUUUUCU
957

AACCAAACGUAACACCAAC
262
AACCAAACGUAACACCAAC
262
GUUGGUGUUACGUUUGGUU
958

AAAGAAAAACCAAACGUAA
263
AAAGAAAAACCAAACGUAA
263
UUACGUUUGGUUUUUCUUU
959

AAAAACCAAACGUAACACC
264
AAAAACCAAACGUAACACC
264
GGUGUUACGUUUGGUUUUU
960

AAGAAAAACCAAACGUAAC
265
AAGAAAAACCAAACGUkAC
265
GUUACGUUUGGUUUUUCUU
961

CAAAGAAAAACCAAACGUA
266
CAAAGAAAAACCAAACGUA
266
UACGUUUGGUUUUUCUUUG
962

ACCCCCGGCGUAGGUCGCG
267
ACCCCCGGCGUAGGUCGCG
267
CGCGACCUACGCCGGGGGU
963

GACCCCCGGCGUAGGUCGC
268
GACCCCCGGCGUAGGUCGC
268
GCGACCUACGCCGGGGGUC
964

CGUUAGUAUGAGUGUCGUG
269
CGUUAGUAUGAGUGUCGUG
269
CACGACACUCAUACUAACG
965

GUUAGUAUGAGUGUCGUGC
270
GUUAGUAUGAGUGUCGUGC
270
GCACGACACUCAUACUAAC
966

UUAGUAUGAGUGUCGUGCA
271
UUAGUAUGAGUGUCGUGCA
271
UGCACGACACUCAUACUAA
967

CCAAACGUAACACCAACCG
272
CCAAACGUAACACCAACCG
272
CGGUUGGUGUUACGUUUGG
968

ACCAAACGUAACACCAACC
273
ACCAAACGUAACACCAACC
273
GGUUGGUGUUACGUUUGGU
969

UUGGGCGUGCCCCCGCGAG
274
UUGGGCGUGCCCCCGCGAG
274
CUCGCGGGGGCACGCCCAA
970

AUUUGGGCGUGCCCCCGCG
275
AUUUGGGCGUGCCCCCGCG
275
CGCGGGGGCACGCCCAAAU
971

UUUGGGCGUGCCCCCGCGA
276
UUUGGGCGUGCCCCCGCGA
276
UCGCGGGGGCACGCCCAAA
972

AAACCAAACGUAACACCAA
277
AAACCAAACGUAACACCAA
277
UUGGUGUUACGUUUGGUUU
973

UGGGCGUGCCCCCGCGAGA
278
UGGGCGUGCCCCCGCGAGA
278
UCUCGCGGGGGCACGCCCA
974

GUCAGAUCGUUGGUGGAGU
279
GUCAGAUCGUUGGUGGAGU
279
ACUCCACCAACGAUCUGAC
975

GUGUCGUGCAGCCUCCAGG
280
GUGUCGUGCAGCCUCCAGG
280
CCUGGAGGCUGCACGACAC
976

GGUCAGAUCGUUGGUGGAG
281
GGUCAGAUCGUUGGUGGAG
281
CUCCACCAACGAUCUGACC
977

AGUGUCGUGCAGCCUCCAG
282
AGUGUCGUGCAGCCUCCAG
282
CUGGAGGCUGCACGACACU
978

GAGUGUCGUGCAGCCUCCA
283
GAGUGUCGUGCAGCCUCCA
283
UGGAGGCUGCACGACACUC
979

UCGUAGACCGUGCACCAUG
284
UCGUAGACCGUGCACCAUG
284
CAUGGUGCACGGUCUACGA
980

GACCGUGCACCAUGAGCAC
285
GACCGUGCACCAUGAGCAC
285
GUGCUCAUGGUGCACGGUC
981

AGUAUGAGUGUCGUGCAGC
286
AGUAUGAGUGUCGUGCAGC
286
GCUGCACGACACUCAUACU
982

UAGUAUGAGUGUCGUGCAG
287
UAGUAUGAGUGUCGUGCAG
287
CUGCACGACACUCAUACUA
983

UCAGAUCGUUGGUGGAGUU
288
UCAGAUCGUUGGUGGAGUU
288
AACUCCACCAACGAUCUGA
984

AGACCGUGCACCAUGAGCA
289
AGACCGUGCACCAUGAGCA
289
UGCUCAUGGUGCACGGUCU
985

AAAACCAAACGUAACACCA
290
AAAACCAAACGUAACACCA
290
UGGUGUUACGUUUGGUUUU
986

GUAGACCGUGCACCAUGAG
291
GUAGACCGUGCACCAUGAG
291
CUCAUGGUGCACGGUCUAC
987

CUCGUAGACCGUGCACCAU
292
CUCGUAGACCGUGCACCAU
292
AUGGUGCACGGUCUACGAG
988

CGUAGACCGUGCACCAUGA
293
CGUAGACCGUGCACCAUGA
293
UCAUGGUGCACGGUCUACG
989

CCUGGGCUCAGCCCGGGUA
294
CCUGGGCUCAGCCCGGGUA
294
UACCCGGGCUGAGCCCAGG
990

UAGACCGUGCACCAUGAGC
295
UAGACCGUGCACCAUGAGC
295
GCUCAUGGUGCACGGUCUA
991

GGUCUCGUAGACCGUGCAC
296
GGUCUCGUAGACCGUGCAC
296
GUGCACGGUCUACGAGACC
992

UCUCGUAGACCGUGCACCA
297
UCUCGUAGACCGUGCACCA
297
UGGUGCACGGUCUACGAGA
993

GUCUCGUAGACCGUGCACC
298
GUCUCGUAGACCGUGCACC
298
GGUGCACGGUCUACGAGAC
994

UUGGGUAAGGUCAUCGAUA
299
UUGGGUAAGGUCAUCGAUA
299
UAUCGAUGACCUUACCCAA
995

UCGCCGACCUCAUGGGGUA
300
UCGCCGACCUCAUGGGGUA
300
UACCCCAUGAGGUCGGCGA
996

CCUCAAAGAAAAACCAAAC
301
CCUCAAAGAAAAACCAAAC
301
GUUUGGUUUUUCUUUGAGG
997

GGGCGUGCCCCCGCGAGAC
302
GGGCGUGCCCCCGCGAGAC
302
GUCUCGCGGGGGCACGCCC
998

GGAUGAACCGGCUGAUAGC
303
GGAUGAACCGGCUGAUAGC
303
GCUAUCAGCCGGUUCAUCC
999

UGGAUGAACCGGCUGAUAG
304
UGGAUGAACCGGCUGAUAG
304
CUAUCAGCCGGUUCAUCCA
1000

CUCAAAGAAAAACCAAACG
305
CUCAAAGAAAAACCAAACG
305
CGUUUGGUUUUUCUUUGAG
1001

AGGAAGACUUCCGAGCGGU
306
AGGAAGACUUCCGAGCGGU
306
ACCGCUCGGAAGUCUUCCU
1002

UCAAAGAAAAACCAAACGU
307
UCAAAGAAAAACCAAACGU
307
ACGUUUGGUUUUUCUUUGA
1003

GGAAGACUUCCGAGCGGUC
308
GGAAGACUUCCGAGCGGUC
308
GACCGCUCGGAAGUCUUCC
1004

CGCCGACCUCAUGGGGUAC
309
CGCCGACCUCAUGGGGUAC
309
GUACCCCAUGAGGUCGGCG
1005

CUUCCGAGCGGUCGCAACC
310
CUUCCGAGCGGUCGCAACC
310
GGUUGCGACCGCUCGGAAG
1006

GGCGUGCCCCCGCGAGACU
311
GGCGUGCCCCCGCGAGACU
311
AGUCUCGCGGGGGCACGCC
1007

UAUGAGUGUCGUGCAGCCU
312
UAUGAGUGUCGUGCAGCCU
312
AGGCUGCACGACACUCAUA
1008

UGCCCCCGCGAGACUGCUA
313
UGCCCCCGCGAGACUGCUA
313
UAGCAGUCUCGCGGGGGCA
1009

CGAGACUGCUAGCCGAGUA
314
CGAGACUGCUAGCCGAGUA
314
UACUCGGCUAGCAGUCUCG
1010

UGAGUGUCGUGCAGCCUCC
315
UGAGUGUCGUGCAGCCUCC
315
GGAGGCUGCACGACACUCA
1011

GCCCCCGCGAGACUGCUAG
316
GCCCCCGCGAGACUGCUAG
316
CUAGCAGUCUCGCGGGGGC
1012

GAGACUGCUAGCCGAGUAG
317
GAGACUGCUAGCCGAGUAG
317
CUACUCGGCUAGCAGUCUC
1013

CCCCCGCGAGACUGCUAGC
318
CCCCCGCGAGACUGCUAGC
318
GCUAGCAGUCUCGCGGGGG
1014

CGCGAGACUGCUAGCCGAG
319
CGCGAGACUGCUAGCCGAG
319
CUCGGCUAGCAGUCUCGCG
1015

GUAUGAGUGUCGUGCAGCC
320
GUAUGAGUGUCGUGCAGCC
320
GGCUGCACGACACUCAUAC
1016

AUGAGUGUCGUGCAGCCUC
321
AUGAGUGUCGUGCAGCCUC
321
GAGGCUGCACGACACUCAU
1017

GCGAGACUGCUAGCCGAGU
322
GCGAGACUGCUAGCCGAGU
322
ACUCGGCUAGCAGUCUCGC
1018

CCCCGCGAGACUGCUAGCC
323
CCCCGCGAGACUGCUAGCC
323
GGCUAGCAGUCUCGCGGGG
1019

CCGCGAGACUGCUAGCCGA
324
CCGCGAGACUGCUAGCCGA
324
UCGGCUAGCAGUCUCGCGG
1020

CCCGCGAGACUGCUAGCCG
325
CCCGCGAGACUGCUAGCCG
325
CGGCUAGCAGUCUCGCGGG
1021

GCGUGCCCCCGCGAGACUG
326
GCGUGCCCCCGCGAGACUG
326
CAGUCUCGCGGGGGCACGC
1022

GACCCCCCCUCCCGGGAGA
327
GACCCCCCCUCCCGGGAGA
327
UCUCCCGGGAGGGGGGGUC
1023

CGGGUCCUUUCUUGGAUCA
328
CGGGUCCUUUCUUGGAUCA
328
UGAUCCAAGAAAGGACCCG
1024

GUGCCCCCGCGAGACUGCU
329
GUGCCCCCGCGAGACUGCU
329
AGCAGUCUCGCGGGGGCAC
1025

CGUGCCCCCGCGAGACUGC
330
CGUGCCCCCGCGAGACUGC
330
GCAGUCUCGCGGGGGCACG
1026

UUCGCCGACCUCAUGGGGU
331
UUCGCCGACCUCAUGGGGU
331
ACCCCAUGAGGUCGGCGAA
1027

CGCCCACAGGACGUCAAGU
332
CGCCCACAGGACGUCAAGU
332
ACUUGACGUCCUGUGGGCG
1028

GCCCACAGGACGUCAAGUU
333
GCCCACAGGACGUCAAGUU
333
AACUUGACGUCCUGUGGGC
1029

ACCCCCCCUCCCGGGAGAG
334
ACCCCCCCUCCCGGGAGAG
334
CUCUCCCGGGAGGGGGGGU
1030

GGACCCCCCCUCCCGGGAG
335
GGACCCCCCCUCCCGGGAG
335
CUCCCGGGAGGGGGGGUCC
1031

CCGGGUCCUUUCUUGGAUC
336
CCGGGUCCUUUCUUGGAUC
336
GAUCCAAGAAAGGACCCGG
1032

CAGGACCCCCCCUCCCGGG
337
CAGGACCCCCCCUCCCGGG
337
CCCGGGAGGGGGGGUCCUG
1033

AGGACGUCAAGUUCCCGGG
338
AGGACGUCAAGUUCCCGGG
338
CCCGGGAACUUGACGUCCU
1034

AGGACCCCCCCUCCCGGGA
339
AGGACCCCCCCUCCCGGGA
339
UCCCGGGAGGGGGGGUCCU
1035

CCACAGGACGUCAAGUUCC
340
CCACAGGACGUCAAGUUCC
340
GGAACUUGACGUCCUGUGG
1036

CAGGACGUCAAGUUCCCGG
341
CAGGACGUCAAGUUCCCGG
341
CCGGGAACUUGACGUCCUG
1037

ACAGGACGUCAAGUUCCCG
342
ACAGGACGUCAAGUUCCCG
342
CGGGAACUUGACGUCCUGU
1038

CACAGGACGUCAAGUUCCC
343
CACAGGACGUCAAGUUCCC
343
GGGAACUUGACGUCCUGUG
1039

CAGUGGAUGAACCGGCUGA
344
CAGUGGAUGAACCGGCUGA
344
UCAGCCGGUUCAUCCACUG
1040

GGGCUCAGCCCGGGUACCC
345
GGGCUCAGCCCGGGUACCC
345
GGGUACCCGGGCUGAGCCC
1041

CCGAGCGGUCGCAACCUCG
346
CCGAGCGGUCGCAACCUCG
346
CGAGGUUGCGACCGCUCGG
1042

CUGGGCUCAGCCCGGGUAC
347
CUGGGCUCAGCCCGGGUAC
347
GUACCCGGGCUGAGCCCAG
1043

AGUGGAUGAACCGGCUGAU
348
AGUGGAUGAACCGGCUGAU
348
AUCAGCCGGUUCAUCCACU
1044

UCCGAGCGGUCGCAACCUC
349
UCCGAGCGGUCGCAACCUC
349
GAGGUUGCGACCGCUCGGA
1045

UGGGCUCAGCCCGGGUACC
350
UGGGCUCAGCCCGGGUACC
350
GGUACCCGGGCUGAGCCCA
1046

GGUACCCUUGGCCCCUCUA
351
GGUACCCUUGGCCCCUCUA
351
UAGAGGGGCCAAGGGUACC
1047

UUCCGAGCGGUCGCAACCU
352
UUCCGAGCGGUCGCAACCU
352
AGGUUGCGACCGCUCGGAA
1048

GGGUACCCUUGGCCCCUCU
353
GGGUACCCUUGGCCCCUCU
353
AGAGGGGCCAAGGGUACCC
1049

GGGUCCUUUCUUGGAUCAA
354
GGGUCCUUUCUUGGAUCAA
354
UUGAUCCAAGAAAGGACCC
1050

CCCACAGGACGUCAAGUUC
355
CCCACAGGACGUCAAGUUC
355
GAACUUGACGUCCUGUGGG
1051

GGUUGCUCUUUCUCUAUCU
356
GGUUGCUCUUUCUCUAUCU
356
AGAUAGAGAAAGAGCAACC
1052

GUGGGCAGGAUGGCUCCUG
357
GUGGGCAGGAUGGCUCCUG
357
CAGGAGCCAUCCUGCCCAC
1053

GGUGGGCAGGAUGGCUCCU
358
GGUGGGCAGGAUGGCUCCU
358
AGGAGCCAUCCUGCCCACC
1054

GUUGCUCUUUCUCUAUCUU
359
GUUGCUCUUUCUCUAUCUU
359
AAGAUAGAGAAAGAGCAAC
1055

GUGGAUGAACCGGCUGAUA
360
GUGGAUGAACCGGCUGAUA
360
UAUCAGCCGGUUCAUCCAC
1056

CCAGGACCCCCCCUCCCGG
361
CCAGGACCCCCCCUCCCGG
361
CCGGGAGGGGGGGUCCUGG
1057

GGGUGGGCAGGAUGGCUCC
362
GGGUGGGCAGGAUGGCUCC
362
GGAGCCAUCCUGCCCACCC
1058

CUUCACGGAGGCUAUGACU
363
CUUCACGGAGGCUAUGACU
363
AGUCAUAGCCUCCGUGAAG
1059

ACCGCCGCCCACAGGACGU
364
ACCGCCGCCCACAGGACGU
364
ACGUCCUGUGGGCGGCGGU
1060

UCCAGGACCCCCCCUCCCG
365
UCCAGGACCCCCCCUCCCG
365
CGGGAGGGGGGGUCCUGGA
1061

AUAUGAUGAUGAACUGGUC
366
AUAUGAUGAUGAACUGGUC
366
GACCAGUUCAUCAUCAUAU
1062

UUCACGGAGGCUAUGACUA
367
UUCACGGAGGCUAUGACUA
367
UAGUCAUAGCCUCCGUGAA
1063

UCACGGAGGCUAUGACUAG
368
UCACGGAGGCUAUGACUAG
368
CUAGUCAUAGCCUCCGUGA
1064

AUGAACCGGCUGAUAGCGU
369
AUGAACCGGCUGAUAGCGU
369
ACGCUAUCAGCCGGUUCAU
1065

GGGAUAUGAUGAUGAACUG
370
GGGAUAUGAUGAUGAACUG
370
CAGUUCAUCAUCAUAUCCC
1066

UGCAGUGGAUGAACCGGCU
371
UGCAGUGGAUGAACCGGCU
371
AGCCGGUUCAUCCACUGCA
1067

GUGCAGUGGAUGAACCGGC
372
GUGCAGUGGAUGAACCGGC
372
GCCGGUUCAUCCACUGCAC
1068

UGAACCGGCUGAUAGCGUU
373
UGAACCGGCUGAUAGCGUU
373
AACGCUAUCAGCCGGUUCA
1069

GGAUAUGAUGAUGAACUGG
374
GGAUAUGAUGAUGAACUGG
374
CCAGUUCAUCAUCAUAUCC
1070

GCUCUUUCUCUAUCUUCCU
375
GCUCUUUCUCUAUCUUCCU
375
AGGAAGAUAGAGAAAGAGC
1071

GGGGGCGACACUCCACCAU
376
GGGGGCGACACUCCACCAU
376
AUGGUGGAGUGUCGCCCCC
1072

GAUGAACCGGCUGAUAGCG
377
GAUGAACCGGCUGAUAGCG
377
CGCUAUCAGCCGGUUCAUC
1073

GAUAUGAUGAUGAACUGGU
378
GAUAUGAUGAUGAACUGGU
378
ACCAGUUCAUCAUCAUAUC
1074

UGGGAUAUGAUGAUGAACU
379
UGGGAUAUGAUGAUGAACU
379
AGUUCAUCAUCAUAUCCCA
1075

UUGCUCUUUCUCUAUCUUC
380
UUGCUCUUUCUCUAUCUUC
380
GAAGAUAGAGAAAGAGCAA
1076

UGGGGGCGACACUCCACCA
381
UGGGGGCGACACUCCACCA
381
UGGUGGAGUGUCGCCCCCA
1077

UGCUCUUUCUCUAUCUUCC
382
UGCUCUUUCUCUAUCUUCC
382
GGAAGAUAGAGAAAGAGCA
1078

GGUCCUUUCUUGGAUCAAC
383
GGUCCUUUCUUGGAUCAAC
383
GUUGAUCCAAGAAAGGACC
1079

AAGACUUCCGAGCGGUCGC
384
AAGACUUCCGAGCGGUCGC
384
GCGACCGCUCGGAAGUCUU
1080

AGCCCGGGUACCCUUGGCC
385
AGCCCGGGUACCCUUGGCC
385
GGCCAAGGGUACCCGGGCU
1081

UUUCUUGGAUCAACCCGCU
386
UUUCUUGGAUCAACCCGCU
386
AGCGGGUUGAUCCAAGAAA
1082

CAGCCCGGGUACCCUUGGC
387
CAGCCCGGGUACCCUUGGC
387
GCCAAGGGUACCCGGGCUG
1083

AGACUUCCGAGCGGUCGCA
388
AGACUUCCGAGCGGUCGCA
388
UGCGACCGCUCGGAAGUCU
1084

UUCUUGGAUCAACCCGCUC
389
UUCUUGGAUCAACCCGCUC
389
GAGCGGGUUGAUCCAAGAA
1085

CCCGGGUACCCUUGGCCCC
390
CCCGGGUACCCUUGGCCCC
390
GGGGCCAAGGGUACCCGGG
1086

GUCCUUUCUUGGAUCAACC
391
GUCCUUUCUUGGAUCAACC
391
GGUUGAUCCAAGAAAGGAC
1087

CUUUCUUGGAUCAACCCGC
392
CUUUCUUGGAUCAACCCGC
392
GCGGGUUGAUCCAAGAAAG
1088

CCUUUCUUGGAUCAACCCG
393
CCUUUCUUGGAUCAACCCG
393
CGGGUUGAUCCAAGAAAGG
1089

UCCUUUCUUGGAUCAACCC
394
UCCUUUCUUGGAUCAACCC
394
GGGUUGAUCCAAGAAAGGA
1090

AAGUUCCCGGGCGGUGGUC
395
AAGUUCCCGGGCGGUGGUC
395
GACCACCGCCCGGGAACUU
1091

GCAGUGGAUGAACCGGCUG
396
GCAGUGGAUGAACCGGCUG
396
CAGCCGGUUCAUCCACUGC
1092

CCGGGUACCCUUGGCCCCU
397
CCGGGUACCCUUGGCCCCU
397
AGGGGCCAAGGGUACCCGG
1093

AGUUCCCGGGCGGUGGUCA
398
AGUUCCCGGGCGGUGGUCA
398
UGACCACCGCCCGGGAACU
1094

CUUGGAUCAACCCGCUCAA
399
CUUGGAUCAACCCGCUCAA
399
UUGAGCGGGUUGAUCCAAG
1095

GGAUCAACCCGCUCAAUGC
400
GGAUCAACCCGCUCAAUGC
400
GCAUUGAGCGGGUUGAUCC
1096

ACUUCCGAGCGGUCGCAAC
401
ACUUCCGAGCGGUCGCAAC
401
GUUGCGACCGCUCGGAAGU
1097

UCUUGGAUCAACCCGCUCA
402
UCUUGGAUCAACCCGCUCA
402
UGAGCGGGUUGAUCCAAGA
1098

UUGGAUCAACCCGCUCAAU
403
UUGGAUCAACCCGCUCAAU
403
AUUGAGCGGGUUGAUCCAA
1099

AACCGCCGCCCACAGGACG
404
AACCGCCGCCCACAGGACG
404
CGUCCUGUGGGCGGCGGUU
1100

GCGUGAACUAUGCAACAGG
405
GCGUGAACUAUGCAACAGG
405
CCUGUUGCAUAGUUCACGC
1101

AUCAACCCGCUCAAUGCCU
406
AUCAACCCGCUCAAUGCCU
406
AGGCAUUGAGCGGGUUGAU
1102

GAUCAACCCGCUCAAUGCC
407
GAUCAACCCGCUCAAUGCC
407
GGCAUUGAGCGGGUUGAUC
1103

CAACCCGCUCAAUGCCUGG
408
CAACCCGCUCAAUGCCUGG
408
CCAGGCAUUGAGCGGGUUG
1104

GCUUCGCCGACCUCAUGGG
409
GCUUCGCCGACCUCAUGGG
409
CCCAUGAGGUCGGCGAAGC
1105

GACUUCCGAGCGGUCGCAA
410
GACUUCCGAGCGGUCGCAA
410
UUGCGACCGCUCGGAAGUC
1106

UCAACCCGCUCAAUGCCUG
411
UCAACCCGCUCAAUGCCUG
411
CAGGCAUUGAGCGGGUUGA
1107

GGCUUCGCCGACCUCAUGG
412
GGCUUCGCCGACCUCAUGG
412
CCAUGAGGUCGGCGAAGCC
1108

UGGAUCAACCCGCUCAAUG
413
UGGAUCAACCCGCUCAAUG
413
CAUUGAGCGGGUUGAUCCA
1109

CGGGCGGUGGUCAGAUCGU
414
CGGGCGGUGGUCAGAUCGU
414
ACGAUCUGACCACCGCCCG
1110

CUUGGCCCCUCUAUGGCAA
415
CUUGGCCCCUCUAUGGCAA
415
UUGCCAUAGAGGGGCCAAG
1111

CCGGGCGGUGGUCAGAUCG
416
CCGGGCGGUGGUCAGAUCG
416
CGAUCUGACCACCGCCCGG
1112

UGGGGUGGGCAGGAUGGCU
417
UGGGGUGGGCAGGAUGGCU
417
AGCCAUCCUGCCCACCCCA
1113

GGAGUUUACCUGUUGCCGC
418
GGAGUUUACCUGUUGCCGC
418
GCGGCAACAGGUAAACUCC
1114

CCUUGGCCCCUCUAUGGCA
419
CCUUGGCCCCUCUAUGGCA
419
UGCCAUAGAGGGGCCAAGG
1115

GUGGAGUUUACCUGUUGCC
420
GUGGAGUUUACCUGUUGCC
420
GGCAACAGGUAAACUCCAC
1116

GGUGGAGUUUACCUGUUGC
421
GGUGGAGUUUACCUGUUGC
421
GCAACAGGUAAACUCCACC
1117

UUCCCGGGCGGUGGUCAGA
422
UUCCCGGGCGGUGGUCAGA
422
UCUGACCACCGCCCGGGAA
1118

UGAACUAUGCAACAGGGAA
423
UGAACUAUGCAACAGGGAA
423
UUCCCUGUUGCAUAGUUCA
1119

AGUUUACCUGUUGCCGCGC
424
AGUUUACCUGUUGCCGCGC
424
GCGCGGCAACAGGUAAACU
1120

GUGAACUAUGCAACAGGGA
425
GUGAACUAUGCAACAGGGA
425
UCCCUGUUGCAUAGUUCAC
1121

UUACCUGUUGCCGCGCAGG
426
UUACCUGUUGCCGCGCAGG
426
CCUGCGCGGCAACAGGUAA
1122

UCCCGGGCGGUGGUCAGAU
427
UCCCGGGCGGUGGUCAGAU
427
AUCUGACCACCGCCCGGGA
1123

GUUCCCGGGCGGUGGUCAG
428
GUUCCCGGGCGGUGGUCAG
428
CUGACCACCGCCCGGGAAC
1124

GCCCGGGUACCCUUGGCCC
429
GCCCGGGUACCCUUGGCCC
429
GGGCCAAGGGUACCCGGGC
