Prion protein (PRNP) IRNA compositions and methods of use thereof

Abstract

The present disclosure relates to double-stranded ribonucleic acid (dsRNAi) agents and compositions that target the prion protein (PRNP) gene, and to methods of using the dsRNAi agents and compositions to inhibit expression of the PRNP gene, as well as to methods of treating subjects having a PRNP-related disease or disorder, e.g., prion disease.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 153,411, filed February 25, 2021, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated by reference in its entirety. The ASCII copy was created on February 17, 2022, is named 121301_13920_SL.txt, and is 185,376 bytes in size. [Background technology]

[0003] Prion diseases, or transmissible spongiform encephalopathies (TSEs), are a group of neurodegenerative disorders that affect both humans and animals. Prion diseases typically progress rapidly with rapid cognitive decline and are fatal within a few months of symptom onset. Prion diseases are caused by the cellular prion protein PrP, which is highly expressed in neurons and glial cells. C but the scrapie prion protein, or PrP, which is insoluble and relatively resistant to degradation by proteases Sc This is caused by PrP misfolding into an autocatalytic self-propagating conformer called PrP. Sc Accumulation of leads to neurodegeneration and / or neuronal damage and loss.

[0004] Prion diseases are classified into three distinct subtypes: sporadic, hereditary, and acquired, depending on how the disease is acquired. Unique mutations in the prion protein gene (PRNP) cause genetic subtypes of prion diseases, such as Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease (GSS), and fatal familial insomnia (FFI). More than 60 PRNP variants have been identified.

[0005] In patients with inherited prion diseases, mutations in the PRNP gene cause cells to carry PrP Sc The acquired subtype of prion disease is caused by the prion protein and includes mad cow disease and kuru. The sporadic subtype of prion disease is caused by unknown factors and accounts for approximately 85% of all prion diseases.

[0006] Previous studies have shown that homozygous deletion of PrP prevents prion infection in mice and goats (Bueler H., Aguzzi A., et al. Cell. 1993;73:1339-1347; Salvesen O., et al. Vet. Res. 2020;51:1), and heterozygous PrP knockout delays disease onset after prion infection (Salvesen O., et al. Vet. Res. 2020;51:1; Bueler H., et al. Mol. Med. 1994;1:19-30; Prusiner SB, et al. Proc. Natl. Acad. Sci. USA 1993;90:10608-10612; Sakaguchi S., et al. et al. J. Virol. 1995;69:7586-7592), and transgenic overexpression of PrP has been shown to enhance it (Fischer M., et al. EMBO J. 1996;15:1255-1264), providing genetic evidence of a dose-response relationship between PrP dose and disease susceptibility. Conditional knockout systems have confirmed that postnatal depletion confers a significant survival benefit (Mallucci G., et al. Science. 2003;302:871-874; Safar JG, et al. J. Gen. Virol. 2005;86:2913-2923). Knockout animals are healthy (Bueler H., et al. Nature. 1992;356:577-582; Richt JA, et al. Nat. Biotechnol. 2007;25:132-138; Benestad SL, et al. Vet. Res. 2012;43:87.) Furthermore, heterozygous inactivating mutations appear to be tolerated in humans (Minikel EV, et al. Sci. Transl. Med. 2016;8:322ra9; Minikel EV, et al. Nature. 2020;581:459-464), minimizing concerns about on-target toxicity of pharmacological PrP lowering.

[0007] Currently, there are no effective treatments for PRNP-related diseases, such as prion diseases, and all available treatments are palliative. Therefore, there remains a need for agents that can selectively and efficiently silence the PRNP gene using the cell's own RNAi machinery, have both high biological activity and in vivo stability, and can effectively inhibit the expression of the target PRNP gene. Summary of the Invention

[0008] The present disclosure provides RNAi agent compositions that effect RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the prion protein (PRNP) gene, which can be in a cell, e.g., in a cell of a subject, such as a human. The present disclosure also provides methods of using the RNAi pharmaceutical compositions of the present disclosure to inhibit expression of the PRNP gene or to treat a subject who would benefit from inhibiting or reducing expression of the PRNP gene, e.g., a subject suffering from or susceptible to a PRNP-related disease, such as a genetic prion disease, e.g., familial Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI); a sporadic prion disease, e.g., sporadic Creutzfeldt-Jakob disease, sporadic fatal insomnia (sFI), and variably proteinase-sensitive prion disease (VPSPr), or an acquired prion disease, e.g., iatrogenic CJD (iCJD), kuru, and variant CJD (vCJD).

[0009] Thus, in one aspect, the disclosure provides a double-stranded ribonucleic acid (RNAi) agent that inhibits expression of the PRNP gene, wherein the dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 by no more than 0, 1, 2, or 3 nucleotides, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO: 5 by no more than 1, 2, or 3 nucleotides, and wherein the sense strand, the antisense strand, or both the sense strand and the antisense strand are conjugated to one or more lipophilic moieties.

[0010] In certain embodiments, the sense strand comprises at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 1, and the antisense strand comprises at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 5. In certain embodiments, the sense strand comprises at least 17 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 1, and the antisense strand comprises at least 17 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 5. In certain embodiments, the sense strand comprises at least 19 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 1, and the antisense strand comprises at least 19 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 5.

[0011] In some embodiments, the nucleotide sequence of the sense strand comprises any one of the sense strand nucleotide sequences in any one of Tables 2-3.

[0012] In another aspect, the present disclosure provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting expression of the PRNP gene, wherein the RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises a region of complementarity to an mRNA encoding PRNP, wherein the region of complementarity comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by no more than 3 nucleotides (i.e., differs by 3, 2, 1, or 0 nucleotides) from any one of the antisense sequences listed in any one of Tables 2-3, and wherein the sense strand, the antisense strand, or both the sense and antisense strands are conjugated to one or more lipophilic moieties.

[0013] In certain embodiments, the antisense strand comprises a region of complementarity comprising at least 15 contiguous nucleotides of any one of the antisense sequences listed in any one of Tables 2-3. In certain embodiments, the antisense strand comprises a region of complementarity comprising at least 17 contiguous nucleotides of any one of the antisense sequences listed in any one of Tables 2-3. In certain embodiments, the antisense strand comprises a region of complementarity comprising at least 19 contiguous nucleotides of any one of the antisense sequences listed in any one of Tables 2-3. In certain embodiments, the antisense strand comprises a region of complementarity comprising at least 20 contiguous nucleotides of any one of the antisense sequences listed in any one of Tables 2-3. In certain embodiments, the antisense strand comprises a region of complementarity comprising at least 21 contiguous nucleotides of any one of the antisense sequences listed in any one of Tables 2-3. In certain embodiments, differences of thymine to uracil or uracil to thymine between aligned (compared) sequences are not counted as different nucleotides between the aligned (compared) sequences.

[0014] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of prion protein (PRNP) in a cell, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the sense strand being selected from nucleotides 502-524, 507-529, 502-529, 540-562, 566-588, 575-597, 576-598, 589-611, 575-611, 593-615, 594-616, 600-622, 593-622, 650-672, 858-880, 976-998, 1100-1122 of SEQ ID NO:1. , 1126-1148, 1220-1242, 1221-1243, 1220-1243, 1304-1326, 1328-1350, 1410-1432, 1445-1467, 1481-1503, 1532-1554, 1610-1632, 1615-1637, 1617 -1639, 1621-1643, 1610-1643, 1610-1639, 1688-1710, 1694-1714, 1830-1852, 1831-1853, 1854-1876, 1830-1853, 1872-1894, 1873-1895, 1938-1960 , 2011-2033, 2015-2037, 2031-2053, 2034-2056, 2069-2091, 2076-2098, 2077-2099, 1872-1895, 2011-2037, 2069-2099, 2079-2101, 2031-2056, 2069 -2098, 2138-2160, 2143-2165, 2158-2180, 2167-2189, 2168-2190, 2170-2192, 2174-2196, 2175-2197, 2177-2199, 2185-2207, 2189-2211, 2196-2218 , 2200-2222, 2202-2224, 2221-2243, 2222-2244, 2223-2245, 2238-2260, 2241-2263, 2242-2264, 2138-2264, 2257-2358, 2174-2245, 2174-2224, 2138 -2196, 2177-2224, 2223-2245, 2138-2189, 2177-2211, 2196-2224, 2257-2279, 2258-2280, 2335-2537, 2336-2358, 2257-2358, 2342-2364, 2397-2419,and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by no more than three nucleotides from any one of the nucleotide sequences 2398-2420, 2399-2421, 2400-2422, 2401-2423, 2404-2426, 2397-2426, 2394-2423, 2397-2421, 2399-2426, and 2398-2421, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 5, and the sense strand, the antisense strand, or both the sense strand and the antisense strand are conjugated to one or more lipophilic moieties.

[0015] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of prion protein (PRNP) in a cell, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the sense strand comprising nucleotides 530-570, 535-565, 539-561, 540-562, 555-605, 560-605, 560-600, 566-588, 567-570, 571-572, 573-574, 575-576, 577-578, 578-579, 579-580, 581-582, 583-584, 585-586, 587-588, 589-590, 589, 568-590, 569-591, 570-592, 575-597, 580-620-585-620, 589-611, 590-612, 591-613, 592-614, 593-615, 594-616, 600-650, 610-650, 613-635, 614-636, 615-637, 616-638, 617-639, 618-640, 640-680, 649-671, 65 at least one that differs by no more than three nucleotides from any one of the nucleotide sequences 0-672, 651-673, 670-700, 674-696, 675-697, 795-830, 804-826, 2325-2375, 2325-2370, 2330-2370, 2335-2357, 2336-2358, 2337-2359, 2338-2360, 2339-2361, or 2340-2362 The sense strand, the antisense strand, or both the sense and antisense strands comprise at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 5, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 5, and the sense strand, the antisense strand, or both the sense strand and the antisense strand are conjugated to one or more lipophilic moieties.

[0016] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of prion protein (PRNP) in a cell, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the sense strand comprising nucleotides 502-524, 540-562, 589-611, 594-616, 650-672, 1445-1467, 1688-1710, 2011-2033, 2031-2053, 2185-2207, 2222-2244, 2336-2358 of SEQ ID NO:1. , 2339-2361, 2399-2421 or 2258-2280, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than three nucleotides from any one of the nucleotide sequences of SEQ ID NO:5, 2339-2361, 2399-2421 or 2258-2280, and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO:5, and the sense strand, the antisense strand, or both the sense strand and the antisense strand are conjugated to one or more lipophilic moieties.

[0017] In some embodiments, the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21 contiguous nucleotides that differ by no more than three nucleotides from any one of the antisense strand nucleotide sequences of a duplex selected from the group consisting of AD-1070511, AD-1070462, AD-1070553, AD-1072048, AD-1070516 and AD-1072050, AD-1071769, AD-1071949, AD-1070444, AD-1071505, AD-1071912, AD-1071789, AD-1071265, AD-1072084, and AD-1071985.

[0018] In some embodiments, the lipophilic moiety is conjugated via a linker or carrier.

[0019] Sense strand, antisense strand, or both sense strand and antisense strand can be conjugated with one or more lipophilic moieties.In some embodiments, lipophilic moieties are conjugated to one or more internal positions in the double-stranded region of dsRNA agent, for example, one or more lipophilic moieties can be conjugated to one or more internal positions on antisense strand.In some embodiments, one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand via linker or carrier.

[0020] In some embodiments, the lipophilicity of the lipophilic moiety is greater than 0, as measured by log Kow.

[0021] In some embodiments, the hydrophobicity of the dsRNA agent is greater than 0.2, as measured by the unbound fraction in a plasma protein binding assay of the dsRNA agent. In some embodiments, the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.

[0022] In some embodiments, internal positions include all but the two most distal positions from each end of the sense or antisense strand, while in other embodiments, internal positions include all but the three most distal positions from each end of the sense or antisense strand.

[0023] In some embodiments, internal positions exclude the cleavage site region of the sense strand, for example, internal positions include all positions except positions 9-12 from the 5' end of the sense strand, or internal positions include all positions except positions 11-13 from the 3' end of the sense strand.

[0024] In some embodiments, internal positions exclude the cleavage site region of the antisense strand. In other embodiments, internal positions include all positions except positions 12-14 counting from the 5' end of the antisense strand. In some embodiments, internal positions include all positions except positions 11-13 on the sense strand counting from the 3' end and positions 12-14 on the antisense strand counting from the 5' end.

[0025] In some embodiments, the one or more lipophilic moieties are conjugated to one or more of the internal positions selected from the group consisting of positions 4-8 and 13-18 on the sense strand and positions 6-10 and 15-18 on the antisense strand, counting from the 5' end of each strand.

[0026] In some embodiments, the one or more lipophilic moieties are conjugated to one or more internal positions selected from the group consisting of positions 5, 6, 7, 15, and 17 on the sense strand and positions 15 and 17 on the antisense strand, counting from the 5' end of each strand.

[0027] In some embodiments, the positions in the double-stranded region exclude the cleavage site region of the sense strand.

[0028] In other embodiments, the sense strand is 21 nucleotides in length, the antisense strand is 23 nucleotides in length, and the lipophilic moiety is conjugated to position 20, 15, 1, 7, 6, or 2 of the sense strand or position 16 of the antisense strand.

[0029] In other embodiments, the sense strand is 21 nucleotides in length, the antisense strand is 23 nucleotides in length, and the lipophilic moiety is conjugated to position 21, 20, 15, 1, 7, 6, or 2 of the sense strand or position 16 of the antisense strand.

[0030] In some embodiments, the lipophilic moiety is an aliphatic compound, an alicyclic compound, or a polyalicyclic compound.

[0031] In some embodiments, the lipophilic moiety is selected from the group consisting of a lipid, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, a heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

[0032] In some embodiments, the lipophilic moiety is selected from the group consisting of cholesterol, lithocholic acid (LCA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), docosanoic acid (DCA), choline, vitamin A, vitamin E, retinoic acid, and alpha-tocopheryl succinate. In some embodiments, the lipophilic moiety is selected from the group consisting of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosanoic acid (DCA). In some embodiments, the lipophilic moiety is not cholesterol.

[0033] In some embodiments, the lipophilic moiety contains a saturated or unsaturated C4-C30 hydrocarbon chain and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

[0034] In some embodiments, the lipophilic moiety contains a saturated or unsaturated C6 to C18 hydrocarbon chain.

[0035] In some embodiments, the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain. In some embodiments, the saturated or unsaturated C16 hydrocarbon chain is conjugated to the 6th position, counting from the 5' end of one chain.

[0036] In some embodiments, the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotides in the internal position or double-stranded region. In some embodiments, the carrier is a cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl, or is an acyclic moiety of a serinol backbone or a diethanolamine backbone system.

[0037] In some embodiments, the lipophilic moiety is conjugated to the dsRNA agent via a linker that contains an ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide linkage, a product of a click reaction, or a carbamate.

[0038] In some embodiments, the lipophilic moiety is conjugated to a nucleobase, a sugar moiety, or an internucleoside linkage.

[0039] In some embodiments, dsRNA agent comprises at least one modified nucleotide.In some embodiments, five or less nucleotides of sense strand and five or less nucleotides of antisense strand are unmodified nucleotides.In other embodiments, all nucleotides of sense strand and all nucleotides of antisense strand comprise modification.

[0040] In some embodiments, at least one of the modified nucleotides is a deoxy-nucleotide, a 3'-terminal deoxy-thymidine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, a 2'-C-alkyl-modified nucleotide, a 2'-hydroxy-modified nucleotide, a 2'-methoxyethyl-modified nucleotide, a 2'-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl ... the terminal nucleotide is selected from the group consisting of: a vinyl-modified nucleotide, a nucleotide containing a 5'-phosphorothioate group, a nucleotide containing a 5'-methylphosphonate group, a nucleotide containing a 5' phosphate or a 5' phosphate mimic, a nucleotide containing a vinyl phosphonate, a glycol nucleic acid (GNA) or a glycol nucleic acid S-isomer (S-GNA) (e.g., a nucleotide containing an adenosine glycol nucleic acid (GNA) or a nucleotide containing a thymidine glycol nucleic acid (GNA) S-isomer), a nucleotide containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, a nucleotide containing 2'-deoxythymidine-3' phosphate, a nucleotide containing 2'-deoxyguanosine-3'-phosphate, and a terminal nucleotide linked to a cholesteryl derivative, and a dodecanoic acid bisdecylamide group, and combinations thereof.

[0041] In other embodiments, the modified nucleotide is selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, 3'-terminal deoxythymidine nucleotides (dT), locked nucleotides, abasic nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, and nucleotides containing unnatural bases.

[0042] In some embodiments, at least one of the modified nucleotides is selected from the group consisting of deoxy-nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, glycol-modified nucleotides (GNAs), and vinyl-phosphonate nucleotides; and combinations thereof.

[0043] In some embodiments, at least one of the modifications in the nucleotide is a thermally destabilizing nucleotide modification, in some embodiments, the thermally destabilizing nucleotide modification is selected from the group consisting of an abasic modification, a mismatch with the opposing nucleotide in a duplex, and a destabilizing sugar modification, a 2'-deoxy modification, an acyclic nucleotide, an unlocked nucleic acid (UNA), and a glycerol nucleic acid (GNA).

[0044] In some embodiments, the modified nucleotides include a short sequence of 3'-terminal deoxythymidine nucleotides (dT).

[0045] In some embodiments, the modifications in the nucleotides are 2'-O-methyl modifications, GNA modifications, and 2' fluoro modifications.

[0046] In some embodiments, the dsRNA agent further comprises at least one phosphorothioate internucleotide linkage. In some embodiments, the dsRNA agent comprises 6 to 8 phosphorothioate internucleotide linkages. In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3'-end of one strand. Optionally, this strand is the antisense strand. In another embodiment, this strand is the sense strand. In a related embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5'-end of one strand. Optionally, this strand is the antisense strand. In another embodiment, this strand is the sense strand. In another embodiment, the phosphorothioate or methylphosphonate internucleotide linkages are at both the 5'-end and the 3'-end of one strand. Optionally, this strand is the antisense strand. In another embodiment, this strand is the sense strand.

[0047] In some embodiments, each strand is 30 nucleotides or less in length.

[0048] In some embodiments, at least one strand comprises a 3' overhang of at least 1 nucleotide or a 3' overhang of at least 2 nucleotides.

[0049] The double-stranded region can be 15 to 30 nucleotide pairs in length, 17 to 23 nucleotide pairs in length, 17 to 25 nucleotide pairs in length, 23 to 27 nucleotide pairs in length, 19 to 21 nucleotide pairs in length, or 21 to 23 nucleotide pairs in length.

[0050] Each strand can be 19-30 nucleotides, 19-23 nucleotides, or 21-23 nucleotides.

[0051] In some embodiments, the dsRNA agent further comprises a targeting ligand that targets liver tissue. In some embodiments, the targeting ligand is a GalNAc conjugate.

[0052] In certain embodiments, the double-stranded RNAi agent further comprises a targeting ligand, eg, a hydrophilic ligand, that targets a receptor that mediates delivery to CNS tissue.

[0053] In certain embodiments, targeting ligand is C16 ligand.In one embodiment, ligand is conjugated at the 2'-position of nucleotide or modified nucleotide in sense strand or antisense strand.For example, C16 ligand can be conjugated as shown in the following structure: [ka] , wherein * represents a bond to an adjacent nucleotide, and B is a nucleobase or nucleobase analog, optionally B is adenine, guanine, cytosine, thymine, or uracil.

[0054] In some embodiments, the lipophilic moiety or targeting ligand is conjugated via a bio-cleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, and functionalized mono- or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

[0055] In some embodiments, the 3' end of the sense strand is protected via an end cap that is an amine-bearing cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.

[0056] In some embodiments, the dsRNA agent further includes a terminal chiral modification occurring at a first internucleotide linkage at the 3'-end of the antisense strand having a linking phosphorus atom in the Sp configuration, a terminal chiral modification occurring at a first internucleotide linkage at the 5'-end of the antisense strand having a linking phosphorus atom in the Rp configuration, and a terminal chiral modification occurring at a first internucleotide linkage at the 5'-end of the sense strand having a linking phosphorus atom in either the Rp or Sp configuration.

[0057] In some embodiments, the dsRNA agent further includes a terminal chiral modification occurring at the first and second internucleotide linkages at the 3'-end of the antisense strand, with the linking phosphorus atom in the Sp configuration; a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the antisense strand, with the linking phosphorus atom in the Rp configuration; and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand, with the linking phosphorus atom in either the Rp or Sp configuration.

[0058] In some embodiments, the dsRNA agent further includes a terminal chiral modification occurring at the first, second, and third internucleotide linkages at the 3'-end of the antisense strand, with the linking phosphorus atom in the Sp configuration; a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the antisense strand, with the linking phosphorus atom in the Rp configuration; and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand, with the linking phosphorus atom in either the Rp or Sp configuration.

[0059] In some embodiments, the dsRNA agent further includes a terminal chiral modification occurring at the first and second internucleotide linkages at the 3'-end of the antisense strand, with the linking phosphorus atom in the Sp configuration; a terminal chiral modification occurring at the third internucleotide linkage at the 3'-end of the antisense strand, with the linking phosphorus atom in the Rp configuration; a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the antisense strand, with the linking phosphorus atom in the Rp configuration; and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand, with the linking phosphorus atom in either the Rp or Sp configuration.

[0060] In some embodiments, the dsRNA agent further includes a terminal chiral modification occurring at the first and second internucleotide linkages at the 3'-end of the antisense strand, with the linking phosphorus atom in the Sp configuration; a terminal chiral modification occurring at the first and second internucleotide linkages at the 5'-end of the antisense strand, with the linking phosphorus atom in the Rp configuration; and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand, with the linking phosphorus atom in either the Rp or Sp configuration.

[0061] In some embodiments, the dsRNA agent further comprises a phosphate or phosphate mimic at the 5'-end of the antisense strand.In some embodiments, the phosphate mimic is 5'-vinyl phosphonate (VP).When the phosphate mimic is 5'-vinyl phosphonate (VP), the 5'-end nucleotide may have the following structure: [ka] where * indicates the position of attachment to the 5' position of the adjacent nucleotide, R is hydrogen, hydroxy, methoxy, fluoro, or another 2'-modification described herein (e.g., hydroxy or methoxy); B is a nucleobase or modified nucleobase, optionally B is adenine, guanine, cytosine, thymine, or uracil.

[0062] In some embodiments, the base pair at position 1 of the 5-terminus of the antisense strand of the duplex is an AU base pair.

[0063] In some embodiments, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

[0064] In one embodiment, the dsRNA agent includes at least one modified nucleotide.

[0065] In one embodiment, substantially all of the nucleotides in the sense strand, substantially all of the nucleotides in the antisense strand contain a modification, or substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand contain a modification.

[0066] In one embodiment, all of the nucleotides in the sense strand comprise a modification, all of the nucleotides in the antisense strand comprise a modification, or all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand comprise a modification.

[0067] In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 3'-terminal deoxythymidine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base containing nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide containing a phosphorothioate group, a nucleotide containing a methylphosphonate group, a nucleotide containing a 5'-phosphate, a nucleotide containing a 5'-phosphate mimic, a thermally destabilized nucleotide, a glycol-modified nucleotide (GNA), and a 2-O-(N-methylacetamide) modified nucleotide, and combinations thereof.

[0068] In one embodiment, the modification to the nucleotide is selected from the group consisting of LNA, glycol nucleic acid (GNA), hexitol nucleic acid (HNA), 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and glycol, and combinations thereof.

[0069] In one embodiment, at least one of the modified nucleotides is selected from the group consisting of deoxy-nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, glycol-modified nucleotides (GNAs), such as Ggn, Cgn, Tgn, or Agn, and vinyl-phosphonate nucleotides, and combinations thereof.

[0070] In another embodiment, at least one of the modifications to the nucleotides is a thermally destabilizing nucleotide modification.

[0071] In one embodiment, the thermally destabilizing nucleotide modification is selected from the group consisting of an abasic modification, a mismatch with the opposing nucleotide in the duplex, and a destabilizing sugar modification, a 2'-deoxy modification, an acyclic nucleotide, an unlocked nucleic acid (UNA), and a glycerol nucleic acid (GNA).

[0072] The double-stranded region can be 19 to 30 nucleotide pairs in length, 19 to 25 nucleotide pairs in length, 19 to 23 nucleotide pairs in length, 23 to 27 nucleotide pairs in length, or 21 to 23 nucleotide pairs in length.

[0073] In one embodiment, each strand is independently 30 nucleotides or less in length.

[0074] In one embodiment, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

[0075] The region of complementarity can be at least 17 nucleotides in length, 19-23 nucleotides in length, or 19 nucleotides in length.

[0076] In one embodiment, at least one strand comprises a 3' overhang of at least 1 nucleotide, hi another embodiment, at least one strand comprises a 3' overhang of at least 2 nucleotides.

[0077] In one embodiment, the dsRNA agent further comprises a ligand.

[0078] In one embodiment, the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent.

[0079] In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0080] In one embodiment, the ligand is one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker.

[0081] In one embodiment, the ligand is: [ka]

[0082] In one embodiment, the dsRNA agent is conjugated to a ligand as shown in the diagram below: [ka] wherein X is O or S.

[0083] In one embodiment, X is O.

[0084] In one embodiment, the dsRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

[0085] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3'-end of one strand, eg, the antisense strand or the sense strand.

[0086] In another embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5'-end of one strand, eg, the antisense strand or the sense strand.

[0087] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkages are at both the 5' and 3' ends of one strand, hi one embodiment, the strand is the antisense strand.

[0088] In one embodiment, the base pair at one position at the 5'-end of the antisense strand of the duplex is an AU base pair.

[0089] The invention also provides cells containing any of the dsRNA agents of the invention, and pharmaceutical compositions comprising any of the dsRNA agents of the invention.

[0090] A pharmaceutical composition of the invention can include a dsRNA agent in an unbuffered solution, e.g., saline or water, or a pharmaceutical composition of the invention can include a dsRNA agent in a buffered solution, e.g., containing acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof, or phosphate buffered saline (PBS).

[0091] In one aspect, the invention provides a method for inhibiting expression of the PRNP gene in a cell, the method comprising contacting the cell with any of the dsRNAs of the invention or any of the pharmaceutical compositions of the invention, thereby inhibiting expression of the PRNP gene in the cell.

[0092] In one embodiment, the cell is in a subject, eg, a human subject, eg, a subject with a PRNP-associated disease.

