Methods and compositions for the specific inhibition of glycolate oxidase (HAO1) by double-stranded RNA

[0001] The present application claims the benefit under 35 U.S.C. §120 of U.S. application Ser. No. 14/583,200, entitled “Methods and Compositions for the Specific Inhibition of Glycolate Oxidase (HAO1) by Double-Stranded RNA” filed on Dec. 26, 2014, which claims priority to, and the benefit under 35 U.S.C. §119(e) of U.S. provisional patent application No. 61/921,181, entitled “Methods and Compositions for the Specific Inhibition of Glycolate Oxidase (HAO1) by Double-Stranded RNA,” filed Dec. 27, 2013, and of U.S. provisional application No. 61/937,838, entitled “Methods and Compositions for the Specific Inhibition of Glycolate Oxidase (HAO1) by Double-Stranded RNA,” filed Feb. 10, 2014. The entire contents of the aforementioned patent applications are incorporated herein by this reference.

[0002] 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 Glycolate Oxidase (HAO1) gene expression and/or activity.

[0003] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is entitled “301937_94015 WO_seq_listing”, was created on Dec. 23, 2014 and is 2.87 MB in size. The information in the electronic format of the Sequence Listing is part of the present application and is incorporated herein by reference in its entirety.

[0004] Primary hyperoxaluria Type 1 (“PH1”) is a rare autosomal recessive inborn error of glyoxylate metabolism, caused by a deficiency of the liver-specific enzyme alanine:glyoxylate aminotransferase. The disorder results in overproduction and excessive urinary excretion of oxalate, causing recurrent urolithiasis and nephrocalcinosis. As glomerular filtration rate declines due to progressive renal involvement, oxalate accumulates leading to systemic oxalosis. The diagnosis is based on clinical and sonographic findings, urine oxalate assessment, enzymology and/or DNA analysis. While early conservative treatment has aimed to maintain renal function, in chronic kidney disease Stages 4 and 5, the best outcomes to date have been achieved with combined liver-kidney transplantation (Cochat et al. Nephrol Dial Transplant 27: 1729-36).

[0005] PH1 is the most common form of primary hyperoxaluria and has an estimated prevalence of 1 to 3 cases per 1 million population and an incidence rate of approximately 1 case per 120,000 live births per year in Europe (Cochat et al. Nephrol Dial Transplant 10 (Suppl 8): 3-7; van Woerden et al. Nephrol Dial Transplant 18: 273-9). It accounts for 1 to 2% of cases of pediatric end-stage renal disease (ESRD), according to registries from Europe, the United States, and Japan (Harambat et al. Clin J Am Soc Nephrol 7: 458-65), but it appears to be more prevalent in countries in which consanguineous marriages are common (with a prevalence of 10% or higher in some North African and Middle Eastern nations; Kamoun and Lakhoua Pediatr Nephrol 10: 479-82; see Cochat and Rumsby N Engl J Med 369(7):649-58).

[0006] Glycolate oxidase (the product of the HAO1, for “hydroxyacid oxidase 1”, gene) is the enzyme responsible for converting glycolate to glyoxylate in the mitochondrial/peroxisomal glycine metabolism pathway in the liver and pancreas. While glycolate is a harmless intermediate of the glycine metabolism pathway, accumulation of glyoxylate (via, e.g., AGT1 mutation) drives oxalate accumulation, which ultimately results in the PH1 disease.

[0007] Double-stranded RNA (dsRNA) agents possessing strand lengths of 25 to 35 nucleotides have been described as effective inhibitors of target gene expression in mammalian cells (Rossi et al., U.S. Pat. No. 8,084,599 and U.S. Patent Application No. 2005/0277610). dsRNA agents of such length are believed to be processed by the Dicer enzyme of the RNA interference (RNAi) pathway, leading such agents to be termed “Dicer substrate siRNA” (“DsiRNA”) agents. Additional modified structures of DsiRNA agents were previously described (Rossi et al., U.S. Patent Application No. 2007/0265220). Effective extended forms of Dicer substrates have also recently been described (Brown, U.S. Pat. No. 8,349,809 and U.S. Pat. No. 8,513,207).

[0008] The present invention is based, at least in part, upon the identification of HAO1 as an attractive target for dsRNA-based knockdown therapies. In particular, provided herein are nucleic acid agents that target and reduce expression of HAO1. Such compositions contain nucleic acids such as double stranded RNA (“dsRNA”), and methods for preparing them. The nucleic acids of the invention are capable of reducing the expression of a target Glycolate Oxidase gene in a cell, either in vitro or in a mammalian subject.

[0009] In one aspect, the invention provides a nucleic acid possessing an oligonucleotide strand of 15-80 nucleotides in length, where the oligonucleotide strand is sufficiently complementary to a target HAO1 mRNA sequence of SEQ ID NOs: 1921-2304 along at least 15 nucleotides of the oligonucleotide strand length to reduce HAO1 target mRNA expression when the nucleic acid is introduced into a mammalian cell.

[0010] Another aspect of the invention provides a nucleic acid possessing an oligonucleotide strand of 19-80 nucleotides in length, where the oligonucleotide strand is sufficiently complementary to a target HAO1 mRNA sequence of SEQ ID NOs: 1921-2304 along at least 19 nucleotides of the oligonucleotide strand length to reduce HAO1 target mRNA expression when the nucleic acid is introduced into a mammalian cell.

[0011] In one embodiment, the oligonucleotide strand is 19-35 nucleotides in length.

[0012] An additional aspect of the invention provides a double stranded nucleic acid (dsNA) possessing first and second nucleic acid strands including RNA, where the first strand is 15-66 nucleotides in length and the second strand of the dsNA is 19-66 nucleotides in length, where the second oligonucleotide strand is sufficiently complementary to a target HAO1 mRNA sequence of SEQ ID NOs: 1921-2304 along at least 15 nucleotides of the second oligonucleotide strand length to reduce HAO1 target mRNA expression when the double stranded nucleic acid is introduced into a mammalian cell.

[0013] Another aspect of the invention provides a double stranded nucleic acid (dsNA) possessing first and second nucleic acid strands, where the first strand is 15-66 nucleotides in length and the second strand of the dsNA is 19-66 nucleotides in length, where the second oligonucleotide strand is sufficiently complementary to a target HAO1 mRNA sequence of SEQ ID NOs: 1921-2304 along at least 19 nucleotides of the second oligonucleotide strand length to reduce HAO1 target mRNA expression when the double stranded nucleic acid is introduced into a mammalian cell.

[0014] A further aspect of the invention provides a double stranded nucleic acid (dsNA) possessing first and second nucleic acid strands, where the first strand is 15-66 nucleotides in length and the second strand of the dsNA is 19-66 nucleotides in length, where the second oligonucleotide strand is sufficiently complementary to a target HAO1 mRNA sequence of SEQ ID NOs: 1921-2304 along at least 19 nucleotides of the second oligonucleotide strand length to reduce HAO1 target mRNA expression, and where, starting from the 5′ end of the HAO1 mRNA sequence of SEQ ID NOs: 1921-2304 (position 1), mammalian Ago2 cleaves the mRNA at a site between positions 9 and 10 of the sequence, when the double stranded nucleic acid is introduced into a mammalian cell.

[0015] Another aspect of the invention provides a dsNA molecule, consisting of: (a) a sense region and an antisense region, where the sense region and the antisense region together form a duplex region consisting of 25-66 base pairs and the antisense region includes a sequence that is the complement of a sequence of SEQ ID NOs: 1921-2304; and (b) from zero to two 3′ overhang regions, where each overhang region is six or fewer nucleotides in length, and where, starting from the 5′ end of the HAO1 mRNA sequence of SEQ ID NOs: 1921-2304 (position 1), mammalian Ago2 cleaves the mRNA at a site between positions 9 and 10 of the sequence, when the double stranded nucleic acid is introduced into a mammalian cell.

[0016] An additional aspect of the invention provides a double stranded nucleic acid (dsNA) possessing first and second nucleic acid strands and a duplex region of at least 25 base pairs, where the first strand is 25-65 nucleotides in length and the second strand of the dsNA is 26-66 nucleotides in length and includes 1-5 single-stranded nucleotides at its 3′ terminus, where the second oligonucleotide strand is sufficiently complementary to a target HAO1 mRNA sequence of SEQ ID NOs: 1921-2304 along at least 19 nucleotides of the second oligonucleotide strand length to reduce HAO1 target gene expression when the double stranded nucleic acid is introduced into a mammalian cell.

[0017] Another aspect of the invention provides a double stranded nucleic acid (dsNA) possessing first and second nucleic acid strands and a duplex region of at least 25 base pairs, where the first strand is 25-65 nucleotides in length and the second strand of the dsNA is 26-66 nucleotides in length and includes 1-5 single-stranded nucleotides at its 3′ terminus, where the 3′ terminus of the first oligonucleotide strand and the 5′ terminus of the second oligonucleotide strand form a blunt end, and the second oligonucleotide strand is sufficiently complementary to a target HAO1 sequence of SEQ ID NOs: 1921-2304 along at least 19 nucleotides of the second oligonucleotide strand length to reduce HAO1 mRNA expression when the double stranded nucleic acid is introduced into a mammalian cell.

[0018] In one embodiment, the first strand is 15-35 nucleotides in length.

[0019] In another embodiment, the second strand is 19-35 nucleotides in length.

[0020] In an additional embodiment, the dsNA possesses a duplex region of at least 25 base pairs; 19-21 base pairs or 21-25 base pairs.

[0021] Optionally, the second oligonucleotide strand includes 1-5 single-stranded nucleotides at its 3′ terminus.

[0022] In one embodiment, the second oligonucleotide strand includes 5-35 single-stranded nucleotides at its 3′ terminus.

[0023] In another embodiment, the single-stranded nucleotides include modified nucleotides. Optionally, the single-stranded nucleotides include 2′-O-methyl, 2′-methoxyethoxy, 2′-fluoro, 2′-allyl, 2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH2-O-2′-bridge, 4′-(CH2)2-O-2′-bridge, 2′-LNA, 2′-amino and/or 2′-O—(N-methlycarbamate) modified nucleotides.

[0024] In certain embodiments, the single-stranded nucleotides include ribonucleotides. Optionally, the single-stranded nucleotides include deoxyribonucleotides.

[0025] In certain embodiments, the 3′ end of the first strand and the 5′ end of the second strand of a dsNA or hybridization complex of the invention are joined by a polynucleotide sequence that includes ribonucleotides, deoxyribonucleotides or both, optionally the polynucleotide sequence includes a tetraloop sequence.

[0026] In one embodiment, the first strand is 25-35 nucleotides in length. Optionally, the second strand is 25-35 nucleotides in length.

[0027] In one embodiment, the second oligonucleotide strand is complementary to target HAO1 cDNA sequence GenBank Accession No. NM_017545.2 along at most 27 nucleotides of the second oligonucleotide strand length.

[0028] In another embodiment, starting from the first nucleotide (position 1) at the 3′ terminus of the first oligonucleotide strand, position 1, 2 and/or 3 is substituted with a modified nucleotide.

[0029] Optionally, the first strand and the 5′ terminus of the second strand form a blunt end.

[0030] In one embodiment, the first strand is 25 nucleotides in length and the second strand is 27 nucleotides in length.

[0031] In another embodiment, starting from the 5′ end of a HAO1 mRNA sequence of SEQ ID NOs: 1921-2304 (position 1), mammalian Ago2 cleaves the mRNA at a site between positions 9 and 10 of the sequence, thereby reducing HAO1 target mRNA expression when the double stranded nucleic acid is introduced into a mammalian cell.

[0032] In one embodiment, the second strand includes a sequence of SEQ ID NOs: 385-768. In an additional embodiment, the first strand includes a sequence of SEQ ID NOs: 1-384.

[0033] In another embodiment, the dsNA of the invention possesses a pair of first strand/second strand sequences of Table 2.

[0034] Optionally, the modified nucleotide residue of the 3′ terminus of the first strand is a deoxyribonucleotide, an acyclonucleotide or a fluorescent molecule.

[0035] In one embodiment, position 1 of the 3′ terminus of the first oligonucleotide strand is a deoxyribonucleotide.

[0036] In another embodiment, the nucleotides of the 1-5 or 5-35 single-stranded nucleotides of the 3′ terminus of the second strand comprise a modified nucleotide.

[0037] In one embodiment, the modified nucleotide of the 1-5 or 5-35 single-stranded nucleotides of the 3′ terminus of the second strand is a 2′-O-methyl ribonucleotide.

[0038] In an additional embodiment, all nucleotides of the 1-5 or 5-35 single-stranded nucleotides of the 3′ terminus of the second strand are modified nucleotides.

[0039] In another embodiment, the dsNA includes a modified nucleotide. Optionally, the modified nucleotide residue is 2′-O-methyl, 2′-methoxyethoxy, 2′-fluoro, 2′-allyl, 2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH2-O-2′-bridge, 4′-(CH2)2-O-2′-bridge, 2′-LNA, 2′-amino or 2′-O—(N-methlycarbamate).

[0040] In one embodiment, the 1-5 or 5-35 single-stranded nucleotides of the 3′ terminus of the second strand are 1-3 nucleotides in length, optionally 1-2 nucleotides in length.

[0041] In another embodiment, the 1-5 or 5-35 single-stranded nucleotides of the 3′ terminus of the second strand is two nucleotides in length and includes a 2′-O-methyl modified ribonucleotide.

[0042] In one embodiment, the second oligonucleotide strand includes a modification pattern of AS-M1 to AS-M96 and AS-M1* to AS-M96*.

[0043] In another embodiment, the first oligonucleotide strand includes a modification pattern of SM1 to SM119.

[0044] In an additional embodiment, each of the first and the second strands has a length which is at least 26 and at most 30 nucleotides.

[0045] In one embodiment, the dsNA is cleaved endogenously in the cell by Dicer.

[0046] In another embodiment, the amount of the nucleic acid sufficient to reduce expression of the target gene is of 1 nanomolar or less, 200 picomolar or less, 100 picomolar or less, 50 picomolar or less, 20 picomolar or less, 10 picomolar or less, 5 picomolar or less, 2, picomolar or less and 1 picomolar or less in the environment of the cell.

[0047] In one embodiment, the dsNA possesses greater potency than a 21mer siRNA directed to the identical at least 19 nucleotides of the target HAO1 mRNA in reducing target HAO1 mRNA expression when assayed in vitro in a mammalian cell at an effective concentration in the environment of a cell of 1 nanomolar or less.

[0048] In an additional embodiment, the nucleic acid or dsNA is sufficiently complementary to the target HAO1 mRNA sequence to reduce HAO1 target mRNA expression by an amount (expressed by %) of at least 10%, at least 50%, at least 80-90%, at least 95%, at least 98%, or at least 99% when the double stranded nucleic acid is introduced into a mammalian cell.

[0049] In one embodiment, the first and second strands are joined by a chemical linker.

[0050] In another embodiment, the 3′ terminus of the first strand and the 5′ terminus of the second strand are joined by a chemical linker.

[0051] In an additional embodiment, a nucleotide of the second or first strand is substituted with a modified nucleotide that directs the orientation of Dicer cleavage.

[0052] In one embodiment, the nucleic acid, dsNA or hybridization complex possesses a deoxyribonucleotide, a dideoxyribonucleotide, an acyclonucleotide, a 3′-deoxyadenosine (cordycepin), a 3′-azido-3′-deoxythymidine (AZT), a 2′,3′-dideoxyinosine (ddI), a 2′,3′-dideoxy-3′-thiacytidine (3TC), a 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T), a monophosphate nucleotide of 3′-azido-3′-deoxythymidine (AZT), a 2′,3′-dideoxy-3′-thiacytidine (3TC) and a monophosphate nucleotide of 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T), a 4-thiouracil, a 5-bromouracil, a 5-iodouracil, a 5-(3-aminoallyl)-uracil, a 2′-O-alkyl ribonucleotide, a 2′-O-methyl ribonucleotide, a 2′-amino ribonucleotide, a 2′-fluoro ribonucleotide, or a locked nucleic acid modified nucleotide.