1125

AAGGAGAUGAAGGCGAAGG
430
AAGGAGAUGAAGGCGAAGG
430
CCUUCGCCUUCAUCUCCUU
1126

AGGAGAUGAAGGCGAAGGC
431
AGGAGAUGAAGGCGAAGGC
431
GCCUUCGCCUUCAUCUCCU
1127

GUUUACCUGUUGCCGCGCA
432
GUUUACCUGUUGCCGCGCA
432
UGCGCGGCAACAGGUAAAC
1128

CUGUUGCCGCGCAGGGGCC
433
CUGUUGCCGCGCAGGGGCC
433
GGCCCCUGCGCGGCAACAG
1129

AACACCAACCGCCGCCCAC
434
AACACCAACCGCCGCCCAC
434
GUGGGCGGCGGUUGGUGUU
1130

GAGUUUACCUGUUGCCGCG
435
GAGUUUACCUGUUGCCGCG
435
CGCGGCAACAGGUAAACUC
1131

UUUACCUGUUGCCGCGCAG
436
UUUACCUGUUGCCGCGCAG
436
CUGCGCGGCAACAGGUAAA
1132

GGGGUGGGCAGGAUGGCUC
437
GGGGUGGGCAGGAUGGCUC
437
GAGCCAUCCUGCCCACCCC
1133

GAAGACUUCCGAGCGGUCG
438
GAAGACUUCCGAGCGGUCG
438
CGACCGCUCGGAAGUCUUC
1134

ACCUGUUGCCGCGCAGGGG
439
ACCUGUUGCCGCGCAGGGG
439
CCCCUGCGCGGCAACAGGU
1135

UACCUGUUGCCGCGCAGGG
440
UACCUGUUGCCGCGCAGGG
440
CCCUGCGCGGCAACAGGUA
1136

UACCUCUUCAACUGGGCAG
441
UACCUCUUCAACUGGGCAG
441
CUGCCCAGUUGAAGAGGUA
1137

CGUGAACUAUGCAACAGGG
442
CGUGAACUAUGCAACAGGG
442
CCCUGUUGCAUAGUUCACG
1i38

ACACCAACCGCCGCCCACA
443
ACACCAACCGCCGCCCACA
443
UGUGGGCGGCGGUUGGUGU
1139

CCCGGGCGGUGGUCAGAUC
444
CCCGGGCGGUGGUCAGAUC
444
GAUCUGACCACCGCCCGGG
1140

ACCUCUUCAACUGGGCAGU
445
ACCUCUUCAACUGGGCAGU
445
ACUGCCCAGUUGAAGAGGU
1141

CUUCGCCGACCUCAUGGGG
446
CUUCGCCGACCUCAUGGGG
446
CCCCAUGAGGUCGGCGAAG
1142

CCUGUUGCCGCGCAGGGGC
447
CCUGUUGCCGCGCAGGGGC
447
GCCCCUGCGCGGCAACAGG
1143

CCAACCGCCGCCCACAGGA
448
CCAACCGCCGCCCACAGGA
448
UCCUGUGGGCGGCGGUUGG
1144

ACCAACCGCCGCCCACAGG
449
ACCAACCGCCGCCCACAGG
449
CCUGUGGGCGGCGGUUGGU
1145

UGGAGUUUACCUGUUGCCG
450
UGGAGUUUACCUGUUGCCG
450
CGGCAACAGGUAAACUCCA
1146

CACCAACCGCCGCCCACAG
451
CACCAACCGCCGCCCACAG
451
CUGUGGGCGGCGGUUGGUG
1147

CAAACGUAACACCAACCGC
452
CAAACGUAACACCAACCGC
452
GCGGUUGGUGUUACGUUUG
1148

CAAGCGGAGACGGCUGGAG
453
CXAGCGGAGACGGCUGGAG
453
CUCCAGCCGUCUCCGCUUG
1149

ACGGAGGCUAUGACUAGGU
454
ACGGAGGCUAUGACUAGGU
454
ACCUAGUCAUAGCCUCCGU
1150

UAACACCAACCGCCGCCCA
455
UAACACCAACCGCCGCCCA
455
UGGGCGGCGGUUGGUGUUA
1151

AUCGUUGGUGGAGUUUACC
456
AUCGUUGGUGGAGUUUACC
456
GGUAAACUCCACCAACGAU
1152

GGGAGACAUAUAUCACAGC
457
GGGAGACAUAUAUCACAGC
457
GCUGUGAUAUAUGUCUCCC
1153

AACCUCGUGGAAGGCGACA
458
AACCUCGUGGAAGGCGACA
458
UGUCGCCUUCCACGAGGUU
1154

GGGGGAGACAUAUAUCACA
459
GGGGGAGACAUAUAUCACA
459
UGUGAUAUAUGUCUCCCCC
1155

AACGUAACACCAACCGCCG
460
AACGUAACACCAACCGCCG
460
CGGCGGUUGGUGUUACGUU
1156

AAACGUAACACCAACCGCC
461
AAACGUAACACCAACCGCC
461
GGCGGUUGGUGUUACGUUU
1157

GGGGAGACAUAUAUCACAG
462
GGGGAGACAUAUAUCACAG
462
CUGUGAUAUAUGUCUCCCC
1158

GAGAUGAAGGCGAAGGCGU
463
GAGAUGAAGGCGAAGGCGU
463
ACGCCUUCGCCUUCAUCUC
1159

AAGCGGAGACGGCUGGAGC
464
AAGCGGAGACGGCUGGAGC
464
GCUCCAGCCGUCUCCGCUU
1160

GUACCCUUGGCCCCUCUAU
465
GUACCCUUGGCCCCUCUAU
465
AUAGAGGGGCCAAGGGUAC
1161

CCUCCAGGACCCCCCCUCC
466
CCUCCAGGACCCCCCCUCC
466
GGAGGGGGGGUCCUGGAGG
1162

CUCCAGGACCCCCCCUCCC
467
CUCCAGGACCCCCCCUCCC
467
GGGAGGGGGGGUCCUGGAG
1163

UACCCUUGGCCCCUCUAUG
468
UACCCUUGGCCCCUCUAUG
468
CAUAGAGGGGCCAAGGGUA
1164

CAACCUCGUGGAAGGCGAC
469
CAACCUCGUGGAAGGCGAC
469
GUCGCCUUCCACGAGGUUG
1165

CGGAGGCUAUGACUAGGUA
470
CGGAGGCUAUGACUAGGUA
470
UACCUAGUCAUAGCCUCCG
1166

GGAGAUGAAGGCGAAGGCG
471
GGAGAUGAAGGCGAAGGCG
471
CGCCUUCGCCUUCAUCUCC
1167

AGAUGAAGGCGAAGGCGUC
472
AGAUGAAGGCGAAGGCGUC
472
GACGCCUUCGCCUUCAUCU
1168

GUAACACCAACCGCCGCCC
473
GUAACACCAACCGCCGCCC
473
GGGCGGCGGUUGGUGUUAC
1169

CGUAACACCAACCGCCGCC
474
CGUAACACCAACCGCCGCC
474
GGCGGCGGUUGGUGUUACG
1170

ACGUAACACCAACCGCCGC
475
ACGUAACACCAACCGCCGC
475
GCGGCGGUUGGUGUUACGU
1171

CACGGAGGCUAUGACUAGG
476
CACGGAGGCUAUGACUAGG
476
CCUAGUCAUAGCCUCCGUG
1172

GUUGGUGGAGUUUACCUGU
477
GUUGGUGGAGUUUACCUGU
477
ACAGGUAAACUCCACCAAC
1173

CGUUGGUGGAGUUUACCUG
478
CGUUGGUGGAGUUUACCUG
478
CAGGUAAACUCCACCAACG
1174

ACCCUUGGCCCCUCUAUGG
479
ACCCUUGGCCCCUCUAUGG
479
CCAUAGAGGGGCCAAGGGU
1175

UUGGUGGAGUUUACCUGUU
480
UUGGUGGAGUUUACCUGUU
480
AACAGGUAAACUCCACCAA
1176

UGGUGGAGUUUACCUGUUG
481
UGGUGGAGUUUACCUGUUG
481
CAACAGGUAAACUCCACCA
1177

UCGUUGGUGGAGUUUACCU
482
UCGUUGGUGGAGUUUACCU
482
AGGUAAACUCCACCAACGA
1178

CGGGUACCCUUGGCCCCUC
483
CGGGUACCCUUGGCCCCUC
483
GAGGGGCCAAGGGUACCCG
1179

GGCUCAGCCCGGGUACCCU
484
GGCUCAGCCCGGGUACCCU
484
AGGGUACCCGGGCUGAGCC
1180

GAUCACUCCCCUGUGAGGA
485
GAUCACUCCCCUGUGAGGA
485
UCCUCACAGGGGAGUGAUC
1181

GGUGGUCAGAUCGUUGGUG
486
GGUGGUCAGAUCGUUGGUG
486
CACCAACGAUCUGACCACC
1182

GAUGAAGGCGAAGGCGUCC
487
GAUGAAGGCGAAGGCGUCC
487
GGACGCCUUCGCCUUCAUC
1183

AGGAUGGCUCCUGUCACCC
488
AGGAUGGCUCCUGUCACCC
488
GGGUGACAGGAGCCAUCCU
1184

CUCAGCCCGGGUACCCUUG
489
CUCAGCCCGGGUACCCUUG
489
CAAGGGUACCCGGGCUGAG
1185

UCAGCCCGGGUACCCUUGG
490
UCAGCCCGGGUACCCUUGG
490
CCAAGGGUACCCGGGCUGA
1186

AUGAAGGCGAAGGCGUCCA
491
AUGAAGGCGAAGGCGUCCA
491
UGGACGCCUUCGCCUUCAU
1187

CGGGGGAGACAUAUAUCAC
492
CGGGGGAGACAUAUAUCAC
492
GUGAUAUAUGUCUCCCCCG
1188

CAGGAUGGCUCCUGUCACC
493
CAGGAUGGCUCCUGUCACC
493
GGUGACAGGAGCCAUCCUG
1189

UGAAGGCGAAGGCGUCCAC
494
UGAAGGCGAAGGCGUCCAC
494
GUGGACGCCUUCGCCUUCA
1190

UGGUCAGAUCGUUGGUGGA
495
UGGUCAGAUCGUUGGUGGA
495
UCCACCAACGAUCUGACCA
1191

GCUCAGCCCGGGUACCCUU
498
GCUCAGCCCGGGUACCCUU
496
AAGGGUACCCGGGCUGAGC
1192

GUGGUCAGAUCGUUGGUGG
497
GUGGUCAGAUCGUUGGUGG
497
CCACCAACGAUCUGACCAC
1193

CAGCCUCCAGGACCCCCCC
498
CAGCCUCCAGGACCCCCCC
498
GGGGGGGUCCUGGAGGCUG
1194

GGCGGUGGUCAGAUCGUUG
499
GGCGGUGGUCAGAUCGUUG
499
CAACGAUCUGACCACCGCC
1195

GCCUCCAGGACCCCCCCUC
500
GCCUCCAGGACCCCCCCUC
500
GAGGGGGGGUCCUGGAGGC
1196

AACCGGCUGAUAGCGUUCG
501
AACCGGCUGAUAGCGUUCG
501
CGAACGCUAUCAGCCGGUU
1197

AGCCUCCAGGACCCCCCCU
502
AGCCUCCAGGACCCCCCCU
502
AGGGGGGGUCCUGGAGGCU
1198

CGGCUUCGCCGACCUCAUG
503
CGGCUUCGCCGACCUCAUG
503
CAUGAGGUCGGCGAAGCCG
1199

GCGGAGACGGCUGGAGCGC
504
GCGGAGACGGCUGGAGCGC
504
GCGCUCCAGCCGUCUCCGC
1200

UCAUGGGGUACAUUCCGCU
505
UCAUGGGGUACAUUCCGCU
505
AGCGGAAUGUACCCCAUGA
1201

GAACCGGCUGAUAGCGUUC
506
GAACCGGCUGAUAGCGUUC
506
GAACGCUAUCAGCCGGUUC
1202

GCGGUGGUCAGAUCGUUGG
507
GCGGUGGUCAGAUCGUUGG
507
CCAACGAUCUGACCACCGC
1203

GGCAGGAUGGCUCCUGUCA
508
GGCAGGAUGGCUCCUGUCA
508
UGACAGGAGCCAUCCUGCC
1204

GCAGGAUGGCUCCUGUCAC
509
GCAGGAUGGCUCCUGUCAC
509
GUGACAGGAGCCAUCCUGC
1205

AUUUGGGUAAGGUCAUCGA
510
AUUUGGGUAAGGUCAUCGA
510
UCGAUGACCUUACCCAAAU
1206

ACCGGCUGAUAGCGUUCGC
511
ACCGGCUGAUAGCGUUCGC
511
GCGAACGCUAUCAGCCGGU
1207

CGGAGACGGCUGGAGCGCG
512
CGGAGACGGCUGGAGCGCG
512
CGCGCUCCAGCCGUCUCCG
1208

GCGGCUUCGCCGACCUCAU
513
GCGGCUUCGCCGACCUCAU
513
AUGAGGUCGGCGAAGCCGC
1209

AAUUUGGGUAAGGUCAUCG
514
AAUUUGGGUAAGGUCAUCG
514
CGAUGACCUUACCCAAAUU
1210

GGGCGGUGGUCAGAUCGUU
515
GGGCGGUGGUCAGAUCGUU
515
AACGAUCUGACCACCGCCC
1211

CAACCGCCGCCCACAGGAC
516
CAACCGCCGCCCACAGGAC
516
GUCCUGUGGGCGGCGGUUG
1212

UGCGGCUUCGCCGACCUCA
517
UGCGGCUUCGCCGACCUCA
517
UGAGGUCGGCGAAGCCGCA
1213

CGGUGGUCAGAUCGUUGGU
518
CGGUGGUCAGAUCGUUGGU
518
ACCAACGAUCUGACCACCG
1214

UUGGGUGUGCGCGCGACUA
519
UUGGGUGUGCGCGCGACUA
519
UAGUCGCGCGCACACCCAA
1215

GUGUGCGCGCGACUAGGAA
520
GUGUGCGCGCGACUAGGAA
520
UUCCUAGUCGCGCGCACAC
1216

GAUGGCUCCUGUCACCCCG
521
GAUGGCUCCUGUCACCCCG
521
CGGGGUGACAGGAGCCAUC
1217

GGAUGGCUCCUGUCACCCC
522
GGAUGGCUCCUGUCACCCC
522
GGGGUGACAGGAGCCAUCC
1218

UGUGCGCGCGACUAGGAAG
523
UGUGCGCGCGACUAGGAAG
523
CUUCCUAGUCGCGCGCACA
1219

UGGGUGUGCGCGCGACUAG
524
UGGGUGUGCGCGCGACUAG
524
CUAGUCGCGCGCACACCCA
1220

GGUGUGCGCGCGACUAGGA
525
GGUGUGCGCGCGACUAGGA
525
UCCUAGUCGCGCGCACACC
1221

GGGUGUGCGCGCGACUAGG
526
GGGUGUGCGCGCGACUAGG
526
CCUAGUCGCGCGCACACCC
1222

CCCCGGCGUAGGUCGCGUA
527
CCCCGGCGUAGGUCGCGUA
527
UACGCGACCUACGCCGGGG
1223

GAAGGCGACAACCUAUCCC
528
GAAGGCGACAACCUAUCCC
528
GGGAUAGGUUGUCGCCUUC
1224

CCCGGCGUAGGUCGCGUAA
529
CCCGGCGUAGGUCGCGUAA
529
UUACGCGACCUACGCCGGG
1225

AGCGGAGACGGCUGGAGCG
530
AGCGGAGACGGCUGGAGCG
530
CGCUCCAGCCGUCUCCGCU
1226

CCCCCGGCGUAGGUCGCGU
531
CCCCCGGCGUAGGUCGCGU
531
ACGCGACCUACGCCGGGGG
1227

AGGCGAAGGCGUCCACAGU
532
AGGCGAAGGCGUCCACAGU
532
ACUGUGGACGCCUUCGCCU
1228

AAGGCGAAGGCGUCCACAG
533
AAGGCGAAGGCGUCCACAG
533
CUGUGGACGCCUUCGCCUU
1229

GUUGGGUGUGCGCGCGACU
534
GUUGGGUGUGCGCGCGACU
534
AGUCGCGCGCACACCCAAC
1230

CUCAUGGGGUACAUUCCGC
535
CUCAUGGGGUACAUUCCGC
535
GCGGAAUGUACCCCAUGAG
1231

GGAAGGCGACAACCUAUCC
536
GGAAGGCGACAACCUAUCC
536
GGAUAGGUUGUCGCCUUCC
1232

GCAAGUUCCUUGCCGACGG
537
GCAAGUUCCUUGCCGACGG
537
CCGUCGGCAAGGAACUUGC
1233

UGCAGCCUCCAGGACCCCC
538
UGCAGCCUCCAGGACCCCC
538
GGGGGUCCUGGAGGCUGCA
1234

GGACUGCACGAUGCUCGUG
539
GGACUGCACGAUGCUCGUG
539
CACGAGCAUCGUGCAGUCC
1235

GAAGGCGAAGGCGUCCACA
540
GAAGGCGAAGGCGUCCACA
540
UGUGGACGCCUUCGCCUUC
1236

GCAACCUCGUGGAAGGCGA
541
GCAACCUCGUGGAAGGCGA
541
UCGCCUUCCACGAGGUUGC
1237

GACGCGGGCUGUGCUUGGU
542
GACGCGGGCUGUGCUUGGU
542
ACCAAGCACAGCCCGCGUC
1238

ACGCGGGCUGUGCUUGGUA
543
ACGCGGGCUGUGCUUGGUA
543
UACCAAGCACAGCCCGCGU
1239

GUGCAGCCUCCAGGACCCC
544
GUGCAGCCUCCAGGACCCC
544
GGGGUCCUGGAGGCUGCAC
1240

GCAGCCUCCAGGACCCCCC
545
GCAGCCUCCAGGACCCCCC
545
GGGGGGUCCUGGAGGCUGC
1241

CGCAACCUCGUGGAAGGCG
546
CGCAACCUCGUGGAAGGCG
546
CGCCUUCCACGAGGUUGCG
1242

UGUCGUGCAGCCUCCAGGA
547
UGUCGUGCAGCCUCCAGGA
547
UCCUGGAGGCUGCACGACA
1243

AUGGCUUGGGAUAUGAUGA
548
AUGGCUUGGGAUAUGAUGA
548
UCAUCAUAUCCCAAGCCAU
1244

CUUGGGAUAUGAUGAUGAA
549
CUUGGGAUAUGAUGAUGAA
549
UUCAUCAUCAUAUCCCAAG
1245

CCCUUGGCCCCUCUAUGGC
550
CCCUUGGCCCCUCUAUGGC
550
GCCAUAGAGGGGCCAAGGG
1246

UGGCUUGGGAUAUGAUGAU
551
UGGCUUGGGAUAUGAUGAU
551
AUCAUCAUAUCCCAAGCCA
1247

CUGUGCAGUGGAUGAACCG
552
CUGUGCAGUGGAUGAACCG
552
CGGUUCAUCCACUGCACAG
1248

AUGACGCGGGCUGUGCUUG
553
AUGACGCGGGCUGUGCUUG
553
CAAGCACAGCCCGCGUCAU
1249

GCUUGGGAUAUGAUGAUGA
554
GCUUGGGAUAUGAUGAUGA
554
UCAUCAUCAUAUCCCAAGC
1250

UAUGACGCGGGCUGUGCUU
555
UAUGACGCGGGCUGUGCUU
555
AAGCACAGCCCGCGUCAUA
1251

UGACGCGGGCUGUGCUUGG
556
UGACGCGGGCUGUGCUUGG
556
CCAAGCACAGCCCGCGUCA
1252

GGCUUGGGAUAUGAUGAUG
557
GGCUUGGGAUAUGAUGAUG
557
CAUCAUCAUAUCCCAAGCC
1253

UGUGCAGUGGAUGAACCGG
558
UGUGCAGUGGAUGAACCGG
558
CCGGUUCAUCCACUGCACA
1254

GCUGUGCAGUGGAUGAACC
559
GCUGUGCAGUGGAUGAACC
559
GGUUCAUCCACUGCACAGC
1255

CUCUUCAACUGGGCAGUAA
560
CUCUUCAACUGGGCAGUAA
560
UUACUGCCCAGUUGAAGAG
1256

CCUCGUGGAAGGCGACAAC
561
CCUCGUGGAAGGCGACAAC
561
GUUGUCGCCUUCCACGAGG
1257

UGUGUCACCCAGACAGUCG
562
UGUGUCACCCAGACAGUCG
562
CGACUGUCUGGGUGACACA
1258

GGCGUGAACUAUGCAACAG
563
GGCGUGAACUAUGCAACAG
563
CUGUUGCAUAGUUCACGCC
1259

CGGCGUGAACUAUGCAACA
564
CGGCGUGAACUAUGCAACA
564
UGUUGCAUAGUUCACGCCG
1260

GUGUCACCCAGACAGUCGA
565
GUGUCACCCAGACAGUCGA
565
UCGACUGUCUGGGUGACAC
1261

CCUCUUCAACUGGGCAGUA
566
CCUCUUCAACUGGGCAGUA
566
UACUGCCCAGUUGAAGAGG
1262

CGUGGAAGGCGACAACCUA
567
CGUGGAAGGCGACAACCUA
567
UAGGUUGUCGCCUUCCACG
1263

UCGUGGAAGGCGACAACCU
568
UCGUGGAAGGCGACAACCU
568
AGGUUGUCGCCUUCCACGA
264

CGGCCUAGUUGGGGCCCCA
569
CGGCCUAGUUGGGGCCCCA
569
UGGGGCCCCAACUAGGCCG
1265

CGACUAGGAAGACUUCCGA
570
CGACUAGGAAGACUUCCGA
570
UCGGAAGUCUUCCUAGUCG
1266

UUUGGGUAAGGUCAUCGAU
571
UUUGGGUAAGGUCAUCGAU
571
AUCGAUGACCUUACCCAAA
1267

GUGGAAGGCGACAACCUAU
572
GUGGAAGGCGACAACCUAU
572
AUAGGUUGUCGCCUUCCAC
1268

ACCUCGUGGAAGGCGACAA
573
ACCUCGUGGAAGGCGACAA
573
UUGUCGCCUUCCACGAGGU
1269

GCGACUAGGAAGACUUCCG
574
GCGACUAGGAAGACUUCCG
574
CGGAAGUCUUCCUAGUCGC
1270

GUCGUGCAGCCUCCAGGAC
575
GUCGUGCAGCCUCCAGGAC
575
GUCCUGGAGGCUGCACGAC
1271

UAGGAAGACUUCCGAGCGG
576
UAGGAAGACUUCCGAGCGG
576
CCGCUCGGAAGUCUUCCUA
1272

ACGGCGUGAACUAUGCAAC
577
ACGGCGUGAACUAUGCAAC
577
GUUGCAUAGUUCACGCCGU
1273

CUCGUGGAAGGCGACAACC
578
CUCGUGGAAGGCGACAACC
578
GGUUGUCGCCUUCCACGAG
1274

GGUCGCAACCUCGUGGAAG
579
GGUCGCAACCUCGUGGAAG
579
CUUCCACGAGGUUGCGACC
1275

CGGUCGCAACCUCGUGGAA
580
CGGUCGCAACCUCGUGGAA
580
UUCCACGAGGUUGCGACCG
1276

GCGCGCGACUAGGAAGACU
581
GCGCGCGACUAGGAAGACU
581
AGUCUUCCUAGUCGCGCGC
1277

GACGGCGUGAACUAUGCAA
582
GACGGCGUGAACUAUGCAA
582
UUGCAUAGUUCACGCCGUC
1278

UAGAUCACUCCCCUGUGAG
583
UAGAUCACUCCCCUGUGAG
583
CUCACAGGGGAGUGAUCUA
1279

AGCGGUCGCAACCUCGUGG
584
AGCGGUCGCAACCUCGUGG
584
CCACGAGGUUGCGACCGCU
1280

UGGAAGGCGACAACCUAUC
585
UGGAAGGCGACAACCUAUC
585
GAUAGGUUGUCGCCUUCCA
1281

CGCGCGACUAGGAAGACUU
586
CGCGCGACUAGGAAGACUU
586
AAGUCUUCCUAGUCGCGCG
1282

CUAGGAAGACUUCCGAGCG
587
CUAGGAAGACUUCCGAGCG
587
CGCUCGGAAGUCUUCCUAG
1283

GUGCGCGCGACUAGGAAGA
588
GUGCGCGCGACUAGGAAGA
588
UCUUCCUAGUCGCGCGCAC
1284

AGAUCACUCCCCUGUGAGG
589
AGAUCACUCCCCUGUGAGG
589
CCUCACAGGGGAGUGAUCU
1285

UGCGCGCGACUAGGAAGAC
590
UGCGCGCGACUAGGAAGAC
590
GUCUUCCUAGUCGCGCGCA
1286

AUAGAUCACUCCCCUGUGA
591
AUAGAUCACUCCCCUGUGA
591
UCACAGGGGAGUGAUCUAU
1287

GAGCGGUCGCAACCUCGUG
592
GAGCGGUCGCAACCUCGUG
592
CACGAGGUUGCGACCGCUC
1288

CACGAACGACUGCUCCAAC
593
CACGAACGACUGCUCCAAC
593
GUUGGAGCAGUCGUUCGUG
1289

GGCAAGUUCCUUGCCGACG
594
GGCAAGUUCCUUGCCGACG
594
CGUCGGCAAGGAACUUGCC
1290

UCGUGCAGCCUCCAGGACC
595
UCGUGCAGCCUCCAGGACC
595
GGUCCUGGAGGCUGCACGA
1291

GUCACGAACGACUGCUCCA
596
GUCACGAACGACUGCUCCA
596
UGGAGCAGUCGUUCGUGAC
1292

GCGGUCGCAACCUCGUGGA
597