[0093] In certain embodiments, the PRNP-associated disease is a prion disease. In certain embodiments, the prion disease is a genetic prion disease selected from the group consisting of familial Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI). In some embodiments, the prion disease is a sporadic prion disease selected from the group consisting of sporadic Creutzfeldt-Jakob disease, sporadic fatal insomnia (sFI), and variably proteinase-sensitive prion disease (VPSPr). In another embodiment, the prion disease is an acquired prion disease selected from the group consisting of iatrogenic CJD (iCJD), kuru, and variant CJD (vCJD).

[0094] In some embodiments, contacting cells with a dsRNA agent inhibits PRNP expression by at least 50%, 60%, 70%, 80%, 90%, or 95%. In some embodiments, inhibiting PRNP expression reduces PRNP protein levels in the subject's serum by at least 50%, 60%, 70%, 80%, 90%, or 95%.

[0095] In one aspect, the invention provides a method of treating a subject having a disorder that would benefit from decreased PRNP expression, comprising administering to the subject a therapeutically effective amount of any one of the dsRNA agents of the invention or any one of the pharmaceutical compositions of the invention, thereby treating the subject having a disorder that would benefit from decreased PRNP expression.

[0096] In another aspect, the invention provides a method for preventing at least one symptom in a subject having a disorder that would benefit from reduced PRNP expression, comprising administering to the subject a prophylactically effective amount of any one of the dsRNA agents of the invention or any one of the pharmaceutical compositions of the invention, thereby preventing at least one symptom in the subject having a disorder that would benefit from reduced PRNP expression.

[0097] In some embodiments, the disorder is a PRNP-associated disease. In some embodiments, the PRNP-associated disease is a prion disease. In some embodiments, the prion disease is a genetic prion disease selected from the group consisting of familial Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI). In some embodiments, the prion disease is a sporadic prion disease selected from the group consisting of sporadic Creutzfeldt-Jakob disease, sporadic fatal insomnia (sFI), and variably proteinase-sensitive prion disease (VPSPr). In another embodiment, the prion disease is an acquired prion disease selected from the group consisting of iatrogenic CJD (iCJD), kuru, and variant CJD (vCJD).

[0098] Another aspect of the present disclosure provides a method for inhibiting expression of PRNP in a subject, the method comprising administering to the subject a therapeutically effective amount of any one of the RNAi agents of the present invention or any one of the pharmaceutical compositions of the present invention, thereby inhibiting expression of PRNP in the subject.

[0099] An additional aspect of the present disclosure provides a method for treating or preventing a PRNP-related disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of any one of the RNAi agents of the present invention or any one of the pharmaceutical compositions of the present invention, thereby treating or preventing a PRNP-related disorder in the subject.

[0100] In certain embodiments, the PRNP-associated disorder is selected from the group consisting of genetic prion diseases, such as familial Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI). In some embodiments, the prion disease is a sporadic prion disease selected from the group consisting of sporadic Creutzfeldt-Jakob disease, sporadic fatal insomnia (sFI), and variably proteinase-sensitive prion disease (VPSPr). In another embodiment, the prion disease is an acquired prion disease selected from the group consisting of iatrogenic CJD (iCJD), kuru, and variant CJD (vCJD).

[0101] In some embodiments, the subject is a human.

[0102] In some embodiments, a dsRNA agent is administered to a subject at a dose of about 0.01 mg / kg to about 50 mg / kg or about 5 mg to 1000 mg.

[0103] In some embodiments, the dsRNA agent is administered to the subject intrathecally.

[0104] In some embodiments, the dsRNA agent is administered to the subject subcutaneously.

[0105] In one embodiment, the method reduces expression of the PRNP gene in brain or spinal tissue, eg, the striatum, cortex, cerebellum, cervical vertebrae, lumbar vertebrae, or thoracic vertebrae.

[0106] In one embodiment, the method reduces expression of the PRNP gene in the brain.

[0107] In some embodiments, the methods of the present invention further comprise administering to the subject an additional agent for the treatment or prevention of a PRNP-related disorder. Exemplary additional therapeutic agents and treatments include, for example, sedatives, antidepressants, clonazepam, sodium valproate, analgesics, antiepileptics, cholinesterase inhibitors, memantine, benzodiazepines, levodopa, COMT inhibitors (e.g., tolcapone and entacapone), dopamine agonists (e.g., bromocriptine, pergolide, pramipexole, ropinirole, piribedil, cabergoline, apomorphine, and lisuride), MAO-B inhibitors (e.g., safinamide, sebelipase alfa, cefotaxime ... Treatment options include: acetaminophen, benzodiazepines (e.g., acetaminophen, benzodiazepine, benzodiazepine), ...

[0108] The invention also provides kits comprising any of the dsRNAs of the invention, or any of the pharmaceutical compositions of the invention, and optionally instructions for use.

[0109] In another embodiment, the RNAi agent is a pharmaceutically acceptable salt thereof. The "pharmaceutically acceptable salt" of each RNAi agent herein includes, but is not limited to, sodium, calcium, lithium, potassium, ammonium, magnesium, and mixtures thereof. Those skilled in the art will understand that when an RNAi agent is provided as a polycationic salt, it will have one cation per free acid group of the optionally modified phosphodiester backbone and / or any other acidic modifications (e.g., a phosphonate group at the 5' end). For example, an oligonucleotide "n" nucleotides in length contains n-1 optionally modified phosphodiesters, such that an oligonucleotide 21 nt in length can be provided as a salt with up to 20 cations (e.g., 20 sodium cations). Similarly, an RNAi agent having a 21 nt sense strand and a 23 nt antisense strand can be provided as a salt with up to 42 cations (e.g., 42 sodium cations). In the foregoing examples, when the RNAi agent also includes a 5'-terminal phosphate group or a 5'-terminal vinylphosphonate group, the RNAi agent can be provided as a salt with up to 44 cations (e.g., 44 sodium cations).

[0110] The present invention is further illustrated by the following detailed description. [Brief explanation of the drawings]

[0111] [Figure 1] Figure 1 is a graph showing PRNP mRNA levels in the right hemisphere of wild-type mice on day 28 after a single intracerebroventricular (ICV) injection of AD-1070516, an exemplary dsRNA agent targeting PRNP, at doses of 50 μg, 150 μg, or 300 μg on day 0, or control artificial CSF (aCSF). DETAILED DESCRIPTION OF THE INVENTION

[0112] The present disclosure provides RNAi compositions that perform RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the PRNP gene. The PRNP gene can be present in a cell, for example, in a subject, such as a human. The use of these iRNAs allows for the targeted degradation of the mRNA of the corresponding gene (PRNP gene) in a mammal.

[0113] The iRNAs of the present invention are designed to target the PRNP gene, including portions of the gene that are conserved in PRNP orthologs of other mammalian species. Without intending to be bound by theory, it is believed that the combination or subcombination of the above features with specific target sites or specific modifications in these iRNAs improves the efficacy, stability, potency, durability, and safety of the iRNAs of the present invention.

[0114] Accordingly, the present disclosure also provides methods of using the RNAi compositions of the present disclosure to inhibit expression of the PRNP gene or to treat a subject having a disorder that would benefit from inhibiting or reducing expression of the PRNP gene, e.g., a PRNP-related disease, e.g., a genetic prion disease, e.g., Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease (GSS), and fatal familial insomnia (FFI); an acquired prion disease, e.g., mad cow disease, iatrogenic CJD (iCJD), kuru, and variant CJD (vCJD); or a sporadic prion disease, e.g., sporadic Creutzfeldt-Jakob disease, sporadic fatal insomnia (sFI), and variably proteinase-sensitive prion disease (VPSPr).

[0115] RNAi agents of the present disclosure can be about 30 nucleotides in length or less, e.g., 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-26, 19-27, 19-28, 19-29, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-26, 19-27, 19-28, 19-25, 19-26, 19-25, 19-26, 19-27, 19-28, 19-29, 19-30 ... In certain embodiments, the RNAi agents of the present disclosure comprise an RNA strand (antisense strand) having a region that is approximately 21-23 nucleotides in length, which is substantially complementary to at least a portion of an mRNA transcript of the PRNP gene.

[0116] In certain embodiments, RNAi agents of the present disclosure include an RNA strand (antisense strand) that can include a longer length, e.g., up to 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides, with a region of at least 19 contiguous nucleotides that is substantially complementary to at least a portion of an mRNA transcript of the PRNP gene. These RNAi agents with longer antisense strand lengths can include a second RNA strand (sense strand) that is, e.g., 20-60 nucleotides in length, where the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.

[0117] The use of these RNAi agents allows for the targeted degradation of PRNP gene mRNA in mammals, and methods and compositions comprising these RNAi agents are therefore useful for treating subjects who would benefit from a reduction in PRNP protein level or activity, such as those with PRNP-related diseases, such as prion diseases.

[0118] The detailed description below discloses methods for making and using compositions containing RNAi agents that inhibit expression of the PRNP gene, as well as compositions and methods for treating subjects with diseases and disorders that would benefit from inhibiting or reducing expression of the PRNP gene.

[0119] I. Definition In order that this disclosure may be more readily understood, certain terms are first defined. Additionally, whenever a value or range of values ​​for a parameter is listed, it is intended that values ​​and ranges intermediate to the listed values ​​are also intended to be part of this disclosure.

[0120] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or to more than one element, e.g., a plurality of elements.

[0121] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to."

[0122] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless context clearly indicates otherwise.

[0123] The term "about" is used herein to mean within a typical tolerance in the art. For example, "about" can be understood as about 2 standard deviations from the mean. In certain embodiments, about means ±10%. In certain embodiments, about means ±5%. When about precedes a series of numbers or ranges, it is understood that "about" can modify each of the consecutive numbers or ranges.

[0124] The terms "at least," "more than," or "or more" preceding a number or series of numbers are understood to include the number adjacent to the term "at least," and all subsequent numbers or integers that may be logically included, if this is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the specified property. When at least precedes a series of numbers or ranges, it is understood that "at least" can modify each of the series of numbers or ranges.

[0125] As used herein, "less than" or "less than" refers to the value adjacent to the phrase and, if logical from the context, to zero, any logically smaller value or integer than the value.For example, a duplex with an overhang of "2 nucleotides or less" has an overhang of 2, 1, or 0 nucleotides.When "less than" is used before a series of numbers or ranges, it is understood that "less than" can modify each of the series of numbers or ranges.As used herein, a range includes both upper and lower limits.

[0126] As used herein, a method of detection can include determining that the amount of analyte present is below the detection level of the method.

[0127] In the event of a conflict between a given target site and the nucleotide sequence for either the sense or antisense strand, the given sequence controls.

[0128] If the chemical structure and chemical name do not match, the chemical structure takes precedence.

[0129] As used herein, the term "prion protein," which is used interchangeably with the term "PRNP," refers to the well-known gene and polypeptide that is also known in the art as AltPrP, ASCR, CD230 antigen, CJD, GSS, kuru, prion protein (p27-30), PRIP, PrP, PrP27-30, PrP33-35C, and PrPc.

[0130] The prion protein gene is highly conserved in mammals and is involved in neural stem cell neurogenesis and differentiation, neuritogenesis, participation in and interaction with signaling pathways, synaptogenesis, neuronal survival through anti- or pro-apoptotic functions, copper binding, redox homeostasis, long-term renewal of hematopoietic stem cells, T cell activation and development, regulation of leukocyte differentiation and phagocytosis, and altered leukocyte recruitment to sites of inflammation (Caughey B, Baron GS. Nature. 2006;443:803-810).

[0131] There are multiple isoforms of the prion protein, two of which cause prion diseases, cellular prion protein (PrP C ), and scrapie prion protein (PrPP Sc ) most related to PrP C and PrP Sc Both isoforms have the same amino acid sequence, but PrP c consists mainly of α-helices, whereas PrP Sc PrP undergoes a three-dimensional structural change, increasing the amount of β-sheet structure. Sc PrP becomes insoluble in detergents and relatively resistant to degradation by proteases. Sc is infectious and PrP C PrP Sc It has been shown to self-replicate by acting as a template to induce conversion to PrP. ScThe abnormal PrP propagates as oligomers, which polymerize to form amyloid fibrils. Sc The protein accumulates in the brain, forming aggregates that damage or destroy neurons. The loss of these cells creates microscopic sponge-like holes (pits) in the brain, which leads to the signs and symptoms of prion disease.

[0132] The term "PRNP" includes human PRNP, the amino acid and nucleotide sequence of which can be found, for example, at GenBank Accession No. NM_000311.5 (GI:1653962152, SEQ ID NO:1); mouse PRNP, the amino acid and nucleotide sequence of which can be found, for example, at GenBank Accession No. NM_001278256.1 (GI:506326230, SEQ ID NO:2); and rat PRNP, the amino acid and nucleotide sequence of which can be found, for example, at GenBank Accession No. XM_006235062.3 (GI:1046878307, SEQ ID NO:3). The term "PRNP" also includes cynomolgus monkey PRNP, the amino acid and nucleotide sequence of which can be found, for example, at GenBank Accession No. XM_005568413.2 (GI:982273619, SEQ ID NO:4).

[0133] Further examples of PRNP mRNA sequences are readily available using, for example, GenBank, UniProt, OMIM, and the Macaca Genome Project website.

[0134] Exemplary PRNP nucleotide sequences can also be found in SEQ ID NOs: 1-4. SEQ ID NOs: 5-8 are the reverse complementary sequences of SEQ ID NOs: 1-4, respectively.

[0135] Further information on PRNP is provided in the NCBI gene database, e.g., www.ncbi.nlm.nih.gov / gene / 5621.

[0136] The entire contents of each of the foregoing GenBank Accession Numbers and Gene Database Numbers are incorporated herein by reference as of the filing date of this application.

[0137] As used herein, the terms "prion protein" and "PRNP" also refer to naturally occurring DNA sequence variations of the PRNP gene. Numerous sequence variations within the PRNP gene have been identified and can be found, for example, in NCBI dbSNP and UniProt (see, for example, www.ncbi.nlm.nih.gov / snp?LinkName=gene_snp&from_uid=5621, the entire contents of which are incorporated herein by reference as of the filing date of this application).

[0138] As used herein, "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of PRNP gene, such as the mRNA that is the product of RNA processing of a primary transcript.In one embodiment, the target portion of the sequence is at least long enough to serve as a substrate for RNAi-dependent cleavage at or near a portion of the nucleotide sequence of an mRNA molecule formed during the transcription of PRNP gene.In one embodiment, the target sequence is located in the protein coding region of PRNP gene.In another embodiment, the target sequence is located in the 3'UTR of PRNP gene.

[0139] The target sequence can be about 9 to 36 nucleotides in length, e.g., about 15 to 30 nucleotides in length. For example, the target sequence can be about 15 to 30 nucleotides, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 The target sequence may be about 19 to about 30 nucleotides in length. In other embodiments, the target sequence is about 19 to about 25 nucleotides in length. In still other embodiments, the target sequence is about 19 to about 23 nucleotides in length. In some embodiments, the target sequence is about 21 to about 23 nucleotides in length. Ranges and lengths between the ranges and lengths listed above are also intended to be part of the present invention.

[0140] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides described by a sequence referenced using standard nucleotide nomenclature.

[0141] " G ", " C ", " A ", " T " and " U " generally refer to the nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as base, respectively, in the context of modified or unmodified nucleotide. However, it is understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides, as described in more detail below, or alternative replacement moieties (see, for example, Table 1). Those skilled in the art are well aware that guanine, cytosine, adenine, thymidine and uracil can be substituted with other moieties without substantially changing the base pairing properties of the oligonucleotide that contains the nucleotide with such replacement moiety. For example, but not limited to, the nucleotide that contains inosine as its base can base pair with the nucleotide that contains adenine, cytosine or uracil. Therefore, the nucleotide that contains uracil, guanine or adenine can be substituted with the nucleotide that contains inosine, for example, in the nucleotide sequence of the dsRNA featured in the present disclosure. In another example, adenine and cytosine anywhere within an oligonucleotide can be substituted with guanine and uracil, respectively, to form GU wobble bases that pair with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods featured in this disclosure.

[0142] As used herein interchangeably, the terms "iRNA", "RNAi agent", "iRNA agent", "RNA interference agent" refer to an agent that contains RNA as defined herein and mediates the targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway.RNA interference (RNAi) is the process that controls the sequence-specific degradation of mRNA.RNAi regulates, for example, inhibits, the expression of PRNP in cells, for example, in cells of a subject, such as a mammalian subject.

[0143] In one embodiment, the RNAi agent of the present disclosure comprises a single-stranded RNAi that interacts with a target RNA sequence, such as a PRNP target mRNA sequence, to cleave the target RNA. Without wishing to be bound by theory, it is believed that long double-stranded RNAs introduced into cells are degraded into double-stranded small interfering RNAs (siRNAs) containing a sense strand and an antisense strand by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). The RNase III-like enzyme Dicer processes these dsRNAs into 19-23 base pair small interfering RNAs with characteristic two-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). These siRNAs are then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, thereby allowing complementary antisense strands to guide target recognition (Nykanen, et al., (2001) Cell 107:309). When bound to the appropriate target mRNA, one or more endonucleases in RISC cleave the target and induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). That is, in one aspect, the present disclosure relates to a single-stranded RNA (ssRNA) (the antisense strand of the siRNA duplex) that is produced in cells and promotes the formation of a RISC complex to silence the target gene, i.e., the PRNP gene. Therefore, the term "siRNA" is used herein to also refer to the RNAi described above.

[0144] In another embodiment, the RNAi agent can be a single-stranded RNA introduced into a cell or organism to inhibit target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15-30 nucleotides and chemically modified. The design and testing of single-stranded RNAs are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are incorporated herein by reference. Any of the antisense nucleotide sequences described herein can be used as the single-stranded siRNA described herein or as the single-stranded siRNA that is chemically modified by the method described in Lima et al., (2012) Cell 150:883-894.

[0145] In another embodiment, the "RNAi agent" used in the compositions and methods of the present disclosure is double-stranded RNA, and is also referred to herein as "double-stranded RNAi agent", "double-stranded RNA (dsRNA) molecule", "dsRNA agent" or "dsRNA". The term "dsRNA" refers to a complex of ribonucleic acid molecules with a duplex structure, comprising two antiparallel, substantially complementary nucleic acid strands, which are referred to as having "sense" or "antisense" orientation with respect to target RNA, i.e., PRNP gene. In some embodiments of the present disclosure, double-stranded RNA (dsRNA) induces the degradation of target RNA, for example, mRNA, through a post-transcriptional gene silencing mechanism, referred to herein as RNA interference or RNAi.

[0146] Generally, dsRNA molecule can comprise ribonucleotide, but as described in detail herein, each strand or both strands can also comprise one or more non-ribonucleotides, for example, deoxyribonucleotide, modified nucleotide.In addition, as used herein, " RNAi agent " can comprise the ribonucleotide with chemical modification, RNAi agent can comprise the substantial modification in multiple nucleotides, and RNAi agent can comprise the substantial modification in multiple nucleotides.

[0147] As used herein, the term "modified nucleotide" refers to the nucleotide that has independently modified sugar moiety, modified internucleotide linkage, or modified nucleobase.Therefore, the term modified nucleotide encompasses the substitution, addition, or removal of, for example, functional group or atom, etc., to internucleoside linkage, sugar moiety, or nucleobase.The modification suitable for use in the agent of the present disclosure includes all types of modification disclosed herein or known in the art.Any such modification used in siRNA type molecules is encompassed by "RNAi agent" for the purpose of this specification and claims.

[0148] In certain embodiments of the present disclosure, the inclusion of deoxy-nucleotides, which are recognized as naturally occurring forms of nucleotides, can be considered to constitute modified nucleotides when present in an RNAi agent.

[0149] The duplex region may be of any length that allows for specific degradation of the desired target RNA via the RISC pathway, and may be about 9 to 36 base pairs in length, e.g., about 15 to 30 base pairs in length, e.g., about 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, or 36 base pairs in length, e.g., about 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, The length may range from 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Ranges and lengths between the ranges and lengths listed above are also intended to be part of the invention.

[0150] The two strands forming the duplex structure can be different portions of a larger RNA molecule, or they can be separate RNA molecules. When the two strands are portions of a larger molecule and are therefore connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the corresponding 5' end of the other strand, the connected RNA strands are called "hairpin loops." A hairpin loop can contain at least one unpaired nucleotide. In some embodiments, a hairpin loop can contain at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23, or more unpaired nucleotides or nucleotides not directed toward the target site of the dsRNA. In some embodiments, a hairpin loop can be 10 or fewer nucleotides. In some embodiments, a hairpin loop can be 8 or fewer unpaired nucleotides. In some embodiments, a hairpin loop can be 4 to 10 unpaired nucleotides. In some embodiments, a hairpin loop can be 4 to 8 nucleotides.

[0151] In certain embodiments, the two strands of a double-stranded oligomeric compound may be linked to each other. The two strands may be linked to each other at both ends or only one end. Linked at one end means that the 5' end of the first strand is linked to the 3' end of the second strand, or the 3' end of the first strand is linked to the 5' end of the second strand. When the two strands are linked to each other at both ends, the 5' end of the first strand is linked to the 3' end of the second strand, and the 3' end of the first strand is linked to the 5' end of the second strand. The two strands may be linked together by an oligonucleotide linker, including, but not limited to, (N)n, where N is independently a modified or unmodified nucleotide, and n is 3 to 23. In some embodiments, n is 3 to 10, e.g., 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the oligonucleotide linker is selected from the group consisting of GNRA, (G)4, (U)4, and (dT)4, where N is a modified or unmodified nucleotide, and R is a modified or unmodified purine nucleotide. Some of the nucleotides in the linker can participate in base pairing interactions with other nucleotides in the linker. The two strands can also be linked together by a non-nucleoside linker, for example, by a linker described herein. Those skilled in the art will understand that any chemical modification or variation of the oligonucleotides described herein can be used in the oligonucleotide linker.

[0152] Hairpin and dumbbell oligomeric compounds will have a duplex region equal to or at least 14, 15, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex region can be up to 200, 100, or 50 nucleotide pairs in length. In some embodiments, the duplex region ranges from 15-30, 17-23, 19-23, and 19-21 nucleotide pairs in length.

[0153] Hairpin oligomeric compounds may have a single-stranded overhang or terminal unpaired region, in some embodiments at the 3' end, and in some embodiments on the antisense side of the hairpin. In some embodiments, the overhang is 1 to 4 nucleotides in length, more generally 2 to 3 nucleotides in length. Hairpin oligomeric compounds capable of inducing RNA interference are also referred to herein as "shRNAs."

[0154] When the two substantially complementary strands of dsRNA are composed of separate RNA molecules, these molecules can, but do not necessarily, be covalently linked.When the two strands are covalently linked by a means other than an uninterrupted chain of nucleotides between the 3'-end of one strand and the 5'-end of the corresponding other strand that form a double-stranded structure, this connecting structure is called a "linker".The RNA strands can have the same or different number of nucleotides.The maximum number of base pairs is the number of nucleotides in the shortest strand of dsRNA minus any overhangs present in the double strand.In addition to the double-stranded structure, RNAi can also include one or more nucleotide overhangs.

[0155] In one embodiment, the RNAi agent of the present invention is a dsRNA, each strand of which is 24-30 nucleotides in length and interacts with a target RNA sequence, e.g., a PRNP target mRNA sequence, to induce cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNA introduced into cells is degraded into siRNAs by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, an RNase III-like enzyme, processes the dsRNA into 19-23 base pair small interfering RNAs with characteristic two-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNA is then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, thereby allowing target recognition to be induced by the complementary antisense strand (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

[0156] In one embodiment, the RNAi agents of the present invention are dsRNA agents, each strand of which contains 19-23 nucleotides that interact with the PRNP RNA sequence to induce cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNAs introduced into cells are degraded into siRNAs by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, an RNase III-like enzyme, processes the dsRNA into 19-23 base pair small interfering RNAs with characteristic two-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNA is then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, thereby allowing target recognition to be induced by the complementary antisense strand (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). In one embodiment, the RNAi agent of the present invention is a 24-30 nucleotide dsRNA that interacts with the PRNP RNA sequence to induce cleavage of the target RNA.

[0157] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of an RNAi agent, such as dsRNA.For example, a nucleotide overhang exists when the 3'-end of one strand of dsRNA extends beyond the 5'-end of the other strand, or vice versa.A dsRNA can comprise an overhang of at least one nucleotide, or the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more.A nucleotide overhang can comprise or consist of a nucleotide / nucleoside analog, such as deoxynucleotide / nucleoside.An overhang can be on the sense strand, on the antisense strand, or any combination thereof.Furthermore, a certain overhanging nucleotide can be present on the 5'-end, 3'-end, or both ends of either the antisense strand or the sense strand of dsRNA.

[0158] In one embodiment, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3' or 5' end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In one embodiment, the sense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3' or 5' end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more of the nucleotides in the overhang are substituted with a nucleoside thiophosphate.

[0159] In certain embodiments, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3' or 5' end, for example, 0 to 3, 1 to 3, 2 to 4, 2 to 5, 4 to 10, 5 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In one embodiment, the sense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3' or 5' end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more of the nucleotides in the overhang are substituted with nucleoside thiophosphates.

[0160] In certain embodiments, the overhang on the sense strand or the antisense strand can comprise an extended length greater than 10 nucleotides, e.g., 1 to 30 nucleotides, 2 to 30 nucleotides, 10 to 30 nucleotides, or 10 to 15 nucleotides in length. In certain embodiments, the extended overhang is on the sense strand of the duplex. In certain embodiments, the extended overhang is on the 3'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is on the 5'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is on the antisense strand of the duplex. In certain embodiments, the extended overhang is on the 3'-end of the antisense strand of the duplex. In certain embodiments, the extended overhang is on the 5'-end of the antisense strand of the duplex. In certain embodiments, one or more of the nucleotides in the overhang are substituted with a nucleoside thiophosphate. In certain embodiments, the overhang comprises a self-complementary portion that enables the overhang to form a stable hairpin structure under physiological conditions.

[0161] In certain embodiments, at least one end of at least one strand extends beyond the duplex targeting region, including structures in which one of the strands comprises a thermodynamically stable tetraloop structure (see, e.g., U.S. Pat. Nos. 8,513,207 and 8,927,705 and WO 2010033225, the contents of each of which are incorporated herein by reference in their entirety). Such structures can include single-stranded extensions (on one or both sides of the molecule) as well as double-stranded extensions.

[0162] In certain embodiments, the 3'-end of the sense strand and the 5'-end of the antisense strand are joined by a polynucleotide sequence comprising ribonucleotides, deoxyribonucleotides, or both, and optionally the polynucleotide sequence comprises a tetraloop sequence. In certain embodiments, the sense strand is 25-35 nucleotides in length.

