Superoxide dismutase 1 (SOD1) iRNA compositions and methods of use thereof for treating or preventing superoxide dismutase 1-(SOD1-) associated neurodegenerative diseases

Abstract

The present disclosure relates to double-stranded ribonucleic acid (dsRNAi) agents and compositions that target the SOD1 gene, and methods for inhibiting expression of the SOD1 gene, as well as methods of using such dsRNA agents and compositions to treat subjects having an SOD1-related neurodegenerative disease or disorder, such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), and Down's syndrome (DS).

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 148,991, filed February 12, 2021, and U.S. Provisional Patent Application No. 63 / 270,176, filed October 21, 2021. The entire contents of each of the foregoing applications 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 herein in its entirety. The ASCII copy was created on February 8, 2022, is named 121301_12720_SL.txt, and is 365,213 bytes in size. [Background technology]

[0003] Superoxide dismutase 1 (SOD1), also known as Cu / Zn superoxide dismutase (Cu / ZnSOD), is a ubiquitously expressed antioxidant enzyme that metabolizes superoxide radicals produced during cellular metabolism into molecular oxygen and hydrogen peroxide, playing a key role in protecting cells from oxidative damage (McCord JM and Fridovich I. (1969) J Bio Chem. 244:6049-6055; Rosen DR (1993) Nature. 364:362; Trist B, et al. (2020) Angew Chem Int Ed Engl. Accepted authors' manuscript). The human SOD1 gene is located on chromosome 21q22.11 and produces a 16-kDa protein that normally forms a 32-kDa homodimer. In mammalian cells, SOD1 is widely distributed in the cytosol, nucleus, lysosomes, peroxisomes, and mitochondrial intermembrane space (IMS) (Fukai T and Ushio-Fukai M. (2011) Antioxid Redox Signal. 15(6):1583-1606; Zhang S, et al. (2015) Free Radic Biol Med. 85:33-44; Huai J and Zhang Z. (2019) Front. Neurol. 10:527). The cytosolic abundance of SOD1 distinguishes it from two other mammalian superoxide dismutases, the mitochondrial tetrameric manganese superoxide dismutase (SOD2) and the extracellular tetrameric Cu / Zn superoxide dismutase (SOD3), which are also involved in superoxide radical metabolism. However, SOD1 activity accounts for approximately 50–80% of total SOD activity in mammalian cells (Mindola P, et al. (2016) Front. Physiol. 7:594).

[0004] The structure of each SOD1 subunit consists of a β-barrel core and seven end loops held together by intramolecular disulfide bonds, a binuclear metal-binding site that holds copper and zinc ions involved in SOD1 catalytic activity, and a global hydrogen-bonding network (Huai J and Zhang Z. (2019) Front. Neurol. 10:527). Previous studies have shown that the stability, structure, and function of SOD1 are controlled by its post-translational modifications, metal ion binding, and disulfide bond state. Loss of metal cofactors and / or disruption of disulfide bonds are often associated with mutations in the SOD1 gene and can result in pathogenic misfolding, aggregation, and / or dysfunction of the SOD1 protein (Huai J and Zhang Z. (2019) Front. Neurol. 10:527).

[0005] Approximately 200 mutations have been documented throughout the coding and non-coding regions of the SOD1 gene. Mutant SOD1 proteins are susceptible to misfolding, impairing protective antioxidant function and potentially leading to the formation of abnormal molecular interactions (e.g., between multiple misfolded SOD1 units (aggregation) and between misfolded SOD1 and other cellular components), thereby contributing to pathology through both loss-of-function effects (e.g., reduced antioxidant activity, impaired nuclear translocation and promoter binding, and impaired SOD1 redox signaling) and gain-of-function effects (e.g., neurotoxicity) (Trist B, et al. (2020) Angew Chem Int Ed Engl. Accepted author's manuscript). SOD1 is thought to be involved in the pathology of various diseases, such as heart failure, cancer (e.g., lung adenocarcinoma, non-small cell lung cancer, and breast cancer), diabetes, amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease, and Down syndrome (DS) (Banks CJ and Anderson JL. (2019) Redox Biol. 26:101270; Trist B, et al. (2020) Angew Chem Int Ed Engl. Accepted authors' manuscript). In the context of neurodegenerative diseases, SOD1 is thought to be involved in initiating and / or accelerating injury pathways in both neurons and surrounding glia, including disruption of proteasome function, microtubule and microfilament disassembly, endoplasmic reticulum stress, and redox dyshomeostasis. More specifically, mutant SOD1 is implicated in familial amyotrophic lateral sclerosis (fALS), and various SOD1 mutations are understood to increase SOD1's aggregation propensity and induce motor neuron death. Furthermore, misfolding and dysfunction of wild-type SOD1 are associated with the death of spinal motor neurons in sporadic ALS (aALS), the death of dopamine neurons in the substantia nigra pars compacta (SNc) in Parkinson's disease, and the death of neurons in the frontal cortex and hippocampus in Alzheimer's disease (Trist B, et al. (2020) Angew Chem Int Ed Engl. Accepted author's manuscript).Furthermore, SOD1 has been implicated in neuronal death by exacerbating other deleterious cellular pathologies, including Aβ plaque formation in Alzheimer's disease and α-synuclein deposition in Parkinson's disease.