GCGGUCGCAACCUCGUGGA
597
UCCACGAGGUUGCGACCGC
1293

GCGCGACUAGGAAGACUUC
598
GCGCGACUAGGAAGACUUC
598
GAAGUCUUCCUAGUCGCGC
1294

GCUAUGACGCGGGCUGUGC
599
GCUAUGACGCGGGCUGUGC
599
GCACAGCCCGCGUCAUAGC
1295

UCACGAACGACUGCUCCAA
600
UCACGAACGACUGCUCCAA
600
UUGGAGCAGUCGUUCGUGA
1296

UCGCAACCUCGUGGAAGGC
601
UCGCAACCUCGUGGAAGGC
601
GCCUUCCACGAGGUUGCGA
1297

CGUGCAGCCUCCAGGACCC
602
CGUGCAGCCUCCAGGACCC
602
GGGUCCUGGAGGCUGCACG
1298

GUCGCAACCUCGUGGAAGG
603
GUCGCAACCUCGUGGAAGG
603
CCUUCCACGAGGUUGCGAC
1299

ACUAGGAAGACUUCCGAGC
604
ACUAGGAAGACUUCCGAGC
604
GCUCGGAAGUCUUCCUAGU
1300

CGCGACUAGGAAGACUUCC
605
CGCGACUAGGAAGACUUCC
605
GGAAGUCUUCCUAGUCGCG
1301

UGGGCGAAGCACAUGUGGA
606
UGGGCGAAGCACAUGUGGA
606
UCCACAUGUGCUUCGCCCA
1302

CCUUGCCUACUAUUCCAUG
607
CCUUGCCUACUAUUCCAUG
607
CAUGGAAUAGUAGGCAAGG
1303

GCCUCAGGAAACUUGGGGU
608
GCCUCAGGAAACUUGGGGU
608
ACCCCAAGUUUCCUGAGGC
1304

UGCUAUGACGCGGGCUGUG
609
UGCUAUGACGCGGGCUGUG
609
CACAGCCCGCGUCAUAGCA
1305

UCGUGCUCGCCACCGCUAC
610
UCGUGCUCGCCACCGCUAC
610
GUAGCGGUGGCGAGCACGA
1306

UGCCUCAGGAAACUUGGGG
611
UGCCUCAGGAAACUUGGGG
611
CCCCAAGUUUCCUGAGGCA
1307

UGUCUCGUGCCCGACCCCG
612
UGUCUCGUGCCCGACCCCG
612
CGGGGUCGGGCACGAGACA
1308

UGUGGCGGCAGGAGAUGGG
613
UGUGGCGGCAGGAGAUGGG
613
CCCAUCUCCUGCCGCCACA
1309

GUCGUGCUCGCCACCGCUA
614
GUCGUGCUCGCCACCGCUA
614
UAGCGGUGGCGAGCACGAC
1310

GAUUUCCACUACGUGACGG
615
GAUUUCCACUACGUGACGG
615
CCGUCACGUAGUGGAAAUC
1311

GGGCCUUGCCUACUAUUCC
616
GGGCCUUGCCUACUAUUCC
616
GGAAUAGUAGGCAAGGCCC
1312

GCCUUGCCUACUAUUCCAU
617
GCCUUGCCUACUAUUCCAU
617
AUGGAAUAGUAGGCAAGGC
1313

GACUAGGAAGACUUCCGAG
618
GACUAGGAAGACUUCCGAG
618
CUCGGAAGUCUUCCUAGUC
1314

GCGGGGGAGACAUAUAUCA
619
GCGGGGGAGACAUAUAUCA
619
UGAUAUAUGUCUCCCCCGC
1315

CGAGCGGUCGCAACCUCGU
620
CGAGCGGUCGCAACCUCGU
620
ACGAGGUUGCGACCGCUCG
1316

GGCCUUGCCUACUAUUCCA
621
GGCCUUGCCUACUAUUCCA
621
UGGAAUAGUAGGCAAGGCC
1317

AUUUCCACUACGUGACGGG
622
AUUUCCACUACGUGACGGG
622
CCCGUCACGUAGUGGAAAU
1318

GGACGUCAAGUUCCCGGGC
623
GGACGUCAAGUUCCCGGGC
623
GCCCGGGAACUUGACGUCC
1319

GAGUGCUAUGACGCGGGCU
624
GAGUGCUAUGACGCGGGCU
624
AGCCCGCGUCAUAGCACUC
1320

GACGUCAAGUUCCCGGGCG
625
GACGUCAAGUUCCCGGGCG
625
CGCCCGGGAACUUGACGUC
1321

UCAGCGACGGGUCUUGGUC
626
UCAGCGACGGGUCUUGGUC
626
GACCAAGACCCGUCGCUGA
1322

UCAAGUUCCCGGGCGGUGG
627
UCAAGUUCCCGGGCGGUGG
627
CCACCGCCCGGGAACUUGA
1323

UCAAGGAGAUGAAGGCGAA
628
UCAAGGAGAUGAAGGCGAA
628
UUCGCCUUCAUCUCCUUGA
1324

CCUAUCCCCAAGGCUCGCC
629
CCUAUCCCCAAGGCUCGCC
629
GGCGAGCCUUGGGGAUAGG
1325

CUUGACCUACCUCAGAUCA
630
CUUGACCUACCUCAGAUCA
630
UGAUCUGAGGUAGGUCAAG
1326

UUUCCACUACGUGACGGGC
631
UUUCCACUACGUGACGGGC
631
GCCCGUCACGUAGUGGAAA
1327

AGUGCUAUGACGCGGGCUG
632
AGUGCUAUGACGCGGGCUG
632
CAGCCCGCGUCAUAGCACU
1328

ACGUCAAGUUCCCGGGCGG
633
ACGUCAAGUUCCCGGGCGG
633
CCGCCCGGGAACUUGACGU
1329

UCUGGAGACAUCGGGCCAG
634
UCUGGAGACAUCGGGCCAG
634
CUGGCCCGAUGUCUCCAGA
1330

GGGCGAAGCACAUGUGGAA
635
GGGCGAAGCACAUGUGGAA
635
UUCCACAUGUGCUUCGCCC
1331

UUGACCUACCUCAGAUCAU
636
UUGACCUACCUCAGAUCAU
636
AUGAUCUGAGGUAGGUCAA
1332

CCAAGCGGAGACGGCUGGA
637
CCAAGCGGAGACGGCUGGA
637
UCCAGCCGUCUCCGCUUGG
1333

ACCAAGCGGAGACGGCUGG
638
ACCAAGCGGAGACGGCUGG
638
CCAGCCGUCUCCGCUUGGU
1334

GGGUGGCUUCAUGCCUCAG
639
GGGUGGCUUCAUGCCUCAG
639
CUGAGGCAUGAAGCCACCC
1335

GUCAAGUUCCCGGGCGGUG
640
GUCAAGUUCCCGGGCGGUG
640
CACCGCCCGGGAACUUGAC
1336

CUCAAGGAGAUGAAGGCGA
641
CUCAAGGAGAUGAAGGCGA
641
UCGCCUUCAUCUCCUUGAG
1337

GACCAAGCGGAGACGGCUG
642
GACCAAGCGGAGACGGCUG
642
CAGCCGUCUCCGCUUGGUC
1338

UCCAGGUCGGGCUCAACCA
643
UCCAGGUCGGGCUCAACCA
643
UGGUUGAGCCCGACCUGGA
1339

CUCUUUCUCUAUCUUCCUC
644
CUCUUUCUCUAUCUUCCUC
644
GAGGAAGAUAGAGAAAGAG
1340

GUCUGGAGACAUCGGGCCA
645
GUCUGGAGACAUCGGGCCA
645
UGGCCCGAUGUCUCCAGAC
1341

GUUGUGACUUGGCCCCCGA
646
GUUGUGACUUGGCCCCCGA
646
UCGGGGGCCAAGUCACAAC
1342

AGACCUGGCUCCAGUCCAA
647
AGACCUGGCUCCAGUCCAA
647
UUGGACUGGAGCCAGGUCU
1343

CUUGCCUACUAUUCCAUGG
646
CUUGCCUACUAUUCCAUGG
648
CCAUGGAAUAGUAGGCXAG
1344

CCCGGUUGCUCUUUCUCUA
649
CCCGGUUGCUCUUUCUCUA
649
UAGAGAAAGAGCAACCGGG
1345

CUUUCUCUAUCUUCCUCUU
650
CUUUCUCUAUCUUCCUCUU
650
AAGAGGAAGAUAGAGAAAG
1346

AGGGUGGCUUCAUGCCUCA
651
AGGGUGGCUUCAUGCCUCA
651
UGAGGCAUGAAGCCACCCU
1347

AAGACCUGGCUCCAGUCCA
652
AAGACCUGGCUCCAGUCCA
652
UGGACUGGAGCCAGGUCUU
1348

CCGGUUGCUCUUUCUCUAU
653
CCGGUUGCUCUUUCUCUAU
653
AUAGAGAAAGAGCAACCGG
1349

CGGUUGCUCUUUCUCUAUC
654
CGGUUGCUCUUUCUCUAUC
654
GAUAGAGAAAGAGCAACCG
1350

UGGGGGAUUUCCACUACGU
655
UGGGGGAUUUCCACUACGU
655
ACGUAGUGGAAAUCCCCCA
1351

AUGUCACGAACGACUGCUC
656
AUGUCACGAACGACUGCUC
656
GAGCAGUCGUUCGUGACAU
1352

GGCCUAGUUGGGGCCCCAC
657
GGCCUAGUUGGGGCCCCAC
657
GUGGGGCCCCAACUAGGCC
1353

UGGACCAAGCGGAGACGGC
658
UGGACCAAGCGGAGACGGC
658
GCCGUCUCCGCUUGGUCCA
1354

UUCCAGGUCGGGCUCAACC
659
UUCCAGGUCGGGCUCAACC
659
GGUUGAGCCCGACCUGGAA
1355

AGCGGGUCGAGUUCCUGGU
660
AGCGGGUCGAGUUCCUGGU
660
ACCAGGAACUCGACCCGCU
1356

CAAGGAGAUGAAGGCGAAG
661
CAAGGAGAUGAAGGCGAAG
661
CUUCGCCUUCAUCUCCUUG
1357

CAUGUCACGAACGACUGCU
662
CAUGUCACGAACGACUGCU
662
AGCAGUCGUUCGUGACAUG
1358

CAGCGGGUCGAGUUCCUGG
663
CAGCGGGUCGAGUUCCUGG
663
CCAGGAACUCGACCCGCUG
1359

UUCCACUACGUGACGGGCA
664
UUCCACUACGUGACGGGCA
664
UGCCCGUCACGUAGUGGAA
1360

UAGGGUGGCUUCAUGCCUC
665
UAGGGUGGCUUCAUGCCUC
665
GAGGCAUGAAGCCACCCUA
1361

UCCAGGACUGCACGAUGCU
666
UCCAGGACUGCACGAUGCU
666
AGCAUCGUGCAGUCCUGGA
1362

UCCACUACGUGACGGGCAU
667
UCCACUACGUGACGGGCAU
667
AUGCCCGUCACGUAGUGGA
1363

AAUAGGGUGGCUUCAUGCC
668
AAUAGGGUGGCUUCAUGCC
668
GGCAUGAAGCCACCCUAUU
1364

GUCUUCACGGAGGCUAUGA
669
GUCUUCACGGAGGCUAUGA
669
UCAUAGCCUCCGUGAAGAC
1365

AUAGGGUGGCUUCAUGCCU
670
AUAGGGUGGCUUCAUGCCU
670
AGGCAUGAAGCCACCCUAU
1366

UCUUCACGGAGGCUAUGAC
671
UCUUCACGGAGGCUAUGAC
671
GUCAUAGCCUCCGUGAAGA
1367

AUGCCUCAGGAAACUUGGG
672
AUGCCUCAGGAAACUUGGG
672
CCCAAGUUUCCUGAGGCAU
1368

ACCGGGACGUGCUCAAGGA
673
ACCGGGACGUGCUCAAGGA
673
UCCUUGAGCACGUCCCGGU
1369

GGGGCUGUGCAGUGGAUGA
674
GGGGCUGUGCAGUGGAUGA
674
UCAUCCACUGCACAGCCCC
1370

AAGCUCCAGGACUGCACGA
675
AAGCUCCAGGACUGCACGA
675
UCGUGCAGUCCUGGAGCUU
1371

GCUCCAGGACUGCACGAUG
676
GCUCCAGGACUGCACGAUG
676
CAUCGUGCAGUCCUGGAGC
1372

UACCGGGACGUGCUCAAGG
677
UACCGGGACGUGCUCAAGG
677
CCUUGAGCACGUCCCGGUA
1373

GGGCUGUGCAGUGGAUGAA
678
GGGCUGUGCAGUGGAUGAA
678
UUCAUCCACUGCACAGCCC
1374

CGUCAAGUUCCCGGGCGGU
679
CGUCAAGUUCCCGGGCGGU
679
ACCGCCCGGGAACUUGACG
1375

UCAAUAGGGUGGCUUCAUG
680
UCAAUAGGGUGGCUUCAUG
680
CAUGAAGCCACCCUAUUGA
1376

AGUCUUCACGGAGGCUAUG
681
AGUCUUCACGGAGGCUAUG
681
CAUAGCCUCCGUGAAGACU
1377

GGACCAAGCGGAGACGGCU
682
GGACCAAGCGGAGACGGCU
682
AGCCGUCUCCGCUUGGUCC
1378

GGCUCCAGUCCAAGCUCCU
683
GGCUCCAGUCCAAGCUCCU
683
AGGAGCUUGGACUGGAGCC
1379

GGCUGUGCAGUGGAUGAAC
684
GGCUGUGCAGUGGAUGAAC
684
GUUCAUCCACUGCACAGCC
1380

CUCCAGGACUGCACGAUGC
685
CUCCAGGACUGCACGAUGC
685
GCAUCGUGCAGUCCUGGAG
1381

GAGUCUUCACGGAGGCUAU
686
GAGUCUUCACGGAGGCUAU
686
AUAGCCUCCGUGAAGACUC
1382

UGGCUCCAGUCCAAGCUCC
687
UGGCUCCAGUCCAAGCUCC
687
GGAGCUUGGACUGGAGCCA
1383

GGGGAUUUCCACUACGUGA
688
GGGGAUUUCCACUACGUGA
688
UCACGUAGUGGAAAUCCCC
1384

CAUGCCUCAGGAAACUUGG
689
CAUGCCUCAGGAAACUUGG
689
CCAAGUUUCCUGAGGCAUG
1385

AUCAAUAGGGUGGCUUCAU
690
AUCAAUAGGGUGGCUUCAU
690
AUGAAGCCACCCUAUUGAU
1386

GCGGGCCUUGCCUACUAUU
691
GCGGGCCUUGCCUACUAUU
691
AAUAGUAGGCAAGGCCCGC
1387

CCGGGACGUGCUCAAGGAG
692
CCGGGACGUGCUCAAGGAG
692
CUCCUUGAGCACGUCCCGG
1388

CCAUGGUGGGGAACUGGGC
693
CCAUGGUGGGGAACUGGGC
693
GCCCAGUUCCCCACCAUGG
1389

CAAUAGGGUGGCUUCAUGC
694
CAAUAGGGUGGCUUCAUGC
694
GCAUGAAGCCACCCUAUUG
1390

AGCUCCAGGACUGCACGAU
695
AGCUCCAGGACUGCACGAU
695
AUCGUGCAGUCCUGGAGCU
1391

CGGGCCUUGCCUACUAUUC
696
CGGGCCUUGCCUACUAUUC
696
GAAUAGUAGGCAAGGCCCG
1392

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

TABLE III

HCV Synthetic Modified siNA Constructs

Target

Seq

Seq

Pos
Target
ID
Cmpd#
Aliases
Sequence
ID

183
GGUCCUUUCUUGGAUCAACCCGC
1393
25237
HCV IRES Loop IIIb (Heptazyme
B GGUCCUUUCUUGGAUCAACCC B
1467

site) as siNA str1 (sense)

183
GGUCCUUUCUUGGAUCAACCCGC
1393
25238
HCV IRES Loop IIIb (Heptazyme
B GGGUUGAUCCAAGAAAGGACC B
1468

site) as siNA str2

(antisense)

183
GGUCCUUUCUUGGAUCAACCCGC
1393
25251
HCV IRES Loop IIIb (Heptazyme
B CCCAACUAGGUUCUUUCCUGG B
1469

site) as sINA str1 (sense)

Inverted Control

183
GGUCCUUUCUUGGAUCAACCCGC
1393
25252
HCV IRES Loop IIIb (Heptazyme
B CCAGGAAAGAACCUAGUUGGG B
1470

site) as siNA str1 (sense)

Inverted Control Compliment

183
GGUCCUUUCUUGGAUCAACCCGC
1393
25814
HCV IRES Loop IIIb (Heptazyme
GGUCCUUUCUUGGAUCAACCCUU
1471

site) as siNA str1 (sense)

+2U overhang

183
GGUCCUUUCUUGGAUCAACCCGC
1393
25815
HCV IRES Loop IIIb (Heptazyme
GGGUUGAUCCAAGAAAGGACCUU
1472

site) as siNA str2

(antisense) +2U overhang

183
GGUCCUUUCUUGGAUCAACCCGC
1393
25834
HCV IRES Loop IlIb (Heptazyme
BGGUCCUUUCUUGGAUCAACCCUUB
1473

site) as siNA str1 (sense)

+2U overhang

183
GGUCCUUUCUUGGAUCAACCCGC
1393
25835
HCV IRES Loop IIIb (Heptazyme
BGGGUUGAUCCAAGAAAGGACCUUB
1474

site) as siNA str2

(antisense) +2U overhang

325
UGCCCCGGGAGGUCUCGUAGACC
1394
28415
HCVa:325U21 sense TT siNA
CCCCGGGAGGUCUCGUAGATT
1475

162
CGGAACCGGUGAGUACACC
54
28416
HCVa:162U21 sense TT siNA
CGGAACCGGUGAGUACACCTT
1476

324
GCCCCGGGAGGUCUCGUAG
1
28417
HCVa:324U21 sense TT siNA
GCCCCGGGAGGUCUCGUAGTT
1477

163
GGAACCGGUGAGUACACCG
53
28418
HCVa:163U21 sense TT siNA
GGAACCGGUGAGUACACCGTT
1478

294
GUGGUACUGCCUGAUAGGG
5
28419
HCVa:294U21 sense TT siNA
GUGGUACUGCCUGAUAGGGTT
1479

293
UGUGGUACUGCCUGAUAGG
2
28420
HCVa:293U21 sense TT siNA
UGUGGUACUGCCUGAUAGGTT
1480

292
UUGUGGUACUGCCUGAUAG
3
28421
HCVa:292U21 sense TT siNA
UUGUGGUACUGCCUGAUAGTT
1481

325
UGCCCCGGGAGGUCUCGUAGACC
1394
28422
HCVa:343L21 antisense TT siNA
UCUACGAGACCUCCCGGGGTT
1482

(325C)

162
CGGAACCGGUGAGUACACC
54
28423
HCVa:180L21 antisense TT siNA
GGUGUACUCACCGGUUCCGTT
1483

(162C)

324
GCCCCGGGAGGUCUCGUAG
1
28424
HCVa:342L21 antisense TT siNA
CUACGAGACCUCCCGGGGCTT
1484

(324C)

163
GGAACCGGUGAGUACACCG
53
28425
HCVa:181L21 antisense TT siNA
CGGUGUACUCACCGGUUCCTT
1485

(163C)

294
GUGGUACUGCCUGAUAGGG
5
28426
HCVa:312L21 antisense TT siNA
CCCUAUCAGGCAGUACCACTT
1486

(294C)

293
UGUGGUACUGCCUGAUAGG
2
28427
HCVa:311L21 antisense TT siNA
CCUAUCAGGCAGUACCACATT
1487

(293C)

292
UUGUGGUACUGCCUGAUAG
3
28428
HCVa:310L21 antisense TT siNA
CUAUCAGGCAGUACCACAATT
1488

(292C)

325
UGCCCCGGGAGGUCUCGUAGACC
1394
28429
HCVa:325U21 sense TT siNA inv
TTAGAUGCUCUGGAGGGCCCC
1489

162
CGGAACCGGUGAGUACACC
54
28430
HCVa:162U21 sense TT siNA inv
TTCCACAUGAGUGGCCAAGGC
1490

324
GCCCCGGGAGGUCUCGUAG
1
28431
HCVa:324U21 sense TT siNA inv
TTGAUGCUCUGGAGGGCCCCG
1491

163
GGAACCGGUGAGUACACCG
53
28432
HCVa:163U21 sense TT siNA inv
TTGCCACAUGAGUGGCCAAGG
1492

294
GUGGUACUGCCUGAUAGGG
5
28433
HCVa:294U21 sense TT siNA inv
TTGGGAUAGUCCGUCAUGGUG
1493

293
UGUGGUACUGCCUGAUAGG
2
28434
HCVa:293U21 sense TT siNA inv
TTGGAUAGUCCGUCAUGGUGU
1494

292
UUGUGGUACUGCCUGAUAG
3
28435
HCVa:292U21 sense TT siNA inv
TTGAUAGUCCGUCAUGGUGUU
1495

325
UGCCCCGGGAGGUCUCGUAGACC
1394
28436
HCVa:343L21 antisense TT siNA
TTGGGGCCCUCCAGAGCAUCU
1496

(325C) inv

162
CGGAACCGGUGAGUACACC
54
28437
HCVa:180L21 antisense TT siNA
TTGCCUUGGCCACUCAUGUGG
1497

(162C) inv

324
GCCCCGGGAGGUCUCGUAG
1
28438
HCVa:342L21 antisense TT siNA
TTCGGGGCCCUCCAGAGCAUC
1498

(324C) inv

163
GGAACCGGUGAGUACACCG
53
28439
HCVa:181L21 antisense TT siNA
TTCCUUGGCCACUCAUGUGGC
1499

(163C) inv

294
GUGGUACUGCCUGAUAGGG
5
28440
HCVa:312L21 antisense TT siNA
TTCACCAUGACGGACUAUCCC
1500

(294C) inv

293
UGUGGUACUGCCUGAUAGG
2
28441
HCVa:311L21 antisense TT siNA
TTACACCAUGACGGACUAUCC
1501

(293C) inv

292
UUGUGGUACUGCCUGAUAG
3
28442
HCVa:310L21 antisense TT siNA
TTAACACCAUGACGGACUAUC
1502

(292C) inv

162
UGCGGAACCGGUGAGUACACCGG
1395
29573
HCVa:162U21 sense siNA
CGGAACCGGUGAGUACACCGG
1503

163
GCGGAACCGGUGAGUACACCGGA
1396
29574
HCVa:163U21 sense siNA
GGAACCGGUGAGUACACCGGA
1504

292
CCUUGUGGUACUGCCUGAUAGGG
1397
29575
HCVa:292U21 sense siNA
UUGUGGUACUGCCUGAUAGGG
1505

293
CUUGUGGUACUGCCUGAUAGGGU
1398
29576
HCVa:293U21 sense siNA
UGUGGUACUGCCUGAUAGGGU
1506

294
UUGUGGUACUGCCUGAUAGGGUG
1399
29577
HCVa:294U21 sense siNA
GUGGUACUGCCUGAUAGGGUG
1507

324
GUGCCCCGGGAGGUCUCGUAGAC
1400
29578
HCVa:324U21 sense siNA
GCCCCGGGAGGUCUCGUAGAC
1508

325
UGCCCCGGGAGGUCUCGUAGACC
1394
29579
HCVa:325U21 sense siNA
CCCCGGGAGGUCUCGUAGACC
1509

162
UGCGGAACCGGUGAGUACACCGG
1395
29580
HCVa:180L21 antisense siNA
GGUGUACUCACCGGUUCCGCA
1510

(162C)

163
GCGGAACCGGUGAGUACACCGGA
1396
29581
HCVa:181L21 antisense siNA
CGGUGUACUCACCGGUUCCGC
1511

(163C)

292
CCUUGUGGUACUGCCUGAUAGGG
1397
29582
HCVa:310L21 antisense siNA
CUAUCAGGCAGUACCACAAGG
1512

(292C)

293
CUUGUGGUACUGCCUGAUAGGGU
1398
29583
HCVa:311L21 antisense siNA
CCUAUCAGGCAGUACCACAAG
1513

(293C)