[0053] In another embodiment, the nucleic acid, dsNA or hybridization complex includes a phosphonate, a phosphorothioate or a phosphotriester phosphate backbone modification.

[0054] In one embodiment, the nucleic acid, dsNA or hybridization complex includes a morpholino nucleic acid or a peptide nucleic acid (PNA).

[0055] In an additional embodiment, the nucleic acid, dsNA or hybridization complex is attached to a dynamic polyconjugate (DPC).

[0056] In one embodiment, the nucleic acid, dsNA or hybridization complex is administered with a DPC, where the dsNA and DPC are optionally not attached.

[0057] In another embodiment, the nucleic acid, dsNA or hybridization complex is attached to a GalNAc moiety, a cholesterol and/or a cholesterol targeting ligand.

[0058] Another aspect of the invention provides a composition of the following:

[0059] a dsNA possessing first and second nucleic acid strands, where the first strand is 15-35 nucleotides in length and the second strand of the dsNA is 19-35 nucleotides in length, where the second oligonucleotide strand is sufficiently complementary to a target HAO1 mRNA sequence of SEQ ID NOs: 1921-2304 along at least 19 nucleotides of the second oligonucleotide strand length to reduce HAO1 target mRNA expression when the double stranded nucleic acid is introduced into a mammalian cell;

[0060] a dsNA possessing first and second nucleic acid strands, where the first strand is 15-35 nucleotides in length and the second strand of the dsNA is 19-35 nucleotides in length, where the second oligonucleotide strand is sufficiently complementary to a target HAO1 mRNA sequence of SEQ ID NOs: 1921-2304 along at least 19 nucleotides of the second oligonucleotide strand length to reduce HAO1 target mRNA expression, and where, starting from the 5′ end of the HAO1 mRNA sequence of SEQ ID NOs: 1921-2304 (position 1), mammalian Ago2 cleaves the mRNA at a site between positions 9 and 10 of the sequence, when the double stranded nucleic acid is introduced into a mammalian cell;

[0061] a dsNA molecule, consisting of: (a) a sense region and an antisense region, where the sense region and the antisense region together form a duplex region consisting of 25-35 base pairs and the antisense region includes a sequence that is the complement of a sequence of SEQ ID NOs: 1921-2304; and (b) from zero to two 3′ overhang regions, where each overhang region is six or fewer nucleotides in length, and where, starting from the 5′ end of the HAO1 mRNA sequence of SEQ ID NOs: 1921-2304 (position 1), mammalian Ago2 cleaves the mRNA at a site between positions 9 and 10 of the sequence, when the double stranded nucleic acid is introduced into a mammalian cell;

[0062] a dsNA possessing first and second nucleic acid strands and a duplex region of at least 25 base pairs, where the first strand is 25-34 nucleotides in length and the second strand of the dsNA is 26-35 nucleotides in length and includes 1-5 single-stranded nucleotides at its 3′ terminus, where the second oligonucleotide strand is sufficiently complementary to a target HAO1 mRNA sequence of SEQ ID NOs: 1921-2304 along at least 19 nucleotides of the second oligonucleotide strand length to reduce HAO1 target gene expression when the double stranded nucleic acid is introduced into a mammalian cell;

[0063] a dsNA possessing first and second nucleic acid strands and a duplex region of at least 25 base pairs, where the first strand is 25-34 nucleotides in length and the second strand of the dsNA is 26-35 nucleotides in length and includes 1-5 single-stranded nucleotides at its 3′ terminus, where the 3′ terminus of the first oligonucleotide strand and the 5′ terminus of the second oligonucleotide strand form a blunt end, and the second oligonucleotide strand is sufficiently complementary to a target HAO1 sequence of SEQ ID NOs: 1921-2304 along at least 19 nucleotides of the second oligonucleotide strand length to reduce HAO1 mRNA expression when the double stranded nucleic acid is introduced into a mammalian cell;

[0064] a nucleic acid possessing an oligonucleotide strand of 19-35 nucleotides in length, where the oligonucleotide strand is sufficiently complementary to a target HAO1 mRNA sequence of SEQ ID NOs: 1921-2304 along at least 19 nucleotides of the oligonucleotide strand length to reduce HAO1 target mRNA expression when the nucleic acid is introduced into a mammalian cell;

[0065] a nucleic acid possessing an oligonucleotide strand of 15-35 nucleotides in length, where the oligonucleotide strand is hybridizable to a target HAO1 mRNA sequence of SEQ ID NOs: 1921-2304 along at least 15 nucleotides of the oligonucleotide strand length;

[0066] a dsNA possessing first and second nucleic acid strands possessing RNA, where the first strand is 15-35 nucleotides in length and the second strand of the dsNA is 19-35 nucleotides in length, where the second oligonucleotide strand is hybridizable to a target HAO1 mRNA sequence of SEQ ID NOs: 1921-2304 along at least 15 nucleotides of the second oligonucleotide strand length;

[0067] a dsNA possessing first and second nucleic acid strands possessing RNA, where the first strand is 15-35 nucleotides in length and the second strand of the dsNA is at least 35 nucleotides in length and optionally includes a sequence of at least 25 nucleotides in length of SEQ ID NOs: 385-768, where the second oligonucleotide strand is sufficiently complementary to a target HAO1 mRNA sequence of SEQ ID NOs: 1921-2304 along at least 15 nucleotides of the second oligonucleotide strand length to reduce HAO1 target mRNA expression when the double stranded nucleic acid is introduced into a mammalian cell;

[0068] a dsNA possessing a first oligonucleotide strand having a 5′ terminus and a 3′ terminus and a second oligonucleotide strand having a 5′ terminus and a 3′ terminus, where the first strand is 25 to 53 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5′ terminus of the first strand, positions 1 to 23 of the first strand are ribonucleotides or modified ribonucleotides; the second strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides or modified ribonucleotides that base pair with the ribonucleotides or ribonucleotides of positions 1 to 23 of the first strand to form a duplex; the 5′ terminus of the first strand and the 3′ terminus of the second strand form a structure of a blunt end, a 1-6 nucleotide 5′ overhang and a 1-6 nucleotide 3′ overhang; the 3′ terminus of the first strand and the 5′ terminus of the second strand form a structure of a blunt end, a 1-6 nucleotide 5′ overhang and a 1-6 nucleotide 3′ overhang; at least one of positions 24 to the 3′ terminal nucleotide residue of the first strand is a deoxyribonucleotide or modified ribonucleotide that optionally base pairs with a deoxyribonucleotide of the second strand; and the second strand is sufficiently complementary to a target HAO1 mRNA sequence of SEQ ID NOs: 1921-2304 along at least 15 nucleotides of the second oligonucleotide strand length to reduce HAO1 target mRNA expression when the double stranded nucleic acid is introduced into a mammalian cell;

[0069] a dsNA possessing a first oligonucleotide strand having a 5′ terminus and a 3′ terminus and a second oligonucleotide strand having a 5′ terminus and a 3′ terminus, where the second strand is 27 to 53 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5′ terminus of the second strand, positions 1 to 23 of the second strand are ribonucleotides or modified ribonucleotides; the first strand is 25 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides or modified ribonucleotides that base pair sufficiently with the ribonucleotides of positions 1 to 23 of the second strand to form a duplex; at least one of positions 24 to the 3′ terminal nucleotide residue of the second strand is a deoxyribonucleotide or modified ribonucleotide, optionally that base pairs with a deoxyribonucleotide or modified ribonucleotide of the first strand; and the second strand is sufficiently complementary to a target HAO1 mRNA sequence of SEQ ID NOs: 1921-2304 along at least 15 nucleotides of the second oligonucleotide strand length to reduce HAO1 target mRNA expression when the double stranded nucleic acid is introduced into a mammalian cell;

[0070] a dsNA possessing a first oligonucleotide strand having a 5′ terminus and a 3′ terminus and a second oligonucleotide strand having a 5′ terminus and a 3′ terminus, where each of the 5′ termini has a 5′ terminal nucleotide and each of the 3′ termini has a 3′ terminal nucleotide, where the first strand (or the second strand) is 25-30 nucleotide residues in length, where starting from the 5′ terminal nucleotide (position 1) positions 1 to 23 of the first strand (or the second strand) include at least 8 ribonucleotides; the second strand (or the first strand) is 36-66 nucleotide residues in length and, starting from the 3′ terminal nucleotide, includes at least 8 ribonucleotides in the positions paired with positions 1-23 of the first strand to form a duplex; where at least the 3′ terminal nucleotide of the second strand (or the first strand) is unpaired with the first strand (or the second strand), and up to 6 consecutive 3′ terminal nucleotides are unpaired with the first strand (or the second strand), thereby forming a 3′ single stranded overhang of 1-6 nucleotides; where the 5′ terminus of the second strand (or the first strand) includes from 10-30 consecutive nucleotides which are unpaired with the first strand (or the second strand), thereby forming a 10-30 nucleotide single stranded 5′ overhang; where at least the first strand (or the second strand) 5′ terminal and 3′ terminal nucleotides are base paired with nucleotides of the second strand (or first strand) when the first and second strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between the first and second strands; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell;

[0071] a dsNA possessing a first oligonucleotide strand having a 5′ terminus and a 3′ terminus and a second oligonucleotide strand having a 5′ terminus and a 3′ terminus, where each of the 5′ termini has a 5′ terminal nucleotide and each of the 3′ termini has a 3′ terminal nucleotide, where the first strand is 25-35 nucleotide residues in length, where starting from the 5′ terminal nucleotide (position 1) positions 1 to 25 of the second strand include at least 8 ribonucleotides; the second strand is 30-66 nucleotide residues in length and, starting from the 3′ terminal nucleotide, includes at least 8 ribonucleotides in the positions paired with positions 1-25 of the first strand to form a duplex; where the 5′ terminus of the second strand includes from 5-35 consecutive nucleotides which are unpaired with the first strand, thereby forming a 5-35 nucleotide single stranded 5′ overhang; where at least the first strand 5′ terminal and 3′ terminal nucleotides are base paired with nucleotides of the second strand when the first and second strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between the first and second strands; and the second strand is sufficiently complementary to a target HAO1 mRNA sequence of SEQ ID NOs: 1921-2304 along at least 15 nucleotides of the second oligonucleotide strand length to reduce HAO1 target mRNA expression when the double stranded nucleic acid is introduced into a mammalian cell;

[0072] a dsNA possessing a first oligonucleotide strand having a 5′ terminus and a 3′ terminus and a second oligonucleotide strand having a 5′ terminus and a 3′ terminus, where each of the 5′ termini has a 5′ terminal nucleotide and each of the 3′ termini has a 3′ terminal nucleotide, where the second strand is 19-30 nucleotide residues in length and optionally 25-30 nucleotide residues in length, where starting from the 5′ terminal nucleotide (position 1) positions 1 to 17 (optionally positions 1 to 23) of the second strand include at least 8 ribonucleotides; the first strand is 24-66 nucleotide residues in length (optionally 30-66 nucleotide residues in length) and, starting from the 3′ terminal nucleotide, includes at least 8 ribonucleotides in the positions paired with positions 1 to 17 (optionally positions 1 to 23) of the second strand to form a duplex; where the 3′ terminus of the first strand and the 5′ terminus of the second strand comprise a structure of a blunt end, a 3′ overhang and a 5′ overhang, optionally where the overhang is 1-6 nucleotides in length; where the 5′ terminus of the first strand includes from 5-35 consecutive nucleotides which are unpaired with the second strand, thereby forming a 5-35 nucleotide single-stranded 5′ overhang; where at least the second strand 5′ terminal and 3′ terminal nucleotides are base paired with nucleotides of the first strand when the first and second strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between the first and second strands; and the second strand is sufficiently complementary to a target HAO1 mRNA sequence of SEQ ID NOs: 1921-2304 along at least 15 nucleotides of the second oligonucleotide strand length to reduce HAO1 target mRNA expression when the double stranded nucleic acid is introduced into a mammalian cell;

[0073] a dsNA possessing a first strand and a second strand, where the first strand and the second strand form a duplex region of 19-25 nucleotides in length, where the first strand includes a 3′ region that extends beyond the first strand-second strand duplex region and includes a tetraloop, and the dsNA further includes a discontinuity between the 3′ terminus of the first strand and the 5′ terminus of the second strand, and the first or second strand is sufficiently complementary to a target HAO1 mRNA sequence of SEQ ID NOs: 1921-2304 along at least 15 nucleotides of the first or second strand length to reduce HAO1 target mRNA expression when the dsNA is introduced into a mammalian cell;

[0074] a dsNA possessing a first oligonucleotide strand having a 5′ terminus and a 3′ terminus and a second oligonucleotide strand having a 5′ terminus and a 3′ terminus, where each of the 5′ termini has a 5′ terminal nucleotide and each of the 3′ termini has a 3′ terminal nucleotide, where the first oligonucleotide strand is 25-53 nucleotides in length and the second is oligonucleotide strand is 25-53 nucleotides in length, and where the dsNA is sufficiently highly modified to substantially prevent dicer cleavage of the dsNA, optionally where the dsNA is cleaved by non-dicer nucleases to yield one or more 19-23 nucleotide strand length dsNAs capable of reducing LDH mRNA expression in a mammalian cell;

[0075] an in vivo hybridization complex within a cell of an exogenous nucleic acid sequence and a target HAO1 mRNA sequence of SEQ ID NOs: 1921-2304; and

[0076] an in vitro hybridization complex within a cell of an exogenous nucleic acid sequence and a target HAO1 mRNA sequence of SEQ ID NOs: 1921-2304.

[0077] In one embodiment, a dsNA of the invention possesses a duplex region of at least 25 base pairs, where the dsNA possesses greater potency than a 21mer siRNA directed to the identical at least 19 nucleotides of the target HAO1 mRNA in reducing target HAO1 mRNA expression when assayed in vitro in a mammalian cell at an effective concentration in the environment of a cell of 1 nanomolar or less.

[0078] In another embodiment, the dsNA of the invention has a duplex region of at least 25 base pairs, where the dsNA possesses greater potency than a 21mer siRNA directed to the identical at least 19 nucleotides of the target HAO1 mRNA in reducing target HAO1 mRNA expression when assayed in vitro in a mammalian cell at a concentration of 1 nanomolar, 200 picomolar, 100 picomolar, 50 picomolar, 20 picomolar, 10 picomolar, 5 picomolar, 2, picomolar and 1 picomolar.

[0079] In an additional embodiment, the dsNA of the invention possesses four or fewer mismatched nucleic acid residues with respect to the target HAO1 mRNA sequence along 15 or 19 consecutive nucleotides of the at least 15 or 19 nucleotides of the second oligonucleotide strand when the 15 or 19 consecutive nucleotides of the second oligonucleotide are aligned for maximum complementarity with the target HAO1 mRNA sequence.

[0080] In another embodiment, the dsNA of the invention possesses three or fewer mismatched nucleic acid residues with respect to the target HAO1 mRNA sequence along 15 or 19 consecutive nucleotides of the at least 15 or 19 nucleotides of the second oligonucleotide strand when the 15 or 19 consecutive nucleotides of the second oligonucleotide are aligned for maximum complementarity with the target HAO1 mRNA sequence.

[0081] Optionally, the dsNA of the invention possesses two or fewer mismatched nucleic acid residues with respect to the target HAO1 mRNA sequence along 15 or 19 consecutive nucleotides of the at least 15 or 19 nucleotides of the second oligonucleotide strand when the 15 or 19 consecutive nucleotides of the second oligonucleotide are aligned for maximum complementarity with the target HAO1 mRNA sequence.

[0082] In certain embodiments, the dsNA of the invention possesses one mismatched nucleic acid residue with respect to the target HAO1 mRNA sequence along 15 or 19 consecutive nucleotides of the at least 15 or 19 nucleotides of the second oligonucleotide strand when the 15 or 19 consecutive nucleotides of the second oligonucleotide are aligned for maximum complementarity with the target HAO1 mRNA sequence.

[0083] In one embodiment, 15 or 19 consecutive nucleotides of the at least 15 or 19 nucleotides of the second oligonucleotide strand are completely complementary to the target HAO1 mRNA sequence when the 15 or 19 consecutive nucleotides of the second oligonucleotide are aligned for maximum complementarity with the target HAO1 mRNA sequence.