[0006] There are currently no effective treatments for superoxide dismutase 1-related neurodegenerative diseases, and even if there are, they are only palliative. Therefore, there remains a need for drugs that have high biological activity and in vivo stability, can effectively inhibit the expression of target SOD1 genes, and can selectively and efficiently silence the SOD1 gene using the cell's own RNAi mechanism. Summary of the Invention [Means for solving the problem]

[0007] The present disclosure provides an RNAi agent composition that performs RNA-induced silencing complex (RISC)-mediated cleavage of an RNA transcript of the superoxide dismutase 1 (SOD1) gene. The SOD1 gene can be present in a cell, e.g., in a subject, such as a human. The present disclosure also provides methods of using the RNAi agent composition of the present disclosure to inhibit expression of the SOD1 gene and / or to treat subjects who would benefit from inhibiting or reducing expression of the SOD1 gene, e.g., subjects suffering from or susceptible to SOD1-related neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), and Down's syndrome (DS).

[0008] Thus, in one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent, or a pharmaceutically acceptable salt thereof, comprising a sense strand and an antisense strand, which form a double-stranded region; a) the nucleotide sequence of the sense strand differs by no more than four bases from the nucleotide sequence 5'-csascuu(Uhd)aaUfCfCfucuauccasgsa-3' (SEQ ID NO: 11), and the nucleotide sequence of the antisense strand differs by no more than four bases from the nucleotide sequence 5'-VPusdCsugdGadTagagdGaUfuaaagugsasg-3' (SEQ ID NO: 12), wherein VP is 5'-vinylphosphonate, (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dC, dG, and dT are 2'-deoxy C, G, and T; and Cf and Uf are 2'-deoxy-2'-fluoro (2'-F) C and U; b) the nucleotide sequence of the sense strand differs by no more than four bases from the nucleotide sequence 5'-csasggu(Chd)cuCfAfCfuuuaauccsusa-3' (SEQ ID NO: 13), and the nucleotide sequence of the antisense strand differs by no more than four bases from the nucleotide sequence 5'-VPusdAsggdAudTaaagdTgAfggaccugscsg-3' (SEQ ID NO: 14), where VP is 5'-vinylphosphonate, (Chd) is 2'-O-hexadecyl-cytidine-3'-phosphate, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA and dT are 2'-deoxy A and T; and Af and Cf are 2'-deoxy-2'-fluoro (2'-F) A and C; c) the nucleotide sequence of the sense strand differs by no more than four bases from the nucleotide sequence 5'-ususcgag(Chd)aGfAfAfggaaaguasasa-3' (SEQ ID NO: 15) and the nucleotide sequence of the antisense strand differs by no more than four bases from the nucleotide sequence 5'-VPusUfsuadCu(Tgn)uccuucUfgCfucgaasasu-3' (SEQ ID NO: 16), where VP is 5'-vinylphosphonate; (Chd) is 2'-O-hexadecyl-cytidine-3'-phosphate, (Tgn) is thymidine-glycol nucleic acid (GNA), S isomer; s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dC is 2'-deoxy C, and Af, Cf, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, G, and U; d) the nucleotide sequence of the sense strand differs by no more than four bases from the nucleotide sequence 5'-gsasaag(Uhd)aaUfGfGfaccagugasasa-3' (SEQ ID NO: 17), and the nucleotide sequence of the antisense strand differs by no more than four bases from the nucleotide sequence 5'-VPusUfsucdAc(Tgn)gguccaUfuAfcuuucscsu-3' (SEQ ID NO: 18), where VP is 5'-vinylphosphonate. where (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate, (Tgn) is thymidine-glycol nucleic acid (GNA), S isomer; s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA is 2'-deoxy A, and Af, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, G, and U; e) the nucleotide sequence