294
UUGUGGUACUGCCUGAUAGGGUG
1399
29584
HCVa:312L21 antisense siNA
CCCUAUCAGGCAGUACCACAA
1514

(294C)

324
GUGCCCCGGGAGGUCUCGUAGAC
1400
29585
HCVa:342L21 antisense siNA
CUACGAGACCUCCCGGGGCAC
1515

(324C)

325
UGCCCCGGGAGGUCUCGUAGACC
1394
29586
HCVa:343L21 antisense siNA
UCUACGAGACCUCCCGGGGCA
1516

(325C)

162
UGCGGAACCGGUGAGUACACCGG
1395
29587
HCVa:162U21 sense siNA inv
GGCCACAUGAGUGGCCAAGGC
1517

163
GCGGAACCGGUGAGUACACCGGA
1396
29588
HCVa:163U21 sense siNA inv
AGGCCACAUGAGUGGCCAAGG
1518

292
CCUUGUGGUACUGCCUGAUAGGG
1397
29589
HCVa:292U21 sense siNA inv
GGGAUAGUCCGUCAUGGUGUU
1519

293
CUUGUGGUACUGCCUGAUAGGGU
1398
29590
HCVa:293U21 sense siNA inv
UGGGAUAGUCCGUCAUGGUGU
1520

294
UUGUGGUACUGCCUGAUAGGGUG
1399
29591
HCVa:294U21 sense siNA inv
GUGGGAUAGUCCGUCAUGGUG
1521

324
GUGCCCCGGGAGGUCUCGUAGAC
1400
29592
HCVa:324U21 sense siNA inv
CAGAUGCUCUGGAGGGCCCCG
1522

325
UGCCCCGGGAGGUCUCGUAGACC
1394
29593
HCVa:325U21 sense siNA inv
CCAGAUGCUCUGGAGGGCCCC
1523

162
UGCGGAACCGGUGAGUACACCGG
1395
29594
HCVa:180L21 antisense siNA
ACGCCUUGGCCACUCAUGUGG
1524

(162C) inv

163
GCGGAACCGGUGAGUACACCGGA
1396
29595
HCVa:181L21 antisensesiNA
CGCCUUGGCCACUCAUGUGGC
1525

(163C) inv

292
CCUUGUGGUACUGCCUGAUAGGG
1397
29596
HCVa:310L21 antisense siNA
GGAACACCAUGACGGACUAUC
1526

(292C) inv

293
CUUGUGGUACUGCCUGAUAGGGU
1398
29597
HCVa:311L21 antisense siNA
GAACACCAUGACGGACUAUCC
1527

(293C) inv

294
UUGUGGUACUGCCUGAUAGGGUG
1399
29598
HCVa:312L21 antisense siNA
AACACCAUGACGGACUAUCCC
1528

(294C) inv

324
GUGCCCCGGGAGGUCUCGUAGAC
1400
29599
HCVa:342L21 antisense siNA
CACGGGGCCCUCCAGAGCAUC
1529

(324C) inv

325
UGCCCCGGGAGGUCUCGUAGACC
1394
29600
HCVa:343L21 antisense siNA
ACGGGGCCCUCCAGAGCAUCU
1530

(325C) inv

325
UGCCCCGGGAGGUCUCGUAGACC
1394
30051
HCVa:325U21 sense siNA 5 5′
BCsCsCsCsGsGGAGGUCUCGUAGAXXB
1531

P = S + 3′ univ. base

2 + 5/3′ invAba

325
UGCCCCGGGAGGUCUCGUAGACC
1394
30052
HCVa:325U21 sense siNA inv
BAsGsAsUsGsCUCUGGAGGGCCCCXXB
1532

5 5′ P = S + 3′ univ. base

2 + 5/3′ invAba

325
UGCCCCGGGAGGUCUCGUAGACC
1394
30053
HCVa:343L21 antisense siNA
UsCsUsAsCsGAGACCUCCCGGGGXXB
1533

(325C) 5 5′ P=S + 3′ univ.

base 2 + 3′ invAba

325
UGCCCCGGGAGGUCUCGUAGACC
1394
30054
HCVa:343L21 antisense siNA
GsGsGsGsCsCCUCCAGAGCAUCUXXB
1534

(325C) inv 5 5′ P = S + 3′

univ. base 2 + 3′ invAba

325
UGCCCCGGGAGGUCUCGUAGACC
1394
30055
HCVa:325U21 sense siNA all
BCsCsCsCsGGGAGGUsCsUsCsGU
1535

Y P = S + 3′ univ. base
sAGAXXB

2 + 5/3′ invAba

325
UGCCCCGGGAGGUCUCGUAGACC
1394
30056
HCVa:325U21 sense siNA inv
BAGAUsGCsUsCsUsGGAGGGCsCs
1536

all Y P = S + 3′ univ. base
CsCsXXB

2 + 5/3′ invAba

325
UGCCCCGGGAGGUCUCGUAGACC
1394
30057
HCVa:343L21 antisense siNA
UsCsUsACsGAGACsCsUsCsCsCs
1537

(325C) all Y P = S + 3′
GGGGXXB

univ. base 2 + 3′ invAba

325
UGCCCCGGGAGGUCUCGUAGACC
1394
30058
HCVa:343L21 antisense siNA
GGGGCsCsCsUsCsCsAGAGCsAUs
1538

(325C) inv all Y P = S + 3′
CsUsXXB

univ. base 2 + 3′ invAba

325
UGCCCCGGGAGGUCUCGUAGACC
1394
30059
HCVa:325U21 sense siNA 4/3
BcscscscsGGGAGGucucGuAsGs
1539

P = S ends + all Y-2′F + 3′
AsXXB

univ. base 2 + 5/3′ invAba

325
UGCCCCGGGAGGUCUCGUAGACC
1394
30060
HCVa:325U21 sense siNA inv
BAsGsAsusGcucuGGAGGGccscs
1540

4/3 P = S ends + all
csXXB

Y-2′F + 3′ univ. base

2 + 5/3′ invAba

325
UGCCCCGGGAGGUCUCGUAGACC
1394
30170
HCVa:325U21 sense siNA all
B ccccGGGAGGucucGuAGAXX B
1541

Y-2′F + 3′ univ. base

2 + 5′/3′ invAba

325
UGCCCCGGGAGGUCUCGUAGACC
1394
30171
HCVa:325U21 sense siNA inv
B AGAuGcucuGGAGGGccccXX B
1542

all Y-2′F + 3′ univ. base

2 + 5/3′ invAba

325
UGCCCCGGGAGGUCUCGUAGACC
1394
30172
HCVa:343L21 antisense siNA
B UsCsUsACsGAGACsCsUsCsCs
1543

(325C) all Y P = S + 3′
CsGGGGXX B

univ. base 2 + 5/3′ invAba

325
UGCCCCGGGAGGUCUCGUAGACC
1394
30173
HCVa:343L19 antisense siNA
ucuAcGAGAccucccGGGG
1544

(325C) all Y-2′F

325
UGCCCCGGGAGGUCUCGUAGACC
1394
30175
HCVa:343L21 antisense siNA
ucuAcGAGAccucccGGGGXX
1545

(325C) all Y-2′F + 3′ univ.

Base 2

325
UGCCCCGGGAGGUCUCGUAGACC
1394
30176
HCVa:343L21 antisense siNA
GGGGcccuccAGAGcAucuXX
1546

(325C) inv all Y-2′F + 3′

univ. Base 2

325
UGCCCCGGGAGGUCUCGUAGACC
1394
30177
HCVa:343L21 antisense siNA
B ucuAcGAGAccucccGGGGXX B
1547

(325C) all Y-2′F + 3′ univ.