[0084] Optionally, a nucleic acid, dsNA or hybridization complex of the invention is isolated.

[0085] Another aspect of the invention provides a method for reducing expression of a target HAO1 gene in a mammalian cell involving contacting a mammalian cell in vitro with a nucleic acid, dsNA or hybridization complex of the invention in an amount sufficient to reduce expression of a target HAO1 mRNA in the cell.

[0086] Optionally, target HAO1 mRNA expression is reduced by an amount (expressed by %) of at least 10%, at least 50% and at least 80-90%.

[0087] In one embodiment, HAO1 mRNA levels are reduced by an amount (expressed by %) of at least 90% at least 8 days after the cell is contacted with the dsNA.

[0088] In another embodiment, HAO1 mRNA levels are reduced by an amount (expressed by %) of at least 70% at least 10 days after the cell is contacted with the dsNA.

[0089] A further aspect of the invention provides a method for reducing expression of a target HAO1 mRNA in a mammal possessing administering a nucleic acid, dsNA or hybridization complex of the invention to a mammal in an amount sufficient to reduce expression of a target HAO1 mRNA in the mammal.

[0090] In one embodiment, the nucleic acid, dsNA or hybridization complex is formulated in a lipid nanoparticle (LNP).

[0091] In another embodiment, the nucleic acid, dsNA or hybridization complex is administered at a dosage of 1 microgram to 5 milligrams per kilogram of the mammal per day, 100 micrograms to 0.5 milligrams per kilogram, 0.001 to 0.25 milligrams per kilogram, 0.01 to 20 micrograms per kilogram, 0.01 to 10 micrograms per kilogram, 0.10 to 5 micrograms per kilogram, and 0.1 to 2.5 micrograms per kilogram.

[0092] In certain embodiments, the nucleic acid, dsNA or hybridization complex possesses greater potency than 21mer siRNAs directed to the identical at least 19 nucleotides of the target HAO1 mRNA in reducing target HAO1 mRNA expression when assayed in vitro in a mammalian cell at an effective concentration in the environment of a cell of 1 nanomolar or less.

[0093] In one embodiment, HAO1 mRNA levels are reduced in a tissue of the mammal by an amount (expressed by %) of at least 70% at least 3 days after the dsNA is administered to the mammal.

[0094] Optionally, the tissue is liver tissue.

[0095] In certain embodiments, the administering step involves intravenous injection, intramuscular injection, intraperitoneal injection, infusion, subcutaneous injection, transdermal, aerosol, rectal, vaginal, topical, oral or inhaled delivery.

[0096] Another aspect of the invention provides a method for treating or preventing a disease or disorder in a subject that involves administering to the subject an amount of a nucleic acid, dsNA or hybridization complex of the invention in an amount sufficient to treat or prevent the disease or disorder in the subject.

[0097] In one embodiment, the disease or disorder is primary hyperoxaluria 1 (PH1).

[0098] Optionally, the subject is human.

[0099] A further aspect of the invention provides a formulation that includes the nucleic acid, dsNA or hybridization complex of the invention, where the nucleic acid, dsNA or hybridization complex is present in an amount effective to reduce target HAO1 mRNA levels when the nucleic acid, dsNA or hybridization complex is introduced into a mammalian cell in vitro by an amount (expressed by %) of at least 10%, at least 50% and at least 80-90%.

[0100] In one embodiment, the effective amount is of 1 nanomolar or less, 200 picomolar or less, 100 picomolar or less, 50 picomolar or less, 20 picomolar or less, 10 picomolar or less, 5 picomolar or less, 2, picomolar or less and 1 picomolar or less in the environment of the cell.

[0101] In another embodiment, the nucleic acid, dsNA or hybridization complex is present in an amount effective to reduce target HAO1 mRNA levels when the nucleic acid, dsNA or hybridization complex is introduced into a cell of a mammalian subject by an amount (expressed by %) of at least 10%, at least 50% and at least 80-90%.

[0102] Optionally, the effective amount is a dosage of 1 microgram to 5 milligrams per kilogram of the subject per day, 100 micrograms to 0.5 milligrams per kilogram, 0.001 to 0.25 milligrams per kilogram, 0.01 to 20 micrograms per kilogram, 0.01 to 10 micrograms per kilogram, 0.10 to 5 micrograms per kilogram, or 0.1 to 2.5 micrograms per kilogram.

[0103] In another embodiment, the nucleic acid, dsNA or hybridization complex possesses greater potency than an 21mer siRNA directed to the identical at least 19 nucleotides of the target HAO1 mRNA in reducing target HAO1 mRNA levels when assayed in vitro in a mammalian cell at an effective concentration in the environment of a cell of 1 nanomolar or less.

[0104] A further aspect of the invention provides a mammalian cell containing the nucleic acid, dsNA or hybridization complex of the invention.

[0105] Another aspect of the invention provides a pharmaceutical composition that includes the nucleic acid, dsNA or hybridization complex of the invention and a pharmaceutically acceptable carrier.

[0106] An additional aspect of the invention provides a kit including the nucleic acid, dsNA or hybridization complex of the invention and instructions for its use.

[0107] A further aspect of the invention provides a composition possessing HAO1 inhibitory activity consisting essentially of a nucleic acid, dsNA or hybridization complex of the invention.

[0121] The present invention is directed to compositions that contain nucleic acids, for example double stranded RNA (“dsRNA”), and methods for preparing them, that are capable of reducing the level and/or expression of the Glycolate Oxidase (HAO1) gene in vivo or in vitro. One of the strands of the dsRNA contains a region of nucleotide sequence that has a length that ranges from 19 to 35 nucleotides that can direct the destruction and/or translational inhibition of the targeted HAO1 transcript.

[0122] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0123] The present invention features one or more DsiRNA molecules that can modulate (e.g., inhibit) Glycolate Oxidase expression. The DsiRNAs of the invention optionally can be used in combination with modulators of other genes and/or gene products associated with the maintenance or development of diseases or disorders associated with Glycolate Oxidase misregulation (e.g., primary hyperoxaluria 1 (PH1) or other disease or disorder of the liver, kidneys, etc.). The DsiRNA agents of the invention modulate Glycolate Oxidase (Hydroxyacid Oxidase 1, HAO1) RNAs such as those corresponding to the cDNA sequences referred to by GenBank Accession Nos. NM_017545.2 (human HAO1) and NM_010403.2 (mouse HAO1), which are referred to herein generally as “Glycolate Oxidase” or “Hydroxyacid Oxidase 1.”

[0124] The below description of the various aspects and embodiments of the invention is provided with reference to exemplary Glycolate Oxidase RNAs, generally referred to herein as Glycolate Oxidase. However, such reference is meant to be exemplary only and the various aspects and embodiments of the invention are also directed to alternate Glycolate Oxidase RNAs, such as mutant Glycolate Oxidase RNAs or additional Glycolate Oxidase splice variants. Certain aspects and embodiments are also directed to other genes involved in Glycolate Oxidase pathways, including genes whose misregulation acts in association with that of Glycolate Oxidase (or is affected or affects Glycolate Oxidase regulation) to produce phenotypic effects that may be targeted for treatment (e.g., glyoxylate and/or oxalate overproduction). DAO and AGT are examples of genes that interact with Glycolate Oxidase. Such additional genes, including those of pathways that act in coordination with Glycolate Oxidase, can be targeted using dsRNA and the methods described herein for use of Glycolate Oxidase-targeting dsRNAs. Thus, the inhibition or and the effects of such inhibition, or up-regulation and the effects of such, of the other genes can be performed as described herein.

[0125] The term “Glycolate Oxidase” refers to nucleic acid sequences encoding a Glycolate Oxidase protein, peptide, or polypeptide (e.g., Glycolate Oxidase transcripts, such as the sequences of Glycolate Oxidase (HAO1) Genbank Accession Nos. NM_017545.2 and NM_010403.2). In certain embodiments, the term “Glycolate Oxidase” is also meant to include other Glycolate Oxidase encoding sequence, such as other Glycolate Oxidase isoforms, mutant Glycolate Oxidase genes, splice variants of Glycolate Oxidase genes, and Glycolate Oxidase gene polymorphisms. The term “Glycolate Oxidase” is also used to refer to the polypeptide gene product of an Glycolate Oxidase gene/transcript, e.g., a Glycolate Oxidase protein, peptide, or polypeptide, such as those encoded by Glycolate Oxidase (HAO1) Genbank Accession Nos. NP_060015.1 and NP_034533.1.

[0126] As used herein, a “Glycolate Oxidase-associated disease or disorder” refers to a disease or disorder known in the art to be associated with altered Glycolate Oxidase expression, level and/or activity. Notably, a “Glycolate Oxidase-associated disease or disorder” or “HAO1-associated disease or disorder” includes diseases or disorders of the kidney, liver, skin, bone, eye and other organs including, but not limited to, primary hyperoxaluria (PH1).

[0127] In certain embodiments, dsRNA-mediated inhibition of a Glycolate Oxidase target sequence is assessed. In such embodiments, Glycolate Oxidase RNA levels can be assessed by art-recognized methods (e.g., RT-PCR, Northern blot, expression array, etc.), optionally via comparison of Glycolate Oxidase levels in the presence of an anti-Glycolate Oxidase dsRNA of the invention relative to the absence of such an anti-Glycolate Oxidase dsRNA. In certain embodiments, Glycolate Oxidase levels in the presence of an anti-Glycolate Oxidase dsRNA are compared to those observed in the presence of vehicle alone, in the presence of a dsRNA directed against an unrelated target RNA, or in the absence of any treatment.

[0128] It is also recognized that levels of Glycolate Oxidase protein can be assessed and that Glycolate Oxidase protein levels are, under different conditions, either directly or indirectly related to Glycolate Oxidase RNA levels and/or the extent to which a dsRNA inhibits Glycolate Oxidase expression, thus art-recognized methods of assessing Glycolate Oxidase protein levels (e.g., Western blot, immunoprecipitation, other antibody-based methods, etc.) can also be employed to examine the inhibitory effect of a dsRNA of the invention.

[0129] An anti-Glycolate Oxidase dsRNA of the invention is deemed to possess “Glycolate Oxidase inhibitory activity” if a statistically significant reduction in Glycolate Oxidase RNA (or when the Glycolate Oxidase protein is assessed, Glycolate Oxidase protein levels) is seen when an anti-Glycolate Oxidase dsRNA of the invention is administered to a system (e.g., cell-free in vitro system), cell, tissue or organism, as compared to a selected control. The distribution of experimental values and the number of replicate assays performed will tend to dictate the parameters of what levels of reduction in Glycolate Oxidase RNA (either as a % or in absolute terms) is deemed statistically significant (as assessed by standard methods of determining statistical significance known in the art). However, in certain embodiments, “Glycolate Oxidase inhibitory activity” is defined based upon a % or absolute level of reduction in the level of Glycolate Oxidase in a system, cell, tissue or organism. For example, in certain embodiments, a dsRNA of the invention is deemed to possess Glycolate Oxidase inhibitory activity if at least a 5% reduction or at least a 10% reduction in Glycolate Oxidase RNA is observed in the presence of a dsRNA of the invention relative to Glycolate Oxidase levels seen for a suitable control. (For example, in vivo Glycolate Oxidase levels in a tissue and/or subject can, in certain embodiments, be deemed to be inhibited by a dsRNA agent of the invention if, e.g., a 5% or 10% reduction in Glycolate Oxidase levels is observed relative to a control.) In certain other embodiments, a dsRNA of the invention is deemed to possess Glycolate Oxidase inhibitory activity if Glycolate Oxidase RNA levels are observed to be reduced by at least 15% relative to a selected control, by at least 20% relative to a selected control, by at least 25% relative to a selected control, by at least 30% relative to a selected control, by at least 35% relative to a selected control, by at least 40% relative to a selected control, by at least 45% relative to a selected control, by at least 50% relative to a selected control, by at least 55% relative to a selected control, by at least 60% relative to a selected control, by at least 65% relative to a selected control, by at least 70% relative to a selected control, by at least 75% relative to a selected control, by at least 80% relative to a selected control, by at least 85% relative to a selected control, by at least 90% relative to a selected control, by at least 95% relative to a selected control, by at least 96% relative to a selected control, by at least 97% relative to a selected control, by at least 98% relative to a selected control or by at least 99% relative to a selected control. In some embodiments, complete inhibition of Glycolate Oxidase is required for a dsRNA to be deemed to possess Glycolate Oxidase inhibitory activity. In certain models (e.g., cell culture), a dsRNA is deemed to possess Glycolate Oxidase inhibitory activity if at least a 50% reduction in Glycolate Oxidase levels is observed relative to a suitable control. In certain other embodiments, a dsRNA is deemed to possess Glycolate Oxidase inhibitory activity if at least an 80% reduction in Glycolate Oxidase levels is observed relative to a suitable control.

[0130] By way of specific example, in Example 2 below, a series of DsiRNAs targeting Glycolate Oxidase were tested for the ability to reduce Glycolate Oxidase mRNA levels in human HeLa or mouse Hepa1-6 cells in vitro, at 1 nM concentrations in the environment of such cells and in the presence of a transfection agent (Lipofectamine™ RNAiMAX, Invitrogen). Within Example 2 below, Glycolate Oxidase inhibitory activity was ascribed to those DsiRNAs that were observed to effect at least a 50% reduction of Glycolate Oxidase mRNA levels under the assayed conditions. It is contemplated that Glycolate Oxidase inhibitory activity could also be attributed to a dsRNA under either more or less stringent conditions than those employed for Example 2 below, even when the same or a similar assay and conditions are employed. For example, in certain embodiments, a tested dsRNA of the invention is deemed to possess Glycolate Oxidase inhibitory activity if at least a 10% reduction, at least a 20% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, at least a 75% reduction, at least an 80% reduction, at least an 85% reduction, at least a 90% reduction, or at least a 95% reduction in Glycolate Oxidase mRNA levels is observed in a mammalian cell line in vitro at 1 nM dsRNA concentration or lower in the environment of a cell, relative to a suitable control.

[0131] Use of other endpoints for determination of whether a double stranded RNA of the invention possesses Glycolate Oxidase inhibitory activity is also contemplated. Specifically, in one embodiment, in addition to or as an alternative to assessing Glycolate Oxidase mRNA levels, the ability of a tested dsRNA to reduce Glycolate Oxidase protein levels (e.g., at 48 hours after contacting a mammalian cell in vitro or in vivo) is assessed, and a tested dsRNA is deemed to possess Glycolate Oxidase inhibitory activity if at least a 10% reduction, at least a 20% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, at least a 75% reduction, at least an 80% reduction, at least an 85% reduction, at least a 90% reduction, or at least a 95% reduction in Glycolate Oxidase protein levels is observed in a mammalian cell contacted with the assayed double stranded RNA in vitro or in vivo, relative to a suitable control. Additional endpoints contemplated include, e.g., assessment of a phenotype associated with reduction of Glycolate Oxidase levels—e.g., reduction of phenotypes associated with elevated levels of glyoxylate-derived deposits (e.g., deposits of calcium oxalate) throughout the body.