of the sense strand differs by no more than four bases from the nucleotide sequence 5'-asgsga(Uhd)gaaGfAfGfaggcaugususa-3' (SEQ ID NO: 19), and the nucleotide sequence of the antisense strand differs by no more than four bases from the nucleotide sequence 5'-VPusAfsacdAu(G2p)ccucucUfuCfauccususu-3' (SEQ ID NO: 20), wherein VP is 5'-vinylphosphonate. (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate, (G2p) is guanosine-2'-phosphate, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA is 2'-deoxy A, and Af, Cf, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, G, and U; f) the nucleotide sequence of the sense strand differs by no more than four bases from the nucleotide sequence 5'-asasgga(Ahd)agUfAfAfuggaccagsusa-3' (SEQ ID NO:21), and the nucleotide sequence of the antisense strand differs by no more than four bases from the nucleotide sequence 5'-VPusdAscudGg(Tgn)ccaudTaCfuuuccuuscsu-3' (SEQ ID NO:22), where VP is 5'-vinylphosphonate, and (Ahd ) is 2'-O-hexadecyl-adenosine-3'-phosphate, (Tgn) is thymidine-glycol nucleic acid (GNA), S isomer; s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA, dG, and dT are 2'-deoxy A, G, T, and Af, Cf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, and U; g) the nucleotide sequence of the sense strand differs by no more than four bases from the nucleotide sequence 5'-asuscaa(Uhd)uuCfGfAfgcagaaggsasa-3' (SEQ ID NO:23), and the nucleotide sequence of the antisense strand differs by no more than four bases from the nucleotide sequence 5'-VPusUfsccdTu(C2p)ugcucgAfaAfuugausgsg-3' (SEQ ID NO:24), wherein VP is 5'-vinylphospho nate, (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate, (C2p) is cytidine-2'-phosphate, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dT is 2'-deoxy T, and Af, Cf, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, G, and U; h) the nucleotide sequence of the sense strand differs by no more than four bases from the nucleotide sequence 5'-cscsuca(Chd)uuUfAfAfuccucuauscsa-3' (SEQ ID NO:25), and the nucleotide sequence of the antisense strand differs by no more than four bases from the nucleotide sequence 5'-VPusdGsaudAg(Agn)ggaudTaAfagugaggsasc-3' (SEQ ID NO:26), where VP is 5'-vinylphosphonate; (C (hd) is 2'-O-hexadecyl-cytidine-3'-phosphate, (Agn) is adenosine-glycol nucleic acid (GNA), S isomer, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA, dG, and dT are 2'-deoxy A, G, and T; Af and Uf are 2'-deoxy-2'-fluoro (2'-F) A and U; i) the nucleotide sequence of the sense strand differs by no more than four bases from the nucleotide sequence 5'-asasgga(Uhd)gaAfGfAfgaggcaugsusa-3' (SEQ ID NO:27), and the nucleotide sequence of the antisense strand differs by no more than four bases from the nucleotide sequence 5'-VPusAfscadTg(C2p)cucucuUfcAfuccuususg-3' (SEQ ID NO:28), wherein VP is 5'-vinylphosphoryl phosphate, (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate, (C2p) is cytidine-2'-phosphate, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dT is 2'-deoxy T, and Af, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, G, and U; or j) the nucleotide sequence of the sense strand differs by no more than four bases from the nucleotide sequence 5'-asasuuu(Chd)gaGfCfAfgaaggaaasgsa-3' (SEQ ID NO:29) and the nucleotide sequence of the antisense strand differs by no more than four bases from the nucleotide sequence 5'-VPusCfsuudTc(C2p)uucugcUfcGfaaauusgsg-3' (SEQ ID NO:30), wherein VP is 5'-vinylphosphonate. where (Chd) is 2'-O-hexadecyl-cytidine-3'-phosphate, (C2p) is cytidine-2'-phosphate; s is a phosphorothioate bond, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dT is 2'-deoxy T, and Af, Cf, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, G, and U.