[0163] The tetraloop can comprise ribonucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof. Typically, a tetraloop has 4 to 5 nucleotides. In some embodiments, the loop comprises a sequence described as GAAA. In some embodiments, at least one of the nucleotides (GAAA) of the loop comprises a nucleotide modification. In some embodiments, the modified nucleotide comprises a 2' modification. In some embodiments, the 2' modification is a modification selected from the group consisting of 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, 2'-aminodiethoxymethanol, 2'-adem, and 2'-deoxy-2'-fioro-d-arabinonucleic acid. In some embodiments, all nucleotides of the loop are modified. In some embodiments, the G in the GAAA sequence comprises a 2'-OH. In some embodiments, each nucleotide in the GAAA sequence comprises a 2'-O-methyl modification. In some embodiments, each A in the GAAA sequence comprises a 2'-OH, and each G in the GAAA sequence comprises a 2'-O-methyl modification. In some embodiments, each A in the GAAA sequence comprises a 2'-O-methoxyethyl (MOE) modification, and each G in the GAAA sequence comprises a 2'-O-methyl modification, or each A in the GAAA sequence comprises a 2'-adem modification, and each G in the GAAA sequence comprises a 2'-O-methyl modification. See, for example, PCT Publication WO 2020 / 206350, the entire contents of which are incorporated herein by reference.

[0164] Exemplary 2' adem modified nucleotides are shown below. [ka]

[0165] In one embodiment of dsRNA, at least one strand comprises a 3' overhang of at least 1 nucleotide.In another embodiment, at least one strand comprises a 3' overhang of at least 2 nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides.In other embodiments, at least one strand of RNAi agent comprises a 5' overhang of at least 1 nucleotide.In certain embodiments, at least one strand comprises a 5' overhang of at least 2 nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides.In still other embodiments, both the 3' end and the 5' end of one strand of RNAi agent comprise an overhang of at least 1 nucleotide.

[0166] In one embodiment, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3' or 5' end, for example, 0 to 3, 1 to 3, 2 to 4, 2 to 5, 4 to 10, 5 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In one embodiment, the sense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3' or 5' end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more of the nucleotides in the overhang are substituted with a nucleoside thiophosphate.

[0167] In certain embodiments, the overhang on the sense strand or the antisense strand, or both, can comprise an extended length greater than 10 nucleotides, e.g., a length of 1 to 30 nucleotides, 2 to 30 nucleotides, 10 to 30 nucleotides, or 10 to 15 nucleotides. In certain embodiments, the extended overhang is on the sense strand of the duplex. In certain embodiments, the extended overhang is on the 3'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is on the 5'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is on the antisense strand of the duplex. In certain embodiments, the extended overhang is on the 3'-end of the antisense strand of the duplex. In certain embodiments, the extended overhang is on the 5'-end of the antisense strand of the duplex. In certain embodiments, one or more of the nucleotides in the overhang are substituted with a nucleoside thiophosphate. In certain embodiments, the overhang comprises a self-complementary portion that enables the overhang to form a stable hairpin structure under physiological conditions.

[0168] The term "blunt" or "blunt-end" when used herein in relation to dsRNA means that there is no unpaired nucleotide or nucleotide analogue at the predetermined end of dsRNA, that is, there is no nucleotide overhang.One end or both ends of dsRNA can be blunt.When both ends of dsRNA are blunt, dsRNA is said to be blunt-ended.For clarity, "blunt-end" dsRNA is the dsRNA that has both ends blunt, that is, there is no nucleotide overhang at either end of the molecule.In most cases, this molecule will be double-stranded throughout its entire length.

[0169] The term "antisense strand" or "guide strand" refers to the strand of an RNAi agent, e.g., dsRNA, that includes a region that is substantially complementary to a target sequence, e.g., PRNP mRNA.

[0170] As used herein, term " complementary region " refers to the region on antisense strand that is substantially complementary to sequence, for example, target sequence, for example, PRNP nucleotide sequence, as defined herein.If complementary region is not completely complementary to target sequence, its mismatch can be in the inner region or terminal region of molecule.Generally, the most tolerable mismatch is in terminal region, for example, within 5, 4, 3 or 2 nucleotides of the 5' or 3' end of RNAi agent.

[0171] In some embodiments, a double-stranded RNA agent of the invention comprises a nucleotide mismatch in the antisense strand. In some embodiments, the antisense strand of a double-stranded RNA agent of the invention comprises four or fewer mismatches with the target mRNA, e.g., the antisense strand comprises 4, 3, 2, 1, or 0 mismatches with the target mRNA. In some embodiments, the antisense strand of a double-stranded RNA agent of the invention comprises four or fewer mismatches with the sense strand, e.g., the antisense strand comprises 4, 3, 2, 1, or 0 mismatches with the sense strand. In some embodiments, a double-stranded RNA agent of the invention comprises a nucleotide mismatch in the sense strand. In some embodiments, the sense strand of a double-stranded RNA agent of the invention comprises four or fewer mismatches with the antisense strand, e.g., the sense strand comprises 4, 3, 2, 1, or 0 mismatches with the antisense strand. In some embodiments, the nucleotide mismatch is within, for example, 5, 4, or 3 nucleotides from the 3' end of the iRNA. In another embodiment, the nucleotide mismatch is within, for example, the 3' terminal nucleotide of the iRNA agent. In some embodiments, the mismatch is not in the seed region.

[0172] Thus, the RNAi agents described herein may contain one or more mismatches to the target sequence. In one embodiment, the RNAi agents described herein contain three or fewer mismatches (i.e., three, two, one, or zero mismatches). In one embodiment, the RNAi agents described herein contain two or fewer mismatches. In one embodiment, the RNAi agents described herein contain one or fewer mismatches. In one embodiment, the RNAi agents described herein contain zero mismatches. In certain embodiments, when the antisense strand of an RNAi agent contains a mismatch to the target sequence, the mismatch can be optionally limited to be within the last five nucleotides from either the 5'-end or the 3'-end of the complementary region. For example, in such an embodiment, in the case of a 23-nucleotide RNAi agent, the strand complementary to a region of the PRNP gene generally does not contain any mismatches within the central 13 nucleotides. Using the methods described herein or known in the art, it can be determined whether an RNAi agent containing a mismatch to the target sequence is effective in inhibiting the expression of the PRNP gene. In particular, if a particular region of complementarity in the PRNP gene is known to have polymorphic sequence variation within the population, it is important to consider the efficacy of mismatched RNAi agents to inhibit expression of PRNP.

[0173] The term "sense strand" or "passenger strand," as used herein, refers to the strand of an RNAi agent that includes a region that is substantially complementary to a region of the antisense strand, as those terms are defined herein.

[0174] As used herein, "substantially all of the nucleotides are modified" means broadly but not entirely modified and may include no more than 5, 4, 3, 2, or 1 or unmodified nucleotides.

[0175] As used herein, the term "cleavage region" refers to the region located directly adjacent to the cleavage site. The cleavage site is the site on the target where cleavage occurs. In some embodiments, the cleavage region comprises three bases directly adjacent to either end of the cleavage site. In some embodiments, the cleavage region comprises two bases directly adjacent to either end of the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bound by nucleotides 10 and 11 of the antisense strand, and this cleavage region comprises nucleotides 11, 12, and 13.

[0176] As used herein, unless otherwise specified, the term "complementary," when used to describe a first nucleotide sequence in the context of a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize to form a duplex structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under specified conditions, as would be understood by one of ordinary skill in the art. Such conditions may be, for example, "stringent conditions," which may include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, at 50°C or 70°C for 12-16 hours, followed by washing (see, e.g., "Molecular Cloning: A Laboratory Manual," Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions that may be encountered in an organism, may be applied. Those skilled in the art can determine the most appropriate set of conditions for testing the complementarity of two sequences depending on the ultimate use of the hybridized nucleotides.

[0177] The complementary sequence in an RNAi agent, for example, in the dsRNA described herein, comprises the base pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence with the oligonucleotide or polynucleotide comprising the second nucleotide sequence throughout the entire length of one or both nucleotide sequences.Such sequences can be referred to herein as "fully complementary" to each other.However, when the first sequence is referred to herein as "substantially complementary" to the second sequence, these two sequences can be completely complementary, or they can form one or more, but generally not more than 5, 4, 3, or 2 mismatched base pairs when hybridizing a duplex of up to 30 base pairs, while maintaining the ability to hybridize under the conditions most relevant to its final use, for example, in vitro or in vivo, inhibiting gene expression.However, if two oligonucleotides are designed to form one or more single-stranded overhangs when hybridizing, these overhangs shall not be considered as mismatches when determining complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, where the longer oligonucleotide comprises a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, may still be referred to as "fully complementary" for purposes described herein.

[0178] "Complementary" sequences, as used herein, may also include or be formed entirely of non-Watson-Crick base pairs or base pairs formed from non-naturally occurring modified nucleotides, such as, but not limited to, G:U wobble base pairs or Hoogsteen base pairs, so long as they satisfy the above requirements regarding their ability to hybridize.

[0179] The terms "complementary," "fully complementary," and "substantially complementary," as used herein, may be used in reference to base matching between two oligonucleotides or polynucleotides, such as between the sense and antisense strands of a dsRNA, or the antisense strand of an RNAi agent and a target sequence, as will be understood from the context of their use.

[0180] As used herein, a polynucleotide that is "substantially complementary to at least a portion of" a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest (e.g., an mRNA encoding PRNP). For example, a polynucleotide is complementary to at least a portion of a PRNP mRNA if the sequence is substantially complementary to an uninterrupted portion of the mRNA encoding PRNP.

[0181] Thus, in some embodiments, the antisense strand polynucleotides disclosed herein are perfectly complementary to the target PRNP sequence.

[0182] In other embodiments, the antisense strand polynucleotides disclosed herein are substantially complementary to the target PRNP sequence and comprise a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary to the equivalent region of the nucleotide sequence of SEQ ID NOs: 1-4 for PRNP, or a fragment of SEQ ID NOs: 1-4, over its entire length.

[0183] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a target PRNP sequence and comprise a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary over its entire length to any one of the sense strand nucleotide sequences in any one of Tables 2-3 or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 2-3.

[0184] In one embodiment, an RNAi agent of the present disclosure comprises a sense strand that is substantially complementary to an antisense polynucleotide that is also identical to a target PRNP sequence, and the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about, or about 99% complementary to the nucleotide sequence of SEQ ID NOs: 5-8 or an equivalent region of a fragment of any one of SEQ ID NOs: 5-8, throughout its entire length.

[0185] In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a fragment of a target PRNP sequence, including nucleotides 502-524, 507-529, 502-529, 540-562, 566-588, 575-597, 576-598, 589-611, 575-611, 593-615, 594-616, 600-622, 593-622, 650-672, 858-880, 976-998, 1100-1122, 1126-1148, 1220-1242, 1221-1243, 1220-1243 of SEQ ID NO:1. , 1304-1326, 1328-1350, 1410-1432, 1445-1467, 1481-1503, 1532-1554, 1610-1632, 1615-1637, 1617-1639, 1621-1643, 1610-1643, 1610-1639, 1688 -1710, 1694-1714, 1830-1852, 1831-1853, 1854-1876, 1830-1853, 1872-1894, 1873-1895, 1938-1960, 2011-2033, 2015-2037, 2031-2053, 2034-2056 , 2069-2091, 2076-2098, 2077-2099, 1872-1895, 2011-2037, 2069-2099, 2079-2101, 2031-2056, 2069-2098, 2138-2160, 2143-2165, 2158-2180, 2167 -2189, 2168-2190, 2170-2192, 2174-2196, 2175-2197, 2177-2199, 2185-2207, 2189-2211, 2196-2218, 2200-2222, 2202-2224, 2221-2243, 2222-2244 , 2223-2245, 2238-2260, 2241-2263, 2242-2264, 2138-2264, 2257-2358, 2174-2245, 2174-2224, 2138-2196, 2177-2224, 2223-2245, 2138-2189, 2177 -2211, 2196-2224, 2257-2279, 2258-2280, 2335-2537, 2336-2358, 2257-2358, 2342-2364, 2397-2419, 2398-2420, 2399-2421, 2400-2422, 2401-2423,The fragment of SEQ ID NO: 1 selected from the group consisting of 2404-2426, 2397-2426, 2394-2423, 2397-2421, 2399-2426, and 2398-2421 comprises a contiguous nucleotide sequence that is at least 80% complementary, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary over its entire length to a fragment of SEQ ID NO: 1 selected from the group consisting of 2404-2426, 2397-2426, 2394-2423, 2397-2421, 2399-2426, and 2398-2421.

[0186] In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a fragment of a target PRNP sequence, including nucleotides 530-570, 535-565, 539-561, 540-562, 555-605, 560-605, 560-600, 566-588, 567-589, 568-570, 569-589, 571-572, 573-574, 575-576, 577-578, 578-579, 579-580, 581-582, 582-583, 583-584, 585-586, 587-588, 589-590, 591-592, 592-593, -590, 569-591, 570-592, 575-597, 580-620-585-620, 589-611, 590-612, 591-613, 592-614, 593-615, 594-616, 600-650, 610-650, 613-635, 614-636, 615-637, 616-638, 617-639, 618-640, 640-680, 649-671, 650-672, 651-673, 670-700, 674-696, 675-697, 795-830, 804-826, 2325-2375, 2325-2370, 2330-2370, 2335-2357, 2336-2358, 2337-2359, 2338-2360 , 2339-2361, or 2340-2362, or a contiguous nucleotide sequence that is at least 80% complementary, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary over its entire length to a fragment of SEQ ID NO: 1 selected from the group consisting of: 2339-2361, or 2340-2362.

[0187] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a fragment of the target PRNP sequence, including nucleotides 502-524, 540-562, 589-611, 594-616, 650-672, 1445-1467, 1688-1710, 2011-2033, 2031-2053, 2185-2207, 2222-2223, 2224-2225, 2226-2227, 2228-2229, 2229-2230, 2229-2231, 2229-2232, 2229-2233, 2229-2234, 2229-2235, 2229-2236, 2230-2237, 2231-2238, 2231-2239, 2231-2236, 2231-2238, 2231-2239 ... 244, 2336-2358, 2339-2361, 2399-2421 or 2258-2280, the fragment of SEQ ID NO: 1 selected from the group consisting of: 244, 2336-2358, 2339-2361, 2399-2421 or 2258-2280.

[0188] In some embodiments, the iRNA of the invention comprises a sense strand that is substantially complementary to an antisense polynucleotide that is also complementary to a target PRNP sequence, and the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary over its entire length to any one of the antisense strand nucleotide sequences in any one of Tables 2-3, or a fragment of any one of the antisense strand nucleotide sequences in any one of Tables 2-3.

[0189] In certain embodiments, the sense and antisense strands are selected from any one of the duplexes AD-1070511, AD-1070462, AD-1070553, AD-1072048, AD-1070516 and AD-1072050, AD-1071769, AD-1071949, AD-1070444, AD-1071505, AD-1071912, AD-1071789, AD-1071265, AD-1072084, and AD-1071985.

[0190] In some embodiments, the double-stranded region of a double-stranded iRNA agent is equal to or at least equal to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotide pairs in length.

[0191] In some embodiments, the antisense strand of the double-stranded iRNA agent is equal to or at least equal to 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0192] In some embodiments, the sense strand of a double-stranded iRNA agent is equal to or at least equal to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0193] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each independently 15 to 30 nucleotides in length.

[0194] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each independently 19 to 25 nucleotides in length.

[0195] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each independently 21 to 23 nucleotides in length.

[0196] In one embodiment, the sense strand of the iRNA agent is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, the strands forming a double-stranded region of 21 contiguous base pairs with a 2-nucleotide long single-stranded overhang at the 3' end.

[0197] In one embodiment of the present invention, an agent for use in the methods and compositions of the present invention is a single-stranded antisense nucleic acid molecule that inhibits a target mRNA via an antisense inhibition mechanism. The single-stranded antisense RNA molecule is complementary to a sequence within the target mRNA. Single-stranded antisense oligonucleotides can inhibit translation stoichiometrically by base pairing with the mRNA and physically interfering with the translation machinery. See Dias, N. et al. (2002) Mol Cancer Ther 1:347-355. The single-stranded antisense RNA molecule may be about 15 to about 30 nucleotides in length and may have a sequence complementary to the target sequence. For example, the single-stranded antisense RNA molecule may comprise a sequence that is at least about 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from any one of the antisense sequences described herein.

[0198] In one embodiment, at least partial suppression of PRNP gene expression is assessed by a decrease in the amount of PRNP mRNA that can be isolated from or detected in a first cell or group of cells in which the PRNP gene is transcribed and which has been treated to inhibit expression of the PRNP gene, compared to a second cell or group of cells (control cells) that is substantially identical to the first cell or group of cells but has not been treated in the same manner as the first cell or group of cells. The degree of inhibition can be expressed as follows:

number

[0199] In one embodiment, inhibition of expression is determined by the dual luciferase method of Example 1, in which the RNAi agent is present at 10 nM.

[0200] As used herein, the phrase " contacting cell with RNAi agent " such as dsRNA includes contacting cell by any possible means.Contacting cell with RNAi agent includes contacting cell with RNAi agent in vitro or contacting cell with RNAi agent in vivo.Contacting can be carried out directly or indirectly.Therefore, for example, RNAi agent can be physically contacted with cell by carrying out a method separately, or RNAi agent can be placed in a situation that can allow or cause it to contact cell afterwards.

[0201] Contacting cells in vitro can be carried out, for example, by incubating cells with RNAi agent.Contacting cells in vivo can be carried out, for example, by injecting RNAi agent into the tissue where cells are located or nearby, or by injecting RNAi agent into another region, for example, into the central nervous system (CNS), optionally via intrathecal injection or other injection, or by injecting RNAi agent into bloodstream or subcutaneous space, so that the agent then reaches the tissue where the cells to be contacted are located.For example, RNAi agent can comprise or be coupled to a ligand that directs or otherwise stabilizes RNAi agent to the target site, for example, in the CNS, for example, lipophilic moiety, as described below and further detailed in, for example, PCT Publication No. WO2019 / 217459, which is incorporated herein by reference. In some embodiments, RNAi agent can contain or bind to a ligand, such as one or more GalNAc derivatives described below, that directs or otherwise stabilizes RNAi agent to target site, for example, liver.In other embodiments, RNAi agent can contain or bind to a lipophilic moiety or one or more GalNAc derivatives.The combination of in vitro and in vivo contact methods is also possible.For example, cell can be contacted with RNAi agent in vitro and then transferred to subject.

[0202] In one embodiment, contacting a cell with an RNAi agent includes "introducing" or "delivering an RNAi agent into a cell" by promoting or performing uptake or absorption into the cell. The absorption or uptake of an RNAi agent can occur by spontaneous diffusive or active cellular processes, or by auxiliary agents or devices. The introduction of an RNAi agent into a cell can be in vitro or in vivo. For example, in the case of in vivo introduction, the RNAi agent can be injected into a tissue site or administered systemically. In vitro introduction into a cell can include methods known in the art, such as electroporation and lipofection. Further approaches are described herein below or known in the art.

[0203] The terms "lipid-soluble" or "lipophilic moiety" refer broadly to any compound or chemical moiety that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is by the octanol-water partition coefficient, log K ow In this case, K ow is the ratio of the concentration of a chemical in the octanol phase to the concentration of the chemical in the aqueous phase of a two-phase system at equilibrium. The octanol-water partition coefficient is a laboratory-measured property of a substance. However, it can also be predicted by using coefficients attributable to the structural components of a chemical calculated using first principles or empirical methods (see, e.g., Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001), incorporated herein by reference in its entirety). It provides a thermodynamic measure of a substance's tendency to prefer a non-aqueous or oily environment rather than water (i.e., its hydrophilic / lipophilic balance). In principle, a chemical can be expressed as its logK ow is greater than 0, the molecule is lipophilic in nature. Typically, a lipophilic moiety has a logK ow For example, the log K of 6-aminohexanol owFor example, the log K of cholesteryl N-(hexan-6-ol)carbamate is predicted to be approximately 0.7. ow is predicted to be 10.7.

[0204] The lipophilicity of a molecule can be changed depending on the functional groups it contains. For example, adding a hydroxyl or amine group to the end of the lipophilic moiety can increase the partition coefficient (e.g., logK ow ) may increase or decrease in value.

[0205] Alternatively, the hydrophobicity of the double-stranded RNAi agent that is conjugated with one or more lipophilic moieties can be measured by its protein binding property.For example, in certain embodiments, if the unbound fraction of the plasma protein binding assay of double-stranded RNAi agent is determined to be positively correlated with the relative hydrophobicity of double-stranded RNAi agent, then it will be positively correlated with the silencing activity of double-stranded RNAi agent.

[0206] In one embodiment, the plasma protein binding assay that is determined is electrophoretic mobility shift assay (EMSA) that uses human serum albumin protein.The exemplary protocol of this binding assay is described in detail, for example, in PCT Publication No. WO2019 / 217459.The hydrophobicity of double-stranded RNAi agent measured by the unbound fraction of siRNA in binding assay is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45 or greater than 0.5 in the case of enhanced siRNA in vivo delivery.

[0207] Thus, conjugating a lipophilic moiety to an internal position of a double-stranded RNAi agent provides optimal hydrophobicity in siRNA for enhanced in vivo delivery.

[0208] The term "lipid nanoparticle" or "LNP" refers to a vesicle comprising a lipid layer that encapsulates a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., a plasmid from which an rNAi or RNAi agent is transcribed. LNPs are described, for example, in U.S. Patent Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference.

[0209] As used herein, a "subject" is an animal, such as a mammal, including primates (e.g., humans, non-human primates, e.g., monkeys and chimpanzees) or non-primates (e.g., cows, sheep, rats, mice, etc.). In one embodiment, the subject is a human, such as a human being treated or evaluated for a disease, disorder, or condition that would benefit from reduced PRNP expression, a human being at risk for a disease, disorder, or condition that would benefit from reduced PRNP expression, a human having a disease, disorder, or condition that would benefit from reduced PRNP expression, or a human being treated for a disease, disorder, or condition that would benefit from reduced PRNP expression as described herein.

[0210] As used herein, the terms "treat" or "treatment" refer to a beneficial or desired result, including, but not limited to, the alleviation or amelioration of one or more signs or symptoms associated with PRNP gene expression or PRNP protein production, e.g., a PRNP-associated disease, e.g., a prion disease such as an inherited prion disease, a sporadic prion disease, or an acquired prion disease, in a subject with such a disease. "Treatment" can also mean prolonging survival compared to expected survival if no treatment is administered.

[0211] The term "lower" in the context of the level of PRNP or a disease marker or symptom in a subject refers to a statistically significant decrease in such level. The decrease can be, for example, at least 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In certain embodiments, the decrease is at least 20%. In certain embodiments, the decrease is at least 50% in a disease marker, e.g., in the protein level or gene expression level. "Lowering" in the context of the level of PRNP in a subject preferably refers to a decrease to a level that is accepted as being within the normal range in an individual without the disorder. In certain embodiments, "lowering" refers to a decrease in the difference between the level of a marker or symptom in a subject suffering from a disease and a level that an individual would consider to be within the normal range, for example, a level of weight loss between an obese individual and an individual whose weight is considered to be within the normal range.

[0212] As used herein, "prevention" or "preventing," when used in reference to a disease, disorder, or condition that would benefit from reduced PRNP gene expression or PRNP protein production, refers to a reduced likelihood that a subject will develop symptoms associated with such disease, disorder, or condition, e.g., symptoms of a PRNP-associated disease. A failure to develop the disease, disorder, or condition, or a reduction in the onset of symptoms associated with such disease, disorder, or condition, e.g., a reduction of at least about 10% by a magnitude clinically acceptable for the disease or disorder, or a delayed onset of symptoms (e.g., a delay of days, weeks, months, or years), is considered effective prevention.

[0213] As used herein, the term "PRNP-associated disease" or "PRNP-associated disorder" refers to any disease or disorder that would benefit from reduced expression or activity of PRNP. Examples of such PRNP-associated diseases include those characterized by the abnormally folded scrapie form of PrP protein (PrP) in brain regions associated with neuronal cell death in diseases such as inherited prion diseases, e.g., Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI), sporadic prion diseases, e.g., sporadic Creutzfeldt-Jakob disease, sporadic fatal insomnia (sFI), and variably proteinase-sensitive prion disease (VPSPr), or acquired prion diseases, e.g., iatrogenic CJD (iCJD), kuru, and variant CJD (vCJD). sc ) deposition.

[0214] In one embodiment, the PRNP-associated disease is a prion disease. Prion diseases are chronic neurodegenerative disorders caused by abnormal prions, microscopic infectious agents made of proteins. Prions cause numerous diseases in various mammals, including bovine spongiform encephalopathy (BSE or mad cow disease) in cattle and scrapie in sheep. The term "spongiform" refers to the characteristic appearance of the infected brain, which becomes porous until it resembles a sponge when viewed under a microscope. Prion diseases are classified into three different subtypes based on how the disease is contracted. Each subtype differs slightly in terms of typical signs, symptoms, and duration of the disease. The subtypes are sporadic, hereditary, and acquired. In some embodiments, the PRNP-associated disease is a hereditary prion disease, such as familial Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI). In some embodiments, the PRNP-associated disease is a sporadic prion disease, such as sporadic Creutzfeldt-Jakob disease or sporadic fatal insomnia. In other embodiments, the PRNP-associated disease is an acquired prion disease, such as iatrogenic Creutzfeldt-Jakob disease, variant Creutzfeldt-Jakob disease, or kuru.

[0215] Prion diseases usually begin slowly and worsen over time. Common symptoms of prion diseases include impaired thinking, memory, and judgment; personality changes such as apathy, agitation, and depression; confusion or disorientation (e.g., getting lost easily); involuntary muscle spasms (myoclonus); loss of coordination (ataxia); sleep problems (insomnia); difficulty speaking, slurring, and impaired vision or blindness. As the condition worsens, people often withdraw from family and society. Gradual loss of bodily functions leads to eventual death.

[0216] Creutzfeldt-Jakob disease (CJD) is the most common of the known human prion diseases. CJD is a rare, degenerative, and fatal brain disorder. It affects approximately 1 in 1 million people annually worldwide, with approximately 350 people affected annually in the United States. CJD usually develops later in life and progresses rapidly. Typical onset of symptoms occurs around age 60, with approximately 70% of people dying within one year. In the early stages of the disease, memory loss, behavioral changes, lack of coordination, and visual impairment may occur. As the disease progresses, mental decline becomes more pronounced, and involuntary movements, blindness, limb weakness, and coma may occur.