[0132] Glycolate Oxidase inhibitory activity can also be evaluated over time (duration) and over concentration ranges (potency), with assessment of what constitutes a dsRNA possessing Glycolate Oxidase inhibitory activity adjusted in accordance with concentrations administered and duration of time following administration. Thus, in certain embodiments, a dsRNA of the invention is deemed to possess Glycolate Oxidase inhibitory activity if at least a 50% reduction in Glycolate Oxidase activity is observed/persists at a duration of time of 2 hours, 5 hours, 10 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or more after administration of the dsRNA to a cell or organism. In additional embodiments, a dsRNA of the invention is deemed to be a potent Glycolate Oxidase inhibitory agent if Glycolate Oxidase inihibitory activity (e.g., in certain embodiments, at least 50% inhibition of Glycolate Oxidase) is observed at a concentration of 1 nM or less, 500 pM or less, 200 pM or less, 100 pM or less, 50 pM or less, 20 pM or less, 10 pM or less, 5 pM or less, 2 pM or less or even 1 pM or less in the environment of a cell, for example, within an in vitro assay for Glycolate Oxidase inihibitory activity as described herein. In certain embodiments, a potent Glycolate Oxidase inhibitory dsRNA of the invention is defined as one that is capable of Glycolate Oxidase inihibitory activity (e.g., in certain embodiments, at least 20% reduction of Glycolate Oxidase levels) at a formulated concentration of 10 mg/kg or less when administered to a subject in an effective delivery vehicle (e.g., an effective lipid nanoparticle formulation). Preferably, a potent Glycolate Oxidase inhibitory dsRNA of the invention is defined as one that is capable of Glycolate Oxidase inihibitory activity (e.g., in certain embodiments, at least 50% reduction of Glycolate Oxidase levels) at a formulated concentration of 5 mg/kg or less when administered to a subject in an effective delivery vehicle. More preferably, a potent Glycolate Oxidase inhibitory dsRNA of the invention is defined as one that is capable of Glycolate Oxidase inihibitory activity (e.g., in certain embodiments, at least 50% reduction of Glycolate Oxidase levels) at a formulated concentration of 5 mg/kg or less when administered to a subject in an effective delivery vehicle. Optionally, a potent Glycolate Oxidase inhibitory dsRNA of the invention is defined as one that is capable of Glycolate Oxidase inhibitory activity (e.g., in certain embodiments, at least 50% reduction of Glycolate Oxidase levels) at a formulated concentration of 2 mg/kg or less, or even 1 mg/kg or less, when administered to a subject in an effective delivery vehicle.

[0133] In certain embodiments, potency of a dsRNA of the invention is determined in reference to the number of copies of a dsRNA present in the cytoplasm of a target cell that are required to achieve a certain level of target gene knockdown. For example, in certain embodiments, a potent dsRNA is one capable of causing 50% or greater knockdown of a target mRNA when present in the cytoplasm of a target cell at a copy number of 1000 or fewer RISC-loaded antisense strands per cell. More preferably, a potent dsRNA is one capable of producing 50% or greater knockdown of a target mRNA when present in the cytoplasm of a target cell at a copy number of 500 or fewer RISC-loaded antisense strands per cell. Optionally, a potent dsRNA is one capable of producing 50% or greater knockdown of a target mRNA when present in the cytoplasm of a target cell at a copy number of 300 or fewer RISC-loaded antisense strands per cell.

[0134] In further embodiments, the potency of a DsiRNA of the invention can be defined in reference to a 19 to 23mer dsRNA directed to the same target sequence within the same target gene. For example, a DsiRNA of the invention that possesses enhanced potency relative to a corresponding 19 to 23mer dsRNA can be a DsiRNA that reduces a target gene by an additional 5% or more, an additional 10% or more, an additional 20% or more, an additional 30% or more, an additional 40% or more, or an additional 50% or more as compared to a corresponding 19 to 23mer dsRNA, when assayed in an in vitro assay as described herein at a sufficiently low concentration to allow for detection of a potency difference (e.g., transfection concentrations at or below 1 nM in the environment of a cell, at or below 100 pM in the environment of a cell, at or below 10 pM in the environment of a cell, at or below 1 nM in the environment of a cell, in an in vitro assay as described herein; notably, it is recognized that potency differences can be best detected via performance of such assays across a range of concentrations—e.g., 0.1 pM to 10 nM—for purpose of generating a dose-response curve and identifying an IC₅₀ value associated with a DsiRNA/dsRNA).

[0135] Glycolate Oxidase inhibitory levels and/or Glycolate Oxidase levels may also be assessed indirectly, e.g., measurement of a reduction of primary hyperoxaluria 1 phenotype(s) in a subject may be used to assess Glycolate Oxidase levels and/or Glycolate Oxidase inhibitory efficacy of a double-stranded nucleic acid of the instant invention.

[0136] In certain embodiments, the phrase “consists essentially of” is used in reference to the anti-Glycolate Oxidase dsRNAs of the invention. In some such embodiments, “consists essentially of” refers to a composition that comprises a dsRNA of the invention which possesses at least a certain level of Glycolate Oxidase inhibitory activity (e.g., at least 50% Glycolate Oxidase inhibitory activity) and that also comprises one or more additional components and/or modifications that do not significantly impact the Glycolate Oxidase inhibitory activity of the dsRNA. For example, in certain embodiments, a composition “consists essentially of” a dsRNA of the invention where modifications of the dsRNA of the invention and/or dsRNA-associated components of the composition do not alter the Glycolate Oxidase inhibitory activity (optionally including potency or duration of Glycolate Oxidase inhibitory activity) by greater than 3%, greater than 5%, greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, or greater than 50% relative to the dsRNA of the invention in isolation. In certain embodiments, a composition is deemed to consist essentially of a dsRNA of the invention even if more dramatic reduction of Glycolate Oxidase inhibitory activity (e.g., 80% reduction, 90% reduction, etc. in efficacy, duration and/or potency) occurs in the presence of additional components or modifications, yet where Glycolate Oxidase inhibitory activity is not significantly elevated (e.g., observed levels of Glycolate Oxidase inhibitory activity are within 10% those observed for the isolated dsRNA of the invention) in the presence of additional components and/or modifications.

[0137] As used herein, the term “nucleic acid” refers to deoxyribonucleotides, ribonucleotides, or modified nucleotides, and polymers thereof in single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs) and unlocked nucleic acids (UNAs; see, e.g., Jensen et al. Nucleic Acids Symposium Series 52: 133-4), and derivatives thereof.

[0138] As used herein, “nucleotide” is used as recognized in the art to include those with 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, e.g., 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, Nucleic Acids Res. 22:2183, 1994. Some of the non-limiting examples of base modifications that can be introduced into nucleic acid molecules include, hypoxanthine, 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., Biochemistry 35:14090, 1996; 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.

[0139] As used herein, “modified nucleotide” refers to a nucleotide that has one or more modifications to the nucleoside, the nucleobase, pentose ring, or phosphate group. For example, modified nucleotides exclude ribonucleotides containing adenosine monophosphate, guanosine monophosphate, uridine monophosphate, and cytidine monophosphate and deoxyribonucleotides containing deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate, and deoxycytidine monophosphate. Modifications include those naturally occurring that result from modification by enzymes that modify nucleotides, such as methyltransferases. Modified nucleotides also include synthetic or non-naturally occurring nucleotides. Synthetic or non-naturally occurring modifications in nucleotides include those with 2′ modifications, e.g., 2′-methoxyethoxy, 2′-fluoro, 2′-allyl, 2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH₂—O-2′-bridge, 4′-(CH₂)₂—O-2′-bridge, 2′-LNA or other bicyclic or “bridged” nucleoside analog, and 2′-O—(N-methylcarbamate) or those comprising base analogs. In connection with 2′-modified nucleotides as described for the present disclosure, by “amino” is meant 2′-NH₂ or 2′-O—NH₂, which can be modified or unmodified. Such modified groups are described, e.g., in Eckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S. Pat. No. 6,248,878. “Modified nucleotides” of the instant invention can also include nucleotide analogs as described above.

[0140] In reference to the nucleic acid molecules of the present disclosure, modifications may exist upon these agents in patterns on one or both strands of the double stranded ribonucleic acid (dsRNA). As used herein, “alternating positions” refers to a pattern where every other nucleotide is a modified nucleotide or there is an unmodified nucleotide (e.g., an unmodified ribonucleotide) between every modified nucleotide over a defined length of a strand of the dsRNA (e.g., 5′-MNMNMN-3′; 3′-MNMNMN-5′; where M is a modified nucleotide and N is an unmodified nucleotide). The modification pattern starts from the first nucleotide position at either the 5′ or 3′ terminus according to a position numbering convention, e.g., as described herein (in certain embodiments, position 1 is designated in reference to the terminal residue of a strand following a projected Dicer cleavage event of a DsiRNA agent of the invention; thus, position 1 does not always constitute a 3′ terminal or 5′ terminal residue of a pre-processed agent of the invention). The pattern of modified nucleotides at alternating positions may run the full length of the strand, but in certain embodiments includes at least 4, 6, 8, 10, 12, 14 nucleotides containing at least 2, 3, 4, 5, 6 or 7 modified nucleotides, respectively. As used herein, “alternating pairs of positions” refers to a pattern where two consecutive modified nucleotides are separated by two consecutive unmodified nucleotides over a defined length of a strand of the dsRNA (e.g., 5′-MMNNMMNNMMNN-3′; 3′-MMNNMMNNMMNN-5′; where M is a modified nucleotide and N is an unmodified nucleotide). The modification pattern starts from the first nucleotide position at either the 5′ or 3′ terminus according to a position numbering convention such as those described herein. The pattern of modified nucleotides at alternating positions may run the full length of the strand, but preferably includes at least 8, 12, 16, 20, 24, 28 nucleotides containing at least 4, 6, 8, 10, 12 or 14 modified nucleotides, respectively. It is emphasized that the above modification patterns are exemplary and are not intended as limitations on the scope of the invention.

[0141] As used herein, “base analog” refers to a heterocyclic moiety which is located at the 1′ position of a nucleotide sugar moiety in a modified nucleotide that can be incorporated into a nucleic acid duplex (or the equivalent position in a nucleotide sugar moiety substitution that can be incorporated into a nucleic acid duplex). In the dsRNAs of the invention, a base analog is generally either a purine or pyrimidine base excluding the common bases guanine (G), cytosine (C), adenine (A), thymine (T), and uracil (U). Base analogs can duplex with other bases or base analogs in dsRNAs. Base analogs include those useful in the compounds and methods of the invention, e.g., those disclosed in U.S. Pat. Nos. 5,432,272 and 6,001,983 to Benner and US Patent Publication No. 20080213891 to Manoharan, which are herein incorporated by reference. Non-limiting examples of bases include hypoxanthine (I), xanthine (X), 3β-D-ribofuranosyl-(2,6-diaminopyrimidine) (K), 3-β-D-ribofuranosyl-(1-methyl-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione) (P), iso-cytosine (iso-C), iso-guanine (iso-G), 1-β-D-ribofuranosyl-(5-nitroindole), 1-β-D-ribofuranosyl-(3-nitropyrrole), 5-bromouracil, 2-aminopurine, 4-thio-dT, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa), 2-amino-6-(2-thienyl)purine (S), 2-oxopyridine (Y), difluorotolyl, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, 3-methyl isocarbostyrilyl, 5-methyl isocarbostyrilyl, and 3-methyl-7-propynyl isocarbostyrilyl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidizopyridinyl, 9-methyl-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-propynyl isocarbostyrilyl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenzyl, tetracenyl, pentacenyl, and structural derivates thereof (Schweitzer et al., J. Org. Chem., 59:7238-7242 (1994); Berger et al., Nucleic Acids Research, 28(15):2911-2914 (2000); Moran et al., J. Am. Chem. Soc., 119:2056-2057 (1997); Morales et al., J. Am. Chem. Soc., 121:2323-2324 (1999); Guckian et al., J. Am. Chem. Soc., 118:8182-8183 (1996); Morales et al., J. Am. Chem. Soc., 122(6):1001-1007 (2000); McMinn et al., J. Am. Chem. Soc., 121:11585-11586 (1999); Guckian et al., J. Org. Chem., 63:9652-9656 (1998); Moran et al., Proc. Natl. Acad. Sci., 94:10506-10511 (1997); Das et al., J. Chem. Soc., Perkin Trans., 1:197-206 (2002); Shibata et al., J. Chem. Soc., Perkin Trans., 1: 1605-1611 (2001); Wu et al., J. Am. Chem. Soc., 122(32):7621-7632 (2000); O'Neill et al., J. Org. Chem., 67:5869-5875 (2002); Chaudhuri et al., J. Am. Chem. Soc., 117:10434-10442 (1995); and U.S. Pat. No. 6,218,108.). Base analogs may also be a universal base.

[0142] As used herein, “universal base” refers to a heterocyclic moiety located at the 1′ position of a nucleotide sugar moiety in a modified nucleotide, or the equivalent position in a nucleotide sugar moiety substitution, that, when present in a nucleic acid duplex, can be positioned opposite more than one type of base without altering the double helical structure (e.g., the structure of the phosphate backbone). Additionally, the universal base does not destroy the ability of the single stranded nucleic acid in which it resides to duplex to a target nucleic acid. The ability of a single stranded nucleic acid containing a universal base to duplex a target nucleic can be assayed by methods apparent to one in the art (e.g., UV absorbance, circular dichroism, gel shift, single stranded nuclease sensitivity, etc.). Additionally, conditions under which duplex formation is observed may be varied to determine duplex stability or formation, e.g., temperature, as melting temperature (Tm) correlates with the stability of nucleic acid duplexes. Compared to a reference single stranded nucleic acid that is exactly complementary to a target nucleic acid, the single stranded nucleic acid containing a universal base forms a duplex with the target nucleic acid that has a lower Tm than a duplex formed with the complementary nucleic acid. However, compared to a reference single stranded nucleic acid in which the universal base has been replaced with a base to generate a single mismatch, the single stranded nucleic acid containing the universal base forms a duplex with the target nucleic acid that has a higher Tm than a duplex formed with the nucleic acid having the mismatched base.

[0143] Some universal bases are capable of base pairing by forming hydrogen bonds between the universal base and all of the bases guanine (G), cytosine (C), adenine (A), thymine (T), and uracil (U) under base pair forming conditions. A universal base is not a base that forms a base pair with only one single complementary base. In a duplex, a universal base may form no hydrogen bonds, one hydrogen bond, or more than one hydrogen bond with each of G, C, A, T, and U opposite to it on the opposite strand of a duplex. Preferably, the universal bases does not interact with the base opposite to it on the opposite strand of a duplex. In a duplex, base pairing between a universal base occurs without altering the double helical structure of the phosphate backbone. A universal base may also interact with bases in adjacent nucleotides on the same nucleic acid strand by stacking interactions. Such stacking interactions stabilize the duplex, especially in situations where the universal base does not form any hydrogen bonds with the base positioned opposite to it on the opposite strand of the duplex. Non-limiting examples of universal-binding nucleotides include inosine, 1-β-D-ribofuranosyl-5-nitroindole, and/or 1-β-D-ribofuranosyl-3-nitropyrrole (US Pat. Appl. Publ. No. 20070254362 to Quay et al.; Van Aerschot et al., An acyclic 5-nitroindazole nucleoside analogue as ambiguous nucleoside. Nucleic Acids Res. 1995 Nov. 11; 23(21):4363-70; Loakes et al., 3-Nitropyrrole and 5-nitroindole as universal bases in primers for DNA sequencing and PCR. Nucleic Acids Res. 1995 Jul. 11; 23(13):2361-6; Loakes and Brown, 5-Nitroindole as an universal base analogue. Nucleic Acids Res. 1994 Oct. 11; 22(20):4039-43).

[0144] As used herein, “loop” refers to a structure formed by a single strand of a nucleic acid, in which complementary regions that flank a particular single stranded nucleotide region hybridize in a way that the single stranded nucleotide region between the complementary regions is excluded from duplex formation or Watson-Crick base pairing. A loop is a single stranded nucleotide region of any length. Examples of loops include the unpaired nucleotides present in such structures as hairpins, stem loops, or extended loops.

[0145] As used herein, “extended loop” in the context of a dsRNA refers to a single stranded loop and in addition 1, 2, 3, 4, 5, 6 or up to 20 base pairs or duplexes flanking the loop. In an extended loop, nucleotides that flank the loop on the 5′ side form a duplex with nucleotides that flank the loop on the 3′ side. An extended loop may form a hairpin or stem loop.