[0009] In one embodiment, a) the nucleotide sequence of the sense strand differs by no more than 3 bases from the nucleotide sequence 5'-csascuu(Uhd)aaUfCfCfucuauccasgsa-3' (SEQ ID NO: 11), and the nucleotide sequence of the antisense strand differs by no more than 3 bases from the nucleotide sequence 5'-VPusdCsugdGadTagagdGaUfuaaagugsasg-3' (SEQ ID NO: 12), wherein VP is 5'-vinylphosphonate, (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dC, dG, and dT are 2'-deoxy C, G, and T; and Cf and Uf are 2'-deoxy-2'-fluoro (2'-F) C and U; b) the nucleotide sequence of the sense strand differs by no more than 3 bases from the nucleotide sequence 5'-csasggu(Chd)cuCfAfCfuuuaauccsusa-3' (SEQ ID NO: 13), and the nucleotide sequence of the antisense strand differs by no more than 3 bases from the nucleotide sequence 5'-VPusdAsggdAudTaaagdTgAfggaccugscsg-3' (SEQ ID NO: 14), where VP is 5'-vinylphosphonate, (Chd) is 2'-O-hexadecyl-cytidine-3'-phosphate, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA and dT are 2'-deoxy A and T; and Af and Cf are 2'-deoxy-2'-fluoro (2'-F) A and C; c) the nucleotide sequence of the sense strand differs by no more than 3 bases from the nucleotide sequence 5'-ususcgag(Chd)aGfAfAfggaaaguasasa-3' (SEQ ID NO: 15) and the nucleotide sequence of the antisense strand differs by no more than 3 bases from the nucleotide sequence 5'-VPusUfsuadCu(Tgn)uccuucUfgCfucgaasasu-3' (SEQ ID NO: 16), where VP is 5'-vinylphosphonate; (Chd) is 2'-O-hexadecyl-cytidine-3'-phosphate, (Tgn) is thymidine-glycol nucleic acid (GNA), S isomer; s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dC is 2'-deoxy C, and Af, Cf, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, G, and U; d) the nucleotide sequence of the sense strand differs by no more than 3 bases from the nucleotide sequence 5'-gsasaag(Uhd)aaUfGfGfaccagugasasa-3' (SEQ ID NO: 17), and the nucleotide sequence of the antisense strand differs by no more than 3 bases from the nucleotide sequence 5'-VPusUfsucdAc(Tgn)gguccaUfuAfcuuucscsu-3' (SEQ ID NO: 18), where VP is 5'-vinylphosphonate. where (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate, (Tgn) is thymidine-glycol nucleic acid (GNA), S isomer; s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA is 2'-deoxy A, and Af, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, G, and U; e) the nucleotide sequence of the sense strand differs by no more than 3 bases from the nucleotide sequence 5'-asgsga(Uhd)gaaGfAfGfaggcaugususa-3' (SEQ ID NO: 19), and the nucleotide sequence of the antisense strand differs by no more than 3 bases from the nucleotide sequence 5'-VPusAfsacdAu(G2p)ccucucUfuCfauccususu-3' (SEQ ID NO: 20), where VP is 5'-vinylphosphonate. (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate, (G2p) is guanosine-2'-phosphate, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA is 2'-deoxy A, Af, Cf, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, G, and U; f) ​​the nucleic acid sequence of the sense strand. The nucleotide sequence differs by no more than three bases from the nucleotide sequence 5'-asasgga(Ahd)agUfAfAfuggaccagsusa-3' (SEQ ID NO:21), and the nucleotide sequence of the antisense strand differs by no more than three bases from the nucleotide sequence 5'-VPusdAscudGg(Tgn)ccaudTaCfuuuccuuscsu-3' (SEQ ID NO:22), where VP is 5'-vinylphosphonate and (Ahd) is , 2'-O-hexadecyl-adenosine-3'-phosphate, (Tgn) is thymidine-glycol nucleic acid (GNA), S isomer; s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA, dG, and dT are 2'-deoxy A, G, and T; Af, Cf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, and U; g) the nucleotide sequence of the sense strand differs by no more than 3 bases from the nucleotide sequence 5'-asuscaa(Uhd)uuCfGfAfgcagaaggsasa-3' (SEQ ID NO:23), and the nucleotide sequence of the antisense strand differs by no more than 3 bases from the nucleotide sequence 5'-VPusUfsccdTu(C2p)ugcucgAfaAfuugausgsg-3' (SEQ ID NO:24), wherein VP is 5'-vinylphospho nate, (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate, (C2p) is cytidine-2'-phosphate, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dT is 2'-deoxy T, and Af, Cf, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, G, and U; h) the nucleotide sequence of the sense strand differs by no more than 3 bases from the nucleotide sequence 5'-cscsuca(Chd)uuUfAfAfuccucuauscsa-3' (SEQ ID NO:25), and the nucleotide sequence of the antisense strand differs by no more than 3 bases from the nucleotide sequence 5'-VPusdGsaudAg(Agn)ggaudTaAfagugaggsasc-3' (SEQ ID NO:26), where VP is 5'-vinylphosphonate; (C (hd) is 2'-O-hexadecyl-cytidine-3'-phosphate, (Agn) is adenosine-glycol nucleic acid (GNA), S isomer, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA, dG, and dT are 2'-deoxy A, G, and T; Af and Uf are 2'-deoxy-2'-fluoro (2'-F) A and U; i) the nucleotide sequence of the sense strand differs by no more than 3 bases from the nucleotide sequence 5'-asasgga(Uhd)gaAfGfAfgaggcaugsusa-3' (SEQ ID NO:27), and the nucleotide sequence of the antisense strand differs by no more than 3 bases from the nucleotide sequence 5'-VPusAfscadTg(C2p)cucucuUfcAfuccuususg-3' (SEQ ID NO:28), wherein VP is 5'-vinylphosphoryl phosphate, (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate, (C2p) is cytidine-2'-phosphate, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dT is 2'-deoxy T, and Af, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, G, and U; or j) the nucleotide sequence of the sense strand differs by no more than 3 bases from the nucleotide sequence 5'-asasuuu(Chd)gaGfCfAfgaaggaaasgsa-3' (SEQ ID NO:29) and the nucleotide sequence of the antisense strand differs by no more than 3 bases from the nucleotide sequence 5'-VPusCfsuudTc(C2p)uucugcUfcGfaaauusgsg-3' (SEQ ID NO:30), wherein VP is 5'-vinylphosphonate. where (Chd) is 2'-O-hexadecyl-cytidine-3'-phosphate, (C2p) is cytidine-2'-phosphate; s is a phosphorothioate bond, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dT is 2'-deoxy T, and Af, Cf, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, G, and U.