[0217] There are three major categories of CJD. In sporadic CJD, the disease develops even if the person has no known risk factors for the disease. This is the most common type of CJD, accounting for at least 85% of cases. In hereditary CJD, patients have a family history of the disease and may test positive for a genetic mutation associated with CJD. Approximately 10–15% of CJD cases in the United States are hereditary. In acquired CJD, the disease is transmitted by exposure to brain or nervous system tissue, usually through certain medical procedures. There is no evidence that CJD is transmitted through casual contact with someone with CJD. Since CJD was first described in 1920, acquired CJD accounts for less than 1% of cases. A type of CJD called variant CJD (or vCJD) can be contracted by eating meat from cattle infected with bovine spongiform encephalopathy (BSE), or a CJD-like disease commonly referred to as "mad cow disease."

[0218] CJD is characterized by rapidly progressive dementia. Early on, patients experience problems with muscle coordination, personality changes (including impaired memory, judgment, and thinking), and visual impairment. People with the disease, especially those with FFI, may also experience insomnia, depression, or unusual sensations. As the disease progresses, people's mental impairment becomes more severe. They often develop involuntary muscle spasms called myoclonus and may become blind. Eventually, they become unable to move or speak and fall into a coma. Pneumonia and other infections often develop in these patients and can be fatal. Variant CJD begins primarily with psychiatric symptoms, strikes younger people than other types of CJD, and typically has a longer time frame between the onset of symptoms and death.

[0219] Some symptoms of CJD are similar to those of other progressive neurological disorders, such as Alzheimer's disease and Huntington's disease. However, CJD causes unique changes in brain tissue that can be seen during autopsy. It also tends to cause a more rapid decline in a person's abilities than Alzheimer's disease or most other types of dementia.

[0220] As used herein, a "therapeutically effective amount" is intended to include an amount of an RNAi agent that, when administered to a subject with a PRNP-related disease, is sufficient to treat the disease (e.g., by reducing, ameliorating, or maintaining an existing disease or one or more symptoms of the disease). A "therapeutically effective amount" can vary depending on the RNAi agent, the method of administration of the agent, the disease and its severity and medical history, age, weight, family history, genetic makeup, type of prior or concurrent treatment, if any, and other personal characteristics of the subject to be treated.

[0221] As used herein, a "prophylactically effective amount" is intended to include an amount of an RNAi agent that, when administered to a subject with a PRNP-related disorder, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Amelioration of the disease includes slowing the progression of the disease or reducing the severity of any future disease. A "prophylactically effective amount" may vary depending on the RNAi agent, the method of administration of the agent, the degree of risk of the disease, and the patient's medical history, age, weight, family history, genetic makeup, type of prior or concurrent treatment, if any, and other personal characteristics of the patient to be treated.

[0222] A "therapeutically effective amount" or a "prophylactically effective amount" also encompasses the amount of an RNAi agent that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The RNAi agent used in the methods of the present disclosure can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0223] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials (including salts), compositions or dosage forms that are suitable for use in contact with the tissues of human and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0224] The phrase "pharmaceutically acceptable carrier," as used herein, means a pharmaceutically acceptable material, composition, or vehicle involved in the transport or transfer of a subject compound from one organ or part of the body to another, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate, or stearic acid), or solvent encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the subject being treated. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium state, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar. (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) pH buffer solutions, (21) polyesters, polycarbonates, or polyanhydrides, (22) bulking agents, such as polypeptides and amino acids, (23) serum components, such as serum albumin, HDL, and LDL, and (22) other non-toxic, compatible substances employed in pharmaceutical formulations.

[0225] The term "sample," as used herein, encompasses similar fluids, cells, or tissues isolated from a subject, as well as collections of fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum, and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, saliva, and the like. Tissue samples can include samples from tissues, organs, or localized regions. For example, samples can be derived from specific organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples can be obtained from the brain (e.g., the whole brain or specific segments of the brain, such as the striatum, or specific types of cells within the brain, such as neurons and glial cells (astrocytes, oligodendrocytes, microglia)). In other embodiments, "a sample obtained from a subject" refers to liver tissue (or a subcomponent thereof) obtained from a subject. In some embodiments, "a sample obtained from a subject" refers to blood obtained from a subject or plasma or serum obtained therefrom. In a further embodiment, "a sample obtained from a subject" refers to brain tissue (or a subcomponent thereof) or retinal tissue (or a subcomponent thereof) obtained from a subject.

[0226] II. RNAi Agents of the Disclosure The present specification describes an RNAi agent that inhibits the expression of the PRNP gene. In one embodiment, the RNAi agent comprises a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of the PRNP gene in a cell, such as a cell in a mammal, of a subject, such as a human, with a PRNP-related disease, for example, a genetic prion disease, such as familial Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI), a sporadic prion disease, such as sporadic Creutzfeldt-Jakob disease, sporadic fatal insomnia (sFI), and variably proteinase-sensitive prion disease (VPSPr), or an acquired prion disease, such as iatrogenic CJD (iCJD), kuru, and variant CJD (vCJD).

[0227] The dsRNA comprises an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed upon expression of the PRNP gene. The region of complementarity is no more than about 15-30 nucleotides in length. When contacted with a cell expressing the PRNP gene, the RNAi agent inhibits expression of the PRNP gene (e.g., human, primate, or non-primate gene) by at least 50%, as assayed, for example, by PCR or branched DNA (bDNA)-based methods, or by protein-based methods, such as immunofluorescence, e.g., using Western blotting or flow cytometry techniques. In certain embodiments, inhibition of expression is at least 50%, as assayed by the Dual-Glo luciferase assay of Example 1, where the siRNA is at a concentration of 10 nM.

[0228] dsRNA comprises two RNA strands, which are complementary and hybridize to form a duplex structure under the conditions in which the dsRNA is used. One strand of the dsRNA (antisense strand) comprises a complementary region that is substantially complementary, and generally completely complementary, to the target sequence. The target sequence can be obtained from the sequence of the mRNA formed during the expression of the PRNP gene. The other strand (sense strand) comprises a region that is complementary to the antisense strand, so that the two strands hybridize to form a duplex structure when combined under suitable conditions. As described elsewhere herein and known in the art, the complementary sequences of dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, such that they are opposite each other on separate oligonucleotides.

[0229] Generally, the duplex structure is 15-30 base pairs in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-22, 19-23, 19-24, 19-25, 19-26, 19-27, 19-28, 19-29, 19-30, 19-31, 19-32, 19-33, 19-34, 19-35, 19-36, 19-37, 19-38, 19-39, 19-40, 19-41, 19-42, 19-43, 19-44, 19-45, 19-46, 19-47, 19-48, 19-49, 19-50, 19-51, 19-52, 19-53, 19-54, 19-55, 19-56, 19-57, 19-58, 19-59, 19-60, 19-61, 19-62, 19-63, 19-64, 19-65, 19-6 9, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. In certain embodiments, the duplex structure is 18 to 25 base pairs in length, e.g., 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 25, 21 to 24, 21 to 23, 21 to 22, 22 to 25, 22 to 24, 22 to 23, 23 to 25, 23 to 24, or 24 to 25 base pairs in length, e.g., 19 to 21 base pairs in length. Ranges and lengths between the ranges and lengths listed above are also intended to be part of this disclosure.

[0230] Similarly, the region of complementarity to the target sequence may be 15 to 30 nucleotides in length, e.g., 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 21-23, or 21-22 nucleotides in length, e.g., 19-23 nucleotides in length or 21-23 nucleotides in length. Ranges and lengths that lie between the ranges and lengths listed above are also intended to be part of this disclosure.

[0231] In some embodiments, the duplex structure is 19-30 base pairs in length. Similarly, the region complementary to the target sequence is 19-30 nucleotides in length.

[0232] In some embodiments, the dsRNA is 15-23 nucleotides in length or 25-30 nucleotides in length. Generally, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNA longer than about 21-23 nucleotides can serve as a substrate for Dicer. As those skilled in the art will recognize, the region of RNA targeted for cleavage will most often be a portion of a longer RNA molecule, often an mRNA molecule. In relevant cases, a "portion" of an mRNA target is a contiguous sequence of the mRNA target that is long enough to allow it to serve as a substrate for RNAi-dependent cleavage (i.e., cleavage via the RISC pathway).

[0233] Those skilled in the art will appreciate that the duplex region is a primary functional portion of the dsRNA, e.g., about 15-36 base pairs, e.g., 15-36, 15-35, 15-34, 15-33, 15-32, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21 , 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs, for example, 19-21 base pairs. That is, in one embodiment, an RNA molecule or complex of RNA molecules having a duplex region of more than 30 base pairs is a dsRNA, as long as it is processed into a functional duplex of, for example, 15-30 base pairs that targets the desired RNA for cleavage. Thus, those skilled in the art will recognize that, in one embodiment, miRNA is a dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In another embodiment, the RNAi agent useful for targeting PRNP expression is not generated in target cells by cleavage of a larger dsRNA.

[0234] The dsRNA described herein can further comprise one or more single-stranded nucleotide overhangs, for example, 1, 2, 3 or 4 nucleotides.Nucleotide overhangs can comprise or consist of nucleotide / nucleoside analogs, such as deoxynucleotides / nucleosides.Overhangs can be on sense strand, antisense strand, or any combination thereof.Furthermore, the nucleotide of a certain overhang can be present on the 5'-end, 3'-end, or both ends of either the antisense strand or the sense strand of dsRNA.In certain embodiments, longer extended overhangs are possible.

[0235] dsRNA can be synthesized by standard methods known in the art, for example, using an automated DNA synthesizer, such as those commercially available from Biosearch, Applied Biosystems, Inc., as discussed further below.

[0236] The iRNA compounds of the present invention can be prepared using a two-step method. First, the individual strands of the double-stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compounds can be prepared using solution phase or solid phase organic synthesis or both. Organic synthesis has the advantage that it is easy to prepare oligonucleotide strands containing unnatural nucleotides or modified nucleotides. The single-stranded oligonucleotides of the present invention can be prepared using solution phase or solid phase organic synthesis or both.

[0237] siRNAs can be produced, e.g., in bulk, by a variety of methods. Exemplary methods include organic synthesis and RNA cleavage, e.g., in vitro cleavage.

[0238] siRNAs can be produced by synthesizing single-stranded RNA molecules, or each strand of a double-stranded RNA molecule separately, and then annealing the component strands.

[0239] Large-scale bioreactors, such as the OligoPilot II from Pharmacia Biotec AB (Uppsala, Sweden), can be used to produce large quantities of specific RNA strands for a given siRNA. The OligoPilot II reactor can efficiently attach nucleotides using only a 1.5 molar excess of phosphoramidite nucleotides. Ribonucleotide amidites are used to create the RNA strands. Using standard cycles of monomer addition, 21- to 23-nucleotide strands of siRNA can be synthesized. Typically, the two complementary strands are generated separately and then annealed, for example, after release from the solid support and deprotection.

[0240] Organic synthesis can be used to produce individual siRNA species.The complementarity of species to PRNP gene can be precisely specified.For example, species can be complementary to the region that contains polymorphism, for example, single nucleotide polymorphism.Furthermore, the position of polymorphism can be precisely specified.In some embodiments, polymorphism is located in the internal region, for example, within at least 4, 5, 7 or 9 nucleotides from one or both ends.

[0241] In one embodiment, the RNA produced is carefully purified, and end siRNA is removed, and for example, using Dicer or equivalent RNAse III-based activity to be cut into siRNA in vitro.For example, dsiRNA can be incubated in an extract from Drosophila in vitro, or with purified components, for example, purified RNAse or RISC complex (RNA-induced silencing complex).For example, see Ketting et al.Genes Dev 2001 Oct 15;15(20):2654-9 and Hammond Science 2001 Aug 10;293(5532):1146-50.

[0242] dsiRNA cleavage typically generates multiple siRNA species, each a specific 21-23 nt fragment of the source dsiRNA molecule. For example, there may be siRNAs that contain sequences complementary to overlapping and flanking regions of the source dsiRNA molecule.

[0243] Regardless of synthesis method, siRNA preparation can be prepared in a solution (for example, aqueous solution or organic solution) that is suitable for formulation.For example, siRNA preparation can be precipitated, redissolved in pure double distilled water, and lyophilized.Then, dried siRNA can be resuspended in a solution that is suitable for the intended formulation process.

[0244] In one embodiment, the dsRNA of the present disclosure comprises at least two nucleotide sequences, a sense strand and an antisense strand. The sense strand sequence for PRNP can be selected from the sequences provided in any one of Tables 2-3, and the corresponding nucleotide sequence of the antisense strand of the sense strand can be selected from the sequences provided in any one of Tables 2-3. In this embodiment, one of the two sequences is complementary to the other of the two sequences, where one of the sequences is substantially complementary to the sequence of mRNA produced upon expression of the PRNP gene. Thus, in this embodiment, the dsRNA comprises two oligonucleotides, one oligonucleotide designated as the sense strand (passenger strand) in any one of Tables 2-3 for PRNP, and the second oligonucleotide designated as the corresponding antisense strand (guide strand) of the sense strand in any one of Tables 2-3.

[0245] In one embodiment, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides, hi another embodiment, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

[0246] Although the sequences provided herein are described as modified or conjugated sequences, it will be understood that the RNA of the RNAi agents of this disclosure, e.g., the dsRNA of this disclosure, can comprise any one of the sequences set forth in any one of Tables 2-3, unmodified, unconjugated, or modified or conjugated differently from those set forth therein. One or more lipophilic ligands or one or more GalNAc ligands can be included at any position in the RNAi agents provided in this application.

[0247] Those skilled in the art are well aware that dsRNAs having duplex structures of approximately 20-23 base pairs, e.g., 21 base pairs, have been hailed as particularly effective in inducing RNA interference (Elbashir et al., (2001) EMBO J., 20:6877-6888). However, others have discovered that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the above-described embodiments, due to the nature of the oligonucleotide sequences provided herein, the dsRNAs described herein can comprise at least one strand that is a minimum of 21 nucleotides in length. It can be reasonably predicted that shorter duplexes, minus only a few nucleotides at one or both ends, can be similarly effective compared to the dsRNAs described above. Thus, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences provided herein and that differ in their ability to inhibit expression of the PRNP gene by no more than 10, 15, 20, 25, or 30% inhibition from dsRNAs containing the complete sequence using in vitro assays with Cos7 and 10 nM concentrations of the RNA agent and the PCR assays provided in the Examples herein are intended to be within the scope of this disclosure.

[0248] In addition, the RNA described herein specifies the site in PRNP transcript that is susceptible to RISC-mediated cleavage.Therefore, the present disclosure also features the RNAi agent that targets within this site.As used herein, if the RNAi agent promotes the cleavage of the transcript at any of specific sites, it is said that the RNAi agent targets within the specific site of RNA transcript.This RNAi agent will generally comprise at least about 15 consecutive nucleotides, for example, at least 19 nucleotides, from one of the sequences provided herein, which is combined with additional nucleotide sequences taken from the region adjacent to the selected sequence in PRNP gene.

[0249] The RNAi agents described herein may contain one or more mismatches to the target sequence. In one embodiment, the RNAi agents described herein contain three or fewer mismatches (i.e., three, two, one, or zero mismatches). In one embodiment, the RNAi agents described herein contain two or fewer mismatches. In one embodiment, the RNAi agents described herein contain one or fewer mismatches. In one embodiment, the RNAi agents described herein contain zero mismatches. In certain embodiments, when the antisense strand of an RNAi agent contains a mismatch to the target sequence, the mismatch can be optionally limited to be within the last five nucleotides from either the 5'-end or the 3'-end of the complementary region. For example, in such an embodiment, in the case of a 23-nucleotide RNAi agent, the strand complementary to a region of the PRNP gene generally does not contain any mismatches within the central 13 nucleotides. Using the methods described herein or known in the art, it can be determined whether an RNAi agent containing a mismatch to the target sequence is effective in inhibiting the expression of the PRNP gene. In particular, if a particular region of complementarity in the PRNP gene is known to have polymorphic sequence variation within the population, it is important to consider the efficacy of mismatched RNAi agents to inhibit expression of PRNP.

[0250] III. Modified RNAi Agents of the Present Disclosure In one embodiment, the RNA of the RNAi agent of the present disclosure, e.g., dsRNA, is unmodified and does not contain, for example, chemical modifications or conjugations known in the art and described herein. In certain embodiments, the RNA of the RNAi agent of the present disclosure, e.g., dsRNA, is chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the present disclosure, substantially all of the nucleotides of the RNAi agent of the present disclosure are modified. In other embodiments of the present disclosure, all of the nucleotides of the RNAi agent of the present disclosure are modified. An RNAi agent of the present disclosure in which "substantially all of the nucleotides are modified" may be widely modified but not entirely modified, and may contain 5, 4, 3, 2, or 1 or less unmodified nucleotides. In yet other embodiments of the present disclosure, an RNAi agent of the present disclosure may contain 5, 4, 3, 2, or 1 or less modified nucleotides.

[0251] Nucleic acids featured in this disclosure can be synthesized or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S. Lett. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, inverted linkage) or 3'-end modifications (conjugation, DNA nucleotides, inverted linkage, etc.), base modifications, such as substitution with stabilizing bases, destabilizing bases, or bases that base-pair with partners in an extended repertoire, base removal (abasic nucleotides) or conjugated bases, sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions, or backbone modifications, including phosphodiester bond modifications or substitutions. Specific examples of RNAi agents useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or lacking natural internucleoside linkages. Among the RNAs with modified backbone, those that do not have phosphorus atom in backbone are included.For the purpose of this specification, and as sometimes referred to in the art, the modified RNAs that do not have phosphorus atom in their internucleoside backbone can also be considered as oligonucleoside.In some embodiments, modified RNAi agent will have phosphorus atom in its internucleoside backbone.

[0252] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and those with reversed polarity, where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.Various salts, such as sodium salts, mixed salts, and free acid forms, are also included.

[0253] Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Patent Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,195, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,711, and the like. No. 7, No. 5,321,131, No. 5,399,676, No. 5,405,939, No. 5,453,496, No. 5,455,233, No. 5,466,677, No. 5,47 No. 6,925, No. 5,519,126, No. 5,536,821, No. 5,541,316, No. 5,550,111, No. 5,563,253, No. 5,571,799, No. 5 , 587,361, 5,625,050, 6,028,188, 6,124,445, 6,160,109, 6,169,170, 6,172,209 No. 6,239,265, No. 6,277,603, No. 6,326,199, No. 6,346,614, No. 6,444,423, No. 6,531,590, No. 6,534, Nos. 6,639, 6,608,035, 6,683,167, 6,858,715, 6,867,294, 6,878,805, 7,015,315, 7,041,816, 7,273,933, 7,321,029, and U.S. Patent No. RE39464, the contents of each of which are incorporated herein by reference in their entirety.

[0254] Modified RNA backbones that do not contain a phosphorus atom in the backbone have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages, including those with morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH2 constituent moieties.

[0255] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Patent Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,64,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, Nos. 5,489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, and 5,677,439, the entire contents of each of which are incorporated herein by reference.

[0256] In another embodiment, RNA mimics suitable for use in RNAi agents are considered, in which both the sugar and internucleoside linkages of nucleotide units, i.e., the backbone, are replaced with novel groups. The base units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an RNA mimic known to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and directly or indirectly linked to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262, the entire contents of each of which are incorporated herein by reference. Additional PNA compounds suitable for use in the RNAi agents of the present disclosure are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0257] Some embodiments featured in this disclosure include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, particularly --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- (known as methylene (methylimino) or MMI backbones), --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- of the above-referenced U.S. Patent No. 5,489,677, and the amide backbones of the above-referenced U.S. Patent No. 5,602,240. In some embodiments, RNAs featured herein have the morpholino backbone structure of the above-referenced U.S. Patent No. 5,034,506. The natural phosphodiester backbone can be represented as -OP(O)(OH)-OCH2-.

[0258] Modified RNAs can also contain one or more substituted sugar moieties. The RNAi agents featured herein, e.g., dsRNAs, can include one of the following at the 2' position: OH, F, O-, S-, or N-alkyl, O-, S-, or N-alkenyl, O-, S-, or N-alkynyl, or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6. 10 Alkyl or C2-C 10 It can be alkenyl or alkynyl. Exemplary suitable modifications include O[(CH) n O] m CH3, O(CH2). n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2, and O(CH2) n ON[(CH2) n CH3)]2, where n and m are from 1 to about 10. In other embodiments, the dsRNA includes one of the following at the 2' position: C1 to C 10The substituents include lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH, OCN, Cl, Br, CN, CF, OCF, SOCH, SOCH, ONO, NO, N, NH, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, interfering substances, groups for improving the pharmacokinetic properties of RNAi agents or groups for improving the pharmacodynamic properties of RNAi agents, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O-CHCHOCH, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is the 2'-dimethylaminooxyethoxy, i.e., O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described herein below in the Examples, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH3)2. Further exemplary modifications include 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides (both R and S isomers within these three families), 2'-alkoxyalkyl, and 2'-NMA (N-methylacetamide).

[0259] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHNH), 2'-O-hexadecyl, and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an RNAi agent, particularly the 3' position of the sugar on the 3'-terminal nucleotide or in a 2'-5'-linked dsRNA and the 5' position of the 5'-terminal nucleotide. RNAi agents can also have sugar mimetics, such as a cyclobutyl moiety in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,514,785, 5,519,134, and 5,56 Nos. 7,811, 5,576,427, 5,591,722, 5,597,909, 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,658,873, 5,670,633, and 5,700,920, some of which are commonly owned with this application, the entire contents of each of the foregoing being incorporated herein by reference.

[0260] The RNAi agent of the present disclosure can also comprise modification or substitution of nucleobase (often simply referred to in the art as "base").As used herein, "unmodified" or "natural" nucleobase includes purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C) and uracil (U).Modified nucleobases include other synthetic nucleobases and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, These include 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine.Additional nucleobases include those disclosed in United States Patent No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P.ed.Wiley-VCH, 2008, those disclosed in The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, JL, ed.John Wiley&Sons, 1990, those disclosed in Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613, and those disclosed in Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993.Some of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the present disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, such as 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y.S., Crooke, S.T., and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and are exemplary base substitutions, especially when combined with 2'-O-methoxyethyl sugar modifications.

[0261] Representative United States patents that teach the preparation of certain of the above modified nucleobases, as well as other modified nucleobases, include, but are not limited to, the above-referenced U.S. Patent Nos. 3,687,808, 4,845,205, 5,130,30, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, 5,552,540, 5,587,469, Nos. 5,594,121, 5,596,091, 5,614,617, 5,681,941, 5,750,692, 6,015,886, 6,147,200, 6,166,197, 6,222,025, 6,235,887, 6,380,368, 6,528,640, 6,639,062, 6,617,438, 7,045,610, 7,427,672, and 7,495,088, the entire contents of each of which are incorporated herein by reference.

[0262] The RNAi agents of the present disclosure can also be modified to include one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by a ring formed by a bridge between two carbon atoms, whether adjacent or non-adjacent. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety that includes a ring formed by bridging two carbon atoms of the sugar ring, whether adjacent or non-adjacent, thereby forming a bicyclic ring system. In certain embodiments, a bridge connects the 4'-carbon and 2'-carbon of the sugar ring, optionally via a 2'-acyclic oxygen atom. Thus, in some embodiments, the agents of the present disclosure can include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety includes an additional bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide containing a bicyclic sugar moiety that includes a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose in the 3'-endo conformation. The addition of a locking nucleic acid to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in the polynucleotides of the present disclosure include, but are not limited to, nucleosides comprising a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agent of the present disclosure includes one or more bicyclic nucleosides comprising a 4' to 2' bridge.

[0263] A locked nucleoside can be represented by this structure (stereochemistry omitted): [ka] wherein B is a nucleobase or modified nucleobase, and L is a linking group connecting the 2'-carbon and 4'-carbon of the ribose ring. Examples of such 4' to 2' bridged bicyclic nucleosides include, but are not limited to, 4'-(CH2)-O-2' (LNA), 4'-(CH2)-S-2', 4'-(CH2)2-O-2' (ENA), 4'-CH(CH3)-O-2' (also referred to as "constrained ethyl" or "cEt"), and 4'-CH(CHOCH3)-O-2' (and analogs thereof, e.g., see U.S. Pat. No. 7,399,844). No. 5), 4'-C(CH3)(CH3)-O-2' (and analogs thereof, see, e.g., U.S. Pat. No. 8,278,283), 4'-CH2-N(OCH3)-2' (and analogs thereof, see, e.g., U.S. Pat. No. 8,278,425), 4'-CH2-ON(CH3)-2' (see, e.g., U.S. Patent Application Publication No. 2004 / 0171570), 4'-CH2-N(R)-O-2', where R is H, C1-C 12 These include alkyl or nitrogen protecting groups (see, e.g., U.S. Pat. No. 7,427,672), 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134), and 4'-CH2-C(=CH2)-2' (and analogs thereof, see, e.g., U.S. Pat. No. 8,278,426). The entire contents of each of the foregoing are incorporated herein by reference.

[0264] Additional representative U.S. patents and publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490, 6,525,191, 6,670,461, 6,770,748, 6,794,499, 6,998,484, 7,053,207, 7,034,133, 7,084,125, 7,399,845, 7,427,672, 7,569,686, 7,741,457, 8,022,193, 8,030,467, 8,278,425, 8,278,426, 8,278,283, No. 2008 / 0039618, and US2009 / 0012281, the entire contents of each of which are incorporated herein by reference.

[0265] Any of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0266] The RNAi agents of the present disclosure can also be modified to contain one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid containing a bicyclic sugar moiety containing a 4'-CH(CH3)-O-2' bridge (L in the preceding structure). In one embodiment, the constrained ethyl nucleotide is in the S conformation and is referred to herein as an "S-cEt."

[0267] The RNAi agent of the present disclosure may also contain one or more "conformation-restricting nucleotides" ("CRNs"). A CRN is a nucleotide analogue with a linker connecting the C2' and C4' carbons of ribose, or the PRNP and -C5' carbons of ribose. The CRN locks the ribose ring into a stable conformation, increasing hybridization affinity to mRNA. The linker is long enough to position the oxygen at an optimal position for stability and affinity, thereby reducing puckering of the ribose ring.

[0268] Representative publications that teach the preparation of certain of the above CRNs include, but are not limited to, US2013 / 0190383 and WO2013 / 036868, the entire contents of each of which are incorporated herein by reference.