[0146] As used herein, “tetraloop” in the context of a dsRNA refers to a loop (a single stranded region) consisting of four nucleotides that forms a stable secondary structure that contributes to the stability of adjacent Watson-Crick hybridized nucleotides. Without being limited to theory, a tetraloop may stabilize an adjacent Watson-Crick base pair by stacking interactions. In addition, interactions among the four nucleotides in a tetraloop include but are not limited to non-Watson-Crick base pairing, stacking interactions, hydrogen bonding, and contact interactions (Cheong et al., Nature 1990 Aug. 16; 346(6285):680-2; Heus and Pardi, Science 1991 Jul. 12; 253(5016):191-4). A tetraloop confers an increase in the melting temperature (Tm) of an adjacent duplex that is higher than expected from a simple model loop sequence consisting of four random bases. For example, a tetraloop can confer a melting temperature of at least 55° C. in 10 mM NaHPO₄ to a hairpin comprising a duplex of at least 2 base pairs in length. A tetraloop may contain ribonucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof. Examples of RNA tetraloops include the UNCG family of tetraloops (e.g., UUCG), the GNRA family of tetraloops (e.g., GAAA), and the CUUG tetraloop. (Woese et al., Proc Natl Acad Sci USA. 1990 November; 87(21):8467-71; Antao et al., Nucleic Acids Res. 1991 Nov. 11; 19(21):5901-5). Examples of DNA tetraloops include the d(GNNA) family of tetraloops (e.g., d(GTTA), the d(GNRA)) family of tetraloops, the d(GNAB) family of tetraloops, the d(CNNG) family of tetraloops, the d(TNCG) family of tetraloops (e.g., d(TTCG)). (Nakano et al. Biochemistry, 41 (48), 14281-14292, 2002; SHINJI et al. Nippon Kagakkai Koen Yokoshu VOL. 78th; NO. 2; PAGE. 731 (2000).)

[0147] As used herein, the term “siRNA” refers to a double stranded nucleic acid in which each strand comprises RNA, RNA analog(s) or RNA and DNA. The siRNA comprises between 19 and 23 nucleotides or comprises 21 nucleotides. The siRNA typically has 2 bp overhangs on the 3′ ends of each strand such that the duplex region in the siRNA comprises 17-21 nucleotides, or 19 nucleotides. Typically, the antisense strand of the siRNA is sufficiently complementary with the target sequence of the Glycolate Oxidase gene/RNA.

[0148] In certain embodiments, an anti-Glycolate Oxidase DsiRNA of the instant invention possesses strand lengths of at least 25 nucleotides. Accordingly, in certain embodiments, an anti-Glycolate Oxidase DsiRNA contains one oligonucleotide sequence, a first sequence, that is at least 25 nucleotides in length and no longer than 35 or up to 50 or more nucleotides. This sequence of RNA can be between 26 and 35, 26 and 34, 26 and 33, 26 and 32, 26 and 31, 26 and 30, and 26 and 29 nucleotides in length. This sequence can be 27 or 28 nucleotides in length or 27 nucleotides in length. The second sequence of the DsiRNA agent can be a sequence that anneals to the first sequence under biological conditions, such as within the cytoplasm of a eukaryotic cell. Generally, the second oligonucleotide sequence will have at least 19 complementary base pairs with the first oligonucleotide sequence, more typically the second oligonucleotide sequence will have 21 or more complementary base pairs, or 25 or more complementary base pairs with the first oligonucleotide sequence. In one embodiment, the second sequence is the same length as the first sequence, and the DsiRNA agent is blunt ended. In another embodiment, the ends of the DsiRNA agent have one or more overhangs.

[0149] In certain embodiments, the first and second oligonucleotide sequences of the DsiRNA agent exist on separate oligonucleotide strands that can be and typically are chemically synthesized. In some embodiments, both strands are between 26 and 35 nucleotides in length. In other embodiments, both strands are between 25 and 30 or 26 and 30 nucleotides in length. In one embodiment, both strands are 27 nucleotides in length, are completely complementary and have blunt ends. In certain embodiments of the instant invention, the first and second sequences of an anti-Glycolate Oxidase DsiRNA exist on separate RNA oligonucleotides (strands). In one embodiment, one or both oligonucleotide strands are capable of serving as a substrate for Dicer. In other embodiments, at least one modification is present that promotes Dicer to bind to the double-stranded RNA structure in an orientation that maximizes the double-stranded RNA structure's effectiveness in inhibiting gene expression. In certain embodiments of the instant invention, the anti-Glycolate Oxidase DsiRNA agent is comprised of two oligonucleotide strands of differing lengths, with the anti-Glycolate Oxidase DsiRNA possessing a blunt end at the 3′ terminus of a first strand (sense strand) and a 3′ overhang at the 3′ terminus of a second strand (antisense strand). The DsiRNA can also contain one or more deoxyribonucleic acid (DNA) base substitutions.

[0150] Suitable DsiRNA compositions that contain two separate oligonucleotides can be chemically linked outside their annealing region by chemical linking groups. Many suitable chemical linking groups are known in the art and can be used. Suitable groups will not block Dicer activity on the DsiRNA and will not interfere with the directed destruction of the RNA transcribed from the target gene. Alternatively, the two separate oligonucleotides can be linked by a third oligonucleotide such that a hairpin structure is produced upon annealing of the two oligonucleotides making up the DsiRNA composition. The hairpin structure will not block Dicer activity on the DsiRNA and will not interfere with the directed destruction of the target RNA.

[0151] The dsRNA molecule can be designed such that every residue of the antisense strand is complementary to a residue in the target molecule. Alternatively, substitutions can be made within the molecule to increase stability and/or enhance processing activity of said molecule. Substitutions can be made within the strand or can be made to residues at the ends of the strand. In certain embodiments, substitutions and/or modifications are made at specific residues within a DsiRNA agent. Such substitutions and/or modifications can include, e.g., deoxy-modifications at one or more residues of positions 1, 2 and 3 when numbering from the 3′ terminal position of the sense strand of a DsiRNA agent; and introduction of 2′-O-alkyl (e.g., 2′-O-methyl) modifications at the 3′ terminal residue of the antisense strand of DsiRNA agents, with such modifications also being performed at overhang positions of the 3′ portion of the antisense strand and at alternating residues of the antisense strand of the DsiRNA that are included within the region of a DsiRNA agent that is processed to form an active siRNA agent. The preceding modifications are offered as exemplary, and are not intended to be limiting in any manner. Further consideration of the structure of preferred DsiRNA agents, including further description of the modifications and substitutions that can be performed upon the anti-Glycolate Oxidase DsiRNA agents of the instant invention, can be found below.

[0152] Where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 4, 3 or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as “fully complementary” for the purposes of the invention.

[0153] The term “double-stranded RNA” or “dsRNA”, as used herein, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary, as defined above, nucleic acid strands. The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where separate RNA molecules, such dsRNA are often referred to as siRNA (“short interfering RNA”) or DsiRNA (“Dicer substrate siRNAs”). Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′ end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop”, “short hairpin RNA” or “shRNA”. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker”. The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, a dsRNA may comprise one or more nucleotide overhangs. In addition, as used herein, “dsRNA” may include chemical modifications to ribonucleotides, internucleoside linkages, end-groups, caps, and conjugated moieties, including substantial modifications at multiple nucleotides and including all types of modifications disclosed herein or known in the art. Any such modifications, as used in an siRNA- or DsiRNA-type molecule, are encompassed by “dsRNA” for the purposes of this specification and claims.

[0154] The phrase “duplex region” refers to the region in two complementary or substantially complementary oligonucleotides that form base pairs with one another, either by Watson-Crick base pairing or other manner that allows for a duplex between oligonucleotide strands that are complementary or substantially complementary. For example, an oligonucleotide strand having 21 nucleotide units can base pair with another oligonucleotide of 21 nucleotide units, yet only 19 bases on each strand are complementary or substantially complementary, such that the “duplex region” consists of 19 base pairs. The remaining base pairs may, for example, exist as 5′ and 3′ overhangs. Further, within the duplex region, 100% complementarity is not required; substantial complementarity is allowable within a duplex region. Substantial complementarity refers to complementarity between the strands such that they are capable of annealing under biological conditions. Techniques to empirically determine if two strands are capable of annealing under biological conditions are well know in the art. Alternatively, two strands can be synthesized and added together under biological conditions to determine if they anneal to one another.

[0155] Single-stranded nucleic acids that base pair over a number of bases are said to “hybridize.” Hybridization is typically determined under physiological or biologically relevant conditions (e.g., intracellular: pH 7.2, 140 mM potassium ion; extracellular pH 7.4, 145 mM sodium ion). Hybridization conditions generally contain a monovalent cation and biologically acceptable buffer and may or may not contain a divalent cation, complex anions, e.g. gluconate from potassium gluconate, uncharged species such as sucrose, and inert polymers to reduce the activity of water in the sample, e.g. PEG. Such conditions include conditions under which base pairs can form.

[0156] Hybridization is measured by the temperature required to dissociate single stranded nucleic acids forming a duplex, i.e., (the melting temperature; Tm). Hybridization conditions are also conditions under which base pairs can form. Various conditions of stringency can be used to determine hybridization (see, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507). Stringent temperature conditions will ordinarily include temperatures of at least about 30° C., more preferably of at least about 37° C., and most preferably of at least about 42° C. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10° C. less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(° C.)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs in length, Tm(° C.)=81.5+16.6(log 10[Na+])+0.41 (% G+C)−(600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for 1×SSC=0.165 M). For example, a hybridization determination buffer is shown in Table 1.

[0157]

	TABLE 1
						To make 50 mL
	final conc.	Vender	Cat#	Lot#	m.w./Stock	solution
NaCl	100	mM	Sigma	S-5150	41K8934	5M	1	mL
KCl	80	mM	Sigma	P-9541	70K0002	74.55	0.298	g
MgCl2	8	mM	Sigma	M-1028	120K8933	1M	0.4	mL
sucrose	2%	w/v	Fisher	BP220-	907105	342.3	1	g
				212
Tris-HCl	16	mM	Fisher	BP1757-	12419	1M	0.8	mL
				500
NaH2PO4	1	mM	Sigma	S-3193	52H-	120.0	0.006	g
					029515
EDTA	0.02	mM	Sigma	E-7889	110K89271	0.5M	2	μL
H2O			Sigma	W-4502	51K2359		to 50	mL
pH = 7.0	adjust with
at 20° C.	HCl

[0158] Useful variations on hybridization conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Antisense to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0159] As used herein, “oligonucleotide strand” is a single stranded nucleic acid molecule. An oligonucleotide may comprise ribonucleotides, deoxyribonucleotides, modified nucleotides (e.g., nucleotides with 2′ modifications, synthetic base analogs, etc.) or combinations thereof. Such modified oligonucleotides can be preferred over native forms because of properties such as, for example, enhanced cellular uptake and increased stability in the presence of nucleases.

[0160] As used herein, the term “ribonucleotide” encompasses natural and synthetic, unmodified and modified ribonucleotides. Modifications include changes to the sugar moiety, to the base moiety and/or to the linkages between ribonucleotides in the oligonucleotide. As used herein, the term “ribonucleotide” specifically excludes a deoxyribonucleotide, which is a nucleotide possessing a single proton group at the 2′ ribose ring position.

[0161] As used herein, the term “deoxyribonucleotide” encompasses natural and synthetic, unmodified and modified deoxyribonucleotides. Modifications include changes to the sugar moiety, to the base moiety and/or to the linkages between deoxyribonucleotide in the oligonucleotide. As used herein, the term “deoxyribonucleotide” also includes a modified ribonucleotide that does not permit Dicer cleavage of a dsRNA agent, e.g., a 2′-O-methyl ribonucleotide, a phosphorothioate-modified ribonucleotide residue, etc., that does not permit Dicer cleavage to occur at a bond of such a residue.

[0162] As used herein, the term “PS-NA” refers to a phosphorothioate-modified nucleotide residue. The term “PS-NA” therefore encompasses both phosphorothioate-modified ribonucleotides (“PS-RNAs”) and phosphorothioate-modified deoxyribonucleotides (“PS-DNAs”).

[0163] As used herein, “Dicer” refers to an endoribonuclease in the RNase III family that cleaves a dsRNA or dsRNA-containing molecule, e.g., double-stranded RNA (dsRNA) or pre-microRNA (miRNA), into double-stranded nucleic acid fragments 19-25 nucleotides long, usually with a two-base overhang on the 3′ end. With respect to certain dsRNAs of the invention (e.g., “DsiRNAs”), the duplex formed by a dsRNA region of an agent of the invention is recognized by Dicer and is a Dicer substrate on at least one strand of the duplex. Dicer catalyzes the first step in the RNA interference pathway, which consequently results in the degradation of a target RNA. The protein sequence of human Dicer is provided at the NCBI database under accession number NP_085124, hereby incorporated by reference.

[0164] Dicer “cleavage” can be determined as follows (e.g., see Collingwood et al., Oligonucleotides 18:187-200 (2008)). In a Dicer cleavage assay, RNA duplexes (100 pmol) are incubated in 20 μL of 20 mM Tris pH 8.0, 200 mM NaCl, 2.5 mM MgCl2 with or without 1 unit of recombinant human Dicer (Stratagene, La Jolla, Calif.) at 37° C. for 18-24 hours. Samples are desalted using a Performa SR 96-well plate (Edge Biosystems, Gaithersburg, Md.). Electrospray-ionization liquid chromatography mass spectroscopy (ESI-LCMS) of duplex RNAs pre- and post-treatment with Dicer is done using an Oligo HTCS system (Novatia, Princeton, N.J.; Hail et al., 2004), which consists of a ThermoFinnigan TSQ7000, Xcalibur data system, ProMass data processing software and Paradigm MS4 HPLC (Michrom BioResources, Auburn, Calif.). In this assay, Dicer cleavage occurs where at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even 100% of the Dicer substrate dsRNA, (i.e., 25-30 bp, dsRNA, preferably 26-30 bp dsRNA) is cleaved to a shorter dsRNA (e.g., 19-23 bp dsRNA, preferably, 21-23 bp dsRNA).

[0165] As used herein, “Dicer cleavage site” refers to the sites at which Dicer cleaves a dsRNA (e.g., the dsRNA region of a DsiRNA agent of the invention). Dicer contains two RNase III domains which typically cleave both the sense and antisense strands of a dsRNA. The average distance between the RNase III domains and the PAZ domain determines the length of the short double-stranded nucleic acid fragments it produces and this distance can vary (Macrae et al. (2006) Science 311: 195-8). As shown in FIG. 1, Dicer is projected to cleave certain double-stranded ribonucleic acids of the instant invention that possess an antisense strand having a 2 nucleotide 3′ overhang at a site between the 21^st and 22^nd nucleotides removed from the 3′ terminus of the antisense strand, and at a corresponding site between the 21^st and 22^nd nucleotides removed from the 5′ terminus of the sense strand. The projected and/or prevalent Dicer cleavage site(s) for dsRNA molecules distinct from those depicted in FIG. 1 may be similarly identified via art-recognized methods, including those described in Macrae et al. While the Dicer cleavage events depicted in FIG. 1 generate 21 nucleotide siRNAs, it is noted that Dicer cleavage of a dsRNA (e.g., DsiRNA) can result in generation of Dicer-processed siRNA lengths of 19 to 23 nucleotides in length. Indeed, in certain embodiments, a double-stranded DNA region may be included within a dsRNA for purpose of directing prevalent Dicer excision of a typically non-preferred 19mer or 20mer siRNA, rather than a 21mer.

[0166] As used herein, “overhang” refers to unpaired nucleotides, in the context of a duplex having one or more free ends at the 5′ terminus or 3′ terminus of a dsRNA. In certain embodiments, the overhang is a 3′ or 5′ overhang on the antisense strand or sense strand. In some embodiments, the overhang is a 3′ overhang having a length of between one and six nucleotides, optionally one to five, one to four, one to three, one to two, two to six, two to five, two to four, two to three, three to six, three to five, three to four, four to six, four to five, five to six nucleotides, or one, two, three, four, five or six nucleotides. “Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the dsRNA, i.e., no nucleotide overhang. For clarity, chemical caps or non-nucleotide chemical moieties conjugated to the 3′ end or 5′ end of an siRNA are not considered in determining whether an siRNA has an overhang or is blunt ended. In certain embodiments, the invention provides a dsRNA molecule for inhibiting the expression of the Glycolate Oxidase target gene in a cell or mammal, wherein the dsRNA comprises an antisense strand comprising a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of the Glycolate Oxidase target gene, and wherein the region of complementarity is less than 35 nucleotides in length, optionally 19-24 nucleotides in length or 25-30 nucleotides in length, and wherein the dsRNA, upon contact with a cell expressing the Glycolate Oxidase target gene, inhibits the expression of the Glycolate Oxidase target gene by at least 10%, 25%, or 40%.