Base 2 + 5′/3′ iB

325
UGCCCCGGGAGGUCUCGUAGACC
1394
30178
HCVa:325U21 sense siNA all
CsCsCsCsGGGAGGUsCsUsCsGUs
1548

Y P = S + 3′ univ. base
AGAXX B

2 + 3′ invAba

325
UGCCCCGGGAGGUCUCGUAGACC
1394
30417
HCVa:325U21 sense siNA w/iB
CCCCGGGAGGUCUCGUAGACC B
1549

325
UGCCCCGGGAGGUCUCGUAGACC
1394
30418
HCVa:325U21 sense siNA w/iB
B CCCCGGGAGGUCUCGUAGACC B
1550

325
UGCCCCGGGAGGUCUCGUAGACC
1394
30419
HCVa:343L21 antisense siNA
UCUACGAGACCUCCCGGGGCA B
1551

(325C) w/iB

325
UGCCCCGGGAGGUCUCGUAGACC
1394
30420
HCVa:343L21 antisense siNA
B UCUACGAGACCUCCCGGGGCA B
1552

(325C) w/iB

325
UGCCCCGGGAGGUCUCGUAGACC
1394
30561
HCVa:325U21 sense siNA
B ccccGGGAGGucucGuAGATT B
1553

Y-2′Ome (stab06) + 5′/3′

invAba

325
UGCCCCGGGAGGUCUCGUAGACC
1394
30562
HCVa:343L21 antisense siNA
ucuAcGAGAccucccGGGGTsT
1554

(325C) Y-2′F, R-2′Ome + TsT

153
AUAGUGGUCUGCGGAACCGGUGA
1401
30649
HCVa:153U21 sense siNA stab07
B AGuGGucuGcGGAAccGGuTT B
1555

159
GUCUGCGGAACCGGUGAGUACAC
1402
30650
HCVa:159U21 sense siNA stab07
B cuGcGGAAccGGuGAGuAcTT B
1556

291
GCCUUGUGGUACUGCCUGAUAGG
1403
30651
HCVa:291U21 sense siNA stab07
B cuuGuGGuAcuGccuGAuATT B
1557

295
UGUGGUACUGCCUGAUAGGGUGC
1404
30652
HCVa:295U21 sense siNA stab07
B uGGuAcuGccuGAuAGGGuTT B
1558

296
GUGGUACUGCCUGAUAGGGUGCU
1405
30653
HCVa:296U21 sense siNA stab07
B GGuAcuGccuGAuAGGGuGTT B
1559

297
UGGUACUGCCUGAUAGGGUGCUU
1406
30654
HCVa:297U21 sense siNA stab07
B GuAcuGccuGAuAGGGuGcTT B
1560

298
GGUACUGCCUGAUAGGGUGCUUG
1407
30655
HCVa:298U21 sense siNA stab07
B uAcuGccuGAuAGGGuGcuTT B
1561

300
UACUGCCUGAUAGGGUGCUUGCG
1408
30656
HCVa:300U21 sense siNA stab07
B cuGccuGAuAGGGuGcuuGTT B
1562

301
ACUGCCUGAUAGGGUGCUUGCGA
1409
30657
HCVa:301U21 sense siNA stab07
B uGccuGAuAGGGuGcuuGcTT B
1563

303
UGCCUGAUAGGGUGCUUGCGAGU
1410
30658
HCVa:303U21 sense siNA stab07
B ccuGAuAGGGuGcuuGcGATT B
1564

306
CUGAUAGGGUGCUUGCGAGUGCC
1411
30659
HCVa:306U21 sense siNA stab07
B GAuAGGGuGcuuGcGAGuGTT B
1565

324
GUGCCCCGGGAGGUCUCGUAGAC
1400
30660
HCVa:324U21 sense siNA stab07
B GccccGGGAGGucucGuAGTT B
1566

153
AUAGUGGUCUGCGGAACCGGUGA
1401
30661
HCVa:171L21 antisense siNA

AccGGuuccGcAGAccAcuTsT
1567

(153C) stab08

159
GUCUGCGGAACCGGUGAGUACAC
1402
30662
HCVa:177L21 antisense siNA

GuAcucAccGGuuccGcAGTsT
1568

(159C) stab08

291
GCCUUGUGGUACUGCCUGAUAGG
1403
30663
HCVa:309L21 antisense siNA
uAucAGGcAGuAccAcAAGTsT
1569

(291C) stab08

295
UGUGGUACUGCCUGAUAGGGUGC
1404
30664
HCVa:313L21 antisense siNA

AcccuAucAGGcAGuAccATsT
1570

(295C) stab08

296
GUGGUACUGCCUGAUAGGGUGCU
1405
30665
HCVa:314L21 antisense siNA
cAcccuAucAGGcAGuAccTsT
1571

(296C) stab08

297
UGGUACUGCCUGAUAGGGUGCUU
1406
30666
HCVa:315L21 antisense siNA

GcAcccuAucAGGcAGuAcTsT
1572

(297C) stab08

298
GGUACUGCCUGAUAGGGUGCUUG
1407
30667
HCVa:316L21 antisense siNA

AGcAcccuAucAGGcAGuATsT
1573

(298C) stab08

300
UACUGCCUGAUAGGGUGCUUGCG
1408
30668
HCVa:318L21 antisense siNA
cAAGcAcccuAucAGGcAGTsT
1574

(300C) stab08

301
ACUGCCUGAUAGGGUGCUUGCGA
1409
30669
HCVa:319L21 antisense siNA

GcAAGcAcccuAucAGGcATsT
1575

(301C) stab08

303
UGCCUGAUAGGGUGCUUGCGAGU
1410
30670
HCVa:321L21 antisense siNA
ucGcAAGcAcccuAucAGGTsT
1576

(303C) stab08

306
CUGAUAGGGUGCUUGCGAGUGCC
1411
30671
HCVa:324L21 antisense siNA
cAcucGcAAGcAcccuAucTsT
1577

(306C) stab08

324
GUGCCCCGGGAGGUCUCGUAGAC
1400
30672
HCVa:342121 antisense siNA
cuAcGAGAccucccGGGGcTsT
1578

(324C) stab08

153
AUAGUGGUCUGCGGAACCGGUGA
1401
30673
HCVa:153U21 sense siNA stab07
B uGGccAAGGcGucuGGuGATT B
1579

inv

159
GUCUGCGGAACCGGUGAGUACAC
1402
30674
HCVa:159U21 sense siNA stab07
B cAuGAGuGGccAAGGcGucTT B
1580

inv

291
GCCUUGUGGUACUGCCUGAUAGG
1403
30675
HCVa:291U21 sense siNA stab07
B AuAGuccGucAuGGuGuucTT B
1581

inv

295
UGUGGUACUGCCUGAUAGGGUGC
1404
30676
HCVa:295U21 sense siNA stab07
B uGGGAuAGuccGucAuGGuTT B
1582

inv

296
GUGGUACUGCCUGAUAGGGUGCU
1405
30677
HCVa:296U21 sense siNA stab07
B GuGGGAuAGuccGucAuGGTT B
1583

inv

297
UGGUACUGCCUGAUAGGGUGCUU
1406
30678
HCVa:297U21 sense siNA stab07
B cGuGGGAuAGuccGucAuGTT B
1584

inv

298
GGUACUGCCUGAUAGGGUGCUUG
1407
30679
HCVa:298U21 sense siNA stab07
B ucGuGGGAuAGuccGucAuTT B
1585

inv

300
UACUGCCUGAUAGGGUGCUUGCG
1408
30680
HCVa:300U21 sense siNA stab07
B GuucGuGGGAuAGuccGucTT B
1586

inv

301
ACUGCCUGAUAGGGUGCUUGCGA
1409
30681
HCVa:301U21 sense siNA stab07
B cGuucGuGGGAuAGuccGuTT B
1587

inv

303
UGCCUGAUAGGGUGCUUGCGAGU
1410
30682
HCVa:303U21 sense siNA stab07
B AGcGuucGuGGGAuAGuccTT B
1588

inv

306
CUGAUAGGGUGCUUGCGAGUGCC
1411
30683
HCVa:306U21 sense siNA stab07
B GuGAGcGuucGuGGGAuAGTT B
1589

inv

324
GUGCCCCGGGAGGUCUCGUAGAC
1400
30684
HCVa:324U21 sense siNA stab07
B GAuGcucuGGAGGGccccGTT B
1590

inv

153
AUAGUGGUCUGCGGAACCGGUGA
1401
30685
HCVa:171L21 antisense siNA
ucAccAGAcGccuuGGccATsT
1591

(153C) stab08 inv

159
GUCUGCGGAACCGGUGAGUACAC
1402
30686
HCVa:177L21 antisense siNA

GacGccuuGGccAcucAuGTsT
1592

(159C) stab08 inv

291
GCCUUGUGGUACUGCCUGAUAGG
1403
30687
HCVa:309L21 antisense siNA

GAAcAccAuGAcGGAcuAuTsT
1593

(291C) stab08 inv

295
UGUGGUACUGCCUGAUAGGGUGC
1404
30688
HCVa:313L21 antisense siNA

AccAuGAcGGAcuAucccATsT
1594

(295C) stab08 inv

296
GUGGUACUGCCUGAUAGGGUGCU
1405
30689
HCVa:314L21 antisense siNA
ccAuGAcGGAcuAucccAcTsT
1595

(296C) stab08 inv

297
UGGUACUGCCUGAUAGGGUGCUU
1406
30690
HCVa:315L21 antisense siNA
cAuGAcGGAcuAucccAcGTsT
1596

(297C) stab08 inv

298
GGUACUGCCUGAUAGGGUGCUUG
1407
30691
HCVa:316L21 antisense siNA

AuGAcGGAcuAucccAcGATsT
1597

(298C) stab08 inv

300
UACUGCCUGAUAGGGUGCUUGCG
1408
30692
HCVa:318L21 antisense siNA

GAcGGAcuAucccAcGAAcTsT
1598

(300C) stab08 inv

301
ACUGCCUGAUAGGGUGCUUGCGA
1409
30693
HCVa:319L21 antisense siNA

AcGGAcuAucccAcGAAcGTsT
1599

(301C) stab08 inv

303
UGCCUGAUAGGGUGCUUGCGAGU
1410
30694
HCVa:321L21 antisense siNA

GGAcuAucccAcGAAcGcuTsT
1600

(303C) stab08 inv

306
CUGAUAGGGUGCUUGCGAGUGCC
1411
30695
HCVa:324L21 antisense siNA
cuAucccAcGAAcGcucAcTsT
1601

(306C) stab08 inv

324
GUGCCCCGGGAGGUCUCGUAGAC
1400
30696
HCVa:342L21 antisense siNA
cGGGGcccuccAGAGcAucTsT
1602

(324C) stab08 inv

325
UGCCCCGGGAGGUCUCGUAGACC
1394
31340
HCVa:325U21 sense siNA
B ccccGGGAGGucucGuAGATT B
1603

stab04

325
UGCCCCGGGAGGUCUCGUAGACC
1394
31341
HCVa:325U21 sense siNA inv
B AGAuGcucuGGAGGGccccTT B
1604

stab04

325
UGCCCCGGGAGGUCUCGUAGACC
1394
31342
HCVa:343121 antisense siNA
ucuAcGAGAccucccGGGGTsT
1605

(325C) stab05

325
UGCCCCGGGAGGUCUCGUAGACC
1394
31343
HCVa:343L21 antisense siNA
GGGGcccuccAGAGcAucuTsT
1606

(325C) inv stab05

325
UGCCCCGGGAGGUCUCGUAGACC
1394
31344
HCVa:325U21 sense siNA
B ccccGGGAGGucucGuAGATT B
1607

stab07

325
UGCCCCGGGAGGUCUCGUAGACC
1394
31345
HCVa:325U21 sense siNA inv
B AGAuGcucuGGAGGGccccTT B
1608

stab07

325
UGCCCCGGGAGGUCUCGUAGACC
1394
31346
HCVa:343L21 antisense siNA

GGGGcccuccAGAGcAucuTsT
1609

(325C) inv stab08

325
UGCCCCGGGAGGUCUCGUAGACC
1394
31347
HCVa:343L21 antisense siNA
ucuAcGAGAccucccGGGGTsT
1610

(325C) stab11

325
UGCCCCGGGAGGUCUCGUAGACC
1394
31348
HCVa:343L21 antisense siNA

GGGGcccuccAGAGcAucuTsT
1611

(325C) inv stab11

153
AUAGUGGUCUGCGGAACCGGUGA
1401
31453
HCVa:153U21 sense siNA
B AGuGGucuGcGGAAccGGuTT B
1612

stab04

159
GUCUGCGGAACCGGUGAGUACAC
1402
31454
HCVa:159U21 sense siNA
B cuGcGGAAccGGuGAGuAcTT B
1613

stab04

287
AAAGGCCUUGUGGUACUGCCUGA
1412
31455
HCVa:287U21 sense siNA
B AGGccuuGuGGuAcuGccuTT B
1614

stab04

291
GCCUUGUGGUACUGCCUGAUAGG
1403
31456
HCVa:291U21 sense siNA
B cuuGuGGuAcuGccuGAuATT B
1615

stab04

295
UGUGGUACUGCCUGAUAGGGUGC
1404
31457
HCVa:295U21 sense siNA
B uGGuAcuGccuGAuAGGGuTT B
1616

stab04

296
GUGGUACUGCCUGAUAGGGUGCU
1405
31458
HCVa:296U21 sense siNA
B GGuAcuGccuGAuAGGGuGTT B
1617

stab04

297
UGGUACUGCCUGAUAGGGUGCUU
1406
31459
HCVa:297U21 sense siNA
B GuAcuGccuGAuAGGGuGcTT B
1618

stab04

298
GGUACUGCOUGAUAGGGUGCUUG
1407
31460
HCVa:298U21 sense siNA
B uAcuGccuGAuAGGGuGcuTT B
1619

stab04

300
UACUGCCUGAUAGGGUGCUUGCG
1408
31461
HCVa:300U21 sense siNA
B cuGccuGAuAGGGuGcuuGTT B
1620

stab04

301
ACUGCCUGAUAGGGUGCUUGCGA
1409
31462
HCVa:301U21 sense siNA
B uGccuGAuAGGGuGcuuGcTT B
1621

stab04

303
UGCCUGAUAGGGUGCUUGCGAGU
1410
31463
HCVa:303U21 sense siNA
B ccuGAuAGGGuGcuuGcGATT B
1622

stab04

306
CUGAUAGGGUGCUUGCGAGUGCC
1411
31464
HCVa:306U21 sense siNA
B GAuAGGGuGcuuGcGAGuGTT B
1623

stab04

153
AUAGUGGUCUGCGGAACCGGUGA
1401
31465
HCVa:171L21 antisense siNA
AccGGuuccGcAGAccAcuTsT
1624

(153C) stab05

159
GUCUGCGGAACCGGUGAGUACAC
1402
31466
HCVa:177L21 antisense siNA
GuAcucAccGGuuccGcAGTsT
1625

(159C) stab05

287
AAAGGCCUUGUGGUACUGCCUGA
1412
31467
HCVa:305L21 antisense siNA
AGGcAGuAccAcMGGccuTsT
1626

(287C) stab05

291
GCCUUGUGGUACUGCCUGAUAGG
1403
31468
HCVa:309L21 antisense siNA

(291C) stab05
uAucAGGcAGuAccAcAAGTsT
1627

295
UGUGGUACUGCCUGAUAGGGUGC
1404
31469
HCVa:313L21 antisense siNA

(295C) stab05
AcccuAucAGGcAGuAccATsT
1628

296
GUGGUACUGCCUGAUAGGGUGCU
1405
31470
HCVa:314L21 antisense siNA

(296C) stab05
cAcccuAucAGGcAGuAccTsT
1629

297
UGGUACUGCCUGAUAGGGUGCUU
1406
31471
HCVa:315L21 antisense siNA

(297C) stab05
GcAcccuAucAGGcAGuAcTsT
1630

298
GGUACUGCCUGAUAGGGUGCUUG
1407
31472
HCVa:316L21 antisense siNA

(298C) stab05
AGcAcccuAucAGGcAGuATsT
1631

300
UACUGCCUGAUAGGGUGCUUGCG
1408
31473
HCVa:318L21 antisense siNA

(300C) stab05
cAAGcAcccuAucAGGcAGTsT
1632

301
ACUGCCUGAUAGGGUGCUUGCGA
1409
31474
HCVa:319L21 antisense siNA

(301C) stabo5
GcAAGcAcccuAucAGGcATsT
1633

303
UGCCUGAUAGGGUGCUUGCGAGU
1410
31475
HCVa:321L21 antisense siNA

(303C) stab05
ucGcAAGcAcccuAucAGGTsT
1634

306
CUGAUAGGGUGCUUGCGAGUGCC
1411
31476
HCVa:324L21 antisense siNA

(306C) stab05
cAcucGcAAGcAcccuAucTsT
1635

153
AUAGUGGUCUGCGGAACCGGUGA
1401
31477
HCVa:153U21 sense siNA inv
B uGGccAAGGcGucuGGuGATT B
1636

stab04

159
GUCUGCGGAACCGGUGAGUACAC
1402
31478
HCVa:159U21 sense siNA inv
B cAuGAGuGGccAAGGcGucTT B
1637

stab04

287
AAAGGCCUUGUGGUACUGCCUGA
1412
31479
HCVa:287U21 sense siNA inv
B uccGucAuGGuGuuccGGATT B
1638

stab04

291
GCCUUGUGGUACUGCCUGAUAGG
1403
31480
HCVa:291U21 sense siNA inv
B AuAGuccGucAuGGuGuucTT B
1639

stab04

295
UGUGGUACUGCCUGAUAGGGUGC
1404
31481
HCVa:295U21 sense siNA inv
B uGGGAuAGuccGucAuGGuTT B
1640

stab04

296
GUGGUACUGCCUGAUAGGGUGCU
1405
31482
HCVa:296U21 sense siNA inv
B GuGGGAuAGuccGucAuGGTT B
1641

stab04

297
UGGUACUGCCUGAUAGGGUGCUU
1406
31483
HCVa:297U21 sense siNA inv
B cGuGGGAuAGuccGucAuGTT B
1642

stab04

298
GGUACUGCCUGAUAGGGUGCUUG
1407
31484
HCVa:298U21 sense siNA inv
B ucGuGGGAuAGuccGucAuTT B
1643

stab04

300
UACUGCCUGAUAGGGUGCUUGCG
1408
31485
HCVa:300U21 sense siNA inv
B GuucGuGGGAuAGuccGucTT B
1644

stab04

301
AGUGCCUGAUAGGGUGCUUGCGA
1409
31486
HCVa:301U21 sense siNA inv
B cGuucGuGGGAuAGuccGuTT B
1645

stab04

303
UGCCUGAUAGGGUGCUUGCGAGU
1410
31487
HCVa:303U21 sense siNA inv
B AGcGuucGuGGGAuAGuccTT B
1646

stab04

306
CUGAUAGGGUGCUUGCGAGUGCC
1411
31488
HCVa:306U21 sense siNA inv
B GuGAGcGuucGuGGGAuAGTT B
1647

stab04

153
AUAGUGGUCUGCGGAACCGGUGA
1401
31489
HCVa:171L21 antisense siNA
ucAccAGAcGccuuGGccATsT
1648

(153C) inv stab05

159
GUCUGCGGAACCGGUGAGUACAC
1402
31490
HCVa:177L21 antisense siNA
GacGccuuGGccAcucAuGTsT
1649

(159C) inv stab05

287
AAAGGCCUUGUGGUACUGCCUGA
1412
31491
HCVa:305L21 antisense siNA
uccGGAAcAccAuGAcGGATsT
1650

(287C) inv stab05

291
GCCUUGUGGUACUGCCUGAUAGG
1403
31492
HCVa:309L21 antisense siNA
GAAcAccAuGAcGGAcuAuTsT
1651

(291C) inv stab05

295
UGUGGUACUGCCUGAUAGGGUGC
1404
31493
HCVa:313L21 antisense siNA
AccAuGAcGGAcuAucccATsT
1652

(295C) inv stab05

296
GUGGUACUGCCUGAUAGGGUGCU
1405
31494
HCVa:314L21 antisense siNA
ccAuGAcGGAcuAucccAcTsT
1653

(296C) inv stab05

297
UGGUACUGCCUGAUAGGGUGCUU
1406
31495
HCVa:315L21 antisense siNA
cAuGAcGGAcuAucccAcGTsT
1654

(297C) inv stab05

298
GGUACUGCCUGAUAGGGUGCUUG
1407
31496
HCVa:316L21 antisense siNA
AuGAcGGAcuAucccAcGATsT
1655

(298C) inv stab05

300
UACUGCCUGAUAGGGUGCUUGCG
1408
31497
HCVa:318L21 antisense siNA
GAcGGAcuAucccAcGAAcTsT
1656

(300C) inv stab05

301
ACUGCCUGAUAGGGUGCUUGCGA
1409
31498
HCVa:319L21 antisense siNA
AcGGAcuAucccAcGAAcGTsT
1657

(301C) inv stab05

303
UGCCUGAUAGGGUGCUUGCGAGU
1410
31499
HCVa:321L21 antisense siNA
GGAcuAucccAcGAAcGcuTsT
1658

(303C) inv stab05

306
CUGAUAGGGUGCUUGCGAGUGCC
1411
31500
HCVa:324L21 antisense siNA
cuAucccAcGAAcGcucAcTsT
1659

(306C) inv stab05

190
GGGUCCUUUCUUGGAUCAACCCG
1413
31659
HCVb:190U21 sense siNA
B GuccuuucuuGGAucAAccTT B
1660

stab04

191
GGUCCUUUCUUGGAUCAACCCGC
1393
31660
HCVb:191U21 sense siNA
B uccuuucuuGGAucAAcccTT B
1661

stab04

189
CGGGUCCUUUCUUGGAUCAACCC
1414
31661
HCVb:189U21 sense siNA
B GGuccuuucuuGGAucAAcTT B
1662

stab04

186
GACCGGGUCCUUUCUUGGAUCAA
1415
31662
HCVb:186U21 sense siNA
B ccGGGuccuuucuuGGAucTT B
1663

stab04

190
GGGUCCUUUCUUGGAUCAACCCG
1413
31663
HCVb:208L21 antisense siNA
GGuuGAuccAAGAAAGGAcTsT
1664

(190C) stab05

191
GGUCCUUUCUUGGAUCAACCCGC
1393
31664
HCVb:209L21 antisense siNA
GGGuuGAuccAAGAAAGGATsT
1665

(191C) stab05

189
CGGGUCCUUUCUUGGAUCAACCC
1414
31665
HCVb:207L21 antisense siNA
GuuGAuccAAGAAAGGAccTsT
1666

(189C) stab05

186
GACCGGGUCCUUUCUUGGAUCAA
1415
31666
HCVb:204L21 antisense siNA
GAucCAAGAAAGGAcccGGTsT
1667

(186C) stab05

190
GGGUCCUUUCUUGGAUCAACCCG
1413
31667
HCVb:190U21 sense siNA inv
B ccAAcuAGGuucuuuccuGTT B
1668

stab04

191
GGUCCUUUCUUGGAUCAACCCGC
1393
31668
HCVb:191U21 sense siNA inv
B cccAAcuAGGuucuuuccuTT B
1669

stab04

189
CGGGUCCUUUCUUGGAUCAACCC
1414
31669
HCVb:189U21 sense siNA inv
B cAAcuAGGuucuuuccuGGTT B
1670

stab04

186
GACCGGGUCCUUUCUUGGAUCAA
1415
31670
HCVb:186U21 sense siNA inv B cuAGGuucuuuccuGGGccTT B
1671

stab04

190
GGGUCCUUUCUUGGAUCAACCCG
1413
31671
HCVb:208L21 antisense siNA
cAGGAAAGAAccuAGuuGGTsT
1672

(190C) inv stab05

191
GGUCCUUUCUUGGAUCAACCCGC
1393
31672
HCVb:209L21 antisense siNA
AGGAAAGAAccuAGuuGGGTsT
1673

(191C) inv stab05

189
CGGGUCCUUUCUUGGAUCAACCC
1414
31673
HCVb:207L21 antisense siNA
cCAGGAAAGAAccuAGuuGTsT
1674

(189C) inv stab05

186
GACCGGGUCCUUUCUUGGAUCAA
1415
31674
HCVb:204L21 antisense siNA
GGcccAGGAAAGAAccuAGTsT
1675

(186C) inv stab05

326
GCCCCGGGAGGUCUCGUAGACCG
1416
31702
HCVa:326U21 sense siNA
B cccGGGAGGucucGuAGAcTT B
1676

stab07

327
CCCCGGGAGGUCUCGUAGACCGU
1417
31703
HCVa:327U21 sense siNA
B ccGGGAGGucucGuAGAccTT B
1677

stab07

328
CCCGGGAGGUCUCGUAGACCGUG
1418
31704
HCVa:328U21 sense siNA
B cGGGAGGucucGuAGAccGTT B
1678

stab07

329
CCGGGAGGUCUCGUAGACCGUGC
1419
31705
HCVa:329U21 sense siNA
B GGGAGGucucGuAGAccGuTT B
1679

stab07

326
GCCCCGGGAGGUCUCGUAGACCG
1416
31706
HCVa:344L21 antisense siNA

GucuAcGAGAccucccGGGTsT
1680

(326C) stab08

327
CCCCGGGAGGUCUCGUAGACCGU
1417
31707
HCVa:345L21 antisense siNA

GGucuAcGAGAccucccGGTsT
1681

(327C) stab08

328
CCCGGGAGGUCUCGUAGACCGUG
1418
31708
HCVa:346L21 antisense siNA
cGGucuAcGAGAccucccGTsT
1682

(328C) stab08

329
CCGGGAGGUCUCGUAGACCGUGC
1419
31709
HCVa:347L21 antisense siNA

AcGGucuAcGAGAccucccTsT
1683

(329C) stab08

326
GCCCCGGGAGGUCUCGUAGACCG
1416
31710
HCVa:326U21 sense siNA inv
B cAGAuGcucuGGAGGGcccTT B
1684

stab07

327
CCCCGGGAGGUCUCGUAGACCGU
1417
31711
HCVa:327U21 sense siNA inv
B ccAGAuGcucuGGAGGGccTT B
1685

stab07

328
CCCGGGAGGUCUCGUAGACCGUG
1418
31712
HCVa:328U21 sense siNA inv
B GccAGAuGcucuGGAGGGcTT B
1686

stab07

329
CCGGGAGGUCUCGUAGACCGUGC
1419
31713
HCVa:329U21 sense siNA inv
B uGccAGAuGcucuGGAGGGTT B
1687

stab07

326
GCCCCGGGAGGUCUCGUAGACCG
1416
31714
HCVa:344L21 antisense siNA

GGGcccuccAGAGcAucuGTsT
1688

(326C) inv stab08

327
CCCCGGGAGGUCUCGUAGACCGU
1417
31715
HCVa:345L21 antisense siNA

GGcccuccAGAGcAucuGGTsT
1689

(327C) inv stab08

328
CCCGGGAGGUCUCGUAGACCGUG
1418
31716
HCVa:346L21 antisense siNA

GcccuccAGAGcAucuGGcTsT
1690

(328C) inv stab08

329
CCGGGAGGUCUCGUAGACCGUGC
1419
31717
HCVa:347L21 antisense siNA
cccuccAGAGcAucuGGcATsT
1691

(329C) inv stab08

291
GCCUUGUGGUACUGCCUGAUAGG
1403
31762
HCVa:291U21 sense siNA
cuuGuGGuAcuGccuGAuATsT
1692

stab08

295
UGUGGUACUGCCUGAUAGGGUGC
1404
31763
HCVa:295U21 sense siNA
uGGuAcuGccuGAuAGGGuTsT
1693

stab08

325
UGCCCCGGGAGGUCUCGUAGACC
1394
31764
HCVa:325U21 sense siNA
ccccGGGAGGucucGuAGATsT
1694

stab08

291
GCCUUGUGGUACUGCCUGAUAGG
1403
31765
HCVa:291U21 sense siNA

AuAGuccGucAuGGuGuucTsT
1695

inv stab08

295
UGUGGUACUGCCUGAUAGGGUGC
1404
31766
HCVa:295U21 sense siNA
uGGGAuAGuccGucAuGGuTsT
1696

inv stab08

325
UGCCCCGGGAGGUCUCGUAGACC
1394
31767
HCVa:325U21 sense siNA

AGAuGcucuGGAGGGccccTsT
1697

inv stab08

327
CCCCGGGAGGUCUCGUAGACCGU
1417
31928
HCVa:327U21 sense siNA
ccGGGAGGucucGuAGAccTsT
1698

stab08

327
CCCCGGGAGGUCUCGUAGACCGU
1417
31929
HCVa:327U21 sense siNA
ccAGAuGcucuGGAGGGccTsT
1699

inv stab08

328
CCCGGGAGGUCUCGUAGACCGUG
1418
31930
HCVa:328U21 sense siNA
cGGGAGGucucGuAGAccGTsT
1700

stab08

328
CCCGGGAGGUCUCGUAGACCGUG
1418
31931
HCVa:328U21 sense siNA

GccAGAuGcucuGGAGGGcTsT
1701

inv stab08

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32007
HCVa:327U21 sense siNA
B ccGGGAGGucucGuAGAccTsT
1702

stab08 + 5′ abasic

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32008
HCVa:327U21 sense siNA
ccGGGAGGucucGuAGAccTsT B
1703

stab08 + 3′ abasic

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32009
HCVa:327U21 sense siNA
B ccGGGAGGucucGuAGAccTsT B
1704

stab08 + 5′ & 3 abasic

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32174
HCVa:327 siNA 3′-classl
UCUCGUAGACCUUGGUCUACGAGACCUCC
1705

10 bp
CGGTT

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32175
HCVa:327 siNA 3-classl
UCGUAGACCUUGGUCUACGAGACCUCCCG
1706

8 bp
GTT

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32176
HCVa:327 siNA 3-classl
GUAGACCUU GGUCUACGAGACCUCCCGG
1707

6 bp
TT

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32177
HCVa:327 siNA 3-classl
AGACCUU GGUCUACGAGACCUCCCGGTT
1708

4 bp

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32178
HCVa:327 siNA 5-classl
GGUCUACGAGACCUCCCGGUUCCGGGAGG
1709

10 bp
UCU

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32179
HCVa:327 siNA 5-classl
GGUCUACGAGACCUCCCGGUUCCGGGAGG
1710

8 bp
U

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32180
HCVa:327 siNA 5-classl
GGUCUACGAGACCUCCCGGUU CCGGGAG
1711

6 bp

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32181
HCVa:327 siNA 5′-classl
GGUCUACGAGACCUCCCGGUU CCGGG
1712

4 bp

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32182
HCVa:327 siNA 3′-gaaa
CUCGUAGACC GAAAGGUCUACGAGACCU
1713

10 bp
CCCGGTT

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32183
HCVa:327 siNA 3′-gaaa
CGUAGACC GAAAGGUCUACGAGACCUCC
1714

8 bp
CGGTT

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32184
HCVa:327 siNA 3′-gaaa
UAGACC GAAAGGUCUACGAGACCUCCCG
1715

6 bp
GTT

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32185
HCVa:327 siNA 3′-gaaa
GACC GAAA GGUCUACGAGACCUCCCGG
1716

4 bp
TT

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32186
HCVa:327 siNA 5′-gaaa
GGUCUACGAGACCUCCCGGUU GAAACCG
1717

10 bp
GGAGGUC

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32187
HCVa:327 siNA 5′-gaaa
GGUCUACGAGACCUCCCGGUU GAAACCG
1718

8 bp
GGAGG

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32188
HCVa:327 siNA 5′-gaaa
GGUCUACGAGACCUCCCGGUU GAAACCG
1719

6 bp
GGA

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32189
HCVa:327 siNA 5′-gaaa
GGUCUACGAGACCUCCCGGUU GAAA CC
1720

4 bp
GG

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32190
HCVa:327 siNA 3′-uuuguguag
CGUAGACCUU UUUGUGUAGGGUCUACGA
1721

10 bp
GACCUCCCGGU

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32191
HCVa:327 siNA 3′-uuuguguag
UAGACCUU UUUGUGUAG
1722

8 bp
GGUCUACGAGACCUCCCGGU

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32192
HCVa:327 siNA 3′-uuuguguag
GACCUU UUUGUGUAG
1723

6 bp GGUCUACGAGACCUCCCGGTT

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32193
HCVa:327 siNA 3′-uuuguguag
CCUU UUUGUGUAG
1724

4 bp
GGUCUACGAGACCUCCCGGTT

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32194
HCVa:327 siNA 5′-uuuguguag
GGUCUACGAGACCUCCCGGUU UUUGUGU
1725

10 bp
AGCCGGGAGGUC

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32195
HCVa:327 siNA 5′-uuuguguag
GGUCUACGAGACCUCCCGGUU UUUGUGU
1726

8 bp
AGCCGGGAGG

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32196
HCVa:327 siNA 5′-uuuguguag
GGUCUACGAGACCUCCCGGUU UUUGUGU
1727

6 bp
AGCCGGGA

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32197
HCVa:327 siNA 5′-uuuguguag
GGUCUACGAGACCUCCCGGUU UUUGUGU
1728

4 bp
AGCCGG

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32198
HCVa:345L21 antisense
GGucuAcGAGAccucccGGTsT
1729

(327C) stab05 siNA

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32199
HCVa:345L21 antisense
pGGucuAcGAGAccucccGGTsT
1730

(327C) stab05 5′p siNA

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32200
HCVa:345L21 antisense
sGGucuAcGAGAccucccGGTsT
1731

(327C) stab05 5′ps siNA

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32201
HCVa:345L21 antisense
GGUCUACGAGACCUCCCGGU
1732

(327C) stab00 siNA

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32202
HCVa:345L21 antisense
pGGUCUACGAGACCUCCCGGTT
1733

(327C) v1 5′p siNA

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32203
HCVa:345L21 antisense
sGGUCUACGAGACCUCCCGGTT
1734

(327C) v1 5′ps siNA

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32204
HCVa:345121 antisense
pGGUCUACGAGACCUCCCGGGGTT
1735

(327C) v2 5′p siNA

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32205
HCVa:345121 antisense
pGGUCUACGAGACCUCCCGG UCUCGUA u B
1736

(327C) v3 5′p siNA

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32206
HCVa:345121 antisense
pGGUCUACGAGACCUCCCGG AGGUCUCGUA
1737

(327C) v4 5′p siNA
uu B

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32207
HCVa:345121 antisense
pGGUCUACGAGACCUCCCGGTT UCUCGUA
1738

(327C) v5 5′p siNA
u B

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32208
HCVa:345121 antisense
pGGUCUACGAGACCUCCCGGTT
1739

(327C) v6 5′p siNA
AGGUCUCGUA u B

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32501
HCVa:327U21 sense siNA
B ccGGGAGGucucGuAGAccTT B
1740

stab04

325
UGCCCCGGGAGGUCUCGUAGACC
1394
32502
HCVa:325U21 sense siNA
B CCCCGGGAGGUCUCGUAGATT B
1741

stab09

326
GCCCCGGGAGGUCUCGUAGACCG
1416
32503
HCVa:326U21 sense siNA
B CCCGGGAGGUCUCGUAGACTT B
1742

stab09

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32504
HCVa:327U21 sense siNA
B CCGGGAGGUCUCGUAGACCTT B
1743

stab09

328
CCCGGGAGGUCUCGUAGACCGUG
1418
32505
HCVa:328U21 sense siNA
B CGGGAGGUCUCGUAGACCGTT B
1744

stab09

329
CCGGGAGGUCUCGUAGACCGUGC
1419
32506
HCVa:329U21 sense siNA
B GGGAGGUCUCGUAGACCGUTT B
1745

stab09

325
UGCCCCGGGAGGUCUCGUAGACC
1394
32507
HCVa:343121 antisense siNA
UCUACGAGACCUCCCGGGGTsT
1746

(325C) stab10

326
GCCCCGGGAGGUCUCGUAGACCG
1416
32508
HCVa:344121 antisense siNA
GUCUACGAGACCUCCCGGGTsT
1747

(326C) stab10

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32509
HCVa:345121 antisense siNA
GGUCUACGAGACCUCCCGGTsT
1748

(327C) stab10

328
CCCGGGAGGUCUCGUAGACCGUG
1418
32510
HCVa:346121 antisense siNA
CGGUCUACGAGACCUCCCGTsT
1749

(328C) stab10

329
CCGGGAGGUCUCGUAGACCGUGC
1419
32511
HCVa:347121 antisense siNA
ACGGUCUACGAGACCUCCCTsT
1750

(329C) stab10

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32512
HCVa:327U21 sense siNA inv
B ccAGAuGcucuGGAGGGccTT B
1751

stab09

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32513
HCVa:345L21 antisense siNA
GGcccuccAGAGcAucuGGTsT
1752

(327C) inv stab05

325
UGCCCCGGGAGGUCUCGUAGACC
1394
32514
HCVa:325U21 sense siNA inv
B AGAUGCUCUGGAGGGCCCCTT B
1753

stab09

326
GCCCCGGGAGGUCUCGUAGACCG
1416
32515
HCVa:326U21 sense siNA inv
B CAGAUGCUCUGGAGGGCCCTT B
1754

stab09

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32516
HCVa:327U21 sense siNA inv
B CCAGAUGCUCUGGAGGGCCTT B
1755

stab09

328
CCCGGGAGGUCUCGUAGACCGUG
1418
32517
HCVa:328U21 sense siNA inv
B GCCAGAUGCUCUGGAGGGCTT B
1756

stab09

329
CCGGGAGGUCUCGUAGACCGUGC
1419
32518
HCVa:329U21 sense siNA inv
B UGCCAGAUGCUCUGGAGGGTT B
1757

stab09

325
UGCCCCGGGAGGUCUCGUAGACC
1394
32519
HCVa:343L21 antisense siNA
GGGGCCCUCCAGAGCAUCUTsT
1758

(325C) inv stab10

326
GCCCCGGGAGGUCUCGUAGACCG
1416
32520
HCVa:344L21 antisense siNA
GGGCCCUCCAGAGCAUCUGTsT
1759

(326C) inv stab10

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32521
HCVa:345L21 antisense siNA
GGCCCUCCAGAGCAUCUGGTsT
1760

(327C) inv stab10

328
CCCGGGAGGUCUCGUAGACCGUG
1418
32522
HCVa:346L21 antisense siNA
GCCCUCCAGAGCAUCUGGCTsT
1761

(328C) inv stab10

329
CCGGGAGGUCUCGUAGACCGUGC
1419
32523
HCVa:347L21 antisense siNA
CCCUCCAGAGCAUCUGGCATsT
1762

(329C) inv stab10

295
UGUGGUACUGCCUGAUAGGGUGC
1404
32714
HCVa:313L21 antisense siNA
pACCCUAUCAGGCAGUACCA
1763

(295C) v1 5′p palindrome
GUACUGCCUGAU B

295
UGUGGUACUGCCUGAUAGGGUGC
1404
32715
HCVa:313L21 antisense siNA
pACCCUAUCAGGCAGUACC
1764

(295C) v2 5′p palindrome
GGUACUGCCUGAU B

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32716
HCVa: 5′p-345L21 antisense
pGGUCUACGAGACCUCCCGG
1765

(327C) v5 5′p palindrome
AGGUCUCGUAGA B

siNA

327
CCCCGGGAGGUCUCGUAGACCGU
1417
32717
HCVa: 5′p-345121 antisense
pGGUCUACGAGACCUCC
1766

(327C) v6 5′p palindrome
GGAGGUCUCGUA B

siNA

291
GCCUUGUGGUACUGCCUGAUAGG
1403
32796
HCVa:309L21 antisense siNA
uAucAgGcaguaccAcaAgTsT
1767

(291C) stab08 mod pair to

#30651

295
UGUGGUACUGCCUGAUAGGGUGC
1404
32797
HCVa:313L21 antisense siNA
acccuaucaggcaguAccaTsT
1768

(295C) stab08 mod pair to

#30652

303
UGCCUGAUAGGGUGCUUGCGAGU
1410
32798
HCVa:321L21 antisense siNA
ucgcaaGcacccuAucaggTsT
1769

(303C) stab08 mod pair to

#30658

306
CUGAUAGGGUGCUUGCGAGUGCC
1411
32799
HCVa:324L21 antisense siNA
cacucgcAagcacccuaucTsT
1770

(306C) stab08 mod A pair to

#30659

306
CUGAUAGGGUGCUUGCGAGUGCC
1411
32800
HCVa:324L21 antisense siNA
cAcucgcAagcacccuaucTsT
1771

(306C) stab08 mod B pair to

#30659

140
UCCCGGGAGAGCCAUAGUGGUCU
1420
33125
HCVa:140U21 sense siNA
B ccGGGAGAGccAuAGuGGuTT B
1772

stab07

141
CCCGGGAGAGCCAUAGUGGUCUG
1421
33126
HCVa:141U21 sense siNA
B cGGGAGAGccAuAGuGGucTT B
1773

stab07

142
CCGGGAGAGCCAUAGUGGUCUGC
1422
33127
HCVa:142U21 sense siNA
B GGGAGAGccAuAGuGGucuTT B
1774

stab07

154
UAGUGGUCUGCGGAACCGGUGAG
1423
33128
HCVa:154U21 sense siNA
B GuGGucuGcGGAAccGGuGTT B
1775

stab07

155
AGUGGUCUGCGGAACCGGUGAGU
1424
33129
HCVa:155U21 sense siNA
B uGGucuGcGGAAccGGuGATT B
1776

stab07

156
GUGGUCUGCGGAACCGGUGAGUA
1425
33130
HCVa:156U21 sense siNA
B GGucuGcGGAAccGGuGAGTT B
1777

stab07

157
UGGUCUGCGGAACCGGUGAGUAC
1426
33131
HCVa:157U21 sense siNA
B GucuGcGGAAccGGuGAGuTT B
1778

stab07

158
GGUCUGCGGAACCGGUGAGUACA
1427
33132
HCVa:158U21 sense siNA
B ucuGcGGAAccGGuGAGuATT B
1779

stab07

160
UCUGCGGAACCGGUGAGUACACC
1428
33133
HCVa:160U21 sense siNA
B uGcGGAAccGGuGAGuAcATT B
1780

stab07

161
CUGCGGAACCGGUGAGUACACCG
1429
33134
HCVa:161U21 sense siNA
B GcGGAAccGGuGAGuAcAcTT B
1781

stab07

164
CGGAACCGGUGAGUACACCGGAA
1430
33135
HCVa:164U21 sense siNA
B GAAccGGuGAGuAcAccGGTT B
1782

stab07

165
GGAACCGGUGAGUACACCGGAAU
1431
33136
HCVa:165U21 sense siNA
B AAccGGuGAGuAcAccGGATT B
1783

stab07

166
GAACCGGUGAGUACACCGGAAUU
1432
33137
HCVa:166U21 sense siNA
B AccGGuGAGuAcAccGGAATT B
1784

stab07

167
AACCGGUGAGUACACCGGAAUUG
1433
33138
HCVa:167U21 sense siNA
B ccGGuGAGuAcAccGGAAuTT B
1785

stab07

282
UCGCGAAAGGCCUUGUGGUACUG
1434
33139
HCVa:282U21 sense siNA
B GcGAAAGGccuuGuGGuAcTT B
1786

stab07

283
CGCGAAAGGCCUUGUGGUACUGC
1435
33140
HCVa:283U21 sense siNA
B cGAAAGGccuuGuGGuAcuTT B
1787

stab07

284
GCGAAAGGCCUUGUGGUACUGCC
1436
33141
HCVa:284U21 sense siNA
B GAAAGGccuuGuGGuAcuGTT B
1788

stab07

285
CGAAAGGCCUUGUGGUACUGCCU
1437
33142
HCVa:285U21 sense siNA
B AAAGGccuuGuGGuAcuGcTT B
1789

stab07

286
GAAAGGCCUUGUGGUACUGCCUG
1438
33143
HCVa:286U21 sense siNA
B AAGGccuuGuGGuAcuGccTT B
1790

stab07

288
AAGGCCUUGUGGUACUGCCUGAU
1439
33144
HCVa:288U21 sense siNA
B GGccuuGuGGuAcuGccuGTT B
1791

stab07

289
AGGCCUUGUGGUACUGCCUGAUA
1440
33145
HCVa:289U21 sense siNA
B GccuuGuGGuAcuGccuGATT B
1792

stab07

290
GGCCUUGUGGUACUGCCUGAUAG
1441
33146
HCVa:290U21 sense siNA
B ccuuGuGGuAcuGccuGAuTT B
1793

stab07

299
GUACUGCCUGAUAGGGUGCUUGC
1442
33147
HCVa:299U21 sense siNA
B AcuGccuGAuAGGGuGcuuTT B
1794

stab07

302
CUGCCUGAUAGGGUGCUUGCGAG
1443
33148
HCVa:302U21 sense siNA
B GccuGAuAGGGuGcuuGcGTT B
1795

stab07

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
33149
HCVa:304U21 sense siNA
B cuGAuAGGGuGcuuGcGAGTT B
1796