[0269] In some embodiments, the RNAi agent of the present disclosure includes one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNAs are unlocked acyclic nucleic acids in which any of their sugar linkages have been removed to form an unlocked "sugar" residue. In one example, UNAs also encompass monomers in which the C1'-C4' bond has been removed (i.e., a covalent carbon-oxygen-carbon bond between the C1' and C4' carbons). In another example, the C2'-PRNP' bond of the sugar has been removed (i.e., a covalent carbon-carbon bond between the C2' and PRNP' carbons) (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which are incorporated herein by reference).

[0270] Representative U.S. publications that teach the preparation of UNAs include, but are not limited to, U.S. Pat. No. 8,314,227, and U.S. Patent Application Publication Nos. 2013 / 0096289, 2013 / 0011922, and 2011 / 0313020, the entire contents of each of which are incorporated herein by reference.

[0271] Potential stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-0-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3'-phosphate, inverted 2'-deoxy-modified ribonucleotides such as inverted dT (idT) and inverted dA (idA), and inverted abasic 2'-deoxyribonucleotides (iAb), among others. Disclosure of these modifications can be found in WO 2011 / 005861.

[0272] In one example, the 3' or 5' end of the oligonucleotide is linked to an inverted 2'-deoxy modified ribonucleotide, such as an inverted dT (idT), an inverted dA (idA), or an inverted abasic 2'-deoxyribonucleotide (iAb). In one particular example, the inverted 2'-deoxy-modified ribonucleotide is linked to the 3' end of the oligonucleotide, such as the 3' end of the sense strand described herein, where the linkage is via a 3'-3' phosphodiester linkage or a 3'-3'-phosphorothioate linkage.

[0273] In another embodiment, the 3' end of the sense strand is linked to an inverted abasic ribonucleotide (iAb) via a 3'-3'-phosphorothioate linkage. In another embodiment, the 3' end of the sense strand is linked to an inverted dA (idA) via a 3'-3'-phosphorothioate linkage.

[0274] In one particular example, the inverted 2'-deoxy-modified ribonucleotide is linked to the 3' end of an oligonucleotide, such as the 3' end of the sense strand described herein, wherein the linkage is via a 3'-3' phosphodiester linkage or a 3'-3'-phosphorothioate linkage.

[0275] In another example, the 3'-terminal nucleotide of the sense strand is an inverted dA (idA) and is joined to the preceding nucleotide via a 3'-3'-linkage (eg, a 3'-3'-phosphorothioate linkage).

[0276] Other modifications of the RNAi agent of the present disclosure include 5' phosphate or 5' phosphate mimic, for example, the 5' terminal phosphate or phosphate mimic on the antisense strand of RNAi agent.Suitable phosphate mimic is disclosed, for example, in US2012 / 0157511, the entire content of which is incorporated herein by reference.

[0277] A. Modified RNAi Agents Comprising Motifs of the Disclosure In certain aspects of the present disclosure, the double-stranded RNAi agent of the present disclosure includes an agent having chemical modifications, such as those disclosed in International Publication No. 2013 / 075035, the entire contents of which are incorporated herein by reference. As shown herein and in International Publication No. 2013 / 075035, one or more motifs of three identical modifications on three consecutive nucleotides can be introduced into the sense or antisense strand of the RNAi agent, particularly at or near the cleavage site. In some embodiments, the sense and antisense strands of the RNAi agent can be otherwise completely modified. The introduction of these motifs interrupts the modification pattern of the sense or antisense strand, if present. The RNAi agent can optionally be conjugated with a lipophilic ligand, for example, a C16 ligand, for example, on the sense strand. The RNAi agent can optionally be modified with an (S)-glycol nucleic acid (GNA) modification, for example, at one or more residues of the antisense strand.

[0278] Thus, the present disclosure provides a double-stranded RNAi agent capable of inhibiting expression of a target gene (i.e., the PRNP gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent can be 15 to 30 nucleotides in length. Each strand can be, for example, 16 to 30 nucleotides in length, 17 to 30 nucleotides in length, 25 to 30 nucleotides in length, 27 to 30 nucleotides in length, 17 to 23 nucleotides in length, 17 to 21 nucleotides in length, 17 to 19 nucleotides in length, 19 to 25 nucleotides in length, 19 to 23 nucleotides in length, 19 to 21 nucleotides in length, 21 to 25 nucleotides in length, or 21 to 23 nucleotides in length. In certain embodiments, each strand is 19 to 23 nucleotides in length.

[0279] The sense and antisense strands typically form a duplex, double-stranded RNA ("dsRNA"), also referred to herein as an "RNAi agent." The duplex region of an RNAi agent can be 15-30 nucleotide pairs in length. For example, the duplex region can be 16-30 nucleotide pairs in length, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another example, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length. In certain embodiments, the duplex region is 19 to 21 nucleotide pairs in length.

[0280] In one embodiment, the RNAi agent may contain one or more overhang regions or capping groups at the 3'-end, 5'-end, or both ends of one or both strands. The overhangs may be 1 to 6 nucleotides in length, e.g., 2 to 6 nucleotides in length, 1 to 5 nucleotides in length, 2 to 5 nucleotides in length, 1 to 4 nucleotides in length, 2 to 4 nucleotides in length, 1 to 3 nucleotides in length, 2 to 3 nucleotides in length, or 1 to 2 nucleotides in length. In certain embodiments, the nucleotide overhang region is 2 nucleotides in length. The overhang may be the result of one strand being longer than the other, or the result of two strands of the same length being twisted. The overhang may form a mismatch with the target mRNA, be complementary to the targeted gene sequence, or be a different sequence. The first and second strands may also be linked by additional bases, for example, to form a hairpin, or by other non-basic linkers.

[0281] In one embodiment, the nucleotides in the overhang region of an RNAi agent can each independently be modified or unmodified nucleotides, including, but not limited to, 2'-sugar modifications, such as 2-F, 2'-O-methyl, thymidine (T), and any combination thereof.

[0282] For example, TT can be an overhang sequence for either end on either strand, which can form a mismatch with the target mRNA, or can be complementary to the targeted gene sequence, or can be another sequence.

[0283] The 5'- or 3'-overhang of the sense strand, antisense strand, or both strands of the RNAi agent can be phosphorylated. In some embodiments, the overhang region contains two nucleotides with a phosphorothioate between them, and the two nucleotides can be the same or different. In one embodiment, the overhang is present at the 3'-end of the sense strand, antisense strand, or both strands. In one embodiment, the 3'-overhang is present in the antisense strand. In one embodiment, the 3'-overhang is present in the sense strand.

[0284] RNAi agents can contain only a single overhang, which can enhance the interference activity of RNAi without affecting its overall stability.For example, the single-stranded overhang can be located at the 3'-end of the sense strand or at the 3'-end of the antisense strand.RNAi can also have a blunt end located at the 5'-end of the antisense strand (i.e., the 3'-end of the sense strand), or vice versa.Generally, the antisense strand of RNAi has a nucleotide overhang at the 3'-end, and the 5'-end is blunt.Without wishing to be bound by theory, the asymmetry between the blunt end at the 5'-end of the antisense strand and the 3'-end overhang of the antisense strand is favorable for the insertion of the guide strand into the RISC process.

[0285] In one embodiment, the RNAi agent is 19 nucleotides in length and double-blunt ended, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 7, 8, and 9 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0286] In another embodiment, the RNAi agent is 20 nucleotides in length and double-blunt ended, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 8, 9, and 10 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0287] In yet another embodiment, the RNAi agent is 21 nucleotides in length and double-blunt ended, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0288] In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, and one end of the RNAi agent is blunt and the other end contains a two-nucleotide overhang. In one example, the two-nucleotide overhang is at the 3' end of the antisense strand. When the two-nucleotide overhang is at the 3' end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the three terminal nucleotides, two of which are overhanging nucleotides, and the third nucleotide is a paired nucleotide adjacent to the overhanging nucleotide. In one embodiment, the RNAi agent further comprises two phosphorothioate internucleotide linkages between the three terminal nucleotides at both the 5'-end of the sense strand and the 5'-end of the antisense strand.In one embodiment, every nucleotide in the sense strand and antisense strand of the RNAi agent, including the nucleotide that is part of a motif, is a modified nucleotide.In one embodiment, each residue is independently modified with 2'-O-methyl or 2'-fluoro, for example, within an alternating motif.Optionally, the RNAi agent further comprises a ligand (e.g., a lipophilic ligand, optionally a C16 ligand).

[0289] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand is 25-30 nucleotide residues in length and, starting from the 5'-terminal nucleotide (position 1), comprises at least 8 ribonucleotides at positions 1-23 of the first strand; and the antisense strand is 36-66 nucleotide residues in length and, starting from the 3'-terminal nucleotide, comprises at least 8 ribonucleotides at positions 1-23 of the sense strand to form a duplex; wherein at least the 3'-terminal nucleotide of the antisense strand is unpaired with the sense strand, and up to 6 consecutive 3'-terminal nucleotides are unpaired with the sense strand, thereby forming a 3' single-stranded overhang of 1-6 nucleotides; and wherein the 5'-end of the antisense strand comprises 10-30 consecutive ribonucleotides that are not paired with the sense strand. the sense strand comprises nucleotides corresponding to the 2'-F modification, thereby forming a single-stranded 5' overhang of 10 to 30 nucleotides, at least the 5'- and 3'-terminal nucleotides of the sense strand base-pair with nucleotides of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplex region between the sense and antisense strands, and the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand length, such that the double-stranded nucleic acid reduces target gene expression when introduced into a mammalian cell, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides, at least one of the motifs occurring at or near the cleavage site. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0290] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, the RNAi agent comprising a first strand having a length of at least 25 nucleotides and at most 29 nucleotides, and a second strand having a length of at most 30 nucleotides with at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, the 3' end of the first strand and the 5' end of the second strand forming a blunt end, the second strand being 1 to 4 nucleotides longer at its 3' end than the first strand, the duplex region being at least 25 nucleotides in length, the second strand being sufficiently complementary to a target mRNA along at least 19 nucleotides of the length of the second strand, the RNAi agent reducing target gene expression when introduced into a mammalian cell, and Dicer cleavage of the RNAi agent resulting in an siRNA comprising the 3' end of the second strand, thereby reducing target gene expression in the mammalian cell. Optionally, the RNAi agent further comprises a ligand.

[0291] In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, one of which motifs occurs at the cleavage site within the sense strand.

[0292] In one embodiment, the antisense strand of the RNAi agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, one of which occurs at or near the cleavage site in the antisense strand.

[0293] For RNAi agents with a duplex region of 17 to 23 nucleotides in length, the cleavage site of the antisense strand is typically approximately 10, 11, and 12 from the 5' end. Thus, three identical modification motifs can occur at positions 9, 10, and 11, 10, 11, and 12, 11, 12, and 13, 12, 13, and 14, or 13, 14, and 15 of the antisense strand, starting from the first nucleotide from the 5' end of the antisense strand, or starting from the first paired nucleotide within the duplex region from the 5' end of the antisense strand. The cleavage site within the antisense strand can also vary depending on the length of the duplex region of the RNAi from the 5' end.

[0294] The sense strand of RNAi agent can contain at least one motif of three identical modifications on three consecutive nucleotides at the cleavage site of its strand, and antisense strand can have at least one motif of three identical modifications on three consecutive nucleotides at or near the cleavage site of strand.When sense strand and antisense strand form dsRNA duplex, sense strand and antisense strand can be aligned so that one motif of three nucleotides on sense strand and one motif of three nucleotides on antisense strand have at least one nucleotide overlap, that is, at least one of the three nucleotides of the motif in sense strand and at least one of the three nucleotides of the motif in antisense strand form base pairing.Alternatively, at least two nucleotides can overlap, or all three nucleotides can overlap.

[0295] In one embodiment, the sense strand of an RNAi agent may contain multiple motifs of three identical modifications on three consecutive nucleotides. The first motif may occur at or near the site of strand cleavage, and the other motifs may be wing modifications. As used herein, the term "wing modification" refers to a motif that occurs in another portion of the strand separated from a motif at or near the site of cleavage of the same strand. The wing modifications may be adjacent to the first motif or separated by at least one or more nucleotides. When the motifs are immediately adjacent to each other, the chemical nature of the motifs is distinct from each other, and when the motifs are separated by one or more nucleotides, the chemical nature may be the same or different. Two or more wing modifications may be present. For example, when two wing modifications are present, each wing modification may occur at one end of the first motif at or near the cleavage site, or on either side of the lead motif.

[0296] Like the sense strand, the antisense strand of an RNAi agent may contain multiple motifs of three identical modifications on three consecutive nucleotides, at least one of which occurs at or near the cleavage site of that strand. The antisense strand may also contain one or more wing modifications in the same alignment as the wing modifications that may be present on the sense strand.

[0297] In one embodiment, wing modifications on the sense or antisense strand of an RNAi agent typically do not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0298] In another embodiment, wing modifications on the sense or antisense strand of an RNAi agent typically do not include the first one or two paired nucleotides in the duplex region at the 3' end, 5' end, or both ends of the strand.

[0299] When the sense and antisense strands of an RNAi agent each contain at least one wing modification, the wing modifications can be at the same end of the duplex region and have an overlap of one, two, or three nucleotides.

[0300] When the sense and antisense strands of an RNAi agent each contain at least two wing modifications, the sense and antisense strands can be aligned such that the two modifications from each single strand are at one end of a duplex region with one, two, or three nucleotide overlap, the two modifications from each single strand are at the other end of the duplex region with one, two, or three nucleotide overlap, and the two modifications from the single strand are on either side of a lead motif within the duplex region with one, two, or three nucleotide overlap.

[0301] In one embodiment, the RNAi agent contains mismatches or combinations thereof in the duplex with the target. Mismatches can occur in the overhang region or the duplex region. Base pairs can be ranked based on their tendency to promote dissociation or melting (e.g., based on the free energy of association or dissociation of a particular pairing; the simplest approach is to examine each pair individually, but adjacent or similar analysis can also be used). In terms of promoting dissociation, A:U is preferred to G:C, G:U is preferred to G:C, and I:C is preferred to G:C (I=inosine). Mismatches, such as non-standard pairings or non-standard pairings (described elsewhere herein), are preferred to standard pairings (A:T, A:U, G:C), and pairings containing universal bases are preferred to standard pairings.

[0302] In one embodiment, the RNAi agent comprises the first 1, 2, 3, 4, or 5 base pairs within the duplex region from the 5' end of the antisense strand independently selected from the group of A:U, G:U, I:C, and at least one mismatch pair, e.g., a non-canonical pairing or a non-canonical pairing or a pairing containing a universal base, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0303] In one embodiment, the nucleotide at position 1 from the 5' end of the antisense strand to the duplex region is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first one, two, or three base pairs from the 5' end of the antisense strand to the duplex region is an AU base pair. For example, the first base pair from the 5' end of the antisense strand to the duplex region is an AU base pair.

[0304] In another embodiment, the nucleotide at the 3' end of the sense strand is deoxythymidine (dT). In another embodiment, the nucleotide at the 3' end of the antisense strand is deoxythymidine (dT). In one embodiment, there is a short sequence of deoxythymidine nucleotides, for example, two dT nucleotides on the 3' end of either the sense strand or the antisense strand.

[0305] In one embodiment, the sense strand sequence has formula (I): 5'n p -N a -(XXX) i -N b -YY YN b -(ZZZ) j -N a -n q 3'(I), During the ceremony, i and j each independently represent 0 or 1; p and q each independently represent 0 to 6; each N a independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b independently represent an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p and n q independently represent overhanging nucleotides, wherein Nb and Y do not have the same modification, and XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. In some embodiments, YYY are all 2'-F modified nucleotides.

[0306] In one embodiment, N a or N b includes alternating pattern modifications.

[0307] In one embodiment, YYY motif occurs at or near the cleavage site of sense strand.For example, when RNAi agent has a double-stranded region of 17-23 nucleotides in length, YYY motif can occur at or near the cleavage site of sense strand (for example, can occur at position 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12, or 11, 12, 13), and this number starts from the first nucleotide from the 5' end, or optionally this number starts from the first paired nucleotide in the double-stranded region from the 5' end.

[0308] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. Thus, the sense strand has the following formula: 5'n p -N a -YYY-N b -ZZZ-N a -n q 3'(Ib), 5'n p -N a -XXX-N b -YYY-N a -n q 3'(Ic), or 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3'(Id).

[0309] When the sense strand is represented by formula (Ib), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides.

[0310] each N a can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0311] When the sense strand is represented by formula (Ic), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0312] When the sense strand is represented by formula (Id), each N b independently represent an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. b is 0, 1, 2, 3, 4, 5, or 6. Each N a can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0313] Each of X, Y and Z may be the same or different from one another.

[0314] In other embodiments, i is 0 and j is 0, and the sense strand has the formula 5'n p -N a -YYY-N a-n q 3'(Ia).

[0315] When the sense strand is represented by formula (Ia), each N a can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0316] In one embodiment, the antisense strand sequence of the RNAi is represented by formula (II): 5'n q’ -N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b '-(X'X'X') l -N' a -n p '3'(II), During the ceremony, k and l each independently represent 0 or 1; p' and q' each independently represent 0 to 6; each N a each independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b ' independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p ' and n q ' independently represent an overhanging nucleotide; In the formula, N b ' and Y' do not have the same modification, and X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides.

[0317] In one embodiment, N a ' or N b ' includes alternating pattern modifications.

[0318] The Y'Y'Y' motif occurs at or near the cleavage site of the antisense strand.For example, when the RNAi agent has a double-stranded region of 17-23 nucleotides in length, the Y'Y'Y' motif can occur at the 9th, 10th, 11th, 10th, 11th, 12th, 11th, 12th, 13th, 12th, 13th, 14th, or 13th, 14th, 15th positions of the antisense strand, and this number starts from the first nucleotide from the 5' end, or optionally this number starts from the first paired nucleotide in the double-stranded region from the 5' end.In another embodiment, the Y'Y'Y' motif occurs at the 11th, 12th, 13th positions.

[0319] In one embodiment, the Y'Y'Y' motif is all 2'-OMe modified nucleotides.

[0320] In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or k and l are both 1.

[0321] Thus, the antisense strand has the formula: 5'n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N a '-n p’ 3'(IIb), 5'n q’ -N a '-Y'Y'Y'-N b '-X'X'X'-n p’ 3'(IIc), or 5'n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N b '-X'X'X'-N a '-n p’ 3'(IId) It can be expressed as:

[0322] When the antisense strand is represented by formula (IIb), N b ’represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0323] When the antisense strand is represented by formula (IIc), N b ' represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0324] When the antisense strand is represented by formula (IId), each N b Each N' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a In another embodiment, N' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. b is 0, 1, 2, 3, 4, 5 or 6.

[0325] In other embodiments, k is 0 and l is 0, and the antisense strand has the formula 5'n p’ -N a’ -Y'Y'Y'-N a’ -n q’ 3'(Ia) It can be expressed as:

[0326] When the antisense strand is represented by formula (IIa), each N a ' independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0327] Each of X', Y' and Z' may be the same as or different from one another.

[0328] Each nucleotide of sense strand and antisense strand can be independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl or 2'-fluoro.For example, each nucleotide of sense strand and antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.Each X, Y, Z, X', Y' and Z' can specifically represent 2'-O-methyl modification or 2'-fluoro modification.

[0329] In one embodiment, the sense strand of the RNAi agent can contain a YYY motif occurring at positions 9, 10, and 11 of the strand when the duplex region is 21 nt, the numbers starting from the first nucleotide from the 5' end, or optionally, the numbers starting from the 5' end with the first paired nucleotide in the duplex region, and Y represents a 2'-F modification. The sense strand can further contain a XXX motif or a ZZZ motif as a wing modification at the opposite end of the duplex region, and XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0330] In one embodiment, the antisense strand can contain a Y'Y'Y' motif occurring at positions 11, 12, or 13 of the strand, where the numbering starts from the first nucleotide from the 5' end, or optionally, the numbering starts from the 5' end with the first paired nucleotide in the duplex region, and Y' represents a 2'-O-methyl modification. The antisense strand can further contain an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the duplex region, and X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0331] The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with the antisense strand represented by any one of the above formulas (IIa), (IIb), (IIc), and (IId), respectively.

[0332] Thus, the RNAi agent used in the methods of the present disclosure can include a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and the RNAi duplex can have the formula (III): Sense:5'n p -N a -(XXX) i -N b -YY YN b -(ZZZ) j -N a -n q 3' Antisense: 3'n p ’ -N a ’ -(X'X'X') k -N b ’ -Y'Y'Y'-N b ’ -(Z'Z'Z') l -N a ’ -n q ’ 5' (III) is expressed as During the ceremony, i, j, k, and l each independently represent 0 or 1; p, p', q and q' each independently represent 0 to 6; each N a and N a ’ independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b and N b ’ independently represent an oligonucleotide sequence comprising 0 to 10 modified nucleotides; During the ceremony, each n p ',n p , n q ' and n q each of which may or may not be present independently represents an overhanging nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides.

[0333] In one embodiment, i is 0 and j is 0, or i is 1 and j is 0, or i is 0 and j is 1, or i and j are both 0, or i and j are both 1. In another embodiment, k is 0 and l is 0, or k is 1 and l is 0, or k is 0 and l is 1, or k and l are both 0, or k and l are both 1.

[0334] An exemplary combination of sense and antisense strands that form an RNAi duplex is of the following formula: 5'n p -N a -YY YN a -n q 3' 3'n p ’ -N a ’ -Y'Y'Y'-N a ’ n q ’ 5' (IIIa) 5'n p -N a -YY YN b -ZZ ZN a -n q 3' 3'n p ’ -N a ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a ’ n q ’ 5' (IIIb) 5'n p -N a -XX XN b -YY YN a-n q 3' 3'n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N a ’ -n q ’ 5' (IIIc) 5'n p -N a -XX XN b -YY YN b -ZZ ZN a -n q 3' 3'n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a -n q ’ 5' (IIId).

[0335] When the RNAi agent is represented by formula (IIIa), each N a independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0336] When the RNAi agent is represented by formula (IIIb), each N b independently represent an oligonucleotide sequence containing 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides. a ' independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0337] When the RNAi agent is represented by formula (IIIc), each N b , N bEach N' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0338] When the RNAi agent is represented by formula (IIId), each N b , N b Each N' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a , N a ’ independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. a , N a ', N b , and N b ’ each independently comprises an alternating pattern of modifications.

[0339] In one embodiment, when the RNAi agent has formula (IIId), N a The modification is a 2'-O-methyl modification or a 2'-fluoro modification. In another embodiment, when the RNAi agent has formula (IIId), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, and p '>0 and at least one n p In yet another embodiment, when the RNAi agent has formula (IIId), N' is linked to the adjacent nucleotide via a phosphorothioate linkage. a The modification is 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p In another embodiment, when the RNAi agent has formula (IIId), N' is linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand is conjugated to one or more C16 (or related thereto) moieties attached via a divalent or trivalent branched linker (described below).a The modification is 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p ' are linked to adjacent nucleotides via phosphorothioate linkages, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more lipophilic moieties, e.g., C16 (or related) moieties, which may be attached via a divalent or trivalent branched linker.

[0340] In one embodiment, when the RNAi agent has formula (IIIa), N a The modification is 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p ' are linked to adjacent nucleotides via phosphorothioate linkages, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more lipophilic moieties, e.g., C16 (or related) moieties, which may be attached via a divalent or trivalent branched linker.

[0341] In one embodiment, the RNAi agent is a multimer containing at least two duplexes represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), and these duplexes are connected by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each duplex may target the same gene or two different genes, or each duplex may target the same gene at two different target sites.

[0342] In one embodiment, the RNAi agent is a multimer containing three, four, five, six or more double strands represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), and the double strands are connected by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each double strand can target the same gene or two different genes, or each double strand can target the same gene at two different target sites.

[0343] In one embodiment, two RNAi agents represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to each other at their 5' ends and optionally conjugated to a ligand at one or both of their 3' ends. The agents can each target the same gene or two different genes, or the agents can each target the same gene at two different target sites.

[0344] Various publications describe the multimeric RNAi agent that can be used in the method of the present disclosure.These publications include International Publication No. 2007 / 091269, International Publication No. 2010 / 141511, International Publication No. 2007 / 117686, International Publication No. 2009 / 014887 and International Publication No. 2011 / 031520 and United States Patent No. 7858769, each of whose entire contents is incorporated herein by reference.

[0345] In certain embodiments, the compositions and methods of the present disclosure include vinyl phosphonate (VP) modifications of RNAi agents as described herein. In exemplary embodiments, a 5'-vinyl phosphonate modified nucleotide of the present disclosure has the following structure: [ka] wherein X is O or S; R is hydrogen, hydroxy, fluoro, or C 1~20 alkoxy (e.g., methoxy or n-hexadecyloxy); R 5 ' is =C(H)-P(O)(OH)2, and the C5' carbon and R 5 The double bond between B is a nucleobase or modified nucleobase, optionally B is adenine, guanine, cytosine, thymine, or uracil.

[0346] In one embodiment, R 5’ is ═C(H)—P(O)(OH) and the double bond between the C5′ carbon and R5′ is in the E configuration. In another embodiment, R is methoxy and R 5’ is ═C(H)—P(O)(OH) and the double bond between the C5′ carbon and R5′ is in the E configuration. In another embodiment, X is S, R is methoxy and R 5’ is =C(H)-P(O)(OH)2, and the double bond between the C5' carbon and R5' is in the E configuration.

[0347] The vinyl phosphonate of the present disclosure can be attached to either the antisense or sense strand of the dsRNA of the present disclosure. In certain embodiments, the vinyl phosphonate of the present disclosure is attached to the antisense strand of the dsRNA, optionally at the 5' end of the antisense strand of the dsRNA.

[0348] Vinyl phosphate modifications are also contemplated for the compositions and methods of the present disclosure. Exemplary vinyl phosphate structures include those previously described, where R 5’ is =C(H)-OP(O)(OH)2, and the C5' carbon and R 5’ The double bond between is in the E or Z configuration (eg, E configuration).

[0349] A. Thermal destabilization modification In certain embodiments, dsRNA molecules can be optimized for RNA interference by incorporating thermally destabilizing modifications into the seed region of antisense strand.As used herein, "seed region" refers to the 2-9 positions of the 5' end of the referenced strand.For example, thermally destabilizing modifications can be incorporated into the seed region of antisense strand to reduce or inhibit off-target gene silencing.

[0350] The term "thermally destabilizing modification" refers to the melting temperature (T m ) lower than the overall melting temperature (T m For example, thermally destabilizing modifications include modifications that result in dsRNAs with a T m can be decreased by 1-4° C., e.g., 1, 2, 3, or 4 degrees Celsius. Also, the term "thermally destabilized nucleotide" refers to a nucleotide that contains one or more thermally destabilizing modifications.