[0167] A dsRNA of the invention comprises two RNA strands that are sufficiently complementary to hybridize to form a duplex structure. One strand of the dsRNA (the antisense strand) comprises a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of the Glycolate Oxidase target gene, the other strand (the sense strand) comprises a region which is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 80, or between 15 and 53, or between 15 and 35, optionally between 25 and 30, between 26 and 30, between 18 and 25, between 19 and 24, or between 19 and 21 base pairs in length. Similarly, the region of complementarity to the target sequence is between 15 and 35, optionally between 18 and 30, between 25 and 30, between 19 and 24, or between 19 and 21 nucleotides in length. The dsRNA of the invention may further comprise one or more single-stranded nucleotide overhang(s). It has been identified that dsRNAs comprising duplex structures of between 15 and 35 base pairs in length can be effective in inducing RNA interference, including DsiRNAs (generally of at least 25 base pairs in length) and siRNAs (in certain embodiments, duplex structures of siRNAs are between 20 and 23, and optionally, specifically 21 base pairs (Elbashir et al., EMBO 20: 6877-6888)). It has also been identified that dsRNAs possessing duplexes shorter than 20 base pairs can be effective as well (e.g., 15, 16, 17, 18 or 19 base pair duplexes). In certain embodiments, the dsRNAs of the invention can comprise at least one strand of a length of 19 nucleotides or more. In certain embodiments, it can be reasonably expected that shorter dsRNAs comprising a sequence complementary to one of the sequences of Tables 4, 6, 8 or 10, minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above and in Tables 2, 3, 5, 7 and 9. Hence, dsRNAs comprising a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides sufficiently complementary to one of the sequences of Tables 4, 6, 8 or 10, and differing in their ability to inhibit the expression of the Glycolate Oxidase target gene in an assay as described herein by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated by the invention. In one embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 5, optionally 1 to 4, in certain embodiments, 1 or 2 nucleotides. Certain dsRNA structures having at least one nucleotide overhang possess superior inhibitory properties as compared to counterparts possessing base-paired blunt ends at both ends of the dsRNA molecule.

[0168] As used herein, the term “RNA processing” refers to processing activities performed by components of the siRNA, miRNA or RNase H pathways (e.g., Drosha, Dicer, Argonaute2 or other RISC endoribonucleases, and RNaseH), which are described in greater detail below (see “RNA Processing” section below). The term is explicitly distinguished from the post-transcriptional processes of 5′ capping of RNA and degradation of RNA via non-RISC- or non-RNase H-mediated processes. Such “degradation” of an RNA can take several forms, e.g. deadenylation (removal of a 3′ poly(A) tail), and/or nuclease digestion of part or all of the body of the RNA by one or more of several endo- or exo-nucleases (e.g., RNase III, RNase P, RNase T1, RNase A (1, 2, 3, 4/5), oligonucleotidase, etc.).

[0169] 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.). Indeed, design and use of the dsRNA agents of the instant invention contemplates the possibility of using such dsRNA agents not only against target RNAs of Glycolate Oxidase possessing perfect complementarity with the presently described dsRNA agents, but also against target Glycolate Oxidase RNAs possessing sequences that are, e.g., only 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% etc. complementary to said dsRNA agents. Similarly, it is contemplated that the presently described dsRNA agents of the instant invention might be readily altered by the skilled artisan to enhance the extent of complementarity between said dsRNA agents and a target Glycolate Oxidase RNA, e.g., of a specific allelic variant of Glycolate Oxidase (e.g., an allele of enhanced therapeutic interest). Indeed, dsRNA agent sequences with insertions, deletions, and single point mutations relative to the target Glycolate Oxidase sequence can also be effective for inhibition. Alternatively, dsRNA agent sequences with nucleotide analog substitutions or insertions can be effective for inhibition.

[0170] Sequence identity may be determined by sequence comparison and alignment algorithms known in the art. To determine the percent identity of two nucleic acid sequences (or of two amino acid sequences), the sequences are aligned for comparison purposes (e.g., gaps can be introduced in the first sequence or second sequence for optimal alignment). The nucleotides (or amino acid residues) at corresponding nucleotide (or amino acid) positions are then compared. When a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # of positions×100), optionally penalizing the score for the number of gaps introduced and/or length of gaps introduced.

[0171] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the alignment generated over a certain portion of the sequence aligned having sufficient identity but not over portions having low degree of identity (i.e., a local alignment). A preferred, non-limiting example of a local alignment algorithm utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the BLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.

[0172] In another embodiment, a gapped alignment, the alignment is optimized by introducing appropriate gaps, and percent identity is determined over the length of the aligned sequences (i.e., a gapped alignment). To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. In another embodiment, a global alignment the alignment is optimized by introducing appropriate gaps, and percent identity is determined over the entire length of the sequences aligned. (i.e., a global alignment). A preferred, non-limiting example of a mathematical algorithm utilized for the global comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0173] Greater than 80% sequence identity, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity, between the dsRNA antisense strand and the portion of the Glycolate Oxidase RNA sequence is preferred. Alternatively, the dsRNA may be defined functionally as a nucleotide sequence (or oligonucleotide sequence) that is capable of hybridizing with a portion of the Glycolate Oxidase RNA (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. hybridization for 12-16 hours; followed by washing). Additional preferred hybridization conditions include hybridization at 70° C. in 1×SSC or 50° C. in 1×SSC, 50% formamide followed by washing at 70° C. in 0.3×SSC or hybridization at 70° C. in 4×SSC or 50° C. in 4×SSC, 50% formamide followed by washing at 67° C. in 1×SSC. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10° C. less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(° C.)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs in length, Tm(° C.)=81.5+16.6(log 10[Na+])+0.41 (% G+C)−(600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for 1×SSC=0.165 M). Additional examples of stringency conditions for polynucleotide hybridization are provided in Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4. The length of the identical nucleotide sequences may be at least 10, 12, 15, 17, 20, 22, 25, 27 or 30 bases.

[0174] 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.

[0175] By “sense region” is meant a nucleotide sequence of a dsRNA molecule having complementarity to an antisense region of the dsRNA molecule. In addition, the sense region of a dsRNA molecule can comprise a nucleic acid sequence having homology with a target nucleic acid sequence.

[0176] By “antisense region” is meant a nucleotide sequence of a dsRNA molecule having complementarity to a target nucleic acid sequence. In addition, the antisense region of a dsRNA molecule comprises a nucleic acid sequence having complementarity to a sense region of the dsRNA molecule.

[0177] As used herein, “antisense strand” refers to a single stranded nucleic acid molecule which has a sequence complementary to that of a target RNA. When the antisense strand contains modified nucleotides with base analogs, it is not necessarily complementary over its entire length, but must at least hybridize with a target RNA.

[0178] As used herein, “sense strand” refers to a single stranded nucleic acid molecule which has a sequence complementary to that of an antisense strand. When the antisense strand contains modified nucleotides with base analogs, the sense strand need not be complementary over the entire length of the antisense strand, but must at least duplex with the antisense strand.

[0179] As used herein, “guide strand” refers to a single stranded nucleic acid molecule of a dsRNA or dsRNA-containing molecule, which has a sequence sufficiently complementary to that of a target RNA to result in RNA interference. After cleavage of the dsRNA or dsRNA-containing molecule by Dicer, a fragment of the guide strand remains associated with RISC, binds a target RNA as a component of the RISC complex, and promotes cleavage of a target RNA by RISC. As used herein, the guide strand does not necessarily refer to a continuous single stranded nucleic acid and may comprise a discontinuity, preferably at a site that is cleaved by Dicer. A guide strand is an antisense strand.

[0180] As used herein, “passenger strand” refers to an oligonucleotide strand of a dsRNA or dsRNA-containing molecule, which has a sequence that is complementary to that of the guide strand. As used herein, the passenger strand does not necessarily refer to a continuous single stranded nucleic acid and may comprise a discontinuity, preferably at a site that is cleaved by Dicer. A passenger strand is a sense strand.

[0181] By “target nucleic acid” is meant a nucleic acid sequence whose expression, level or activity is to be modulated. The target nucleic acid can be DNA or RNA. For agents that target Glycolate Oxidase, in certain embodiments, the target nucleic acid is Glycolate Oxidase (HAO1) RNA, e.g., in certain embodiments, Glycolate Oxidase (HAO1) mRNA. Glycolate Oxidase RNA target sites can also interchangeably be referenced by corresponding cDNA sequences. Levels of Glycolate Oxidase may also be targeted via targeting of upstream effectors of Glycolate Oxidase, or the effects of modulated or misregulated Glycolate Oxidase may also be modulated by targeting of molecules downstream of Glycolate Oxidase in the Glycolate Oxidase signalling pathway.

[0182] 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. 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). “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 dsRNA molecule of the invention comprises 19 to 30 (e.g., 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.

[0183] As used herein, a dsNA, e.g., DsiRNA or siRNA, having a sequence “sufficiently complementary” to a target RNA or cDNA sequence (e.g., glycolate oxidase (HAO1) mRNA) means that the dsNA has a sequence sufficient to trigger the destruction of the target RNA (where a cDNA sequence is recited, the RNA sequence corresponding to the recited cDNA sequence) by the RNAi machinery (e.g., the RISC complex) or process. For example, a dsNA that is “sufficiently complementary” to a target RNA or cDNA sequence to trigger the destruction of the target RNA by the RNAi machinery or process can be identified as a dsNA that causes a detectable reduction in the level of the target RNA in an appropriate assay of dsNA activity (e.g., an in vitro assay as described in Example 2 below), or, in further examples, a dsNA that is sufficiently complementary to a target RNA or cDNA sequence to trigger the destruction of the target RNA by the RNAi machinery or process can be identified as a dsNA that produces at least a 5%, at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 55%, at least a 60%, at least a 65%, at least a 70%, at least a 75%, at least a 80%, at least a 85%, at least a 90%, at least a 95%, at least a 98% or at least a 99% reduction in the level of the target RNA in an appropriate assay of dsNA activity. In additional examples, a dsNA that is sufficiently complementary to a target RNA or cDNA sequence to trigger the destruction of the target RNA by the RNAi machinery or process can be identified based upon assessment of the duration of a certain level of inhibitory activity with respect to the target RNA or protein levels in a cell or organism. For example, a dsNA that is sufficiently complementary to a target RNA or cDNA sequence to trigger the destruction of the target RNA by the RNAi machinery or process can be identified as a dsNA capable of reducing target mRNA levels by at least 20% at least 48 hours post-administration of said dsNA to a cell or organism. Preferably, a dsNA that is sufficiently complementary to a target RNA or cDNA sequence to trigger the destruction of the target RNA by the RNAi machinery or process is identified as a dsNA capable of reducing target mRNA levels by at least 40% at least 72 hours post-administration of said dsNA to a cell or organism, by at least 40% at least four, five or seven days post-administration of said dsNA to a cell or organism, by at least 50% at least 48 hours post-administration of said dsNA to a cell or organism, by at least 50% at least 72 hours post-administration of said dsNA to a cell or organism, by at least 50% at least four, five or seven days post-administration of said dsNA to a cell or organism, by at least 80% at least 48 hours post-administration of said dsNA to a cell or organism, by at least 80% at least 72 hours post-administration of said dsNA to a cell or organism, or by at least 80% at least four, five or seven days post-administration of said dsNA to a cell or organism.

[0184] In certain embodiments, a nucleic acid of the invention (e.g., a DsiRNA or siRNA) possesses a sequence “sufficiently complementary to hybridize” to a target RNA or cDNA sequence, thereby achieving an inhibitory effect upon the target RNA. Hybridization, and conditions available for determining whether one nucleic acid is sufficiently complementary to another nucleic acid to allow the two sequences to hybridize, is described in greater detail below.

[0185] As will be clear to one of ordinary skill in the art, “sufficiently complementary” (contrasted with, e.g., “100% complementary”) allows for one or more mismatches to exist between a dsNA of the invention and the target RNA or cDNA sequence (e.g., glycolate oxidase (HAO1) mRNA), provided that the dsNA possesses complementarity sufficient to trigger the destruction of the target RNA by the RNAi machinery (e.g., the RISC complex) or process. In certain embodiments, a “sufficiently complementary” dsNA of the invention can harbor one, two, three or even four or more mismatches between the dsNA sequence and the target RNA or cDNA sequence (e.g., in certain such embodiments, the antisense strand of the dsRNA harbors one, two, three, four, five or even six or more mismatches when aligned with the target RNA or cDNA sequence for maximum complementarity). Additional consideration of the preferred location of such mismatches within certain dsRNAs of the instant invention is considered in greater detail below.

[0186] In one embodiment, dsRNA molecules of the invention that down regulate or reduce Glycolate Oxidase gene expression are used for treating, preventing or reducing Glycolate Oxidase-related diseases or disorders (e.g., PH1) in a subject or organism.

[0187] In one embodiment of the present invention, each sequence of a DsiRNA molecule of the invention is independently 25 to 35 nucleotides in length, in specific embodiments 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides in length. In another embodiment, the DsiRNA duplexes of the invention independently comprise 25 to 30 base pairs (e.g., 25, 26, 27, 28, 29, or 30). In another embodiment, one or more strands of the DsiRNA molecule of the invention independently comprises 19 to 35 nucleotides (e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35) that are complementary to a target (Glycolate Oxidase) nucleic acid molecule. In certain embodiments, a DsiRNA molecule of the invention possesses a length of duplexed nucleotides between 25 and 66 nucleotides, optionally between 25 and 49 nucleotides in length (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66 nucleotides in length; optionally, all such nucleotides base pair with cognate nucleotides of the opposite strand). In related embodiments, a dsNA of the invention possesses strand lengths that are, independently, between 19 and 66 nucleotides in length, optionally between 25 and 53 nucleotides in length, e.g., 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 or 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66 nucleotides in length. In certain embodiments, one strand length is 19-35 nucleotides in length, while the other strand length is 30-66 nucleotides in length and at least one strand has a 5′ overhang of at least 5 nucleotides in length relative to the other strand. In certain related embodiments, the 3′ end of the first strand and the 5′ end of the second strand form a structure that is a blunt end or a 1-6 nucleotide 3′ overhang, while the 5′ end of the first strand forms a 5-35 nucleotide overhang with respect to the 3′ end of the second strand. Optionally, between one and all nucleotides of the 5-35 nucleotide overhang are modified nucleotides (optionally, deoxyribonucleotides and/or modified ribonucleotides).

[0188] In some embodiments, a dsNA of the invention has a first or second strand that has at least 8 contiguous ribonucleotides. In certain embodiments, a dsNA of the invention has 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or more (e.g., 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 26, or more, up to the full length of the strand) ribonucleotides, optionally including modified ribonucleotides (2′-O-methyl ribonucleotides, phosphorothioate linkages, etc.). In certain embodiments, the ribonucleotides or modified ribonucleotides are contiguous.

[0189] In certain embodiments of the invention, tetraloop- and modified nucleotide-containing dsNAs are contemplated as described, e.g., in US 2011/0288147. In certain such embodiments, a dsNA of the invention possesses a first strand and a second strand, where the first strand and the second strand form a duplex region of 19-25 nucleotides in length, wherein the first strand comprises a 3′ region that extends beyond the first strand-second strand duplex region and comprises a tetraloop, and the dsNA comprises a discontinuity between the 3′ terminus of the first strand and the 5′ terminus of the second strand. Optionally, the discontinuity is positioned at a projected dicer cleavage site of the tetraloop-containing dsNA. It is contemplated that, as for any of the other duplexed oligonucleotides of the invention, tetraloop-containing duplexes of the invention can possess any range of modifications disclosed herein or otherwise known in the art, including, e.g., 2′-O-methyl, 2′-fluoro, inverter basic, GalNAc moieties, etc.