stab07

305
CCUGAUAGGGUGCUUGCGAGUGC
1445
33150
HCVa:305U21 sense siNA
B uGAuAGGGuGcuuGcGAGuTT B
1797

stab07

307
UGAUAGGGUGCUUGCGAGUGCCC
1446
33151
HCVa:307U21 sense siNA
B AuAGGGuGcuuGcGAGuGcTT B
1798

stab07

308
GAUAGGGUGCUUGCGAGUGCCCC
1447
33152
HCVa:308U21 sense siNA
B uAGGGuGcuuGcGAGuGccTT B
1799

stab07

310
UAGGGUGCUUGCGAGUGCCCCGG
1448
33153
HCVa:310U21 sense siNA
B GGGuGcuuGcGAGuGccccTT B
1800

stab07

311
AGGGUGCUUGCGAGUGCCCCGGG
1449
33154
HCVa:311U21 sense siNA
B GGuGcuuGcGAGuGccccGTT B
1801

stab07

314
GUGCUUGCGAGUGCCCCGGGAGG
1450
33155
HCVa:314U21 sense siNA
B GcuuGcGAGuGccccGGGATT B
1802

stab07

315
UGCUUGCGAGUGCCCCGGGAGGU
1451
33156
HCVa:315U21 sense siNA
B cuuGcGAGuGccccGGGAGTT B
1803

stab07

316
GCUUGCGAGUGCCCCGGGAGGUC
1452
33157
HCVa:316U21 sense siNA
B uuGcGAGuGccccGGGAGGTT B
1804

stab07

317
CUuGCGAGUGCCCCGGGAGGUCU
1453
33158
HCVa:317U21 sense siNA
B uGcGAGuGccccGGGAGGuTT B
1805

stab07

318
UUGCGAGUGCCCCGGGAGGUCUC
1454
33159
HCVa:318U21 sense siNA
B GcGAGuGccccGGGAGGucTT B
1806

stab07

319
UGCGAGUGCCCCGGGAGGUCUCG
1455
33160
HCVa:319U21 sense siNA
B cGAGuGccccGGGAGGucuTT B
1807

stab07

320
GCGAGUGCCCCGGGAGGUCUCGU
1456
33161
HCVa:320U21 sense siNA
B GAGuGccccGGGAGGucucTT B
1808

stab07

322
GAGUGCCCCGGGAGGUCUCGUAG
1457
33162
HCVa:322U21 sense siNA
B GuGccccGGGAGGucucGuTT B
1809

stab07

323
AGUGCCCCGGGAGGUCUCGUAGA
1458
33163
HCVa:323U21 sense siNA
B uGccccGGGAGGucucGuATT B
1810

stab07

330
CGGGAGGUCUCGUAGACCGUGCA
1459
33164
HCVa:330U21 sense siNA
B GGAGGucucGuAGAccGuGTT B
1811

stab07

140
UCCCGGGAGAGCCAUAGUGGUCU
1420
33165
HCVa:158L21 antisense siNA

AccAcuAuGGcucucccGGTsT
1812

(140C) stab08

141
CCCGGGAGAGCCAUAGUGGUCUG
1421
33166
HCVa:159L21 antisense siNA

GaccAcuAuGGcucucccGTsT
1813

(141C) stab08

142
CCGGGAGAGCCAUAGUGGUCUGC
1422
33167
HCVa:160L21 antisense siNA

AGAccAcuAuGGcucucccTsT
1814

(142C) stab08

154
UAGUGGUCUGCGGAACCGGUGAG
1423
33168
HCVa:172L21 antisense siNA
cAccGGuuccGcAGAccAcTsT
1815

(154C) stab08

155
AGUGGUCUGCGGAACCGGUGAGU
1424
33169
HCVa:173L21 antisense siNA
ucAccGGuuccGcAGAccATsT
1816

(155C) stab08

156
GUGGUCUGCGGAACCGGUGAGUA
1425
33170
HCVa:174L21 antisense siNA
cucAccGGuuccGcAGAccTsT
1817

(156C) stab08

157
UGGUCUGCGGAACCGGUGAGUAC
1426
33171
HCVa:175L21 antisense siNA

AcucAccGGuuccGcAGAcTsT
1818

(157C) stab08

158
GGUCUGCGGAACCGGUGAGUACA
1427
33172
HCVa:176L21 antisense siNA
uAcucAccGGuuccGcAGATsT
1819

(158C) stab08

160
UCUGCGGAACCGGUGAGUACACC
1428
33173
HCVa:178L21 antisense siNA
uGuAcucAccGGuuccGcATsT
1820

(160C) stab08

161
CUGCGGAACCGGUGAGUACACCG
1429
33174
HCVa:179L21 antisense siNA

GuGuAcucAccGGuuccGcTsT
1821

(161C) stab08

164
CGGAACCGGUGAGUACACCGGAA
1430
33175
HCVa:182L21 antisense siNA
ccGGuGuAcucAccGGuucTsT
1822

(164C) stab08

165
GGAACCGGUGAGUACACCGGAAU
1431
33176
HCVa:183L21 antisense siNA
uccGGuGuAcucAccGGuuTsT
1823

(165C) stab08

166
GAACCGGUGAGUACACCGGAAUU
1432
33177
HCVa:184L21 antisense siNA
uuccGGuGuAcucAccGGuTsT
1824

(166C) stab08

167
AACCGGUGAGUACACCGGAAUUG
1433
33178
HCVa:185L21 antisense siNA

AuuccGGuGuAcucAccGGTsT
1825

(167C) stab08

282
UCGCGAAAGGCCUUGUGGUACUG
1434
33179
HCVa:300L21 antisense siNA

GuAccAcAAGGccuuucGcTsT
1826

(282C) stab08

283
CGCGAAAGGCCUUGUGGUACUGC
1435
33180
HCVa:301L21 antisense siNA

AGuAccAcAAGGccuuucGTsT
1827

(283C) stab08

284
GCGAAAGGCCUUGUGGUACUGCC
1436
33181
HCVa:302L21 antisense siNA
cAGuAccAcAAGGccuuucTsT
1828

(284C) stab08

285
CGAAAGGCCUUGUGGUACUGCCU
1437
33182
HCVa:303L21 antisense siNA

GcAGuAccAcAAGGccuuuTsT
1829

(285C) stab08

286
GAAAGGCCUUGUGGUACUGCCUG
1438
33183
HCVa:304L21 antisense siNA

GGCAGuAccAcAAGGccuuTsT
1830

(286C) stab08

288
AAGGCCUUGUGGUACUGCCUGAU
1439
33184
HCVa:306L21 antisense siNA
cAGGcAGuAccAcAAGGccTsT
1831

(288C) stab08

289
AGGCCUUGUGGUACUGCCUGAUA
1440
33185
HCVa:307L21 antisense siNA
ucAGGcAGuAccAcAAGGcTsT
1832

(289C) stab08

290
GGCCUUGUGGUACUGCCUGAUAG
1441
33186
HCVa:308L21 antisense siNA

AucAGGcAGuAccAcAAGGTsT
1833

(290C) stab08

299
GUACUGCCUGAUAGGGUGCUUGC
1442
33187
HCVa:317L21 antisense siNA

AAGcAcccuAucAGGcAGuTsT
1834

(299C) stab08

302
CUGCCUGAUAGGGUGCUUGCGAG
1443
33188
HCVa:320L21 antisense siNA
cGcAAGcAcccuAucAGGcTsT
1835

(302C) stab08

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
33189
HCVa:322L21 antisense siNA
cucGcAAGcAcccuAucAGTsT
1836

(304C) stab08

305
CCUGAUAGGGUGCUUGCGAGUGC
1445
33190
HCVa:323L21 antisense siNA

AcucGcAAGcAcccuAucATsT
1837

(305C) stab08

307
UGAUAGGGUGCUUGCGAGUGCCC
1446
33191
HCVa:325L21 antisense siNA

GcAcucGcAAGcAcccuAuTsT
1838

(307C) stab08

308
GAUAGGGUGCUUGCGAGUGCCCC
1447
33192
HCVa:326L21 antisense siNA

GGcAcucGcAAGcAcccuATsT
1839

(308C) stab08

310
UAGGGUGCUUGCGAGUGCCCCGG
1448
33193
HCVa:328L21 antisense siNA

GGGGcAcucGcAAGcAcccTsT
1840

(310C) stab08

311
AGGGUGCUUGCGAGUGCCCCGGG
1449
33194
HCVa:329L21 antisense siNA
cGGGGcAcucGcAAGcAccTsT
1841

(311C) stab08

314
GUGCUUGCGAGUGCCCCGGGAGG
1450
33195
HCVa:332L21 antisense siNA
ucccGGGGcAcucGcAAGcTsT
1842

(314C) stab08

315
UGCUUGCGAGUGCCCCGGGAGGU
1451
33196
HCVa:333L21 antisense siNA
cucccGGGGcAcucGcAAGTsT
1843

(315C) stab08

316
GCUUGCGAGUGCCCCGGGAGGUC
1452
33197
HCVa:334L21 antisense siNA
ccucccGGGGcAcucGcAATsT
1844

(316C) stab08

317
CUUGCGAGUGCCCCGGGAGGUCU
1453
33198
HCVa:335L21 antisense siNA

AccucccGGGGcAcucGcATsT
1845

(317C) stab08

318
UUGCGAGUGCCCCGGGAGGUCUC
1454
33199
HCVa:336L21 antisense siNA

GAccucccGGGGcAcucGcTsT
1846

(318C) stab08

319
UGCGAGUGCCCCGGGAGGUCUCG
1455
33200
HCVa:337L21 antisense siNA

AGAccucccGGGGcAcucGTsT
1847

(319C) stab08

320
GCGAGUGCCCCGGGAGGUCUCGU
1456
33201
HCVa:338L21 antisense siNA

GAGAccucccGGGGcAcucTsT
1848

(320C) stab08

322
GAGUGCCCCGGGAGGUCUCGUAG
1457
33202
HCVa:340L21 antisense siNA

AcGAGAccucccGGGGcAcTsT
1849

(322C) stab08

323
AGUGCCCCGGGAGGUCUCGUAGA
1458
33203
HCVa:341L21 antisense siNA
uAcGAGAccucccGGGGcATsT
1850

(323C) stab08

330
CGGGAGGUCUCGUAGACCGUGCA
1459
33204
HCVa:348L21 antisense siNA
cAcGGucuAcGAGAccuccTsT
1851

(330C) stab08

303
UGCCUGAUAGGGUGCUUGCGAGU
1410
33329
HCVa:321L21 antisense siNA
pucGcAAGcAcccuAucAGGTsT
1852

(303C) stab08 + 5′ P

303
UGCCUGAUAGGGUGCUUGCGAGU
1410
33330
HCVa:321L21 antisense siNA
pucGcAAGcAcccuAucAGGTsT
1853

(303C) stab05 + 5′ P

295
UGUGGUACUGCCUGAUAGGGUGC
1404
33331
HCVa:313L21 antisense siNA
AcccuAucAGGcAGuAccATsT
1854

(295C) stab05 + 5′ P

295
UGUGGUACUGCCUGAUAGGGUGC
1404
33332
HCVa:313L21 antisense siNA
pAcccuAucAGGcAGuAccATsT
1855

(295C) stab08 + 5′ P

306
CUGAUAGGGUGCUUGCGAGUGCC
1411
33333
HCVa:324L21 antisense siNA
pcAcucGcAAGcAcccuAucTsT
1856

(306C) stab08 + 5′ P

327
CCCCGGGAGGUCUCGUAGACCGU
1417
33334
HCVa:345L21 antisense siNA
pGGucuAcGAGAccucccGGTsT
1857

(327C) stab08 + 5′ P

303
UGCCUGAUAGGGUGCUUGCGAGU
1410
33346
HCVa:321L21 antisense siNA
L ucGcAAGcAcccuAucAGGTsT
1858

(303C) stab08 + 5′ aminoL

303
UGCCUGAUAGGGUGCUUGCGAGU
1410
33347
HCVa:321L21 antisense siNA
L ucGcAAGcAcccuAucAGGTsT
1859

(303C) stab05 + 5′ aminoL

295
UGUGGUACUGCCUGAUAGGGUGC
1404
33348
HCVa:313L21 antisense siNA
LAcccuAucAGGcAGuAccATsT
1860

(295C) stab05 + 5′ aminoL

295
UGUGGUACUGCCUGAUAGGGUGC
1404
33349
HCVa:313L21 antisense siNA
L AcccuAucAGGcAGuAccATsT
1861

(295C) stab08 + 5′ aminoL

306
CUGAUAGGGUGCUUGCGAGUGCC
1411
33350
HCVa:324L21 antisense siNA
L cAcucGcAAGcAcccuAucTsT
1862

(306C) stab08 + 5′ aminoL

327
CCCCGGGAGGUCUCGUAGACCGU
1417
33351
HCVa:345L21 antisense siNA
L GGucuAcGAGAccucccGGTsT
1863

(327C) stab08 + 5′ aminoL

327
CCCCGGGAGGUCUCGUAGACCGU
1417
34024
HCVa:327U21 sense siNA
B ccGAGAGGucGcGuAGuccTT B
1864

inact1 stab07

327
CCCCGGGAGGUCUCGUAGACCGU
1417
34025
HCVa:327U21 sense siNA
B ccGAGAGGucGcGucGAucTT B
1865

inact2 stab07

327
CCCCGGGAGGUCUCGUAGACCGU
1417
34026
HCVa:327U21 sense siNA
B ccGGuAGGucccGuGGAcATT B
1866

inact3 stab07

327
CCCCGGGAGGUCUCGUAGACCGU
1417
34027
HCVa:345L21 antisense siNA

GGAcuAcGcGAccucucGGTsT
1867

(327C) inact1 stab08

327
CCCCGGGAGGUCUCGUAGACCGU
1417
34028
HCVa:345L21 antisense siNA

GAucGAcGcGAccucucGGTsT
1868

(327C) inact2 stab08

327
CCCCGGGAGGUCUCGUAGACCGU
1417
34029
HCVa:345L21 antisense siNA
uGuccAcGGGAccuAccGGTsT
1869

(327C) inact3 stab08

282
UCGCGAAAGGCCUUGUGGUACUG
1434
34030
HCVa:282U21 sense siNA
B GcuAAAGGcGuuGuGGcAcTT B
1870

inact1 stab07

282
UCGCGAAAGGCCUUGUGGUACUG
1434
34031
HCVa:282U21 sense siNA
B GcGuAAGGcccuGuGGuAATT B
1871

inact2 stab07

282
UCGCGAAAGGCCUUGUGGUACUG
1434
34032
HCVa:282U21 sense siNA
B GAGAAAcGccuGGuGGuucTT B
1872

inact3 stab07

283
CGCGAAAGGCCUUGUGGUACUGC
1435
34033
HCVa:283U21 sense siNA
B cGuAAGGcAuuGuGGcAcuTT B
1873

inact1 stab07

283
CGCGAAAGGCCUUGUGGUACUGC
1435
34034
HCVa:283U21 sense siNA
B cGAGAGGcAuuGuGcuAcuTT B
1874

inact2 stab07

283
CGCGAAAGGCCUUGUGGUACUGC
1435
34035
HCVa:283U21 sense siNA
B ccAAAGGucuuGAGGuGcuTT B
1875

inact3 stab07

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
34036
HCVa:304U21 sense siNA
B cGGAuAGGcuGcuuGuGAGTT B
1876

inact1 stab07

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
34037
HCVa:304U21 sense siNA
B cuGcuAGGGuAcuuGGGAGTT B
1877

inact2 stab07

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
34038
HCVa:304U21 sense siNA
B ccGAuAuGGuGAuuGcGGGTT B
1878

inact3 stab07

307
UGAUAGGGUGCUUGCGAGUGCCC
1446
34039
HCVa:307U21 sense siNA
B AuuGGGuGcuGGcGAGuAcTT B
1879

inact1 stab07

307
UGAUAGGGUGCUUGCGAGUGCCC
1446
34040
HCVa:307U21 sense siNA
B AuAuGGuGccuGcGAGuGGTT B
1880

inact2 stab07

307
UGAUAGGGUGCUUGCGAGUGCCC
1446
34041
HCVa:307U21 sense siNA
B AGAGGGuAcuuGcGcGuGuTT B
1881

inact3 stab07

282
UCGCGAAAGGCCUUGUGGUACUG
1434
34042
HCVa:300L21 antisense siNA

GuGccAcAAcGccuuuAGcTsT
1882

(282C) inact1 stab08

282
UCGCGAAAGGCCUUGUGGUACUG
1434
34043
HCVa:300L21 antisense siNA
uuAccAcAGGGccuuAcGcTsT
1883

(282C) inact2 stab08

282
UCGCGAAAGGCCUUGUGGUACUG
1434
34044
HCVa:300L21 antisense siNA

GAAccAccAGGcGuuucucTsT
1884

(282C) inact3 stab08

283
CGCGAAAGGCCUUGUGGUACUGC
1435
34045
HCVa:301L21 antisense siNA

AguGccAcAAuGccuuAcGTsT
1885

(283C) inact1 stab08

283
CGCGAAAGGCCUUGUGGUACUGC
1435
34046
HCVa:301121 antisense siNA

AguAGcAcAAuGccucucGTsT
1886

(283C) inact2 stab08

283
CGCGAAAGGCCUUGUGGUACUGC
1435
34047
HCVa:301121 antisense siNA

AGcAccucAAGAccuuuGGTsT
1887

(283C) inact3 stab08

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
34048
HCVa:322121 antisense siNA
cucAcAAGcAGccuAuccGTsT
1888

(304C) inact1 stab08

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
34049
HCVa:322121 antisense siNA
cucccAAGuAcccuAGcAGTsT
1889

(304C) inact2 stab08

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
34050
HCVa:322121 antisense siNA
cccGcAAucAccAuAucGGTsT
1890

(304C) inact3 stab08

307
UGAUAGGGUGCUUGCGAGUGCCC
1446
34051
HCVa:325121 antisense siNA

GuAcucGccAGcAcccAAuTsT
1891

(307C) inact1 stab08

307
UGAUAGGGUGCUUGCGAGUGCCC
1446
34052
HCVa:325121 antisense siNA
ccAcucGcAGGcAccAuAuTsT
1892

(307C) inact2 stab08

307
UGAUAGGGUGCUUGCGAGUGCCC
1446
34053
HCVa:325121 antisense siNA

AcAcGcGcAAGuAcccucuTsT
1893

(307C) inact3 stab08

282
UCGCGAAAGGCCUUGUGGUACUG
1434
34054
HCVa:282U21 sense siNA inv
B cAuGGuGuuccGGAAAGcGTT B
1894

stab07

283
CGCGAAAGGCCUUGUGGUACUGC
1435
34055
HCVa:283U21 sense siNA inv
B ucAuGGuGuuccGGAAAGcTT B
1895

stab07

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
34056
HCVa:304U21 sense siNA inv
B GAGcGuucGuGGGAuAGucTT B
1896

stab07

307
UGAUAGGGUGCUUGCGAGUGCCC
1446
34057
HCVa:307U21 sense siNA inv
B cGuGAGcGuucGuGGGAuATT B
1897

stab07

282
UCGCGAAAGGCCUUGUGGUACUG
1434
34058
HCVa:300L21 antisense siNA
cGcuuuccGGAAcAccAuGTsT
1898

(282C) inv stab08

283
CGCGAAAGGCCUUGUGGUACUGC
1435
34059
HCVa:301L21 antisense siNA

GcuuuccGGAAcAccAuGATsT
1899

(283C) inv stab08

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
34060
HCVa:322L21 antisense siNA

GAcuAucccAcGAAcGcucTsT
1900

(304C) inv stab08

307
UGAUAGGGUGCUUGCGAGUGCCC
1446
34061
HCVa:325L21 antisense siNA
uAucccAcGAAcGcucAcGTsT
1901

(307C) inv stab08

82
UAGCCAUGGCGUUAGUAUGAGUG
1460
34128
HCVb:100L18 (82C) 5′p
pUCAUACUAACGCCAUGGC GUUAGUAUGAB
1902

palindrome siNA

82
UAGCCAUGGCGUUAGUAUGAGUG
1460
34129
HCVb:100L17 (82C) 5′p
pCAUACUAACGCCAUGGC GUUAGUAUGB
1903

palindrome siNA

82
UAGCCAUGGCGUUAGUAUGAGUG
1460
34130
HCVb:100L16 (82C) 5′p
pAUACUAACGCCAUGGC GUUAGUAUB
1904

palindrome siNA

82
UAGCCAUGGCGUUAGUAUGAGUG
1460
34131
HCVb:100L15 (82C) 5′p
pUACUAACGCCAUGGC GUUAGUAB
1905

palindrome siNA

126
CCCUCCCGGGAGAGCCAUAGUGG
1461
34132
HCVb:144L19 (126C) 5′p
pACUAUGGCUCUCCCGGGAG AGCCAUAGUB
1906

palindrome siNA

126
CCCUCCCGGGAGAGCCAUAGUGG
1461
34133
HCVb:144L18 (126C) 5′p
pCUAUGGCUCUCCCGGGAG AGCCAUAGB
1907

palindrome siNA

126
CCCUCCCGGGAGAGCCAUAGUGG
1461
34134
HCVb:144L17 (126C) 5′p
pUAUGGCUCUCCCGGGAG AGCCAUAB
1908

palindrome siNA

126
CCCUCCCGGGAGAGCCAUAGUGG
1461
34135
HCVb:144L16 (126C) 5′p
pAUGGCUCUCCCGGGAG AGCCAUB
1909

palindrome siNA

126
CCCUCCCGGGAGAGCCAUAGUGG
1461
34136
HCVb:144L15 (126C) 5′p
pUGGCUCUCCCGGGAG AGCCAB
1910

palindrome siNA

155
GAACCGGUGAGUACACCGGAAUU
1432
34137
HCVb:171L17 (155C) 5′p
pCCGGUGUACUCACCGGU GAGUACACCGGB
1911

palindrome siNA

155
GAACCGGUGAGUACACCGGAAUU
1432
34138
HCVb:170L16 (155C) 5′p
pCGGUGUACUCACCGGU GAGUACACCGB
1912

palindrome siNA

155
GAACCGGUGAGUACACCGGAAUU
1432
34139
HCVb:169L15 (155C) 5′p
pGGUGUACUCACCGGU GAGUACACCB
1913

palindrome siNA

315
GCCCCGGGAGGUCUCGUAGACCG
1416
34140
HCVb:331L17 (315C) 5′p
pCUACGAGACCUCCCGGG AGGUCUCGUAGB
1914

palindrome siNA

315
GCCCCGGGAGGUCUCGUAGACCG
1416
34141
HCVb:330L16 (315C) 5′p
pUACGAGACCUCCCGGG AGGUCUCGUAB
1915

palindrome siNA

315
GCCCCGGGAGGUCUCGUAGACCG
1416
34142
HCVb:329L15 (315C) 5′p
pACGAGACCUCCCGGG AGGUCUCGUB
1916

palindrome siNA

327
CCCCGGGAGGUCUCGUAGACCGU
1417
34494
HCVa:345L21 antisense siNA
GGucuAcGAGAccucccGGTT B
1917

(327C) stab19

327
CCCCGGGAGGUCUCGUAGACCGU
1417
34495
HCVa:345L21 antisense siNA
GGcccuccAGAGcAucuGGTT B
1918

(327C) inv stab19

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
34496
HCVa:322L21 antisense siNA
cucGcAAGcAcccuAucAGTT B
1919

(304C) stab19

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
34499
HCVa:322L21 antisense siNA
GAcuAucccAcGAAcGcucTT B
1920