[0351] It has been discovered that dsRNAs having an antisense strand containing at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end of the antisense strand have reduced off-target gene silencing activity. Thus, in some embodiments, the antisense strand contains at least one (e.g., one, two, three, four, five, or more) thermally destabilizing modification of the duplex within the first 9 nucleotide positions of the 5' region of the antisense strand. In some embodiments, the one or more thermally destabilizing modifications of the duplex are located at positions 2-9, e.g., positions 4-8, from the 5' end of the antisense strand. In some further embodiments, the thermally destabilizing modification of the duplex is located at positions 6, 7, or 8 from the 5' end of the antisense strand. In even further embodiments, the thermally destabilizing modification of the duplex is located at position 7 from the 5' end of the antisense strand. In some embodiments, the thermally destabilizing modification of the duplex is located at positions 2, 3, 4, 5, or 9 from the 5' end of the antisense strand.

[0352] Thermally destabilizing modifications can include, but are not limited to, abasic modifications, mismatches with the opposing nucleotide in the opposing strand, and sugar modifications such as 2'-deoxy modifications, acyclic nucleotides such as unlocked nucleic acids (UNAs) or glycol nucleic acids (GNAs), and 2'-5'-linked ribonucleotides ("3'-RNAs").

[0353] Exemplary abasic modifications include, but are not limited to, the following: [ka] wherein R=H, Me, Et or OMe, R'=H, Me, Et or OMe, R"=H, Me, Et or OMe [ka] wherein B is a modified or unmodified nucleobase.

[0354] Exemplary sugar modifications include, but are not limited to, the following: [ka] wherein B is a modified or unmodified nucleobase.

[0355] In some embodiments, the thermally destabilizing modification of the duplex is selected from the group consisting of: [ka] where B is a modified or unmodified nucleobase, and the asterisk on each structure represents either R, S, or racemic.

[0356] In some embodiments, the thermally destabilizing modification of the duplex is selected from the group consisting of: [ka] wherein B is a modified or unmodified nucleobase and the asterisk represents either R, S or racemic (e.g., S).

[0357] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar, for example, one in which any of the bonds of the ribose carbon ring (e.g., C1'-C2', C2'-PRNP', PRNP'-C4', C4'-O4', or C1'-O4') is absent, or at least one of the ribose carbons or oxygens (e.g., C1', C2', PRNP', C4', or O4'), independently or in combination, is absent from the nucleotide. In some embodiments, an acyclic nucleotide is [ka] B is a modified or unmodified nucleobase; R 1 and R 2 are independently H, halogen, OR3, or alkyl, and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. The term "UNA" refers to an acyclic unlocked nucleic acid in which any of the sugar bonds has been removed to form an unlocked "sugar" residue. In one example, UNA also encompasses monomers in which the C1'-C4' bond has been removed (i.e., a carbon-oxygen-carbon covalent bond between the C1' and C4' carbons). In another example, the C2'-PRNP' bond of the sugar has been removed (i.e., a carbon-carbon covalent bond between the C2' and PRNP' carbons) (see Mikhailov et al., Tetrahedron Letters, 26(17):2059 (1985), and Fluiter et al., Mol. Biosyst., 10:1039 (2009), which are incorporated by reference in their entireties). Acyclic derivatives offer greater backbone flexibility without affecting Watson-Crick pairing. Acyclic nucleotides can be linked via 2'-5' or 3'-5' linkages.

[0358] The term "GNA" refers to glycol nucleic acid, which is a polymer similar to DNA or RNA, but differs in the composition of its "backbone" in that it is made up of repeating glycerol units linked by phosphodiester bonds. [ka]

[0359] The thermally destabilizing modification of the duplex can be a mismatch (i.e., non-complementary base pair) between the thermally destabilizing nucleotide and the opposite nucleotide in the opposite strand of the dsRNA duplex. Exemplary mismatch base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or a combination thereof. Other mismatch base pairings known in the art are also contemplated by the present invention. Mismatches can occur between nucleotides that are either naturally occurring or modified nucleotides, i.e., mismatch base pairing can occur between the nucleobases derived from each nucleotide, regardless of the modification on the ribose sugar of the nucleotide. In certain embodiments, the dsRNA molecule contains at least one nucleobase in the mismatch pairing that is a 2'-deoxynucleobase, for example, the 2'-deoxynucleobase is in the sense strand.

[0360] In some embodiments, the thermally destabilizing modifications of the duplex in the seed region of the antisense strand include nucleotides, such as modified nucleobases, that disrupt Watson-Crick hydrogen bonding with complementary bases on the target mRNA. [ka]

[0361] Many examples of abasic nucleotides, acyclic nucleotide modifications (including UNA and GNA) and mismatch modifications are described in detail in WO 2011 / 133876, which is incorporated herein by reference in its entirety.

[0362] Thermally destabilizing modifications can also include universal base and phosphate modifications that have reduced or eliminated ability to form hydrogen bonds with opposing bases.

[0363] In some embodiments, the thermal destabilizing modification of duplex comprises the nucleotide with non-standard base, for example but not limited to, the nucleobase modification that the ability to form hydrogen bond with the base in opposite strand is impaired or completely lost.These nucleobase modifications have been evaluated for the destabilization of the central region of dsRNA duplex, as described in International Publication No. 2010 / 0011895, the entire contents of which are incorporated herein by reference.Exemplary nucleobase modifications include: [ka]

[0364] In some embodiments, the thermally destabilizing modifications of the duplex in the seed region of the antisense strand include one or more α-nucleotides that are complementary to bases on the target mRNA, such as: [ka] wherein R is H, OH, OCH3, F, NH2, NHMe, NMe2 or O-alkyl.

[0365] Exemplary phosphate modifications that have been shown to decrease the thermal stability of dsRNA duplexes, relative to natural phosphodiester linkages, include the following: [ka]

[0366] The alkyl R group can be a C1-C6 alkyl. Specific alkyl R groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, pentyl, and hexyl.

[0367] As those skilled in the art will recognize, considering that the functional role of nucleobase defines the specificity of the RNAi agent of the present disclosure, nucleobase modification can be carried out in various ways as described herein, for example, for the purpose of enhancing on-target effect against off-target effect, for example, introducing destabilizing modification into the RNAi agent of the present disclosure, but generally the range of modification that exists on the RNAi agent of the present disclosure tends to be much larger than the non-nucleobase modification, for example, the modification of the sugar group or phosphate backbone of polyribonucleotide.Such modification will be described in more detail in other sections of the present disclosure, and is expressly intended for the RNAi agent of the present disclosure that has either natural nucleobase or modified nucleobase as described above or elsewhere herein.

[0368] In addition to the antisense strand containing a thermally destabilizing modification, the dsRNA may also contain one or more stabilizing modifications. For example, the dsRNA may contain at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stabilizing modifications. Without limitation, all of the stabilizing modifications may be present in one strand. In some embodiments, both the sense and antisense strands contain at least two stabilizing modifications. The stabilizing modifications may occur at any nucleotide in either the sense strand or the antisense strand. For example, the stabilizing modifications may occur at any nucleotide in the sense strand or the antisense strand, and each stabilizing modification may occur in an alternating pattern on the sense strand or the antisense strand, or the sense strand or the antisense strand may contain both stabilizing modifications in an alternating pattern. The alternating pattern of stabilizing modifications on the sense strand may be the same as or different from that of the antisense strand, and the alternating pattern of stabilizing modifications on the sense strand may be shifted relative to the alternating pattern of stabilizing modifications on the antisense strand.

[0369] In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stabilizing modifications. Without limitation, the stabilizing modifications within the antisense strand can be located at any position. In some embodiments, the antisense strand comprises stabilizing modifications at positions 2, 6, 8, 9, 14, and 16 from the 5' end. In some other embodiments, the antisense strand comprises stabilizing modifications at positions 2, 6, 14, and 16 from the 5' end. In yet some other embodiments, the antisense strand comprises stabilizing modifications at positions 2, 14, and 16 from the 5' end.

[0370] In some embodiments, the antisense strand comprises at least one stabilizing modification adjacent to the destabilizing modification.For example, the stabilizing modification can be at the 5'-end or 3'-end of the destabilizing modification, i.e., at the -1 or +1 position from the position of the destabilizing modification, nucleotide.In some embodiments, the antisense strand comprises a stabilizing modification at each of the 5'-end and 3'-end of the destabilizing modification, i.e., at the -1 and +1 positions from the position of the destabilizing modification.

[0371] In some embodiments, the antisense strand comprises at least two stabilizing modifications at the 3' end of the destabilizing modification, ie, at positions +1 and +2 from the position of the destabilizing modification.

[0372] In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stabilizing modifications. Without limitation, the stabilizing modifications within the sense strand can be located at any position. In some embodiments, the sense strand comprises stabilizing modifications at positions 7, 10, and 11 from the 5' end. In some other embodiments, the sense strand comprises stabilizing modifications at positions 7, 9, 10, and 11 from the 5' end. In some embodiments, the sense strand comprises stabilizing modifications at positions opposite or complementary to positions 11, 12, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some other embodiments, the sense strand comprises stabilizing modifications at positions opposite or complementary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some embodiments, the sense strand comprises blocks of two, three, or four stabilizing modifications.

[0373] In some embodiments, the sense strand does not contain a stabilizing modification at a position opposite or complementary to a thermally destabilizing modification of the duplex in the antisense strand.

[0374] Exemplary thermally stabilizing modifications include, but are not limited to, 2'-fluoro modifications. Other thermally stabilizing modifications include, but are not limited to, LNA.

[0375] In some embodiments, the dsRNA of the present disclosure contains at least four (e.g., four, five, six, seven, eight, nine, ten, or more) 2'-fluoro nucleotides. Without limitation, all of the 2'-fluoro nucleotides can be present in one strand. In some embodiments, both the sense and antisense strands contain at least two 2'-fluoro nucleotides. The 2'-fluoro modification can occur on either the sense strand or the antisense strand's nucleotide. For example, the 2'-fluoro modification can occur on any nucleotide on the sense strand or the antisense strand, and each 2'-fluoro modification can occur in an alternating pattern on the sense strand or the antisense strand, or both the sense strand and the antisense strand contain 2'-fluoro modifications in an alternating pattern. The alternating pattern of 2'-fluoro modifications on the sense strand can be the same as or different from that of the antisense strand, and the alternating pattern of 2'-fluoro modifications on the sense strand can be shifted relative to the alternating pattern of 2'-fluoro modifications on the antisense strand.

[0376] In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) 2'-fluoro nucleotides. Without limitation, the 2'-fluoro modifications in the antisense strand can be present at any position. In some embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 8, 9, 14, and 16 from the 5' end. In some other embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5' end. In yet some other embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 14, and 16 from the 5' end.

[0377] In some embodiments, antisense strand comprises at least one 2'-fluoro nucleotide adjacent to destabilizing modification.For example, 2'-fluoro nucleotide can be at the 5'-end or 3'-end of destabilizing modification, that is, at the -1 position or +1 position from the position of destabilizing modification, nucleotide.In some embodiments, antisense strand comprises 2'-fluoro nucleotide at each of the 5'-end and 3'-end of destabilizing modification, that is, at the -1 and +1 positions from the position of destabilizing modification.

[0378] In some embodiments, the antisense strand comprises at least two 2'-fluoro nucleotides at the 3' end of the destabilizing modification, ie, at positions +1 and +2 from the position of the destabilizing modification.

[0379] In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) 2'-fluoro nucleotides. Without limitation, the 2'-fluoro modifications within the sense strand can be present at any position. In some embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 7, 10, and 11 from the 5'-end. In some other embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 7, 9, 10, and 11 from the 5'-end. In some embodiments, the sense strand comprises 2'-fluoro nucleotides at positions opposite or complementary to positions 11, 12, and 15 of the antisense strand, counting from the 5'-end of the antisense strand. In some other embodiments, the sense strand comprises 2'-fluoro nucleotides at positions opposite or complementary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5'-end of the antisense strand. In some embodiments, the sense strand comprises two, three, or four blocks of 2'-fluoro nucleotides.

[0380] In some embodiments, the sense strand does not contain a 2'-fluoro nucleotide at a position opposite or complementary to a thermally destabilizing modification of the duplex in the antisense strand.

[0381] In some embodiments, a dsRNA molecule of the disclosure comprises a 21-nucleotide (nt) sense strand and a 23-nucleotide (nt) antisense strand, wherein the antisense strand contains at least one thermally destabilized nucleotide, wherein the at least one thermally destabilized nucleotide occurs within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), one end of the dsRNA is blunt while the other end comprises a 2-nt overhang, and the dsRNA optionally comprises at least one (e.g., one, two, three, four, five, six, or all seven) of the following characteristics: The antisense strand further comprises the following features: (i) the antisense strand comprises 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand comprises 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2'-fluoro modifications; and (vii) the dsRNA comprises a blunt end at the 5' end of the antisense strand. In another embodiment, a dinucleotide overhang is at the 3' end of the antisense strand.

[0382] In some embodiments, a dsRNA molecule of the present disclosure comprises a sense strand and an antisense strand, wherein the sense strand is 25 to 30 nucleotide residues in length and, starting from the 5'-terminal nucleotide (position 1), comprises at least 8 ribonucleotides at positions 1 to 23 of the sense strand; the antisense strand is 36 to 66 nucleotide residues in length and, starting from the 3'-terminal nucleotide, at least 8 ribonucleotides at these positions are paired with positions 1 to 23 of the sense strand to form a duplex; at least the 3'-terminal nucleotide of the antisense strand is not paired with the sense strand, and up to 6 consecutive 3'-terminal nucleotides are not paired with the sense strand, thereby forming a 1-6 nucleotide 3' single-stranded overhang; and the 5'-end of the antisense strand is separated by 10 to 30 consecutive nucleotides that are not paired with the sense strand. the antisense strand is composed of at least 19 ribonucleotides of a target RNA, thereby forming a single-stranded 5' overhang of 10 to 30 nucleotides, at least the 5'- and 3'-terminal nucleotides of the sense strand base-pair with nucleotides of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplex region between the sense and antisense strands, and the antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the length of the antisense strand to reduce target gene expression when the double-stranded nucleic acid is introduced into a mammalian cell, and the antisense strand contains at least one thermally destabilizing nucleotide, the at least one thermally destabilizing nucleotide being within the seed region of the antisense strand (i.e., at positions 2 to 9 of the 5'-end of the antisense strand).For example, the thermally destabilizing nucleotide occurs between positions 14-17 opposite or complementary to positions 14-17 of the 5' end of the sense strand, and the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, or all seven) of the following features: (i) the antisense strand contains two, three, four, five, or six 2'-fluoro modifications; (ii) the antisense strand contains one, two, three, four, or five phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand contains two, three, four, or five 2'-fluoro modifications; (v) the sense strand contains one, two, three, four, or five phosphorothioate internucleotide linkages; and (vi) the dsRNA contains at least four 2'-fluoro modifications; and (vii) the dsRNA contains a duplex region 12-30 nucleotide pairs in length.

[0383] In some embodiments, a dsRNA molecule of the present disclosure comprises a sense strand and an antisense strand, the dsRNA molecule comprising a sense strand having a length of at least 25 nucleotides and at most 29 nucleotides, and an antisense strand having a length of at most 30 nucleotides, wherein the sense strand comprises a modified nucleotide at position 11 from the 5' end that is susceptible to enzymatic degradation, the 3' end of the sense strand and the 5' end of the antisense strand form a blunt end, and the antisense strand is 1 to 4 nucleotides longer at its 3' end than the sense strand, the duplex region is at least 25 nucleotides in length, and the antisense strand is sufficiently complementary to a target mRNA along at least 19 nucleotides of the antisense strand, such that the dsRNA molecule reduces target gene expression when introduced into a mammalian cell, and Dicer cleavage of the dsRNA results in an siRNA comprising the 3' end of the antisense strand, thereby inhibiting target gene expression in mammalian cells. The antisense dsRNA optionally further comprises at least one thermally destabilized nucleotide within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally further comprises at least one of the following features (e.g., one, two, three, four, five, six, or all seven): (i) the antisense dsRNA comprises two, three, four, five, or six 2'-fluoro modifications; (ii) the antisense dsRNA comprises two, three, four, five, or six 2'-fluoro modifications; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand contains two, three, four, or five 2'-fluoro modifications; (v) the sense strand contains one, two, three, four, or five phosphorothioate internucleotide linkages; and (vi) the dsRNA contains at least four 2'-fluoro modifications; and (vii) the dsRNA has a duplex region 12 to 29 nucleotide pairs in length.

[0384] In some embodiments, any nucleotide in the sense strand and antisense strand of dsRNA molecule can be modified.Each nucleotide can be modified with the same or different modifications, and these modifications can include one or both of non-linked phosphate oxygen or one or more of linked phosphate oxygen, modifying the ribose sugar component, for example, modifying the 2' hydroxyl on the ribose sugar, replacing phosphate moiety extensively with " dephosphorylation " linker, modifying or replacing naturally occurring base, and replacing or replacing ribose phosphate backbone.

[0385] Because nucleic acids are polymers of subunits, many modifications occur at positions that are repeated within nucleic acids, such as modifications of bases or phosphate moieties, or unlinked Os in phosphate moieties.In some cases, modifications occur at all target positions in nucleic acids, but in many cases, they do not occur.For example, modifications can occur only at the 3'-end or 5'-end positions, or only in terminal regions, such as at the terminal nucleotide position of the chain or at the last 2, 3, 4, 5, or 10 nucleotides of the chain.Modifications can occur in double-stranded regions, single-stranded regions, or both.Modifications can occur only in the double-stranded region of RNA, or only in the single-stranded region of RNA.For example, phosphorothioate modifications at unlinked O positions can occur only at one or both ends, or only in terminal regions, such as at the terminal nucleotide position of the chain or within the last 2, 3, 4, 5, or 10 nucleotides, or in double-stranded and single-stranded regions, especially at the ends. The 5' end can be phosphorylated.

[0386] This may, for example, enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes in the single-stranded overhang, for example, in the 5' overhang or 3' overhang, or in both overhangs.For example, it may be desirable to include purine nucleotides in the overhang.In some embodiments, all or part of the bases in the 3' overhang or 5' overhang can be modified, for example, with the modifications described herein.Modifications may include, for example, the use of modifications at the 2' position of the ribose sugar with modifications known in the art, for example, the use of deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl, instead of the ribosugar of the nucleic acid base, and modifications at the phosphate group, for example, phosphorothioate modifications.The overhang does not need to be homologous to the target sequence.

[0387] In some embodiments, each residue of the sense strand and the antisense strand is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, or 2'-fluoro. These strands may contain two or more modifications. In some embodiments, each residue of the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluoro. It should be understood that these modifications are in addition to at least one thermally destabilizing modification of the duplex present in the antisense strand.

[0388] At least two different modifications are typically present on the sense strand and the antisense strand. These modifications may be 2'-deoxy, 2'-O-methyl, or 2'-fluoro modifications, acyclic nucleotides, etc. In some embodiments, the sense strand and the antisense strand each contain two differently modified nucleotides selected from 2'-O-methyl or 2'-deoxy. In some embodiments, each residue in the sense strand and the antisense strand is independently modified with a 2'-O-methyl nucleotide, a 2'-deoxy nucleotide, a 2'-deoxy-2'-fluoro nucleotide, a 2'-ON-methylacetamide (2'-O-NMA, 2'O-CHC(O)N(Me)H) nucleotide, a 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotide, a 2'-O-aminopropyl (2'-O-AP) nucleotide, or a 2'-ara-F nucleotide. Again, it is understood that these modifications are in addition to at least one thermally destabilizing modification of the duplex present in the antisense strand.

[0389] In some embodiments, the dsRNA molecules of the present disclosure comprise an alternating pattern of modifications. The term "alternating motif" or "alternating pattern," as used herein, refers to a motif with one or more modifications, each modification occurring on alternating nucleotides in a single strand. The alternating nucleotides may refer to one every other nucleotide, one every three nucleotides, or similar patterns. For example, if A, B, and C each represent a type of modification to a nucleotide, the alternating motif may be "ABABABABABAB...," "AABBAABBAABB...," "AABAABAABAAB...," "AAABAAABAAAB...," "AAABBBAAABBB...," or "ABCABCABCABC...," etc.

[0390] The types of modifications contained within an alternating motif can be the same or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, the alternation pattern, i.e., the modifications on every other nucleotide, can be the same, but each of the sense or antisense strands can be selected from several possible modifications within the alternating motif, such as "ABABAB...," "ACACAC...," "BDBDBD...," or "CDCDCD...," etc.

[0391] In some embodiments, dsRNA molecules of the present disclosure include an alternating motif modification pattern on the sense strand that is shifted relative to the alternating motif modification pattern on the antisense strand. The shift can be such that modified groups on nucleotides in the sense strand correspond to differently modified groups on nucleotides in the antisense strand, or vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA duplex, the alternating motif in the sense strand can begin with "ABABAB" from the 5'-3' end of the strand, and the alternating motif in the antisense strand can begin with "BABABA" from the 3'-5' end of the strand in the duplex region. As another example, the alternating motif in the sense strand can begin with "AABBAABB" from the 5'-3' end of the strand, and the alternating motif in the antisense strand can begin with "BBAABBAA" from the 3'-5' end of the strand in the duplex region, resulting in a complete or partial shift in the modification pattern between the sense and antisense strands.

[0392] In one particular example, the alternating motif in the sense strand is "ABABAB" from 5'-3' of the strand, where each A is an unmodified ribonucleotide and each B is a 2'-O methyl modified nucleotide.

[0393] In one particular example, the alternating motif in the sense strand is "ABABAB" from 5'-3' of the strand, where each A is a 2'-deoxy-2'-fluoro modified nucleotide and each B is a 2'-O methyl modified nucleotide.

[0394] In another specific example, the alternating motif in the antisense strand is "BABABA" from 3'-5' of the strand, where each A is a 2'-deoxy-2'-fluoro modified nucleotide and each B is a 2'-O methyl modified nucleotide.

[0395] In one particular example, the alternating motif in the sense strand is "ABABAB" from 5'-3' of that strand and the alternating motif in the antisense strand is "BABABA" from 3'-5' of that strand, where each A is an unmodified ribonucleotide and each B is a 2'-O methyl modified nucleotide.

[0396] In one particular example, the alternating motif in the sense strand is "ABABAB" from 5'-3' of that strand and the alternating motif in the antisense strand is "BABABA" from 3'-5' of that strand, where each A is a 2'-deoxy-2'-fluoro modified nucleotide and each B is a 2'-O methyl modified nucleotide.

[0397] The dsRNA molecule of the present disclosure can further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage.Phosphorothioate or methylphosphonate internucleotide linkage modification can occur on the nucleotide of either sense strand or antisense strand or both strands at any position of the strand.For example, internucleotide linkage modification can occur on any nucleotide on sense strand or antisense strand, each internucleotide linkage modification can occur in an alternating pattern on sense strand or antisense strand, or sense strand or antisense strand contains both internucleotide linkage modifications in an alternating pattern.The alternating pattern of internucleotide linkage modification on sense strand can be the same or different from that of antisense strand, and the alternating pattern of internucleotide linkage modification on sense strand can be shifted relative to the alternating pattern of internucleotide linkage modification on antisense strand.

[0398] In some embodiments, the dsRNA molecule comprises phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region.For example, the overhang region comprises two nucleotides, and the two nucleotides comprise two nucleotides with phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides.Internucleotide linkage modification can also be made to link the overhang nucleotide to the terminal paired nucleotide in the double-stranded region.For example, at least 2, 3, 4 or all of the overhang nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide linkage, and optionally there can be an additional phosphorothioate or methylphosphonate internucleotide linkage connecting the overhang nucleotide to the paired nucleotide adjacent to the overhang nucleotide.For example, there can be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, such that two of the three nucleotides are overhang nucleotides and the third is the paired nucleotide adjacent to the overhang nucleotide.In one embodiment, these terminal three nucleotides can be at the 3'-end of the antisense strand.

[0399] In some embodiments, the sense strand of the dsRNA molecule comprises 1 to 10 blocks of two to ten phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0400] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0401] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of three phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, one of the phosphorothioate or methylphosphonate internucleotide linkages being positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0402] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of four phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0403] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of five phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0404] In some embodiments, the antisense strand of the dsRNA molecule contains two blocks of six phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand containing either phosphorothioate or methylphosphonate or phosphate linkages.

[0405] In some embodiments, the antisense strand of the dsRNA molecule contains two blocks of seven phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, or 8 phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand containing either phosphorothioate or methylphosphonate or phosphate linkages.

[0406] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of eight phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, or 6 phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0407] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate internucleotide linkages separated by one, two, three, or four phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand containing any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand containing either phosphorothioate or methylphosphonate or phosphate linkages.

[0408] In some embodiments, the dsRNA molecules of this disclosure further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within 1 to 10 nucleotides at the termini of the sense or antisense strand, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides can be linked via phosphorothioate or methylphosphonate internucleotide linkages at one or both termini of the sense or antisense strand.

[0409] In some embodiments, the dsRNA molecules of the present disclosure further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within 1 to 10 nucleotides of the inner region of each duplex of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides can be linked via phosphorothioate or methylphosphonate internucleotide linkage modifications at positions 8 to 16 of the duplex region, counting from the 5' end of the sense strand, and the dsRNA molecule can optionally further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1 to 10 of that end.

[0410] In some embodiments, dsRNA molecules of the disclosure further comprise one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the sense strand, and one to two phosphorothioate or methylphosphonate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and one to five within positions 18-23 of the antisense strand.

[0411] In some embodiments, dsRNA molecules of the disclosure further comprise one phosphorothioate internucleotide linkage modification within the range of positions 1-5 (counting from the 5' end) and one phosphorothioate or methylphosphonate internucleotide linkage modification within the range of positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 or 2 (counting from the 5' end) and two phosphorothioate or methylphosphonate internucleotide linkage modifications within the range of positions 18-23 of the antisense strand.

[0412] In some embodiments, dsRNA molecules of the disclosure further comprise two phosphorothioate internucleotide linkage modifications within the range of positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the range of positions 18-23 of the antisense strand.

[0413] In some embodiments, dsRNA molecules of the disclosure further comprise two phosphorothioate internucleotide linkage modifications within the range of positions 1 to 5 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the range of positions 18 to 23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the range of positions 18 to 23 of the antisense strand.

[0414] In some embodiments, dsRNA molecules of the disclosure further comprise two phosphorothioate internucleotide linkage modifications within the range of positions 1-5 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the range of positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within the range of positions 18-23 of the antisense strand (counting from the 5' end).