[0190] In certain embodiments, a dsNA comprising a first strand and a second strand, each strand, independently, having a 5′ terminus and a 3′ terminus, and having, independently, respective strand lengths of 25-53 nucleotides in length, is sufficiently highly modified (e.g., at least 10% or more, at least 20% or more, at least 30% or more, at least 40% or more, at least 50% or more, at least 60% or more, at least 70% or more, at least 80% or more, at least 90% or more, at least 95% or more residues of one and/or both strands are modified such that dicer cleavage of the dsNA is prevented (optionally, modified residues occur at and/or flanking one or all predicted dicer cleavage sites of the dsNA). Such non-dicer-cleaved dsNAs retain glycolate oxidase (HAO1) inhibition activity and are optionally cleaved by non-dicer nucleases to yield, e.g., 15-30, or in particular embodiments, 19-23 nucleotide strand length dsNAs capable of inhibiting glycolate oxidase (HAO1) in a mammalian cell. In certain related embodiments, dsNAs possessing sufficiently extensive modification to block dicer cleavage of such dsNAs optionally possess regions of unmodified nucleotide residues (e.g., one or two or more consecutive nucleotides, forming a “gap” or “window” in a modification pattern) that allow for and/or promote cleavage of such dsNAs by non-Dicer nucleases. In other embodiments, Dicer-cleaved dsNAs of the invention can include extensive modification patterns that possess such “windows” or “gaps” in modification such that Dicer cleavage preferentially occurs at such sites (as compared to heavily modified regions within such dsNAs).

[0191] In certain embodiments of the present invention, an oligonucleotide is provided (optionally, as a free antisense oligonucleotide or as an oligonucleotide of a double-stranded or other multiple-stranded structure) that includes a sequence complementary to the HAO1 target as described elsewhere herein and that is 15 to 80 nucleotides in length, e.g., in specific embodiments 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 nucleotides in length.

[0192] In certain additional embodiments of the present invention, each oligonucleotide of a DsiRNA molecule of the invention is independently 25 to 53 nucleotides in length, in specific embodiments 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, 50, 51, 52 or 53 nucleotides in length. For DsiRNAs possessing a strand that exceeds 30 nucleotides in length, available structures include those where only one strand exceeds 30 nucleotides in length (see, e.g., U.S. Pat. No. 8,349,809), or those where both strands exceed 30 nucleotides in length (see, e.g., WO 2010/080129). Stabilizing modifications (e.g., 2′-O-Methyl, phosphorothioate, deoxyribonucleotides, including dNTP base pairs, 2′-F, etc.) can be incorporated within any double stranded nucleic acid of the invention, and can be used in particular, and optionally in abundance, especially within DsiRNAs possessing one or both strands exceeding 30 nucleotides in length. While the guide strand of a double stranded nucleic acid of the invention must possess a sequence of, e.g., 15, 16, 17, 18 or 19 nucleotides that are complementary to a target RNA (e.g., mRNA), additional sequence(s) of the guide strand need not be complementary to the target RNA. The end structures of double stranded nucleic acids possessing at least one strand length in excess of 30 nucleotides can also be varied while continuing to yield functional dsNAs—e.g. the 5′ end of the guide strand and the 3′ end of the passenger strand may form a 5′-overhang, a blunt end or a 3′ overhang (for certain dsNAs, e.g., “single strand extended” dsNAs, the length of such a 5′ or 3′ overhang can be 1-4, 1-5, 1-6, 1-10, 1-15, 1-20 or even 1-25 or more nucleotides); similarly, the 3′ end of the guide strand and the 5′ end of the passenger strand may form a 5′-overhang, a blunt end or a 3′ overhang (for certain dsNAs, e.g., “single strand extended” dsNAs, the length of such a 5′ or 3′ overhang can be 1-4, 1-5, 1-6, 1-10, 1-15, 1-20 or even 1-25 or more nucleotides). In certain embodiments, the 5′ end of the passenger strand includes a 5′-overhang relative to the 3′ end of the guide strand, such that a one to fifteen or more nucleotide single strand extension exists. Optionally, such single strand extensions of the dsNAs of the invention (whether present on the passenger or the guide strand) can be modified, e.g., with phosphorothioate (PS), 2′-F, 2′-O-methyl and/or other forms of modification contemplated herein or known in the art, including conjugation to, e.g., GalNAc moieties, inverted abasic residues, etc. In some embodiments, the guide strand of a single-strand extended duplex of the invention is between 35 and 50 nucleotides in length, while the duplex presents a single-strand extension that is seven to twenty nucleotides in length. In certain embodiments, the guide strand of a duplex is between 37 and 42 nucleotides in length and the duplex possesses a 5′ single-stranded overhang of the passenger strand that is approximately five to fifteen single-stranded nucleotides in length (optionally, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotides in length). In related embodiments, the passenger strand of the duplex is about 25 to about 30 nucleotides in length. In certain embodiments, the extended region of the duplex can optionally be extensively modified, e.g., with one or more of 2′-O-methyl, 2′-Fluoro, GalNAc moieties, phosphorothioate internucleotide linkages, inverted abasic residues, etc. In certain embodiments, the length of the passenger strand is 31-49 nucleotides while the length of the guide strand is 31-53 nucleotides, optionally while the 5′ end of the guide strand forms a blunt end (optionally, a base-paired blunt end) with the 3′ end of the passenger strand, optionally, with the 3′ end of the guide strand and the 5′ end of the passenger strand forming a 3′ overhang of 1-4 nucleotides in length. Exemplary “extended” Dicer substrate structures are set forth, e.g., in US 2010/0173974 U.S. Pat. No. 8,513,207 and U.S. Pat. No. 8,349,809, both of which are incorporated herein by reference. In certain embodiments, one or more strands of the dsNA molecule of the invention independently comprises 19 to 35 nucleotides (e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35) that are complementary to a target (Glycolate Oxidase) nucleic acid molecule. In certain embodiments, a DsiRNA molecule of the invention possesses a length of duplexed nucleotides between 25 and 49 nucleotides in length (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 nucleotides in length; optionally, all such nucleotides base pair with cognate nucleotides of the opposite strand).

[0193] In a further embodiment of the present invention, each oligonucleotide of a DsiRNA molecule of the invention is independently 19 to 66 nucleotides in length, in specific embodiments 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 or 66 nucleotides in length. For dsNAs possessing a strand that exceeds 30 nucleotides in length, available structures include those where only one strand exceeds 30 nucleotides in length (see, e.g., U.S. Pat. No. 8,349,809, where an exemplary double stranded nucleic acid possesses a first oligonucleotide strand having a 5′ terminus and a 3′ terminus and a second oligonucleotide strand having a 5′ terminus and a 3′ terminus, where each of the 5′ termini has a 5′ terminal nucleotide and each of the 3′ termini has a 3′ terminal nucleotide, where the first strand (or the second strand) is 25-30 nucleotide residues in length, where starting from the 5′ terminal nucleotide (position 1) positions 1 to 23 of the first strand (or the second strand) include at least 8 ribonucleotides; the second strand (or the first strand) is 36-66 nucleotide residues in length and, starting from the 3′ terminal nucleotide, includes at least 8 ribonucleotides in the positions paired with positions 1-23 of the first strand to form a duplex; where at least the 3′ terminal nucleotide of the second strand (or the first strand) is unpaired with the first strand (or the second strand), and up to 6 consecutive 3′ terminal nucleotides are unpaired with the first strand (or the second strand), thereby forming a 3′ single stranded overhang of 1-6 nucleotides; where the 5′ terminus of the second strand (or the first strand) includes from 10-30 consecutive nucleotides which are unpaired with the first strand (or the second strand), thereby forming a 10-30 nucleotide single stranded 5′ overhang; where at least the first strand (or the second strand) 5′ terminal and 3′ terminal nucleotides are base paired with nucleotides of the second strand (or first strand) when the first and second strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between the first and second strands; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell), or those where both strands exceed 30 nucleotides in length (see, e.g., U.S. Pat. No. 8,513,207, where an exemplary double stranded nucleic acid (dsNA) possesses a first oligonucleotide strand having a 5′ terminus and a 3′ terminus and a second oligonucleotide strand having a 5′ terminus and a 3′ terminus, where the first strand is 31 to 49 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5′ terminus of the first strand, positions 1 to 23 of the first strand are ribonucleotides; the second strand is 31 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that base pair with the ribonucleotides of positions 1 to 23 of the first strand to form a duplex; the 5′ terminus of the first strand and the 3′ terminus of said second strand form a blunt end or a 1-4 nucleotide 3′ overhang; the 3′ terminus of the first strand and the 5′ terminus of said second strand form a duplexed blunt end, a 5′ overhang or a 3′ overhang; optionally, at least one of positions 24 to the 3′ terminal nucleotide residue of the first strand is a deoxyribonucleotide, optionally, that base pairs with a deoxyribonucleotide of said second strand; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell).

[0194] In certain embodiments, an active dsNA of the invention can possess a 5′ overhang of the first strand (optionally, the passenger strand) with respect to the second strand (optionally, the guide strand) of 2-50 nucleotides (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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) or more in length. In related embodiments, the duplex region formed by the first and second strands of such a dsNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more base pairs in length. The 5′ overhang “extended” region of the first strand is optionally modified at one or more residues (optionally, at alternating residues, all residues, or any other selection of residues).

[0195] In certain embodiments, an active dsNA of the invention can possess a 3′ overhang of the first strand (optionally, the passenger strand) with respect to the second strand (optionally, the guide strand) of 2-50 nucleotides (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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) or more in length. In related embodiments, the duplex region formed by the first and second strands of such a dsNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more base pairs in length. The 3′ overhang “extended” region of the first strand is optionally modified at one or more residues (optionally, at alternating residues, all residues, or any other selection of residues).

[0196] In additional embodiments, an active dsNA of the invention can possess a 5′ overhang of the second strand (optionally, the guide strand) with respect to the first strand (optionally, the passenger strand) of 2-50 nucleotides (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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) or more in length. In related embodiments, the duplex region formed by the first and second strands of such a dsNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more base pairs in length. The 5′ overhang “extended” region of the second strand is optionally modified at one or more residues (optionally, at alternating residues, all residues, or any other selection of residues).

[0197] In further embodiments, an active dsNA of the invention can possess a 3′ overhang of the second strand (optionally, the guide strand) with respect to the first strand (optionally, the passenger strand) of 2-50 nucleotides (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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) or more in length. In related embodiments, the duplex region formed by the first and second strands of such a dsNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more base pairs in length. The 3′ overhang “extended” region of the second strand is optionally modified at one or more residues (optionally, at alternating residues, all residues, or any other selection of residues).

[0198] In another embodiment of the present invention, each sequence of a DsiRNA molecule of the invention is independently 25 to 35 nucleotides in length, in specific embodiments 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides in length. In another embodiment, the DsiRNA duplexes of the invention independently comprise 25 to 30 base pairs (e.g., 25, 26, 27, 28, 29, or 30). In another embodiment, one or more strands of the DsiRNA molecule of the invention independently comprises 19 to 35 nucleotides (e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35) that are complementary to a target (HAO1) nucleic acid molecule. In certain embodiments, a DsiRNA molecule of the invention possesses a length of duplexed nucleotides between 25 and 34 nucleotides in length (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 nucleotides in length; optionally, all such nucleotides base pair with cognate nucleotides of the opposite strand). (Exemplary DsiRNA molecules of the invention are shown in FIG. 1, and below.)

[0199] In certain embodiments, at least 10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more of the nucleotide residues of a nucleic acid of the instant invention are modified residues. For a dsNA of the invention, at least 10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more of the nucleotide residues of the first strand are modified residues. Additionally and/or alternatively for a dsNA of the invention, at least 10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more of the nucleotide residues of the second strand are modified residues. For the dsNAs of the invention, modifications of both duplex (double-stranded) regions and overhang (single-stranded) regions are contemplated. Thus, in certain embodiments, at least 10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more (e.g., all) duplex nucleotide residues are modified residues. Additionally and/or alternatively, at least 10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more (e.g., all) overhang nucleotide residues of one or both strands are modified residues. Optionally, the modifications of the dsNAs of the invention do not include an inverted abasic (e.g., inverted deoxy abasic) or inverted dT end-protecting group. Alternatively, a dsNA of the invention includes a terminal cap moiety (e.g., an inverted deoxy abasic and/or inverted dT end-protecting group). Optionally, such a terminal cap moiety is located at the 5′ end, at the 3′ end, or at both the 5′ end and the 3′ end of the first strand, of the second strand, or of both first and second strands.

[0200] 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. Within certain aspects, the term “cell” refers specifically to mammalian cells, such as human cells, that contain one or more isolated dsRNA molecules of the present disclosure. In particular aspects, a cell processes dsRNAs or dsRNA-containing molecules resulting in RNA interference of target nucleic acids, and contains proteins and protein complexes required for RNAi, e.g., Dicer and RISC.

[0201] In certain embodiments, dsRNAs of the invention are Dicer substrate siRNAs (“DsiRNAs”). DsiRNAs can possess certain advantages as compared to inhibitory nucleic acids that are not dicer substrates (“non-DsiRNAs”). Such advantages include, but are not limited to, enhanced duration of effect of a DsiRNA relative to a non-DsiRNA, as well as enhanced inhibitory activity of a DsiRNA as compared to a non-DsiRNA (e.g., a 19-23mer siRNA) when each inhibitory nucleic acid is suitably formulated and assessed for inhibitory activity in a mammalian cell at the same concentration (in this latter scenario, the DsiRNA would be identified as more potent than the non-DsiRNA). Detection of the enhanced potency of a DsiRNA relative to a non-DsiRNA is often most readily achieved at a formulated concentration (e.g., transfection concentration of the dsRNA) that results in the DsiRNA eliciting approximately 30-70% knockdown activity upon a target RNA (e.g., a mRNA). For active DsiRNAs, such levels of knockdown activity are most often achieved at in vitro mammalian cell DsiRNA transfection concentrations of 1 nM or less of as suitably formulated, and in certain instances are observed at DsiRNA transfection concentrations of 200 pM or less, 100 pM or less, 50 pM or less, 20 pM or less, 10 pM or less, 5 pM or less, or even 1 pM or less. Indeed, due to the variability among DsiRNAs of the precise concentration at which 30-70% knockdown of a target RNA is observed, construction of an IC₅₀ curve via assessment of the inhibitory activity of DsiRNAs and non-DsiRNAs across a range of effective concentrations is a preferred method for detecting the enhanced potency of a DsiRNA relative to a non-DsiRNA inhibitory agent.

[0202] In certain embodiments, a DsiRNA (in a state as initially formed, prior to dicer cleavage) is more potent at reducing Glycolate Oxidase target gene expression in a mammalian cell than a 19, 20, 21, 22 or 23 base pair sequence that is contained within it. In certain such embodiments, a DsiRNA prior to dicer cleavage is more potent than a 19-21mer contained within it. Optionally, a DsiRNA prior to dicer cleavage is more potent than a 19 base pair duplex contained within it that is synthesized with symmetric dTdT overhangs (thereby forming a siRNA possessing 21 nucleotide strand lengths having dTdT overhangs). In certain embodiments, the DsiRNA is more potent than a 19-23mer siRNA (e.g., a 19 base pair duplex with dTdT overhangs) that targets at least 19 nucleotides of the 21 nucleotide target sequence that is recited for a DsiRNA of the invention (without wishing to be bound by theory, the identity of a such a target site for a DsiRNA is identified via identification of the Ago2 cleavage site for the DsiRNA; once the Ago2 cleavage site of a DsiRNA is determined for a DsiRNA, identification of the Ago2 cleavage site for any other inhibitory dsRNA can be performed and these Ago2 cleavage sites can be aligned, thereby determining the alignment of projected target nucleotide sequences for multiple dsRNAs). In certain related embodiments, the DsiRNA is more potent than a 19-23mer siRNA that targets at least 20 nucleotides of the 21 nucleotide target sequence that is recited for a DsiRNA of the invention. Optionally, the DsiRNA is more potent than a 19-23mer siRNA that targets the same 21 nucleotide target sequence that is recited for a DsiRNA of the invention. In certain embodiments, the DsiRNA is more potent than any 21mer siRNA that targets the same 21 nucleotide target sequence that is recited for a DsiRNA of the invention. Optionally, the DsiRNA is more potent than any 21 or 22mer siRNA that targets the same 21 nucleotide target sequence that is recited for a DsiRNA of the invention. In certain embodiments, the DsiRNA is more potent than any 21, 22 or 23mer siRNA that targets the same 21 nucleotide target sequence that is recited for a DsiRNA of the invention. As noted above, such potency assessments are most effectively performed upon dsRNAs that are suitably formulated (e.g., formulated with an appropriate transfection reagent) at a concentration of 1 nM or less. Optionally, an IC₅₀ assessment is performed to evaluate activity across a range of effective inhibitory concentrations, thereby allowing for robust comparison of the relative potencies of dsRNAs so assayed.