(304C) inv stab19

282
UCGCGAAAGGCCUUGUGGUACUG
1434
34581
HCVa:282U21 sense siNA
GCGAAAGGCCUUGUGGUACTT
1921

stab00

283
CGCGAAAGGCCUUGUGGUACUGC
1435
34582
HCVa:283U21 sense siNA
CGAAAGGCCUUGUGGUACUTT
1922

stab00

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
34583
HCVa:304U21 sense siNA
CUGAUAGGGUGCUUGCGAGTT
1923

stab00

307
UGAUAGGGUGCUUGCGAGUGCCC
1446
34584
HCVa:307U21 sense siNA
AUAGGGUGCUUGCGAGUGCTT
1924

stab00

327
CCCCGGGAGGUCUCGUAGACCGU
1417
34585
HCVa:327U21 sense siNA
CCGGGAGGUCUCGUAGACCTT
1925

stab00

282
UCGCGAAAGGCCUUGUGGUACUG
1434
34586
HCVa:300L21 antisense siNA
GUACCACAAGGCCUUUCGCTT
1926

(282C) stab00

283
CGCGAAAGGCCUUGUGGUACUGC
1435
34587
HCVa:301L21 antisense siNA
AGUACCACAAGGCCUUUCGTT
1927

(283C) stab00

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
34588
HCVa:322L21 antisense siNA
CUCGCAAGCACCCUAUCAGTT
1928

(304C) stab00

307
UGAUAGGGUGCUUGCGAGUGCCC
1446
34589
HCVa:325L21 antisense siNA
GCACUCGCAAGCACCCUAUTT
1929

(307C) stab00

282
UCGCGAAAGGCCUUGUGGUACUG
1434
34590
HCVa:282U21 sense siNA inv
CAUGGUGUUCCGGAAAGCGTT
1930

stab00

283
CGCGAAAGGCCUUGUGGUACUGC
1435
34591
HCVa:283U21 sense siNA inv
UCAUGGUGUUCCGGAAAGCTT
1931

stab00

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
34592
HCVa:304U21 sense siNA inv
GAGCGUUCGUGGGAUAGUCTT
1932

stab00

307
UGAUAGGGUGCUUGCGAGUGCCC
1446
34593
HCVa:307U21 sense siNA inv
CGUGAGCGUUCGUGGGAUATT
1933

stab00

327
CCCCGGGAGGUCUCGUAGACCGU
1417
34594
HCVa:327U21 sense siNA inv
CCAGAUGCUCUGGAGGGCCTT
1934

stab00

282
UCGCGAAAGGCCUUGUGGUACUG
1434
34595
HCVa:300L21 antisense siNA
CGCUUUCCGGAACACCAUGTT
1935

(282C) inv stab00

283
CGCGAAAGGCCUUGUGGUACUGC
1435
34596
HCVa:301L21 antisense siNA
GCUUUCCGGAACACCAUGATT
1936

(283C) inv stab00

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
34597
HCVa:322L21 antisense siNA
GACUAUCCCACGAACGCUCTT
1937

(304C) inv stab00

307
UGAUAGGGUGCUUGCGAGUGCCC
1446
34598
HCVa:325L21 antisense siNA
UAUCCCACGAACGCUCACGTT
1938

(307C) inv stab00

327
CCCCGGGAGGUCUCGUAGACCGU
1417
34599
HCVa:345L21 antisense siNA
GGCCCUCCAGAGCAUCUGGTT
1939

(327C) inv stab00

327
CCCCGGGAGGUCUCGUAGACCGU
1417
35173
HCVa:327U21 sense siNA
B ccGGGAGGucucGUAGACCTT B
1940

stab07 N1

327
CCCCGGGAGGUCUCGUAGACCGU
1417
35174
HCVa:345L21 antisense siNA

GGUCUAcGAGAccucccGGTsT
1941

(327C) stab08 N1

327
CCCCGGGAGGUCUCGUAGACCGU
1417
35175
HCVa:345L21 antisense siNA

GGUcuAcGAGAccucccGGTsT
1942

(327C) stab25

327
CCCCGGGAGGUCUCGUAGACCGU
1417
35176
HCVa:345L21 antisense siNA

GGucuAcGAGAccucccGGTsT
1943

(327C) stab08 N3

327
CCCCGGGAGGUCUCGUAGACCGU
1417
35177
HCVa:345L21 antisense siNA

GGucuAcGAGAccucccGGTsT
1944

(327C) stab24

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
35178
HCVa:304U21 sense siNA
B cuGAuAGGGuGcuUGCGAGTT B
1945

stab07 N1

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
35179
HCVa:322L21 antisense siNA
CUCGCAAGcAcccuAucAGTsT
1946

(304C) stab08 N1

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
35180
HCVa:322L21 antisense siNA
CUCGcAAGcAcccuAucAGTsT
1947

(304C) stab25

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
35181
HCVa:322L21 antisense siNA
CUcGcAAGcAcccuAucAGTsT
1948

(304C) stab08 N3

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
35182
HCVa:322L21 antisense siNA
CucGcAAGcAcccuAucAGTsT
1949

(304C) stab24

327
CCCCGGGAGGUCUCGUAGACCGU
1417
35225
HCVa:327 siNA stab0/0
GGUCUACGAGACCUCCCGG
1950

Pal01
CCGGGAGGUCUCGUAGACC

327
CCCCGGGAGGUCUCGUAGACCGU
1417
35226
HCVa:327 siNA stab0/0 PaI02
GGUCUACGAGACCUCCCGG
1951

CCGGGAGGUCUCGUAGACCTT

327
CCCCGGGAGGUCUCGUAGACCGU
1417
35227
HCVa:327 siNA stab0/0 PaI03
GGUCUACGAGACCUCCCG
1952

CGGGAGGUCUCGUAGACC

327
CCCCGGGAGGUCUCGUAGACCGU
1417
35228
HCVa:327 siNA stab0/0 PaI04
GGUCUACGAGACCUCCCG
1953

CGGGAGGUCUCGUAGACCTT

327
CCCCGGGAGGUCUCGUAGACCGU
1417
35229
HCVa:327 siNA stab0/0 PaI05
GGUCUACGAGACCUCCC
1954

GGGAGGUCUCGUAGACC

327
CCCCGGGAGGUCUCGUAGACCGU
1417
35230
HCVa:327 siNA stab0/0 PaI06
GGUCUACGAGACCUCCC
1955

GGGAGGUCUCGUAGACCTT

327
CCCCGGGAGGUCUCGUAGACCGU
1417
35231
HCVa:327 siNA stab0/0 PaI07
GGUCUACGAGACCUCC
1956

GGAGGUCUCGUAGACC

327
CCCCGGGAGGUCUCGUAGACCGU
1417
35232
HCVa:327 siNA stab0/0 PaI08
GGUCUACGAGACCUCC
1957

GGAGGUCUCGUAGACCTT

327
CCCCGGGAGGUCUCGUAGACCGU
1417
35235
HCVa:327 siNA stab0/0 PaI11
GUCUACGAGACCUCCCGG
1958

GAGGUCUCGUAGAC

327
CCCCGGGAGGUCUCGUAGACCGU
1417
35236
HCVa:327 siNA stab0/0 PaI12
GUCUACGAGACCUCCCGG
1959

GAGGUCUCGUAGACTT

327
CCCCGGGAGGUCUCGUAGACCGU
1417
35237
HCVa:327 siNA stab0/0 PaI13
UCUACGAGACCUCCCGG
1960

GAGGUCUCGUAGA

327
CCCCGGGAGGUCUCGUAGACCGU
1417
35238
HCVa:327 siNA stab0/0 PaI14
UCUACGAGACCUCCCGG
1961

GAGGUCUCGUAGATT

327
CCCCGGGAGGUCUCGUAGACCGU
1417
35239
HCVa:327 siNA stab0/0 PaI15
CUACGAGACCUCCCGG
1962

GAGGUCUCGUAG

327
CCCCGGGAGGUCUCGUAGACCGU
1417
35240
HCVa:327 siNA stab0/0 PaI16
CUACGAGACCUCCCGG
1963

GAGGUCUCGUAGTT

327
CCCCGGGAGGUCUCGUAGACCGU
1417
35241
HCVa:327 siNA stab0/0 PaI17
GGUCUACGAGACCUCCAGG
1964

UCUCGUAGACC

327
CCCCGGGAGGUCUCGUAGACCGU
1417
35242
HCVa:327 siNA stab0/0 PaI18
GGUCUACGAGACCUCCAGG
1965

UCUCGUAGACCTT

327
CCCCGGGAGGUCUCGUAGACCGU
1417
35243
HCVa:327 siNA stab0/0 PaI19
GGUCUACGAGACCUCGAGG
1966

UCUCGUAGACC

327
CCCCGGGAGGUCUCGUAGACCGU
1417
35244
HCVa:327 siNA stab0/0 PaI20
GGUCUACGAGACCUCGAGG
1967

UCUCGUAGACCTT

327
CCCCGGGAGGUCUCGUAGACCGU
1417
35245
HCVa:327 siNA stab0/0 PaI21
GGUCUACGAGACCUGCAGG
1968

UCUCGUAGACC

327
CCCCGGGAGGUCUCGUAGACCGU
1417
35246
HCVa:327 siNA stab0/0 PaI22
GGUCUACGAGACCUGCAGG
1969

UCUCGUAGACCTT

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
35247
HCVa:304 siNA stab0/0 PaI01
GACUAUCCCACGAACGCUC
1970

GAGCGUUCGUGGGAUAGUCTT

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
35248
HCVa:304 siNA stab0/0 PaI02
GACUAUCCCACGAACGCUC
1971

GAGCGUUCGUGGGAUAGUC

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
35249
HCVa:304 siNA stab0/0 PaI03
GACUAUCCCACGAACGCGU
1972

UCGUGGGAUAGUCTT

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
35250
HCVa:304 siNA stab0/0 PaI04
GACUAUCCCACGAACGCGU
1973

UCGUGGGAUAGUC

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
35251
HCVa:304 siNA stab0/0 PaI05
GACUAUCCCACGAACGUUC
1974

GUGGGAUAGUCTT

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
35252
HCVa:304 siNA stab0/0 PaI06
GACUAUCCCACGAACGUUC
1975

GUGGGAUAGUC

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
35253
HCVa:304 siNA stab0/0 PaI07
ACUAUCCCACGAACGUUC
1976

GUGGGAUAGUTT

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
35254
HCVa:304 siNA stab0/0 PaI08
ACUAUCCCACGAACGUUC
1977

GUGGGA

327
CCCCGGGAGGUCUCGUAGACCGU
1462
36414
HCVa bf-L-21 siNA stab00
CCGGGAGGUCUCGUAGACCTT L
1978

UCGCGAAAGGCCUUGUGGUACUG

[HCVa:327U21 sense o18S
GCGAAAGGCCUUGUGGUACTT

HCVa:282U21 sense]

327
CCCCGGGAGGUCUCGUAGACCGU
1463
36415
HCVa bf-L-22 siNA stab00
CCGGGAGGUCUCGUAGACCTT L
1979

UGAUAGGGUGCUUGCGAGUGCCC

[HCVa:327U21 sense o18S
AUAGGGUGCUUGCGAGUGCTT

HCVa:307U21 sense]

307
UGAUAGGGUGCUUGCGAGUGCCC
1464
36430
HCVa bf-L-20 siNA stab00
AUAGGGUGCUUGCGAGUGCTT L
1980

UCGCGAAAGGCCUUGUGGUACUG

[HCVa:307U21 sense o18S
GCGAAAGGCCUUGUGGUACTT

HCVa:282U21 sense]

307
UGAUAGGGUGCUUGCGAGUGCCC
1446
36438
HCVa:307U21 sense siNA stab00
AUAGGGUGCUUGCGAGUGCTT
1924

307
UGAUAGGGUGCUUGCGAGUGCCC
1446
36446
HCVa:325L21 antisense siNA
GCACUCGCAAGCACCCUAUTT
1929

(307C) stab00

327
CCCCGGGAGGUCUCGUAGACCGU
1417
36447
HCVa:345L21 antisense siNA
GGUCUACGAGACCUCCCGGTT
1732

(327C) stab00

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
36727
HCVa:304U21 sense siNA stab09
B CUGAUAGGGUGCUUGCGAGTT B
1981

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
36728
HCVa:322L21 antisense siNA
CUCGCAAGCACCCUAUCAGTsT
1982

(304C) stab10

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
37010
HCVa:304U21 sense siNA stab04
B cuGAuAGGGuGcuuGcGAGTT B
1983

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
37011
HCVa:322L21 antisense siNA
cucGcAAGcAcccuAucAGTsT
1984

(304C) stab05

307
CCCCGGGAGGUCUCGUAGACCGU
1463
37781
HCVa bf-L-22 siNA stab07
B ccGGGAGGucucGuAGAccTT L
1985

UGAUAGGGUGCUUGCGAGUGCCC

[HCVa:327U21 sense o18S
AuAGGGuGcuuGcGAGuGcTT B

HCVa:307U21 sense]

307
UGAUAGGGUGCUUGCGAGUGCCC
1446
37790
HCVa:325L21 antisense siNA

GCAcucGcAAGcAcccuAuTT
1986

(307C) stab26

327
CCCCGGGAGGUCUCGUAGACCGU
1417
37791
HCVa:345L21 antisense siNA

GGUcuAcGAGAccucccGGTT
1987

(327C) stab26

282
UCGCGAAAGGCCUUGUGGUACUG
1434
38279
HCVa:300L21 antisense siNA

GUAccAcAAGGccuuucGcTsT
1988

(282C) stab25

283
CGCGAAAGGCCUUGUGGUACUGC
1435
38280
HCVa:301L21 antisense siNA

AGUAccAcAAGGccuuucGTsT
1989

(283C) stab25

307
UGAUAGGGUGCUUGCGAGUGCCC
1446
38281
HCVa:325L21 antisense siNA

GCAcucGcAAGcAcccuAuTsT
1990

(307C) stab25

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
38283
HCVa:322L21 antisense siNA
CUCGcAAGcAcccuAucAGTT
1991

(304C) stab26

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
38284
HCVa:322L21 antisense siNA
CUCGcAAGcAcccuAucAGTTB
1992

(304C) stab27

282
UCGCGAAAGGCCUUGUGGUACUG
1434
38293
HCVa:300L21 antisense siNA

GuAccAcAAGGccuuucGcTT B
1993

(282C) stab19

282
UCGCGAAAGGCCUUGUGGUACUG
1434
38294
HCVa:300L21 antisense siNA

GUAccAcAAGGccuuucGcTT
1994

(282C) stab26

282
UCGCGAAAGGCCUUGUGGUACUG
1434
38295
HCVa:300L21 antisense siNA

GUAccAcAAGGccuuucGcTT B
1995

(282C) stab27

282
UCGCGAAAGGCCUUGUGGUACUG
1434
38296
HCVa:300L21 antisense siNA

GuAccAcAAGGccuuucGcTsT
1998

(282C) stab29

282
UCGCGAAAGGCCUUGUGGUACUG
1434
38297
HCVa:300L21 antisense siNA

GuAccAcAAGGccuuucGcTT
1997

(282C) stab30

282
UCGCGAAAGGCCUUGUGGUACUG
1434
38298
HCVa:300L21 antisense siNA

GuAccAcAAGGccuuucGcTT B
1998

(282C) stab31

282
UCGCGAAAGGCCUUGUGGUACUG
1434
38299
HCVa:300L21 antisense siNA

GuAccAcAAGGccuuucGcTT
1999

(282C) stab32

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
38300
HCVa:322L21 antisense siNA
cucGcAAGcAcccuAucAGTT
2000

(304C) stab32

327
CCCCGGGAGGUCUCGUAGACCGU
1417
38301
HCVa:345L21 antisense siNA

GGUcuAcGAGAccucccGGTT B
2001

(327C) stab27

327
CCCCGGGAGGUCUCGUAGACCGU
1417
38302
HCVa:345L21 antisense siNA

GGucuAcGAGAccucccGGTT
2002

(327C) stab30

327
CCCCGGGAGGUCUCGUAGACCGU
1417
38303
HCVa:345L21 antisense siNA

GGucuAcGAGAccucccGGTT B
2003

(327C) stab31

327
CCCCGGGAGGUCUCGUAGACCGU
1417
38304
HCVa:345L21 antisense siNA

GGucuAcGAGAccucccGGTT
2004

(327C) stab32

304
CCCCGGGAGGUCUCGUAGACCGU
1465
38310
HCV bf-L-23 siNA stab00
CCGGGAGGUCUCGUAGACCU L
2005

GCCUGAUAGGGUGCUUGCGAGUG

[HCV:327U21 sense o18S
CUGAUAGGGUGCUUGCGAGTT

HCV:304U21 sense]

282
GCCUGAUAGGGUGCUUGCGAGUG
1466
38311
HCV bf-L-24 siNA stab00
CUGAUAGGGUGCUUGCGAGTT L
2006

UCGCGAAAGGCCUUGUGGUACUG

[HCV:304U21 sense o18S
GCGAAAGGCCUUGUGGUACTT

HCV:282U21 sense]

304
CCCCGGGAGGUCUCGUAGACCGU
1465
38312
HCV bf-L-23 siNA stab07
B ccGGGAGGucucGuAGAccTT L
2007

GCCUGAUAGGGUGCUUGCGAGUG

[HCV:327U21 sense o18S
cuGAuAGGGuGcuuGcGAGTT B

HCV:304U21 sense]

282
CCCCGGGAGGUCUCGUAGACCGU
1462
38313
HCV bf-L-21 siNA stab07
B ccGGGAGGucucGuAGAccTT L
2008

UCGCGAAAGGCCUUGUGGUACUG

[HCVa:327U21 sense o18S

GcGAAAGGccuuGuGGuAcTT B

HCVa:282U21 sense]

282
GCCUGAUAGGGUGCUUGCGAGUG
1466
38314
HCV bf-L-24 siNA stab07
B cuGAuAGGGuGcuuGcGAGTT L
2009

UCGCGAAAGGCCUUGUGGUACUG

[HCV:304U21 sense o18S

GcGAAAGGccuuGuGGuAcTT B

HCV:282U21 sense]

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
38758
HCVa:322L21 siRNA (304C)
CUCGcAAGcAcccuAucAGUU
2010

stab26

327
CCCCGGGAGGUCUCGUAGACCGU
1417
38759
HCVa:345L21 siRNA (327C)
GGUcuAcGAGAccucccGGUU
2011

stab26

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
46211
HCVa:322L21 siRNA (304C)
CUCGcAAGcAcccuAucAGGC
2012

stab26

327
CCCCGGGAGGUCUCGUAGACCGU
1417
46214
HCVa:345L21 siRNA (327C)
GGUcuAcGAGAccucccGGGC
2013

stab26

292
CUGAUAGGGCGCUUGCGAG
2014
46672
HCV-JFH-1::(292)U23 siRNA
B cuGAuAGGGcGcuuGcGAGTT B
2034

stab07 active

292
CUGAUAGGGCGCUUGCGAG
2014
46673
HCV-JFH-1::(292)L21 siRNA
CUCGcAAGcGcccuAucAGTsT
2035

stab25 active

46746
HCVa:(293)U23 siRNA
B cuGAuAcccAcGAuGcGAGTT B 2036

stab07 control01

46747
HCVa:(316)U23 siRNA
B ccGGGAccAGAGcuAGAccTT B
2037

stab07 control01

46748
HCVa:(271)U23 siRNA
B GcGAAAccGGAAcuGGuAcTT B
2038

stab07 control01

46751
HCVa:(293)L21 siRNA
CUCGcAucGuGGGuAucAGTsT
2039

stab25 control01

46752
HCVa:(316)L21 siRNA
GGUcuAGcucuGGucccGGTsT
2040

stab25 control01

46753
HCVa:(271)L21 siRNA
GUAccAGuuccGGuuucGcTsT
2041

stab25 control01

46755
HCVa:(316)L21 siRNA
GGucuAGcucuGGucccGGTsT
2042

stab29 control01

46756
HCVa:(271)U21 siRNA
GuAccAGuuccGGuuucGcTsT
2043

stab29 control01

46757
HCVa:(293)L21 siRNA
cucGcAucGuGGGuAucAGTsT
2044

stab08 control01

293
GCCUGAUAGGGUGCUUGCGAGUG
1444
47023
HCVa:(293)L21 siRNA
GACuAucccAcGAAcGcucTsT
2045

stab25 invert

293
GCCUGAUAGGGUGCUUGCGAGUG
1444
47024
HCVa:(293)L21 siRNA

GAcuAucccAcGAAcGCUCTsT
2046

stab25 invert

293
GCCUGAUAGGGUGCUUGCGAGUG
1444
47025
HCVa:(293)U22 siRNA
B cuGAuAGGGuGcuuGcGAGTT
2047

stab07 3′ n-1

293
GCCUGAUAGGGUGCUUGCGAGUG
1444
47026
HCVa:(293)U21 siRNA
B cuGAuAGGGuGcuuGcGAGT
2048

stab07 3′ n-2

293
GCCUGAUAGGGUGCUUGCGAGUG
1444
47027
HCVa:(293)U22 siRNA
cuGAuAGGGuGcuuGcGAGTT B
2049

stab07 5′ n-1

293
GCCUGAUAGGGUGCUUGCGAGUG
1444
47028
HCVa:(293)U21 siRNA
uGAuAGGGuGcuuGcGAGTT B
2050

stab07 5′-2

293
GCCUGAUAGGGUGCUUGCGAGUG
1444
47029
HCVa:(293)U20 siRNA

GAuAGGGuGcuuGcGAGTT B
2051

stab07 5′-3

293
GCCUGAUAGGGUGCUUGCGAGUG
1444
47030
HCVa:(293)L20 siRNA
cucGcAAGcAcccuAucAGT
2052

stab08 3′-1

293
GCCUGAUAGGGUGCUUGCGAGUG
1444
47031
HCVa:(293)L19 siRNA
cucGcAAGcAcccuAucAG
2053

stab08 3′-2

293
GCCUGAUAGGGUGCUUGCGAGUG
1444
47032
HCVa:(293)L18 siRNA
cucGcAAGcAcccuAucA
2054

3′-3 stab08

293
GCCUGAUAGGGUGCUUGCGAGUG
1444
47033
HCVa:(293)L20 siRNA
ucGcAAGcAcccuAucAGTsT
2055

5′-1 stab08

293
GCCUGAUAGGGUGCUUGCGAGUG
1444
47034
HCVa:(293)L19 siRNA
cGcAAGcAcccuAucAGTsT
2056

5′-2 stab08

293
GCCUGAUAGGGUGCUUGCGAGUG
1444
47035
HCVa:(293)L18 siRNA

GcAAGcAcccuAucAGTsT
2057

5′-3 stab08

293
GCCUGAUAGGGUGCUUGCGAGUG
1444
47036
HCVa:(293)L20 siRNA
CUCGcAAGcAcccuAucAGT
2058

3′-1 stab25

293
UGAUAGGGUGCUUGCGAG
2015
47038
HCVa:(293)L18 siRNA
CUCGcAAGcAcccuAucA
2059

3′ n-3 stab25

293
GCCUGAUAGGGUGCUUGCGAGUG
1444
47039
HCVa:(293)L20 siRNA
UCGcAAGcAcccuAucAGTsT
2060

5′-1 stab25

293
AACUGAUAGGGUGCUUGCG
2016
47040
HCVa:(293)L19 siRNA
CGcAAGcAcccuAucAGTsT
2061

5′-2 stab25

73
GUCUUCACGCAGAAAGCGUCUAG
2017
47208
HCVa:(73)U23 siRNA
B cuucAcGcAGAAAGcGucuTT B
2062

stab07

86
AAGCGUCUAGCCAUGGCGUUAGU
2018
47209
HCVa:(86)U23 siRNA
B GcGucuAGccAuGGcGuuATT B
2063

stab07

172
GUGAGUACACCGGAAUUGCCAGG
2019
47212
HCVa:(172)U23 siRNA
B GAGuAcAccGGAAuuGccATT B
2064

stab07

195
ACGACCGGGUCCUUUCUUGGAUC
2020
47213
HCVa:(195)U23 siRNA
B GAccGGGuccuuucuuGGATT B
2065

stab07

220
CCCGCUCAAUGCCUGGAGAUUUG
2021
47214
HCVa:(220)U23 siRNA
B cGcucAAuGccuGGAGAuuTT B
2066

stab07

250
CCCCGCGAGACUGCUAGCCGAGU
2022
47215
HCVa:(250)U23 siRNA
B ccGcGAGAcuGcuAGccGATT B
2067

stab07

252
CCGCGAGACUGCUAGCCGAGUAG
2023
47216
HCVa:(252)U23 siRNA
B GcGAGAcuGcuAGccGAGuTT B
2068

stab07

267
CCGAGUAGUGUUGGGUCGCGAAA
2024
47217
HCVa:(267)U23 siRNA
B GAGuAGuGuuGGGucGcGATT B
2069

stab07

274
GUGUUGGGUCGCGAAAGGCCUUG
2025
47218
HCVa:(274)U23 siRNA
B GuuGGGucGcGAAAGGccuTT B
2070

stab07

277
UUGGGUCGCGAAAGGCCUUGUGG
2026
47219
HCVa:(277)U23 siRNA
B GGGucGcGAAAGGccuuGuTT B
2071

stab07

73
GUCUUCACGCAGAAAGCGUCUAG
2017
47222
HCVa:(73)L21 siRNA
AGAcGcuuucuGcGuGAAGTsT
2072

stab25

86
AAGCGUCUAGCCAUGGCGUUAGU
2018
47223
HCVa:(86)L21 siRNA
UAAcGccAuGGcuAGAcGcTsT
2073

stab25

140
UCCCGGGAGAGCCAUAGUGGUCU
1420
47224
HCVa:(140)L21 siRNA
ACCAcuAuGGcucucccGGTsT
2074

stab25

157
UGGUCUGCGGAACCGGUGAGUAC
1426
47225
HCVa:(157)L21 siRNA
ACUcAccGGuuccGcAGAcTsT
2075

stab25

172
GUGAGUACACCGGAAUUGCCAGG
2019
47226
HCVa:(172)L21 siRNA
UGGcAAuuccGGuGuAcucTsT
2076

stab25

195
ACGACCGGGUCCUUUCUUGGAUC
2020
47227
HCVa:(195)L21 siRNA
UCCAAGAAAGGAcccGGucTsT
2077

stab25

220
CCCGCUCAAUGCCUGGAGAUUUG
2021
47228
HCVa:(220)L21 siRNA
AAUcuccAGGcAuuGAGcGTsT
2078

stab25

250
CCCCGCGAGACUGCUAGCCGAGU
2022
47229
HCVa:(250)L21 siRNA
UCGGcuAGcAGucucGcGGTsT
2079

stab25

252
CCGCGAGACUGCUAGCCGAGUAG
2023
47230
HCVa:(252)L21 siRNA
ACUcGGcuAGcAGucucGcTsT
2080

stab25

267
CCGAGUAGUGUUGGGUCGCGAAA
2024
47231
HCVa:(267)L21 siRNA
UCGcGAcccAAcAcuAcucTsT
2081

stab25

274
GUGUUGGGUCGCGAAAGGCCUUG
2025
47232
HCVa:(274)L21 siRNA
AGGccuuucGcGAcccAAcTsT
2082

stab25

277
UUGGGUCGCGAAAGGCCUUGUGG
2026
47233
HCVa:(277)L21 siRNA
ACAAGGccuuucGcGAcccTsT
2083

stab25

303
UGCCUGAUAGGGUGCUUGCGAGU
1410
47234
HCVa:(303)L21 siRNA
UCGcAAGcAcccuAucAGGTsT
2084

stab25

329
CCGGGAGGUCUCGUAGACCGUGC
1419
47235
HCVa:(329)L21 siRNA
ACGGucuAcGAGAccucccTsT
2085

stab25

47354
HCVa:(293)U23 siRNA
B GAGcGuucGuGGGAuAGucTT B
2086

stab04 invert

47527
HCVa:(316)L21 siRNA

GGcccuccAGAGcAucuGGTsT
2087

stab29A invert

GAAAGGAUUUGGCUACAAA
2027
47654
HCVa/PPIB:(300)U25
UUUGUAGCCAAAUCCUUUCUGGUAC
2088

bifunctional stab00 active

AAGGAUUUGGCUACAAAAA
2028
47655
HCVa/PPIB:( )U27 bifunctional
UUUUUGUAGCCAAAUCCUUUGUGGUAC
2089

stab00 active

AAGGACUUCAUGAUCCAGG
2029
47663
HCVa/PPIB:( )U27 bifunctional
CCUGGAUCAUGAAGUCCUUUGUGGUAC
2090

stab00 active

GAGAGCACCAAGACAGACA
2030
47664
HCVa/PPIB:( )U27 bifunctional
UGUCUGUCUUGGUGCUCUCCUUGCGAG
2091

stab00 active

AAAGACUGUUCCAAAAACA
2031
47665
HCVa/PPIB:( )U24 bifunctional
UGUUUUUGGAACAGUCUUUGCGAG
2092