[0415] In some embodiments, dsRNA molecules of the disclosure further comprise one phosphorothioate internucleotide linkage modification within the range of positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18-23 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within the range of positions 18-23 of the antisense strand (counting from the 5' end).

[0416] In some embodiments, dsRNA molecules of the disclosure further comprise one phosphorothioate internucleotide linkage modification within the range of positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18-23 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18-23 of the antisense strand.

[0417] In some embodiments, dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification within the range of positions 1 to 5 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within the range of positions 18 to 23 (counting from the 5' end) of the antisense strand.

[0418] In some embodiments, dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications within the range of positions 1 to 5 (counting from the 5' end) of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within the range of positions 18 to 23 (counting from the 5' end) of the antisense strand.

[0419] In some embodiments, dsRNA molecules of the disclosure further comprise two phosphorothioate internucleotide linkage modifications within the range of positions 1 to 5 (counting from the 5' end) and one within the range of positions 18 to 23 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18 to 23 of the antisense strand.

[0420] In some embodiments, dsRNA molecules of the disclosure further comprise two phosphorothioate internucleotide linkage modifications within the range of positions 1 to 5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18 to 23 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within the range of positions 18 to 23 of the antisense strand (counting from the 5' end).

[0421] In some embodiments, dsRNA molecules of the disclosure further comprise two phosphorothioate internucleotide linkage modifications within the range of positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the range of positions 18-23 of the antisense strand.

[0422] In some embodiments, the dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 and one at position 21 (counting from the 5' end) of the antisense strand.

[0423] In some embodiments, dsRNA molecules of the disclosure further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 (counting from the 5' end) of the antisense strand.

[0424] In some embodiments, dsRNA molecules of the disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the antisense strand.

[0425] In some embodiments, dsRNA molecules of the disclosure further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 (counting from the 5' end) of the antisense strand.

[0426] In some embodiments, dsRNA molecules of the disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 22 and 23 of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the antisense strand.

[0427] In some embodiments, the dsRNA molecules of this disclosure further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 23 and 23 (counting from the 5' end) of the antisense strand.

[0428] In some embodiments, the compounds of the present disclosure comprise a pattern of backbone chiral centers. In some embodiments, the regular pattern of backbone chiral centers comprises at least 5 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 6 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 7 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 8 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 9 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 10 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 11 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 12 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 13 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 14 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 15 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 16 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 17 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 18 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 19 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises no more than 8 internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises no more than 7 internucleotide linkages in the Rp configuration.In some embodiments, the regular pattern of backbone chiral centers comprises 6 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 5 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 4 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 3 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 2 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 1 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 8 or fewer non-chiral internucleotide linkages (phosphodiesters as a non-limiting example). In some embodiments, the regular pattern of backbone chiral centers comprises 7 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises 6 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises 5 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises 4 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises 3 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises 2 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises 1 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises at least 10 internucleotide linkages in the Sp configuration and 8 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises at least 11 internucleotide linkages in the Sp configuration and 7 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises at least 12 internucleotide linkages in the Sp configuration and 6 or fewer non-chiral internucleotide linkages.In some embodiments, the regular pattern of backbone chiral centers comprises at least 13 internucleotide linkages in the Sp configuration and no more than 6 non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises at least 14 internucleotide linkages in the Sp configuration and no more than 5 non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises at least 15 internucleotide linkages in the Sp configuration and no more than 4 non-chiral internucleotide linkages. In some embodiments, the internucleotide linkages in the Sp configuration may be contiguous or non-contiguous. In some embodiments, the internucleotide linkages in the Rp configuration may be contiguous or non-contiguous. In some embodiments, the non-chiral internucleotide linkages may be contiguous or non-contiguous.

[0429] In some embodiments, compounds of the present disclosure include blocks that are stereochemical blocks. In some embodiments, the blocks are Rp blocks, in that each internucleotide linkage of the block is Rp. In some embodiments, the 5'-block is an Rp block. In some embodiments, the 3'-block is an Rp block. In some embodiments, the blocks are Sp blocks, in that each internucleotide linkage of the block is Sp. In some embodiments, the 5'-block is an Sp block. In some embodiments, the 3'-block is an Sp block. In some embodiments, provided oligonucleotides include both Rp blocks and Sp blocks. In some embodiments, provided oligonucleotides include one or more Rp blocks but no Sp blocks. In some embodiments, provided oligonucleotides include one or more Sp blocks but no Rp blocks. In some embodiments, provided oligonucleotides include one or more natural phosphate-linked (PO) blocks, in that each internucleotide linkage is a natural phosphate linkage.

[0430] In some embodiments, compounds of the present disclosure include a 5'-block that is an Sp block in which each sugar moiety comprises a 2'-F modification. In some embodiments, the 5'-block is an Sp block in which each internucleotide linkage is a modified internucleotide linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 5'-block is an Sp block in which each internucleotide linkage is a phosphorothioate linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 5'-block comprises four or more nucleoside units. In some embodiments, the 5'-block comprises five or more nucleoside units. In some embodiments, the 5'-block comprises six or more nucleoside units. In some embodiments, the 5'-block comprises seven or more nucleoside units. In some embodiments, the 3'-block is an Sp block in which each sugar moiety comprises a 2'-F modification. In some embodiments, the 3'-block is an Sp block in which each internucleotide linkage is a modified internucleotide linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 3'-block is an Sp block, in which each of the internucleotide linkages is a phosphorothioate linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 3'-block comprises 4 or more nucleoside units. In some embodiments, the 3'-block comprises 5 or more nucleoside units. In some embodiments, the 3'-block comprises 6 or more nucleoside units. In some embodiments, the 3'-block comprises 7 or more nucleoside units.

[0431] In some embodiments, compounds of the disclosure include a region of nucleosides or oligonucleotides followed by a particular type of internucleotide linkage, such as a natural phosphate linkage, a modified internucleotide linkage, an Rp chiral internucleotide linkage, an Sp chiral internucleotide linkage, etc. In some embodiments, A is followed by Sp. In some embodiments, A is followed by Rp. In some embodiments, A is followed by a natural phosphate linkage (PO). In some embodiments, U is followed by Sp. In some embodiments, U is followed by Rp. In some embodiments, U is followed by a natural phosphate linkage (PO). In some embodiments, C is followed by Sp. In some embodiments, C is followed by Rp. In some embodiments, C is followed by a natural phosphate linkage (PO). In some embodiments, G is followed by Sp. In some embodiments, G is followed by Rp. In some embodiments, G is followed by a natural phosphate linkage (PO). In some embodiments, C and U are followed by Sp. In some embodiments, C and U are followed by Rp. In some embodiments, C and U are followed by a natural phosphate linkage (PO). In some embodiments, A and G are followed by Sp. In some embodiments, A and G are followed by Rp.

[0432] In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotides 21 and 22 and between nucleotides 22 and 23, the antisense strand contains at least one thermally destabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven, or all eight) of the following features: (i) the antisense strand contains two, three, four, five, or six 2'-fluoro modifications; (ii) the antisense strand comprises three, four, or five phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (v) the sense strand comprises one, two, three, four, or five phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2'-fluoro modifications; (vii) the dsRNA comprises a duplex region 12 to 40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0433] In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, the antisense strand contains at least one thermally destabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven, or all eight) of the following features: (i) the antisense strand comprises two, (ii) the sense strand is conjugated to a ligand; (iii) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (iv) the sense strand comprises one, two, three, four, or five phosphorothioate internucleotide linkages; (v) the dsRNA comprises at least four 2'-fluoro modifications; (vi) the dsRNA comprises a duplex region 12 to 40 nucleotide pairs in length; (vii) the dsRNA comprises a duplex region 12 to 40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0434] In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, and the antisense strand contains at least one thermally destabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven, or all eight) of the following features: (i) the antisense strand comprises two, three, four, five, or six 2'-fluoro modifications; (ii) the antisense strand comprises one, two, three, four, or five phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (v) the sense strand comprises three, four, or five phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2'-fluoro modifications; (vii) the dsRNA comprises a duplex region 12 to 40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0435] In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, and the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, the antisense strand containing at least one thermally destabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally has at least one of the following features (e.g., (e.g., one, two, three, four, five, six, or all seven): (i) the antisense strand comprises two, three, four, five, or six 2'-fluoro modifications; (ii) the sense strand is conjugated to a ligand; (iii) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (iv) the sense strand comprises three, four, or five phosphorothioate internucleotide linkages; (v) the dsRNA comprises at least two 2'-fluoro modifications; (vi) the dsRNA comprises a duplex region 12 to 40 nucleotide pairs in length; and (vii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0436] In some embodiments, the dsRNA molecule of the present disclosure comprises mismatches in the duplex with the target, or a combination thereof. Mismatches can occur in the overhang region or the duplex region. Base pairs can be ranked based on their tendency to promote dissociation or melting (for example, based on the free energy of association or dissociation of a particular pairing; the simplest approach is to examine each pair individually, but then adjacent or similar analysis can also be used). In terms of promoting dissociation, A:U is more preferable than G:C, G:U is more preferable than G:C, and I:C is more preferable than G:C (I=inosine). Mismatches, such as non-standard pairings or non-standard pairings (described elsewhere herein), are more preferable than standard pairings (A:T, A:U, G:C), and pairings that include universal bases are more preferable than standard pairings.

[0437] In some embodiments, the dsRNA molecules of the present disclosure comprise at least one mismatch pair within the duplex region from the 5' end of the antisense strand, which may be independently selected from the group of A:U, G:U, I:C, and a non-canonical or non-standard or universal base pair, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0438] In some embodiments, the nucleotide at position 1 from the 5' end of the antisense strand to the duplex region is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first one, two, or three base pairs from the 5' end of the antisense strand to the duplex region is an AU base pair. For example, the first base pair from the 5' end of the antisense strand to the duplex region is an AU base pair.

[0439] It has been found that the introduction of a 4'- or 5'-modified nucleotide at the 3' end of a nucleotide phosphodiester (PO), phosphorothioate (PS) or phosphorodithioate (PS2) linkage at any position in a single- or double-stranded oligonucleotide can exert a steric effect on the internucleotide linkage, thereby protecting or stabilizing it against nucleases.

[0440] In some embodiments, 5'-modified nucleotide is introduced at the 3' end of dinucleotide at any position in single-stranded or double-stranded siRNA.For example, 5'-alkylated nucleotide can be introduced at the 3' end of dinucleotide at any position in single-stranded or double-stranded siRNA.The alkyl group at the 5' position of ribose sugar can be racemic or chiral pure R or S isomer.Exemplary 5'-alkylated nucleoside includes 5'-methyl nucleotide.5'-methyl can be racemic or chiral pure R or S isomer.

[0441] In some embodiments, 4'-modified nucleotides are introduced at the 3' end of dinucleotides at any position in single-stranded or double-stranded siRNA. For example, 4'-alkylated nucleotides can be introduced at the 3' end of dinucleotides at any position in single-stranded or double-stranded siRNA. The alkyl group at the 4' position of the ribose sugar can be racemic or chirally pure R- or S-isomer. Exemplary 4'-alkylated nucleotides include 4'-methyl nucleosides. 4'-methyl can be racemic or chirally pure R- or S-isomers. Alternatively, 4'-O-alkylated nucleotides can be introduced at the 3' end of dinucleotides at any position in single-stranded or double-stranded siRNA. The 4'-O-alkyl group at the ribose sugar can be racemic or chirally pure R- or S-isomers. Exemplary 4'-O-alkylated nucleotides include 4'-O-methyl nucleosides. The 4'-O-methyl can be either racemic or chirally pure R or S isomers.

[0442] In some embodiments, 5'-alkylated nucleotide is introduced at any position of the sense strand or antisense strand of dsRNA, and this modification maintains or improves the efficacy of dsRNA.5'-alkyl can be either racemic or chiral pure R or S isomer.Exemplary 5'-alkylated nucleotide includes 5'-methyl nucleotide.5'-methyl can be either racemic or chiral pure R or S isomer.

[0443] In some embodiments, 4'-alkylated nucleotide is introduced at any position of the sense strand or antisense strand of dsRNA, and this modification maintains or improves the efficacy of dsRNA.4'-alkyl can be either racemic or chiral pure R or S isomer.Exemplary 4'-alkylated nucleotide includes 4'-methyl nucleoside.4'-methyl can be either racemic or chiral pure R or S isomer.

[0444] In some embodiments, 4'-O-alkylated nucleotide is introduced at any position of the sense strand or antisense strand of dsRNA, and this modification maintains or improves the efficacy of dsRNA.5'-Alkyl can be either racemic or chiral pure R or S isomer.Exemplary 4'-O-alkylated nucleotide includes 4'-O-methyl nucleotide.4'-O-methyl can be either racemic or chiral pure R or S isomer.

[0445] In some embodiments, dsRNA molecules of the present disclosure may contain 2'-5' linkages (having 2'-H, 2'-OH, and 2'-OMe, and also having P=O or P=S). For example, 2'-5' linkage modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5' end of the sense strand to prevent activation of the sense strand by RISC.

[0446] In another embodiment, the dsRNA molecule of the present disclosure can comprise L sugar (for example, L-ribose, L-arabinose, with 2'-H, 2'-OH and 2'-OMe).For example, these L sugar modifications can be used to promote nuclease resistance, or to inhibit the binding of sense strand to antisense strand, or can be used at the 5' end of sense strand to prevent the activation of sense strand by RISC.

[0447] Multimeric siRNA has been described in various publications, and all of them can be used with the dsRNA of the present disclosure.These publications include WO2007 / 091269, US Patent No. 7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520, and each of them is incorporated herein by reference in its entirety.

[0448] As described in more detail below, RNAi agents containing one or more carbohydrate moieties conjugated to them can optimize one or more properties of the RNAi agent. In many cases, the carbohydrate moiety will be attached to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, such as a non-carbohydrate (preferably cyclic) carrier to which a carbohydrate ligand is attached. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose-replacement modified subunit (PRMS). The cyclic carrier can be a carbon-cyclic system, i.e., all ring atoms are carbon atoms, or a heterocyclic system, i.e., one or more ring atoms can be heteroatoms, such as nitrogen, oxygen, or sulfur. The cyclic carrier can be a monocyclic ring system or can contain two or more rings, e.g., fused rings. The cyclic carrier can be a fully saturated ring system or can contain one or more double bonds.

[0449] The ligand can be attached to the polynucleotide via a carrier. The carrier comprises (i) at least one "backbone attachment point," e.g., two "backbone attachment points," and (ii) at least one "tethering attachment point." As used herein, a "backbone attachment point" refers to a functional group, e.g., a hydroxyl group, or generally a bond that is available and suitable for incorporating the carrier into the backbone of a ribonucleic acid, e.g., a phosphate or a sulfur-containing modified phosphate backbone. In some embodiments, a "tethering attachment point" (TAP) refers to a ring atom, e.g., a carbon atom or heteroatom (other than the atom that provides the backbone attachment point), of the cyclic carrier to which the selected moiety is attached. This moiety can be, for example, a carbohydrate, e.g., a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is attached to the cyclic carrier by an intervening tether. Thus, cyclic carriers often contain a functional group, e.g., an amino group, or generally a bond, that provides a linkage suitable for the incorporation or tethering of another chemical entity, e.g., a ligand, to the constituent ring.

[0450] RNAi agent can be conjugated to ligand through carrier, and said carrier can be cyclic or acyclic group.For example, cyclic group can be selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and decalin.For example, acyclic group can be selected from serinol skeleton or diethanolamine skeleton.

[0451] In certain embodiments, the RNAi agent used in the methods of the disclosure is an agent selected from the group of agents listed in any one of Tables 2-3. These agents may further comprise a ligand, such as one or more lipophilic moieties, one or more GalNAc derivatives, or both one or more lipophilic moieties and one or more GalNAc derivatives.

[0452] IV. iRNA conjugated to a ligand Another modification of the iRNA of the invention involves chemically linking the iRNA to one or more ligands, moieties, or conjugates that enhance the activity, cellular distribution, or cellular uptake, for example, of the iRNA. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acids Sci. USA, 1989, 86:6553-6556), cholic acid (Manoharan et al., Bior. Med. Chem. Let., 1994, 4:1053-1060), thioethers such as beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Bior. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), fatty chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), phospholipids such as dihexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), palmityl moieties (Mishra et al., Biochim.Biophys. Acta, 1995, 1264:229-237), or octadecylamine or hexylamino-carbonyloxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0453] In certain embodiments, the ligand changes the distribution, targeting or life span of the iRNA agent into which it is incorporated.In some embodiments, the ligand enhances the affinity of selected target, for example, molecule, cell or cell type, compartment, tissue, organ or region of body, such as cell or organ compartment, for example, when compared with the species in which such ligand does not exist.Normal ligand does not participate in double-stranded pairing in double-stranded nucleic acid.

[0454] Ligands can include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL) or globulins), carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid), or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, e.g., synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryl acid), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helical peptides.

[0455] The ligand can also include a targeting group, such as a cell or tissue targeting agent, such as a lectin, a glycoprotein, lipid, or protein, such as an antibody, that binds to a specific cell type, such as a kidney cell.The targeting group can be thyroid stimulating hormone, melanocyte stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine polyvalent mannose, polyvalent fucose, glycosylated polyamino acid, polyvalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, biotin, or RGD peptide or RGD peptide mimic.In certain embodiments, the ligand is a polyvalent galactose, such as N-acetyl-galactosamine.

[0456] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules, such as cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutanoic acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithophosphate, and the like. Examples of suitable cleavage inhibitors include oleic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

[0457] Ligands can be proteins, e.g., glycoproteins or peptides, e.g., molecules with specific affinity for a co-ligand, or antibodies, e.g., antibodies that bind to specific cell types, such as cancer cells, endothelial cells, or bone cells. Ligands can also include hormones and hormone receptors. They can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose. Ligands can be, for example, lipopolysaccharides, activators of p38 MAP kinase, or activators of NF-κB.

[0458] The ligand can be a substance, e.g., a drug, that can increase the uptake of an iRNA agent into a cell, for example, by disrupting the cytoskeleton of the cell, e.g., by disrupting the cell's microtubules, microfilaments, or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0459] In some embodiments, the ligands binding to the iRNAs described herein act as pharmacokinetic modulators (PK modulators). PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. Oligonucleotides containing several phosphorothioate linkages have also been shown to bind to serum proteins, and therefore short oligonucleotides containing multiple phosphorothioate linkages in the backbone, e.g., oligonucleotides of about 5, 10, 15, or 20 bases, are also suitable as ligands (e.g., as PK-modulating ligands) for the present invention. Furthermore, aptamers that bind serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0460] The ligand-conjugated iRNAs of the invention can be synthesized by using oligonucleotides bearing reactive functional pendant side chains, such as those derived from the attachment of a linking molecule onto the oligonucleotide (described below). The reactive oligonucleotides can be reacted directly with commercially available ligands, synthesized ligands bearing any of a variety of protecting groups, or ligands bearing a linking moiety attached thereto.

[0461] The oligonucleotides used in the conjugates of the present invention can be conveniently and routinely prepared by the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors, including, for example, Applied Biosystems® (Foster City, California). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives.

[0462] In the ligand-conjugated oligonucleotides and sequence-specific linked nucleosides bearing ligand molecules of the present invention, the oligonucleotides and oligonucleosides can be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors already bearing a linking moiety, ligand-nucleotide or ligand-nucleoside conjugate precursors already bearing a ligand molecule, or non-nucleoside ligand-bearing building blocks.

[0463] When using a nucleotide-conjugate precursor that already possesses a linking moiety, typically, synthesis of the sequence-specifically linked nucleoside is completed, and then a ligand molecule is reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the invention are synthesized by automated synthesizers using phosphoramidites derived from ligand-nucleoside conjugates, in addition to commercially available standard and non-standard phosphoramidites routinely used in oligonucleotide synthesis.

[0464] A. Lipid Conjugates In certain embodiments, the ligand or conjugate is lipid or lipid-based molecule.This lipid or lipid-based molecule can usually bind serum protein, for example, human serum albumin (HSA).HSA-binding ligand allows the distribution of conjugate to target tissue in the body, for example, to target tissue other than the kidney.For example, the target tissue can be the liver, including the liver parenchymal cells.Other molecules that can bind HSA can also be used as ligand.For example, naproxen or aspirin can be used.Lipid or lipid-based ligand can (a) increase the resistance of conjugate to degradation, (b) increase targeting or transport into target cell or cell membrane, or (c) be used to adjust the binding to serum protein, for example, HSA.

[0465] Lipid-based ligands can be used to regulate, for example, control (for example, inhibit) the binding of conjugate to target tissue.For example, lipid or lipid-based ligands that bind more strongly to HSA are less likely to target the kidney, and therefore less likely to be eliminated from the body.A lipid or lipid-based ligand that binds less strongly to HSA can be used so that the conjugate targets the kidney.

[0466] In certain embodiments, the lipid-based ligand binds HSA. For example, the ligand can bind to HSA with sufficient affinity, thereby enhancing the distribution of the conjugate to non-renal tissues. However, this affinity is usually not so strong that the HSA-ligand binding is irreversible.

[0467] In certain embodiments, the lipid-based ligand binds weakly or not at all to HSA, thereby enhancing distribution of the conjugate to the kidney. Other moieties that target kidney cells can be used instead of or in addition to the lipid-based ligand.

[0468] In another aspect, the ligand is a moiety, for example, a vitamin, that is taken up by target cells, for example, proliferating cells.These are particularly useful for treating disorders characterized by unwanted cell proliferation, for example, malignant or non-malignant, for example, cancer cells.Exemplary vitamins include vitamins A, E and K.Other exemplary vitamins include vitamin B, for example, folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients that are taken up by cancer cells.Also included are HSA and low-density lipoprotein (LDL).

[0469] B. Cell-penetrating agents In another aspect, the ligand is a cell-penetrating agent, such as a helical cell-penetrating agent. In certain embodiments, the cell-penetrating agent is amphipathic. Exemplary cell-penetrating agents include peptides such as tat or antennapedia. When the cell-penetrating agent is a peptide, it can be modified, including peptidyl mimics, invertomers, non-peptide or pseudopeptide linkages, and the use of D-amino acids. Helical agents are usually α-helical agents, and can have lipophilic and lipophobic phases.

[0470] The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules that can fold into defined three-dimensional structures similar to natural peptides. Attachment of peptides and peptidomimetics to iRNA agents can affect the pharmacokinetic distribution of iRNAs, such as by enhancing cellular recognition and uptake. The peptide or peptidomimetic portion can be about 5 to 50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0471] The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., composed primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. Alternatively, the peptide moiety can contain a hydrophobic membrane transport sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 9). An RFGF analog containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 10)) can also be a targeting moiety. The peptide moiety can be a "delivery" peptide, capable of transporting large polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 11)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 12)) have been shown to function as delivery peptides. Peptides or peptidomimetics can be encoded by random sequences of DNA, such as peptides identified from phage display libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature, 354:82-84, 1991). Peptides or peptidomimetics that are typically tethered to dsRNA agents via incorporated monomeric units include cell-targeting peptides, such as arginine-glycine-aspartic acid (RGD)-peptides or RGD mimetics. The peptide portion can range in length from about 5 amino acids to about 40 amino acids. The peptide portion can have structural modifications, such as those that increase stability or affect conformational properties. Any of the structural modifications described below can be utilized.

[0472] The RGD peptides used in the compositions and methods of the present invention can be linear or cyclic, and can be modified, for example, glycosylated or methylated, to facilitate targeting to specific tissues.RGD-containing peptides and peptidiomimetics can contain D-amino acids and synthetic RGD mimics.In addition to RGD, other moieties can be used to target integrin ligands, such as PECAM-1 or VEGF.

[0473] RGD peptide moieties can be used to target specific cell types, such as tumor cells, e.g., endothelial tumor cells or breast cancer tumor cells (Zitzmann et al., Cancer Res., 62:5139-43, 2002). RGD peptides can facilitate targeting of dsRNA agents to tumors in a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Typically, RGD peptides will facilitate targeting of iRNA agents to the kidney. RGD peptides can be linear or cyclic and can be modified to facilitate targeting to specific tissues, e.g., glycosylated or methylated. For example, glycosylated RGD peptides can be used to target α- V iRNA agents can be delivered to tumor cells that express β3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).

[0474] A "cell-penetrating peptide" is capable of penetrating cells, such as microbial cells, e.g., bacterial or fungal cells, or mammalian cells, e.g., human cells. Microbial cell-penetrating peptides can be, for example, α-helical linear peptides (e.g., LL-37 or Ceropin P1), disulfide bond-containing peptides (e.g., α-defensins, β-defensins, or bactenecins), or peptides containing only one or two dominant amino acids (e.g., PR-39 or indolicidin). Cell-penetrating peptides can also contain a nuclear localization signal (NLS). For example, a cell-penetrating peptide can be a bipartite amphipathic peptide, such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0475] C. Carbohydrate conjugates In some embodiments of the compositions and methods of the present invention, the iRNA further comprises a carbohydrate. Carbohydrate-conjugated iRNAs are advantageous for in vivo delivery of nucleic acids, as described herein, and are suitable for in vivo therapeutic use. As used herein, "carbohydrate" refers to a compound that is either a carbohydrate itself (which may be linear, branched, or cyclic) composed of one or more monosaccharide units having at least six carbon atoms with an oxygen, nitrogen, or sulfur atom attached to each carbon atom, or a compound having as part thereof a carbohydrate moiety (which may be linear, branched, or cyclic) composed of one or more monosaccharide units, each having at least six carbon atoms with an oxygen, nitrogen, or sulfur atom attached to each carbon atom. Exemplary carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides, such as starch, glycogen, cellulose, and polysaccharide resins. Particular monosaccharides include C5 and above (e.g., C5, C6, C7 or C8) sugars, and disaccharides and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7 or C8).

[0476] In certain embodiments, the carbohydrate conjugate comprises a monosaccharide.

[0477] In certain embodiments, the monosaccharide is N-acetylgalactosamine (GalNAc). GalNAc conjugates containing one or more N-acetylgalactosamine (GalNAc) derivatives are described, for example, in U.S. Patent No. 8,106,022, the entire contents of which are incorporated herein by reference. In some embodiments, the GalNAc conjugate functions as a ligand that targets iRNA to specific cells. In some embodiments, the GalNAc conjugate targets iRNA to liver cells, for example, by functioning as a ligand for the asialoglycoprotein receptor of liver cells (e.g., hepatocytes).