[0203] The dsRNA molecules of the invention are added directly, or can be complexed with lipids (e.g., 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 direct dermal application, transdermal application, or injection, with or without their incorporation in biopolymers. In particular embodiments, the nucleic acid molecules of the invention comprise sequences shown in FIG. 1, and the below exemplary structures. Examples of such nucleic acid molecules consist essentially of sequences defined in these figures and exemplary structures. Furthermore, where such agents are modified in accordance with the below description of modification patterning of DsiRNA agents, chemically modified forms of constructs described in FIG. 1, and the below exemplary structures can be used in all uses described for the DsiRNA agents of FIG. 1, and the below exemplary structures.

[0204] In another aspect, the invention provides mammalian cells containing one or more dsRNA molecules of this invention. The one or more dsRNA molecules can independently be targeted to the same or different sites.

[0205] By “RNA” is meant a molecule comprising at least one, and preferably at least 4, 8 and 12 ribonucleotide residues. The at least 4, 8 or 12 RNA residues may be contiguous. 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 dsRNA 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.

[0206] 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 dsRNA agents of the invention can be administered. A subject can be a mammal or mammalian cells, including a human or human cells.

[0207] The phrase “pharmaceutically acceptable carrier” refers to a carrier for the administration of a therapeutic agent. Exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract. The pharmaceutically acceptable carrier of the disclosed dsRNA compositions may be micellar structures, such as a liposomes, capsids, capsoids, polymeric nanocapsules, or polymeric microcapsules.

[0208] Polymeric nanocapsules or microcapsules facilitate transport and release of the encapsulated or bound dsRNA into the cell. They include polymeric and monomeric materials, especially including polybutylcyanoacrylate. A summary of materials and fabrication methods has been published (see Kreuter, 1991). The polymeric materials which are formed from monomeric and/or oligomeric precursors in the polymerization/nanoparticle generation step, are per se known from the prior art, as are the molecular weights and molecular weight distribution of the polymeric material which a person skilled in the field of manufacturing nanoparticles may suitably select in accordance with the usual skill.

[0209] Various methodologies of the instant invention include step that involves comparing a value, level, feature, characteristic, property, etc. to a “suitable control”, referred to interchangeably herein as an “appropriate control”. A “suitable control” or “appropriate control” is a control or standard familiar to one of ordinary skill in the art useful for comparison purposes. In one embodiment, a “suitable control” or “appropriate control” is a value, level, feature, characteristic, property, etc. determined prior to performing an RNAi methodology, as described herein. For example, a transcription rate, mRNA level, translation rate, protein level, biological activity, cellular characteristic or property, genotype, phenotype, etc. can be determined prior to introducing an RNA silencing agent (e.g., DsiRNA) of the invention into a cell or organism. In another embodiment, a “suitable control” or “appropriate control” is a value, level, feature, characteristic, property, etc. determined in a cell or organism, e.g., a control or normal cell or organism, exhibiting, for example, normal traits. In yet another embodiment, a “suitable control” or “appropriate control” is a predefined value, level, feature, characteristic, property, etc.

[0210] The term “in vitro” has its art recognized meaning, e.g., involving purified reagents or extracts, e.g., cell extracts. The term “in vivo” also has its art recognized meaning, e.g., involving living cells, e.g., immortalized cells, primary cells, cell lines, and/or cells in an organism.

[0211] “Treatment”, or “treating” as used herein, is defined as the application or administration of a therapeutic agent (e.g., a dsRNA agent or a vector or transgene encoding same) to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disorder with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, or symptoms of the disease or disorder. The term “treatment” or “treating” is also used herein in the context of administering agents prophylactically. The term “effective dose” or “effective dosage” is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term “therapeutically effective dose” is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The term “patient” includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.

Structures of Anti-Glycolate Oxidase DsiRNA Agents

[0212] In certain embodiments, the anti-Glycolate Oxidase DsiRNA agents of the invention can have the following structures:

[0213] In one such embodiment, the DsiRNA comprises:

[0214]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA and “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers. In a related embodiment, the DsiRNA comprises:

[0215]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, and “D”=DNA. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand.

[0216] DsiRNAs of the invention can carry a broad range of modification patterns (e.g., 2′-O-methyl RNA patterns, e.g., within extended DsiRNA agents). Certain modification patterns of the second strand of DsiRNAs of the invention are presented below.

[0217] In one embodiment, the DsiRNA comprises:

[0218]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. In a related embodiment, the DsiRNA comprises:

[0219]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand.

[0220] In another such embodiment, the DsiRNA comprises:

[0221]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. In a related embodiment, the DsiRNA comprises:

[0222]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand.

[0223] In another such embodiment, the DsiRNA comprises:

[0224]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. In a related embodiment, the DsiRNA comprises:

[0225]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand.

[0226] In further embodiments, the DsiRNA comprises:

[0227]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. In a related embodiment, the DsiRNA comprises:

[0228]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0229]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. In a further related embodiment, the DsiRNA comprises:

[0230]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M7” or “M7” modification pattern.

[0231] In additional embodiments, the DsiRNA comprises:

[0232]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. In a related embodiment, the DsiRNA comprises:

[0233]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0234]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0235]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M6” or “M6” modification pattern.

[0236] In other embodiments, the DsiRNA comprises:

[0237]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In a related embodiment, the DsiRNA comprises:

[0238]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0239]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0240]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M5” or “M5” modification pattern.

[0241] In further embodiments, the DsiRNA comprises:

[0242]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In a related embodiment, the DsiRNA comprises:

[0243]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0244]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0245]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M4” or “M4” modification pattern.

[0246] In additional embodiments, the DsiRNA comprises:

[0247]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In a related embodiment, the DsiRNA comprises:

[0248]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0249]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0250]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M8” or “M8” modification pattern.

[0251] In other embodiments, the DsiRNA comprises:

[0252]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0253]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0254]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0255]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M3” or “M3” modification pattern.

[0256] In additional embodiments, the DsiRNA comprises:

[0257]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In a related embodiment, the DsiRNA comprises:

[0258]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0259]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0260]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M2” or “M2” modification pattern.

[0261] In further embodiments, the DsiRNA comprises:

[0262]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In a related embodiment, the DsiRNA comprises:

[0263]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0264]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0265]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M1” or “M1” modification pattern.

[0266] In additional embodiments, the DsiRNA comprises:

[0267]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0268]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0269]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0270]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M9” or “M9” modification pattern.

[0271] In other embodiments, the DsiRNA comprises:

[0272]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In a related embodiment, the DsiRNA comprises:

[0273]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0274]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0275]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M10” or “M10” modification pattern.

[0276] In further embodiments, the DsiRNA comprises:

[0277]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0278]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0279]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0280]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M11” or “M11” modification pattern.

[0281] In additional embodiments, the DsiRNA comprises:

[0282]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0283]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0284]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0285]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M12” or “M12” modification pattern.

[0286] In further embodiments, the DsiRNA comprises:

[0287]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0288]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0289]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0290]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M13” or “M13” modification pattern.

[0291] In other embodiments, the DsiRNA comprises:

[0292]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0293]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0294]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0295]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M21” or “M21” modification pattern.

[0296] In further embodiments, the DsiRNA comprises:

[0297]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0298]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0299]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0300]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M14” or “M14” modification pattern.

[0301] In additional embodiments, the DsiRNA comprises:

[0302]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0303]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0304]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0305]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M15” or “M15” modification pattern.

[0306] In further embodiments, the DsiRNA comprises:

[0307]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In a related embodiment, the DsiRNA comprises:

[0308]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0309]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0310]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M16” or “M16” modification pattern.

[0311] In additional embodiments, the DsiRNA comprises:

[0312]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0313]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0314]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0315]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M17” or “M17” modification pattern.

[0316] In further embodiments, the DsiRNA comprises:

[0317]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0318]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0319]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0320]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M18” or “M18” modification pattern.

[0321] In additional embodiments, the DsiRNA comprises:

[0322]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0323]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0324]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0325]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M19” or “M19” modification pattern.

[0326] In further embodiments, the DsiRNA comprises:

[0327]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0328]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0329]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0330]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M20” or “M20” modification pattern.

[0331] In additional embodiments, the DsiRNA comprises:

[0332]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0333]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0334]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0335]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M22” or “M22” modification pattern.

[0336] In further embodiments, the DsiRNA comprises:

[0337]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0338]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0339]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0340]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M24” or “M24” modification pattern.

[0341] In additional embodiments, the DsiRNA comprises:

[0342]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0343]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0344]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0345]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M25” or “M25” modification pattern.

[0346] In further embodiments, the DsiRNA comprises:

[0347]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0348]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0349]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0350]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M26” or “M26” modification pattern.

[0351] In additional embodiments, the DsiRNA comprises:

[0352]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0353]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0354]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0355]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M27” or “M27” modification pattern.

[0356] In further embodiments, the DsiRNA comprises:

[0357]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0358]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0359]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0360]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M28” or “M28” modification pattern.

[0361] In further embodiments, the DsiRNA comprises:

[0362]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0363]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0364]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0365]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M29” or “M29” modification pattern.

[0366] In additional embodiments, the DsiRNA comprises:

[0367]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0368]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M30” or “M30” modification pattern.

[0369] In further embodiments, the DsiRNA comprises:

[0370]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0371]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M31” or “M31” modification pattern.

[0372] In additional embodiments, the DsiRNA comprises:

[0373]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0374]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M32” or “M32” modification pattern.

[0375] In further embodiments, the DsiRNA comprises:

[0376]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0377]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0378]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0379]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M34” or “M34” modification pattern.

[0380] In additional embodiments, the DsiRNA comprises:

[0381]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0382]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0383]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0384]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M35” or “M35” modification pattern.

[0385] In further embodiments, the DsiRNA comprises:

[0386]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0387]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0388]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0389]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M37” or “M37” modification pattern.

[0390] In additional embodiments, the DsiRNA comprises:

[0391]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0392]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0393]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0394]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M38” or “M38” modification pattern.

[0395] In further embodiments, the DsiRNA comprises:

[0396]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0397]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0398]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0399]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M40” or “M40” modification pattern.

[0400] In additional embodiments, the DsiRNA comprises:

[0401]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0402]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0403]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0404]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M41” or “M41” modification pattern.

[0405] In further embodiments, the DsiRNA comprises:

[0406]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0407]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0408]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0409]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M36” or “M36” modification pattern.

[0410] In additional embodiments, the DsiRNA comprises:

[0411]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0412]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0413]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0414]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M42” or “M42” modification pattern.

[0415] In further embodiments, the DsiRNA comprises:

[0416]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0417]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0418]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0419]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M43” or “M43” modification pattern.

[0420] In additional embodiments, the DsiRNA comprises:

[0421]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0422]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0423]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0424]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M44” or “M44” modification pattern.

[0425] In further embodiments, the DsiRNA comprises:

[0426]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0427]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0428]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0429]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M45” or “M45” modification pattern.

[0430] In additional embodiments, the DsiRNA comprises:

[0431]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0432]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0433]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0434]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M46” or “M46” modification pattern.

[0435] In further embodiments, the DsiRNA comprises:

[0436]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0437]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0438]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0439]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M47” or “M47” modification pattern.

[0440] In additional embodiments, the DsiRNA comprises:

[0441]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0442]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0443]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0444]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M48” or “M48” modification pattern.

[0445] In further embodiments, the DsiRNA comprises:

[0446]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0447]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0448]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0449]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M52” or “M52” modification pattern.

[0450] In further embodiments, the DsiRNA comprises:

[0451]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0452]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0453]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0454]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M54” or “M54” modification pattern.

[0455] In additional embodiments, the DsiRNA comprises:

[0456]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0457]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0458]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0459]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M55” or “M55” modification pattern.

[0460] In further embodiments, the DsiRNA comprises:

[0461]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0462]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0463]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0464]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M56” or “M56” modification pattern.

[0465] In additional embodiments, the DsiRNA comprises:

[0466]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0467]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0468]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0469]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M57” or “M57” modification pattern.

[0470] In further embodiments, the DsiRNA comprises:

[0471]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0472]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0473]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0474]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M58” or “M58” modification pattern.

[0475] In additional embodiments, the DsiRNA comprises:

[0476]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0477]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0478]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0479]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M59” or “M59” modification pattern.

[0480] In further embodiments, the DsiRNA comprises:

[0481]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0482]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0483]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0484]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M60” or “M60” modification pattern.

[0485] In additional embodiments, the DsiRNA comprises:

[0486]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0487]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0488]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0489]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M61” or “M61” modification pattern.

[0490] In further embodiments, the DsiRNA comprises:

[0491]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0492]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0493]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0494]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M62” or “M62” modification pattern.

[0495] In additional embodiments, the DsiRNA comprises:

[0496]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0497]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0498]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0499]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M63” or “M63” modification pattern.

[0500] In further embodiments, the DsiRNA comprises:

[0501]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0502]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0503]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0504]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M64” or “M64” modification pattern.

[0505] In additional embodiments, the DsiRNA comprises:

[0506]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0507]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0508]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0509]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M65” or “M65” modification pattern.

[0510] In further embodiments, the DsiRNA comprises:

[0511]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0512]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0513]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0514]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M66” or “M66” modification pattern.

[0515] In additional embodiments, the DsiRNA comprises:

[0516]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0517]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0518]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0519]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M67” or “M67” modification pattern.

[0520] In further embodiments, the DsiRNA comprises:

[0521]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0522]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0523]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0524]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M68” or “M68” modification pattern.

[0525] In additional embodiments, the DsiRNA comprises:

[0526]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0527]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0528]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0529]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M69” or “M69” modification pattern.

[0530] In further embodiments, the DsiRNA comprises:

[0531]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0532]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0533]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0534]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M70” or “M70” modification pattern.

[0535] In additional embodiments, the DsiRNA comprises:

[0536]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0537]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0538]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0539]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M71” or “M71” modification pattern.

[0540] In further embodiments, the DsiRNA comprises:

[0541]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0542]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0543]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0544]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M72” or “M72” modification pattern.

[0545] In additional embodiments, the DsiRNA comprises:

[0546]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers and underlined residues are 2′-O-methyl RNA monomers. The top strand is the sense strand, and the bottom strand is the antisense strand. In one related embodiment, the DsiRNA comprises:

[0547]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an overhang domain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNA monomers, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0548]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0549]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M73” or “M73” modification pattern.

[0550] In additional embodiments, the DsiRNA comprises:

[0551]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. In a further related embodiment, the DsiRNA comprises:

[0552]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M7*” or “M7*” modification pattern.

[0553] In further embodiments, the DsiRNA comprises:

[0554]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0555]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M6*” or “M6*” modification pattern.

[0556] In other embodiments, the DsiRNA comprises:

[0557]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another related embodiment, the DsiRNA comprises:

[0558]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M5*” or “M5*” modification pattern.

[0559] In further embodiments, the DsiRNA comprises:

[0560]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0561]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M4*” or “M4*” modification pattern.

[0562] In additional embodiments, the DsiRNA comprises:

[0563]

	5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In a further related embodiment, the DsiRNA comprises:

[0564]

	5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
	3′-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, and “D”=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. This modification pattern is also referred to herein as the “AS-M8*” or “M8*” modification pattern.