stab00 active

GAGAGCACCAAGACAGACA
2030
47666
HCVa/PPIB:( )U26 bifunctional
UGUCUGUCUUGGUGCUCUCGUAGACC
2093

stab00 active

AGAUGGCACAGGAGGAAAG
2032
47667
HCVa/PPIB:( )U27 bifunctional
CUUUCCUCCUGUGCCAUCUCGUAGACC
2094

stab00 active

GCGAAAGGCCUUGUGGUAC
8
47668
HCVa/PPIB:( )L25 bifunctional
GUACCACAAGGCCUUUCGCUACAAA
2095

stab00 active

GCGAAAGGCCUUGUGGUAC
8
47669
HCVa/PPIB:( )L27 bifunctional
GUACCACAAGGCCUUUCGCUACAAAAA
2096

stab00 active

GCGAAAGGCCUUGUGGUAC
8
47670
HCVa/PPIB:( )L27 bifunctional
GUACCACAAGGCCUUUCGCGAUCCAGG
2097

stab00 active

CUGAUAGGGUGCUUGCGAG
2033
47671
HCVa/PPIB:( )L27 bifunctional
CUCGCAAGCACCCUAUCAGGACAGACA
2098

stab00 active

CUGAUAGGGUGCUUGCGAG
2033
47672
HCVa/PPIB:( )L24 bifunctional
CUCGCAAGCACCCUAUCAGAAACA
2099

stab00 active

CCGGGAGGUCUCGUAGACC
38
47673
HCVa/PPIB:( )L26 bifunctional
GGUCUACGAGACCUCCCGGACAGACA
2100

stab00 active

CCGGGAGGUCUCGUAGACC
38
47674
HCVa/PPIB:( )L27 bifunctional
GGUCUACGAGACCUCCCGGGAGGAAAG
2101

stab00 active

293
GCCUGAUAGGGUGCUUGCGAGUG
1444
47677
HCVa:(293)L21 siRNA stab36
CUCGcAAGcAcccuAucAGGC
2102

active

316
CCCCGGGAGGUCUCGUAGACCGU
1417
38756
HCVa:334L21 siRNA (316C) stab36
GGUcuAcGAGAccucccGGGG
2103

282
UCGCGAAAGGCCUUGUGGUACUG
1434
47786
HCV:(282)U23 siRNA stab07B
B GcGAAAGGCCuuGuGGuAcTT B
2104

active

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
47787
HCV:(304)U23 siRNA stab07B
B cuGAuAGGGUGcuuGcGAGTT B
2105

active

327
CCCCGGGAGGUCUCGUAGACCGU
1417
47788
HCV:(327)U23 siRNA stab07B
B ccGGGAGGUCucGuAGAccTT B
2106

active

316
CCCCGGGAGGUCUCGUAGACCGU
1417
47791
HCVa:(316)L21 siRNA stab35
GGUcuAGcucuGGucccGGUU
2107

active

282
UCGCGAAAGGCCUUGUGGUACUG
1434
47855
HCVa:(282)U21 siRNA stab07C
B GcGAAAGGCCuuGuGGuAc B
2108

active

293
GCCUGAUAGGGUGCUUGCGAGUG
1444
47856
HCVa:(293)U21 siRNA stab07C
B cuGAuAGGGUGcuuGcGAG B
2109

active

316
CCCCGGGAGGUCUCGUAGACCGU
1417
47857
HCVa:(316)U21 siRNA stab07C
B ccGGGAGGUCucGuAGAcc B
2110

active

282
UCGCGAAAGGCCUUGUGGUACUG
1434
47860
HCVa:(282)U20 siRNA stab07D
B GcGAAAGGCCuuGuGGuAc
2111

active

304
GCCUGAUAGGGUGCUUGCGAGUG
1444
47861
HCV:(304)U20 siRNA stab07D
B cuGAuAGGGUGcuuGcGAG
2112

active

316
CCCCGGGAGGUCUCGUAGACCGU
1417
47862
HCVa:(316)U20 siRNA stab07
B ccGGGAGGUCucGuAGAcc
2113

3′-3

73
GUCUUCACGCAGAAAGCGUCUAG
2017
47878
HCVa:(73)U23 siRNA stab07B
B cuucAcGcAGAAAGcGucuTT B
2114

active

86
AAGCGUCUAGCCAUGGCGUUAGU
2018
47879
HCVa:(86)U23 siRNA stab07B
B GcGucuAGCCAuGGcGuuATT B
2115

active

140
UCCCGGGAGAGCCAUAGUGGUCU
1420
47880
HCVa:(140)U23 siRNA stab07B
B ccGGGAGAGCcAuAGuGGuTT B
2116

active

157
UGGUCUGCGGAACCGGUGAGUAC
1426
47881
HCVa:(157)U23 siRNA stab07B
B GucuGcGGAAccGGuGAGuTT B
2117

active

303
UGCCUGAUAGGGUGCUUGCGAGU
1410
47882
HCVa:(303)U23 siRNA stab07B
B ccuGAuAGGGuGcuuGcGATT B
2118

active

329
CCGGGAGGUCUCGUAGACCGUGC
1419
47883
HCVa:(329)U23 siRNA stab07B
B GGGAGGucUCGuAGAccGuTT B
2119

active

73
GUCUUCACGCAGAAAGCGUCUAG
2017
47884
HCVa:(73)U23 siRNA stab09
B CUUCACGCAGAAAGCGUCUTT B
2120

active

86
AAGCGUCUAGCCAUGGCGUUAGU
2018
47885
HCVa:(86)U23 siRNA stab09
B GCGUCUAGCCAUGGCGUUATT B
2121

active

140
UCCCGGGAGAGCCAUAGUGGUCU
1420
47886
HCVa:(140)U23 siRNA stab09
B CCGGGAGAGCCAUAGUGGUTT B
2122

active

157
UGGUCUGCGGAACCGGUGAGUAC
1426
47887
HCVa:(157)U23 siRNA stab09
B GUCUGCGGAACCGGUGAGUTT B
2123

active

303
UGCCUGAUAGGGUGCUUGCGAGU
1410
47888
HCVa:(303)U23 siRNA stab09
B CCUGAUAGGGUGCUUGCGATT B
2124

active

329
CCGGGAGGUCUCGUAGACCGUGC
1419
47889
HCVa:(329)U23 siRNA stab09
B GGGAGGUCUCGUAGACCGUTT B
2125

active

73
GUCUUCACGCAGAAAGCGUCUAG
2017
47890
HCVa:(73)L21 siRNA stab10
AGACGCUUUCUGCGUGAAGTsT
2126

active

86
AAGCGUCUAGCCAUGGCGUUAGU
2018
47891
HCVa:(86)L21 siRNA stab10
UAACGCCAUGGCUAGACGCTsT
2127

active

140
UCCCGGGAGAGCCAUAGUGGUCU
1420
47892
HCVa:(140)L21 siRNA stab10
ACCACUAUGGCUCUCCCGGTsT
2128

active

157
UGGUCUGCGGAACCGGUGAGUAC
1426
47893
HCVa:(157)L21 siRNA stab10
ACUCACCGGUUCCGCAGACTsT
2129

active

303
UGCCUGAUAGGGUGCUUGCGAGU
1410
47894
HCVa:(303)L21 siRNA stab10
UCGCAAGCACCCUAUCAGGTsT
2130

active

329
CCGGGAGGUCUCGUAGACCGUGC
1419
47895
HCVa:(329)L21 siRNA stab10
ACGGUCUACGAGACCUCCCTsT
2131

active

73
GUCUUCACGCAGAAAGCGUCUAG
2017
47896
HCVa:(73)L21 siRNA stab25
AGAcGcuuucuGcGuGAAGTsT
2132

active

86
AAGCGUCUAGCCAUGGCGUUAGU
2018
47897
HCVa:(86)L21 siRNA stab25
uAAcGccAuGGcuAGAcGcTsT
2133

active

140
UCCCGGGAGAGCCAUAGUGGUCU
1420
47898
HCVa:(140)L21 siRNA stab25
AccAcuAuGGcucucccGGTsT
2134

active

157
UGGUCUGCGGAACCGGUGAGUAC
1426
47899
HCVa:(157)L21 siRNA stab25
AcucAccGGuuccGcAGAcTsT
2135

active

303
UGCCUGAUAGGGUGCUUGCGAGU
1410
47900
HCVa:(303)L21 siRNA stab25
ucGcAAGcAcccuAucAGGTsT
2136

active

329
CCGGGAGGUCUCGUAGACCGUGC
1419
47901
HCVa:(329)L21 siRNA stab25
AcGGucuAcGAGAccucccTsT
2137

active

316
CCCCGGGAGGUCUCGUAGACCGU
1417
50721
HCVa:(316)U23 siRNA
B ccGGGAGGucucGuAGAcBTT B
2138

stab07iB19 active

293
GCCUGAUAGGGUGCUUGCGAGUG
1444
50722
HCVa:(293)U23 siRNA
B cuGAuAGGGuGcuuGcGABTT B
2139

stab07iB19 active

316
CCCCGGGAGGUCUCGUAGACCGU
1417
50723
HCVa:(316)L21 siRNA
GGUcuAcGAGAccucccGBGG
2140

stab36iB19 active

293
GCCUGAUAGGGUGCUUGCGAGUG
1444
50724
HCVa:(293)L21 siRNA
CUCGcAAGcAcccuAucABGC
2141

stab36iB19 active

316
CCCCGGGAGGUCUCGUAGACCGU
1417
50725
HCVa:(316)U23 siRNA
B BcGGGAGGucucGuAGAccTT B
2142

stab07iB1 active

293
GCCUGAUAGGGUGCUUGCGAGUG
1444
50726
HCVa:(293)U23 siRNA
B BuGAuAGGGuGcuuGcGAGTT B
2143

stab07iB1 active

316
CCCCGGGAGGUCUCGUAGACCGU
1417
50727
HCVa:(316)L21 siRNA
B GUcuAcGAGAccucccGGGG
2144

stab36iB1 active

293
GCCUGAUAGGGUGCUUGCGAGUG
1444
50728
HCVa:(293)L21 siRNA
B UCGcAAGcAcccuAucAGGC
2145

stab36iB1 active

316
CCCCGGGAGGUCUCGUAGACCGU
1417
50729
HCVa:(316)U23 siRNA
B ccGGGAGGBcucGuAGAccTT B
2146

stab07iB9 active

293
GCCUGAUAGGGUGCUUGCGAGUG
1444
50730
HCVa:(293)U23 siRNA
B cuGAuAGGBuGcuuGcGAGTT B
2147

stab07iB9 active

316
CCCCGGGAGGUCUCGUAGACCGU
1417
50731
HCVa:(316)L21 siRNA
GGUcuAcGBGAccucccGGGG
2148

stab36iB9 active

293
GCCUGAUAGGGUGCUUGCGAGUG
1444
50732
HCVa:(293)L21 siRNA
CUCGcAAGBAcccuAucAGGC
2149

stab36iB9 active

Uppercase = ribonucleotide

u = 2′-deoxy-2′-fluoro uridine

c = 2′-deoxy-2′-fluoro cytidine

g = 2′-deoxy-2′-fluoro guanosine

a = 2′-deoxy-2′-fluoro adenosine

T = thymidine

B = inverted deoxy abasic

s = phosphorothioate linkage

A = deoxy Adenosine

G = deoxy Guanosine

U = deoxy Uridine

C = deoxy Cytidine

G = 2′-O-methyl Guanosine

A = 2′-O-methyl Adenosine

C = 2′-O-methyl Cytidine

U = 2′-O-methyl Uridine

L = hegS = hexethelyne glycol spacer; spacer-18 (Glen Research 10-1918-xx)

p = terminal phosphate

TABLE IV

Non-limiting examples of Stabilization Chemistries for chemically modified

siNA constructs

Chemistry
pyrimidine
Purine
cap
p = S
Strand

“Stab 00”
Ribo
Ribo
TT at 3′-ends

S/AS

“Stab 1”
Ribo
Ribo
—
5 at 5′-end
S/AS

1 at 3′-end

“Stab 2”
Ribo
Ribo
—
All linkages
Usually AS

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

4 at 3′-end

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

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

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

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

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

Methyl

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

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

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

“Stab 12”
2′-fluoro
LNA
5′ and 3′-ends

Usually S

“Stab 13”
2′-fluoro
LNA

1 at 3′-end
Usually AS

“Stab 14”
2′-fluoro
2′-deoxy

2 at 5′-end
Usually AS

1 at 3′-end

“Stab 15”
2′-deoxy
2′-deoxy

2 at 5′-end
Usually AS

1 at 3′-end

“Stab 16”
Ribo
2′-O-
5′ and 3′-

Usually S

Methyl
ends

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

Usually S

Methyl
ends

“Stab 18”
2′-fluoro
2′-O-
5′ and 3′-

Usually S

Methyl
ends

“Stab 19”
2′-fluoro
2′-O-
3′-end

S/AS

Methyl

“Stab 20”
2′-fluoro
2′-deoxy
3′-end

Usually AS

“Stab 21”
2′-fluoro
Ribo
3′-end

Usually AS

“Stab 22”
Ribo
Ribo
3′-end

Usually AS

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

Usually S

ends

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

Methyl*

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

Methyl*

“Stab 26”
2′-fluoro*
2′-O-
—

S/AS

Methyl*

“Stab 27”
2′-fluoro*
2′-O-
3′-end

S/AS

Methyl*

“Stab 28”
2′-fluoro*
2′-O-
3′-end

S/AS

Methyl*

“Stab 29”
2′-fluoro*
2′-O-

1 at 3′-end
S/AS

Methyl*

“Stab 30”
2′-fluoro*
2′-O-

S/AS

Methyl*

“Stab 31”
2′-fluoro*
2′-O-
3′-end

S/AS

Methyl*

“Stab 32”
2′-fluoro
2′-O-

S/AS

Methyl

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

ends

“Stab 34”
2′-fluoro
2′-O-
5′ and 3′-

Usually S

Methyl*
ends

“Stab 35”
2′-fluoro**
2′-O-

Usually AS

Methyl**

“Stab 36”
2′-fluoro**
2′-O-

Usually AS

Methyl**

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

4 at 3′-end

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

ends

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

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

ends

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

Methyl

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

“Stab 12F”
2′-OCF3
LNA
5′ and 3′-

Usually S

ends

“Stab 13F”
2′-OCF3
LNA

1 at 3′-end
Usually AS

“Stab 14F”
2′-OCF3
2′-deoxy

2 at 5′-end
Usually AS

1 at 3′-end

“Stab 15F”
2′-OCF3
2′-deoxy

2 at 5′-end
Usually AS

1 at 3′-end

“Stab 18F”
2′-OCF3
2′-O-
5′ and 3′-

Usually S

Methyl
ends

“Stab 19F”
2′-OCF3
2′-O-
3′-end

S/AS

Methyl

“Stab 20F”
2′-OCF3
2′-deoxy
3′-end

Usually AS

“Stab 21F”
2′-OCF3
Ribo
3′-end

Usually AS

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

Usually S

ends

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

Methyl*

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

Methyl*

“Stab 26F”
2′-OCF3*
2′-O-
—

S/AS

Methyl*

“Stab 27F”
2′-OCF3*
2′-O-
3′-end

S/AS

Methyl*

“Stab 28F”
2′-OCF3*
2′-O-
3′-end

S/AS

Methyl*

“Stab 29F”
2′-OCF3*
2′-O-

1 at 3′-end
S/AS

Methyl*

“Stab 30F”
2′-OCF3*
2′-O-

S/AS

Methyl*

“Stab 31F”
2′-OCF3*
2′-O-
3′-end

S/AS

Methyl*

“Stab 32F”
2′-OCF3
2′-O-

S/AS

Methyl

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

ends

“Stab 34F”
2′-OCF3
2′-O-
5′ and 3′-

Usually S

Methyl*
ends

“Stab 35F”
2′-OCF3*†
2′-O-

Usually AS

Methyl*†

“Stab 36F”
2′-OCF3*†
2′-O-

Usually AS

Methyl*†

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

All Stab 00-34 chemistries can comprise 3′-terminal thymidine (TT) residues

All Stab 00-34 chemistries typically comprise about 21 nucleotides, but can vary as described herein.

All Stab 00-36 chemistries can also include a single ribonucleotide in the sense or passenger strand at the 11^thbase paired position of the double stranded nucleic acid duplex as determined from the 5′-end of the antisense or guide strand (see FIG. 6C)

S = sense strand

AS = antisense strand

*Stab 23 has a single ribonucleotide adjacent to 3′-CAP

*Stab 24 and Stab 28 have a single ribonucleotide at 5′-terminus

*Stab 25, Stab 26, Stab 27, Stab 35 and Stab 36 have three ribonucleotides at 5′-terminus

*Stab 29, Stab 30, Stab 31, Stab 33, and Stab 34 any purine at first three nucleotide positions from 5′-terminus are ribonucleotides

p = phosphorothioate linkage

†Stab 35 has 2′-O-methyl U at 3′-overhangs and three ribonucleotides at 5′-terminus

†Stab 36 has 2′-O-methyl overhangs that are complementary to the target sequence (naturually occurring overhangs) and three ribonucleotides at 5′-terminus

TABLE V

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

A. 2.5 μmol Synthesis Cycle ABI 394 Instrument

Phosphoramidites
6.5
163 μL
45 sec
2.5 min
7.5 min

S-Ethyl Tetrazole
23.8
238 μL
45 sec
2.5 min
7.5 min

Acetic Anhydride
100
233 μL
5 sec
5 sec
5 sec

N-Methyl
186
233 μL
5 sec
5 sec
5 sec

Imidazole

TCA
176
2.3 mL
21 sec
21 sec
21 sec

Iodine
11.2
1.7 mL
45 sec
45 sec
45 sec

Beaucage
12.9
645 μL
100 sec
300 sec
300 sec

Acetonitrile
NA
6.67 mL
NA
NA
NA

B. 0.2 μmol Synthesis Cycle ABI 394 Instrument

Phosphoramidites
15
31 μL
45 sec
233 sec
465 sec

S-Ethyl Tetrazole
38.7
31 μL
45 sec
233 min
465 sec

Acetic Anhydride
655
124 μL
5 sec
5 sec
5 sec

N-Methyl
1245
124 μL
5 sec
5 sec
5 sec

Imidazole

TCA
700
732 μL
10 sec
10 sec
10 sec

Iodine
20.6
244 μL
15 sec
15 sec
15 sec

Beaucage
7.7
232 μL
100 sec
300 sec
300 sec

Acetonitrile
NA
2.64 mL
NA
NA
NA

C. 0.2 μmol Synthesis Cycle 96 well Instrument

Equivalents: DNA/
Amount: DNA/2′-O-

Wait Time* 2′-O-

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

Phosphoramidites
22/33/66
40/60/120 μL
60 sec
180 sec
360 sec

S-Ethyl Tetrazole
70/105/210
40/60/120 μL
60 sec
180 min
360 sec

Acetic Anhydride
265/265/265
50/50/50 μL
10 sec
10 sec
10 sec

N-Methyl
502/502/502
50/50/50 μL
10 sec
10 sec
10 sec

Imidazole

TCA
238/475/475
250/500/500 μL
15 sec
15 sec
15 sec

Iodine
6.8/6.8/6.8
80/80/80 μL
30 sec
30 sec
30 sec

Beaucage
34/51/51
80/120/120
100 sec
200 sec
200 sec

Acetonitrile
NA
1150/1150/1150 μL
NA
NA
NA

Wait time does not include contact time during delivery.

Tandem synthesis utilizes double coupling of linker molecule

TABLE VI

Lipid Nanoparticle (LNP) Formulations

Formulation

#
Composition
Molar Ratio

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

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

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

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

Cholesterol

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

DSPC/Chol/PEG-n-DMG

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

2KPEG-Chol

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

2KPEG-DMG

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

2KPEG-DMG

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

2KPEG-Chol

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

DMG/Linoleyl alcohol

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

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

ratio of 5

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

DMG

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

N/P ratio of 3

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

N/P ratio of 4

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

ratio of 3

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

Cholesterol

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

Cholesterol, N/P ratio of 5

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

Cholesterol

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

cholesterol/Linoleyl alcohol

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

ratio of 2

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

ratio of 3

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

ratio of 1.5

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

ratio of 2

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

Chol, N/P ratio of 2

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

N/P ratio of 1.5

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

N/P ratio of 1.5

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

ratio of 1.5

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

ratio of 1.5

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

N/P ratio of 2

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

N/P ratio of 2

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

N/Pratio of 2

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

ratio of 2

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

CLinDMA Structure

pCLinDMA Structure

eCLinDMA Structure

PEG-n-DMG Structure

DMOBA Structure

DMLBA Structure

DOBA Structure

DSPC

Cholesterol

2KPEG-Cholesterol

2KPEG-DMG

TABLE VII

Description of pattern
Pattern #
Score

G or C at position 1
1
5

A or U at position 19
2
10

A/U rich between position 15-19
3
10

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

G/C rich between position 1-5
5
10

A or U at position 18
6
5

A or U at position 10
7
10

G at position 13 (not preferred)
8
−3

A at position 13
9
3

G at position 9 (not preferred)
10
−3

A at position 9
11
3

A or U at position 14
12
10

Sirna algorithm describing patterns with their relative score for predicting hyper-active siNAs. All the positions given are for the sense strand of 19-mer siNA.

	Number	Date	Country
Parent	11311826	Dec 2005	US
Child	11510872		US
Parent	10942560	Sep 2004	US
Child	11311826		US
Parent	10667271	Sep 2003	US
Child	10942560		US
Parent	PCT/US03/05043	Feb 2003	US
Child	10667271		US
Parent	PCT/US02/09187	Mar 2002	US
Child	PCT/US03/05043		US

RNA inteference mediated inhibition of hepatitis C virus (HVC) gene expression using short interfering nucleic acid (siNA)

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

US Classifications

International Classifications

Abstract

Description

Claims

Parent Case Info

Provisional Applications (1)

Continuation in Parts (5)