[0478] In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives can be attached via a linker, e.g., via a bivalent or trivalent branched linker. In some embodiments, the GalNAc conjugate is conjugated to the 3' end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 3' end of the sense strand) via a linker, e.g., via a linker described herein. In some embodiments, the GalNAc conjugate is conjugated to the 5' end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 5' end of the sense strand) via a linker, e.g., via a linker described herein.

[0479] In certain embodiments of the invention, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a monovalent linker. In some embodiments, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a bivalent linker. In yet other embodiments of the invention, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a trivalent linker. In other embodiments of the invention, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a tetravalent linker.

[0480] In certain embodiments, a double-stranded RNAi agent of the invention comprises one GalNAc or GalNAc derivative attached to an iRNA agent, hi certain embodiments, a double-stranded RNAi agent of the invention comprises multiple (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each of which is independently attached to multiple nucleotides of the double-stranded RNAi agent via multiple monovalent linkers.

[0481] In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of a larger molecule connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the corresponding 5' end of the other strand, forming a hairpin loop containing multiple unpaired nucleotides, each unpaired nucleotide in the hairpin loop can independently comprise a GalNAc or GalNAc derivative linked via a monovalent linker. This hairpin loop can also be formed by an extended overhang in one strand of the duplex.

[0482] In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of a larger molecule connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the corresponding 5' end of the other strand, forming a hairpin loop containing multiple unpaired nucleotides, each unpaired nucleotide in the hairpin loop can independently comprise a GalNAc or GalNAc derivative linked via a monovalent linker. This hairpin loop can also be formed by an extended overhang in one strand of the duplex.

[0483] In some embodiments, the GalNAc conjugate is [ka] Formula II is.

[0484] In some embodiments, the RNAi agent is attached to the carbohydrate conjugate via a linker as shown in the following schematic diagram, where X is O or S: [ka]

[0485] In some embodiments, the RNAi agent is conjugated to L96, as defined in Table 1 and shown below. [ka]

[0486] In certain embodiments, the carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of: [ka] Formula II, [ka] Formula III, [ka] Formula IV, [ka] Formula V, [ka] Formula VI, [ka] Formula VII, [ka] Formula VIII, [ka] Formula IX, [ka] Formula X, [ka] Formula XI, [ka] Formula XII, [ka] Formula XIII, [ka] Formula XIV, [ka] Formula XV, [ka] Formula XVI, [ka] Formula XVII, [ka] Formula XVIII, [ka] Formula XIX, [ka] Formula XX, [ka] Formula XXI, [ka] Formula XXII, [ka] Formula XXIII, [ka] , wherein Y is O or S and n is 3 to 6 (Formula XXIV), [ka] , wherein Y is O or S and n is 3 to 6 (Formula XXV), [ka] Formula XXVI, [ka] , wherein X is O or S (Formula XXVII), [ka] [ka] [ka] [ka] Formula XXXIV.

[0487] In certain embodiments, the carbohydrate conjugate used in the compositions and methods of the present invention is a monosaccharide. In certain embodiments, the monosaccharide is N-acetylgalactosamine, e.g., [ka] Formula II is.

[0488] Other exemplary carbohydrate conjugates for use in the embodiments described herein include, but are not limited to: [ka] (formula XXXVI), When one of X or Y is an oligonucleotide, the other is hydrogen.

[0489] In some embodiments, suitable ligands are those disclosed in WO 2019 / 055633, the entire contents of which are incorporated herein by reference. In one embodiment, the ligand comprises the following structure: [ka]

[0490] In certain embodiments, the RNAi agents of the present disclosure may include GalNAc ligands, even though such GalNAc ligands are currently expected to be of limited value for the intrathecal / CNS delivery routes of the present disclosure.

[0491] In certain embodiments of the invention, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a monovalent linker. In some embodiments, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a bivalent linker. In yet other embodiments of the invention, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a trivalent linker. In other embodiments of the invention, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a tetravalent linker.

[0492] In certain embodiments, a double-stranded RNAi agent of the invention comprises one GalNAc or GalNAc derivative attached to an iRNA agent, e.g., at the 5' end of the sense strand of a dsRNA agent, or at the 5' end of one or both sense strands of a dual-targeting RNAi agent described herein. In certain embodiments, a double-stranded RNAi agent of the invention comprises multiple (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each of which is independently attached to multiple nucleotides of the double-stranded RNAi agent via multiple monovalent linkers.

[0493] In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of a single larger molecule connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the corresponding 5' end of the other strand, forming a hairpin loop containing multiple unpaired nucleotides, each unpaired nucleotide in the hairpin loop can independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker.

[0494] In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator or a cell-penetrating peptide.

[0495] Further carbohydrate conjugates and linkers suitable for use in the present invention include those described in WO 2014 / 179620 and WO 2014 / 179627, the entire contents of each of which are incorporated herein by reference.

[0496] D. Linker In some embodiments, the conjugates or ligands described herein can be attached to the iRNA oligonucleotide using a variety of linkers, which may or may not be cleavable.

[0497] The term "linker" or "linking group" means an organic moiety that connects two parts of a compound, e.g., covalently bonds the two parts of a compound.Typically, a linker is a direct bond or an atom such as oxygen or sulfur, a unit such as NR, C(O), C(O)NH, SO, SO, SONH, or a chain of atoms, including, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylhetero ...alkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkynylarylalkynyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkynylarylalkynyl, alkylheteroarylalkynyl, alkylhet alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclyl and the like, wherein one or more methylenes can be interrupted or terminated by O, S, S(O), SO, N(R), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, where R is hydrogen, acyl, aliphatic, or substituted aliphatic.In certain embodiments, the linker is about 1 to 24 atoms, 2 to 24, 3 to 24, 4 to 24, 5 to 24, 6 to 24, 6 to 18, 7 to 18, 8 to 18 atoms, 7 to 17, 8 to 17, 6 to 16, 7 to 16, or 8 to 16 atoms.

[0498] A cleavable linking group is one that is sufficiently stable outside a cell, but that, once inside a target cell, is cleaved to release the two moieties held together by the linker. In another embodiment, the cleavable linking group is cleaved at a rate that is at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or more, or at least about 100-fold faster in the target cell or under first reference conditions (which may, for example, be selected to mimic or represent intracellular conditions) than in the subject's blood or under second reference conditions (which may, for example, be selected to mimic or represent conditions found in blood or serum).

[0499] Cleavable linking groups are susceptible to cleaving agents, such as pH, redox potential, or the presence of degradable molecules. Cleavage agents are generally more widespread or found at higher levels or activity inside cells than in serum or blood. Examples of such degrading agents include redox agents that are selective for a specific substrate or have no substrate specificity, e.g., oxidases or reductases or reducing agents present in cells, such as mercaptans, that can degrade redox-cleavable linking groups by reduction; esterases; reagents that can create an endosomal or acidic environment, e.g., reagents that result in a pH of 5 or less; and enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as general acids, peptidases (which may be substrate-specific), and phosphatases.

[0500] Cleavable linking groups, such as disulfide bonds, can be pH-sensitive. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some linkers have cleavable linking groups that are cleaved at a selected pH, thereby releasing the cationic lipid from the ligand inside the cell or into a desired compartment of the cell.

[0501] Linker can contain a cleavable linking group that can be cleaved by specific enzyme.The type of cleavable linking group incorporated into linker can depend on the target cell.For example, liver targeting ligand can be linked to cationic lipid through a linker that contains ester group.Hepatocytes are rich in esterase, so linker will be cleaved more efficiently in hepatocytes than in cell types that are not rich in esterase.Other cell types that are rich in esterase include lung, renal cortex and testicular cells.

[0502] When targeting cell types rich in peptidases, such as hepatocytes and synoviocytes, linkers containing peptide bonds can be used.

[0503] Generally, the suitability of a candidate cleavable linking group can be evaluated by testing the ability (or conditions) of a degrading agent to cleave the candidate linking group. It may also be desirable to test the candidate cleavable linking group for its ability to resist cleavage in blood or when in contact with other non-target tissues. Thus, the relative susceptibility to cleavage between first and second conditions can be determined, where the first condition is selected to exhibit cleavage in target cells, and the second condition is selected to exhibit cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be performed in a cell-free system, in cells, in cell culture, in organ or tissue culture, or in whole animals. It may be useful to perform initial evaluation in cell-free or culture conditions and confirm by further evaluation in whole animals. In certain embodiments, useful candidate compounds are cleaved at a rate that is at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0504] i. Redox-cleavable linking group In certain embodiments, the cleavable linking group is a redox-cleavable linking group that is cleaved upon reduction or oxidation. An example of a reductively cleavable linking group is a disulfide linking group (-SS-). To determine whether a candidate cleavable linking group is a suitable "reductively cleavable linking group" or is suitable for use with, for example, a particular iRNA moiety and a particular targeting agent, one can refer to the methods described herein. For example, candidates can be evaluated in cells by incubating with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate that would be observed in target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In some conditions, the candidate compound is cleaved at a maximum of about 10% in blood. In other embodiments, useful candidate compounds are degraded at a rate at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of the candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic intracellular media and compared to conditions selected to mimic extracellular media.

[0505] ii. Phosphate-based cleavable linkers In certain embodiments, the cleavable linker comprises a phosphate-based cleavable linking group. The phosphate-based cleavable linking group is cleaved by a reagent that decomposes or hydrolyzes the phosphate group. An example of a reagent that cleaves the phosphate group within a cell is an enzyme such as a phosphatase within the cell. Examples of phosphate-based linking groups include -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-, where Rk in each occurrence can independently be C1-C20 alkyl, C1-C20 haloalkyl, C6-C10 aryl, or C7-C12 aralkyl. Exemplary embodiments include -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. In certain embodiments, the phosphate-based linking group is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0506] iii. Acid-cleavable linking group In certain embodiments, the cleavable linker comprises an acid-cleavable linking group. An acid-cleavable linking group is a linking group that is cleaved under acidic conditions. In other embodiments, the acid-cleavable linking group is cleaved in an acidic environment with a pH of about 6.5 or less (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0 or less), or by an agent, such as an enzyme, that can act as a general acid. Within a cell, certain low-pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for the acid-cleavable linking group. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and esters of amino acids. Acid-cleavable groups can have the general formula -C=NN-, C(O)O, or -OC(O). Exemplary embodiments include allyl groups, substituted alkyl groups, or tertiary alkyl groups, such as dimethylpentyl or t-butyl, when the carbon is bonded to the oxygen of the ester (alkoxy group). These candidates can be evaluated using methods similar to those described above.

[0507] iv. Ester-based cleavable linking groups In certain embodiments, the cleavable linker comprises an ester-based cleavable linking group. Ester-based cleavable linking groups are cleaved in cells by enzymes such as esterases and amidases. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable linking groups have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0508] v. Peptide-based cleavable linking groups In yet another embodiment, the cleavable linker comprises a peptide-based cleavable linking group. Peptide-based cleavable linking groups are cleaved intracellularly by enzymes such as peptidases and proteases. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (—C(O)NH—). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. Peptide-based cleaving groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to yield peptides and proteins, and do not include all amide functionalities. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)—, where R and R are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0509] In some embodiments, the iRNA of the present invention is conjugated to a carbohydrate via a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the present invention include, but are not limited to: [ka] (formula XXXVII), [ka] (formula XXXVIII), [ka] (formula XXXIX), [ka] (formula XL), [ka] (formula XLI), [ka] (formula XLII), [ka] (Formula XLIII), and [ka] (Formula XLIV), and when one of X or Y is an oligonucleotide, the other is hydrogen.

[0510] In certain embodiments of the compositions and methods of the present invention, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives attached via a bivalent or trivalent branched linker.

[0511] In certain embodiments, the dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formulas (XLV)-(XLVI): [ka] During the ceremony, q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C each independently represent 0 to 20, and the repeat units may be the same or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5Cis, independently at each occurrence, absent, CO, NH, O, S, OC(O), NHC(O), CH, CHNH, or CHO; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C is independently at each occurrence absent, alkylene, or substituted alkylene, wherein one or more methylenes are selected from O, S, S(O), SO, N(R N ), C(R')=C(R''), C≡C or C(O), R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C are each independently absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, [ka] or heterocyclyl, L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B , and L 5C represents a ligand, i.e., each occurrence independently represents a monosaccharide (e.g., GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and R a is H or an amino acid side chain. Trivalent conjugated GalNAc derivatives are useful in part for use with RNAi agents to inhibit expression of target genes, such as those of formula (XLIX): [ka] In the formula, L 5A , L 5B , and L 5C represents a monosaccharide, such as a ...

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent or a pharma- ceutical acceptable salt thereof for inhibiting expression of a prion protein (PRNP) in a cell, comprising: said dsRNA agent or a pharma- ceutically acceptable salt thereof comprising a sense strand and an antisense strand which form a double-stranded region; (a) the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from a portion of the nucleotide sequence of SEQ ID NO:1 by no more than 3 nucleotides, and the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides that differ from a corresponding portion of the nucleotide sequence of SEQ ID NO:5 by no more than 3 nucleotides; the sense strand, the antisense strand, or both the sense strand and the antisense strand are conjugated to one or more lipophilic moieties; (b) the antisense strand comprises a region complementary to an mRNA encoding PRNP, and the region complementary to an mRNA encoding PRNP is selected from the group consisting of those shown in Tables 2 to 3 below. Table 2. Unmodified sense and antisense strand sequences of PRNP dsRNA agents 【Table 1-1】 【Table 1-2】 【Table 1-3】 【Table 1-4】 【Table 1-5】 【Table 1-6】 【Table 1-7】 【Table 1-8】 【Table 1-9】 Table 3. Modified sense and antisense strand sequences of PRNP dsRNA agents. 【Table 2-1】 【Table 2-2】 【Table 2-3】 【Table 2-4】 【Table 2-5】 【Table 2-6】 【Table 2-7】 【Table 2-8】 【Table 2-9】 【Table 2-10】 comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense nucleotide sequences in any one of the sequences, the sense strand, the antisense strand, or both the sense strand and the antisense strand are conjugated to one or more lipophilic moieties; (c) the sense strand is selected from the group consisting of nucleotides 858-880, 2031-2056, 2031-2053, 2034-2056, 502-524, 507-529, 502-529, 540-562, 566-588, 575-597, 576-598, 589-611, 575-611, 593-615, 594-616, 600-622, 593-622, 650-672, 976-998, 1100-1122, 1126-1148, 1220-1242, 1221-1243, 1220-1243, 1304-1326, and 1328-1350 of SEQ ID NO:1; , 1410-1432, 1445-1467, 1481-1503, 1532-1554, 1610-1632, 1615-1637, 1617-1639, 1621-1643, 1610-1643, 1610-1639, 1688-1710, 1694-1714, 1830 -1852, 1831-1853, 1854-1876, 1830-1853, 1872-1894, 1873-1895, 1938-1960, 2011-2033, 2015-2037, 2069-2091, 2076-2098, 2077-2099, 1872-1895 , 2011-2037, 2069-2099, 2079-2101, 2069-2098, 2138-2160, 2143-2165, 2158-2180, 2167-2189, 2168-2190, 2170-2192, 2174-2196, 2175-2197, 2177 -2199, 2185-2207, 2189-2211, 2196-2218, 2200-2222, 2202-2224, 2221-2243, 2222-2244, 2223-2245, 2238-2260, 2241-2263, 2242-2264, 2138-2264 , 2257-2358, 2174-2245, 2174-2224, 2138-2196, 2177-2224, 2223-2245, 2138-2189, 2177-2211, 2196-2224, 2257-2279, 2258-2280, 2335-2537, 2336 -2358, 2257-2358, 2342-2364, 2397-2419, 2398-2420, 2399-2421, 2400-2422, 2401-2423, 2404-2426, 2397-2426, 2394-2423, 2397-2421, 2399-2426,and 2398-2421, wherein the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than three nucleotides from any one of the nucleotide sequences of SEQ ID NO:5; the sense strand, the antisense strand, or both the sense strand and the antisense strand are conjugated to one or more lipophilic moieties; or (d) the sense strand is selected from the group consisting of nucleotides 530-570, 535-565, 539-561, 540-562, 555-605, 560-605, 560-600, 566-588, 567-589, 568-590, 569-591, 570-592, 575-597, 580- 620-585-620, 589-611, 590-612, 591-613, 592-614, 593-615, 594-616, 600-650, 610-650, 613-635, 614-636, 615-637, 616-638, 617-639, 618-640, 640-680, 649-671, 650-672, 651-673, 670-700, 674-696, 675-697, 795-830, 804-826, 2325-2375, 2325-2370, 2330-2370, 2335-2357, 2336-2358, 2337-2359, 2338-2360, 2339-2361, or 2340-2362, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than three nucleotides from any one of the nucleotide sequences of SEQ ID NO:5; the sense strand, the antisense strand, or both the sense strand and the antisense strand are conjugated to one or more lipophilic moieties; A dsRNA agent or a pharma- ceutically acceptable salt thereof.

2. 2. The dsRNA agent, or a pharma- ceutically acceptable salt thereof, of claim 1, wherein said one or more lipophilic moieties are conjugated to one or more interior positions in said double-stranded region of said dsRNA agent, or a pharma- ceutically acceptable salt thereof.

3. 3. The dsRNA agent of claim 1 or 2, or a pharma- ceutically acceptable salt thereof, wherein the one or more lipophilic moieties are conjugated to one or more interior positions in at least one strand via a linker or carrier.

4. 4. The dsRNA agent of any one of claims 1-3, or a pharma- ceutically acceptable salt thereof, wherein said one or more lipophilic moieties are conjugated to one or more of said internal positions selected from the group consisting of positions 4-8 and 13-18 on the sense strand and positions 6-10 and 15-18 on the antisense strand, counting from the 5' end of each strand.

5. 5. The dsRNA agent of claim 4, or a pharma- ceutically acceptable salt thereof, wherein said one or more lipophilic moieties are conjugated to one or more of the internal positions selected from the group consisting of positions 5, 6, 7, 15, and 17 on the sense strand and positions 15 and 17 on the antisense strand, counting from the 5' end of each strand.

6. 6. The dsRNA agent of any one of claims 1 to 5, or a pharma- ceutically acceptable salt thereof, wherein said lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound.

7. 7. The dsRNA agent of any one of claims 1-6, or a pharma- ceutically acceptable salt thereof, wherein said lipophilic moiety contains a saturated or unsaturated C4-C30 hydrocarbon chain and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

8. 8. The dsRNA agent of claim 7, or a pharma- ceutically acceptable salt thereof, wherein said lipophilic moiety contains a saturated or unsaturated C6-C18 hydrocarbon chain.

9. 9. The dsRNA agent of claim 8, or a pharma- ceutically acceptable salt thereof, wherein said lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain.

10. 10. The dsRNA agent of claim 9, or a pharma- ceutically acceptable salt thereof, wherein said saturated or unsaturated C16 hydrocarbon chain is conjugated to the 6-position counting from the 5'-end of said chain.

11. 11. The dsRNA agent of any one of claims 1-10, or a pharma- ceutically acceptable salt thereof, wherein said lipophilic moiety is conjugated via a carrier that replaces one or more nucleotides in said internal position or said double-stranded region.

12. 12. The dsRNA agent of any one of claims 1-11, or a pharma- ceutically acceptable salt thereof, wherein the lipophilic moiety is conjugated to the dsRNA agent via a linker that contains an ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide linkage, a product of a click reaction, or a carbamate.

13. 13. The dsRNA agent of any one of claims 1-12, or a pharma- ceutically acceptable salt thereof, wherein said lipophilic moiety is conjugated to a nucleobase, a sugar moiety, or an internucleoside linkage.

14. 14. The dsRNA agent, or a pharma- ceutically acceptable salt thereof, of any one of claims 1-13, wherein at least one nucleotide of the dsRNA agent, or a pharma- ceutically acceptable salt thereof, comprises a nucleotide modification.

15. 15. The dsRNA agent of claim 14, or a pharma- ceutically acceptable salt thereof, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a nucleotide modification.

16. At least one of the nucleotide modifications is a deoxy-nucleotide modification, a 3'-terminal deoxythymidine (dT) nucleotide modification, a 2'-O-methyl nucleotide modification, a 2'-fluoro nucleotide modification, a 2'-deoxy nucleotide modification, a locked nucleotide modification, an unlocked nucleotide modification, a conformationally restricted nucleotide modification, a constrained ethyl nucleotide modification, an abasic nucleotide modification, a 2'-amino nucleotide modification, a 2'-O-allyl-nucleotide modification, a 2'-C-alkyl-nucleotide modification, a 2'-hydroxy-nucleotide modification, a 2'-methoxyethyl nucleotide modification, a 2'-O-alkyl-nucleotide modification, a morpholino nucleotide modification, a phosphoramidate modification, a nucleotide modification comprising a non-natural base, a tetrahydropyran nucleotide modification, a 1,5-anhydrohexitol nucleotide modification, a cyclohexenyl nucleotide modification, a nucleotide modification comprising a 5'-phosphorothioate group, a nucleotide modification comprising a 5'-methylphosphonate group, a nucleotide modification comprising a 5' phosphate or a 5' phosphate mimic, a vinyl phosphate, or a nucleotide modification comprising a 5' phosphate or a 5' phosphate mimic. Nucleotide modifications including phosphates, nucleotide modifications including adenosine-glycol nucleic acid (GNA), nucleotide modifications including thymidine-glycol nucleic acid (GNA) S-isomers, nucleotide modifications including 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotide modifications including 2'-deoxythymidine-3' phosphate, nucleotide modifications including 2'-deoxyguanosine-3'-phosphate, and terminal nucleotide modifications linked to cholesteryl derivatives, dodecanoic acid bisdecylamide group modifications, cytidine-2'- 16. The dsRNA agent of claim 14 or 15, or a pharma- ceutically acceptable salt thereof, selected from the group of: a phosphate modification, a guanosine-2'-phosphate modification, a uridine-2'-phosphate modification, an adenosine-2'-phosphate modification, a 2'-O-hexadecyl-adenosine-3'-phosphate modification, a 2'-O-hexadecyl-cytidine-3'-phosphate modification, a 2'-O-hexadecyl-guanosine-3'-phosphate modification, and a 2'-O-hexadecyl-uridine-3'-phosphate modification, and combinations thereof.

17. 17. The dsRNA agent of any one of claims 1 to 16, or a pharma- ceutically acceptable salt thereof, further comprising at least one phosphorothioate internucleotide linkage.

18. 20. The dsRNA agent of claim 17, or a pharma- ceutically acceptable salt thereof, wherein the dsRNA agent comprises 6 to 8 phosphorothioate internucleotide linkages.

19. 19. The dsRNA agent of any one of claims 1-18, or a pharma- ceutically acceptable salt thereof, wherein at least one strand comprises a 3' overhang of at least 1 nucleotide, or at least one strand comprises a 3' overhang of at least 2 nucleotides.

20. 20. The dsRNA agent of any one of claims 1 to 19, or a pharma- ceutically acceptable salt thereof, wherein said double-stranded region is 15 to 30 nucleotide pairs in length.

21. 21. The dsRNA agent of any one of claims 1-20, or a pharma- ceutically acceptable salt thereof, wherein said lipophilic moiety or targeting ligand is conjugated via a biocleavable linker selected from the group consisting of DNA, RNA, disulfides, amides, and functionalized mono- or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

22. 22. The dsRNA agent of any one of claims 1-21, or a pharma- ceutically acceptable salt thereof, wherein the 3' end of the sense strand is protected via an end cap that is a cyclic group having an amine, the cyclic group being selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.

23. 23. The dsRNA agent of any one of claims 1-22, or a pharma- ceutically acceptable salt thereof, further comprising a phosphate or a phosphate mimetic at the 5'-end of the antisense strand.

24. 23. A cell comprising the dsRNA agent of any one of claims 1 to 22, or a pharma- ceutically acceptable salt thereof.

25. A pharmaceutical composition for inhibiting expression of a gene encoding a prion protein (PRNP), comprising the dsRNA agent of any one of claims 1 to 22, or a pharma- ceutically acceptable salt thereof.

26. 26. An in vitro method of inhibiting expression of a prion protein (PRNP) gene in a cell, said method comprising contacting said cell with a dsRNA agent of any one of claims 1-22 or a pharma- ceutically acceptable salt thereof, or the pharmaceutical composition of claim 25, thereby inhibiting expression of said PRNP gene in said cell.

27. 27. The method of claim 26, wherein contacting the cell with the dsRNA agent or a pharma- ceutically acceptable salt thereof inhibits expression of PRNP by at least 50%, 60%, 70%, 80%, 90%, or 95%.

28. A pharmaceutical composition for treating a subject having a disorder that would benefit from a decrease in PRNP expression, comprising a therapeutically effective amount of a dsRNA agent of any one of claims 1 to 22 or a pharma- ceutically acceptable salt thereof, or the pharmaceutical composition of claim 25.

29. A pharmaceutical composition for preventing at least one symptom in a subject having a disorder that would benefit from a decrease in PRNP expression, comprising a prophylactically effective amount of a dsRNA agent according to any one of claims 1 to 22 or a pharma- ceutically acceptable salt thereof, or the pharmaceutical composition according to claim 25.

30. 30. The pharmaceutical composition of claim 28 or 29, wherein the disorder is a PRNP-related disease.

31. The pharmaceutical composition of claim 30, wherein the PRNP-related disease is a prion disease.

32. 32. The pharmaceutical composition of claim 31, wherein the prion disease is an inherited prion disease selected from the group consisting of familial Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI).

33. 32. The pharmaceutical composition of claim 31, wherein the prion disease is a sporadic prion disease selected from the group consisting of sporadic Creutzfeldt-Jakob disease, sporadic fatal insomnia (sFI), and variably proteinase-sensitive prion disease (VPSPr).

34. 32. The pharmaceutical composition of claim 31, wherein the prion disease is an acquired prion disease selected from the group consisting of iatrogenic CJD (iCJD), kuru, and variant CJD (vCJD).

35. The pharmaceutical composition according to any one of claims 28 to 34, wherein the subject is a human.

36. 36. The pharmaceutical composition of any one of claims 28-35, wherein said dsRNA agent or a pharma- ceutically acceptable salt thereof is administered at a dose of about 0.01 mg / kg to about 50 mg / kg or about 5 mg to 1000 mg.

37. The pharmaceutical composition of any one of claims 28 to 36, wherein the dsRNA agent, or a pharma- ceutically acceptable salt thereof, is administered intrathecally.

38. The pharmaceutical composition according to any one of claims 28 to 37, further comprising administering an additional agent for the treatment or prevention of a PRNP-associated disorder.

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