[0010] In one embodiment, a) the nucleotide sequence of the sense strand differs by no more than two bases from the nucleotide sequence 5'-csascuu(Uhd)aaUfCfCfucuauccasgsa-3' (SEQ ID NO: 11), and the nucleotide sequence of the antisense strand differs by no more than two bases from the nucleotide sequence 5'-VPusdCsugdGadTagagdGaUfuaaagugsasg-3' (SEQ ID NO: 12), where VP is 5'-vinylphosphonate, (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dC, dG, and dT are 2'-deoxy C, G, and T; and Cf and Uf are 2'-deoxy-2'-fluoro (2'-F) C and U; b) the nucleotide sequence of the sense strand differs by no more than two bases from the nucleotide sequence 5'-csasggu(Chd)cuCfAfCfuuuaauccsusa-3' (SEQ ID NO: 13), and the nucleotide sequence of the antisense strand differs by no more than two bases from the nucleotide sequence 5'-VPusdAsggdAudTaaagdTgAfggaccugscsg-3' (SEQ ID NO: 14), where VP is 5'-vinylphosphonate, (Chd) is 2'-O-hexadecyl-cytidine-3'-phosphate, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA and dT are 2'-deoxy A and T; and Af and Cf are 2'-deoxy-2'-fluoro (2'-F) A and C; c) the nucleotide sequence of the sense strand differs by no more than two bases from the nucleotide sequence 5'-ususcgag(Chd)aGfAfAfggaaaguasasa-3' (SEQ ID NO: 15) and the nucleotide sequence of the antisense strand differs by no more than two bases from the nucleotide sequence 5'-VPusUfsuadCu(Tgn)uccuucUfgCfucgaasasu-3' (SEQ ID NO: 16), where VP is 5'-vinylphosphonate; (Chd) is 2'-O-hexadecyl-cytidine-3'-phosphate, (Tgn) is thymidine-glycol nucleic acid (GNA), S isomer; s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dC is 2'-deoxy C, and Af, Cf, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, G, and U; d) the nucleotide sequence of the sense strand differs by no more than two bases from the nucleotide sequence 5'-gsasaag(Uhd)aaUfGfGfaccagugasasa-3' (SEQ ID NO: 17), and the nucleotide sequence of the antisense strand differs by no more than two bases from the nucleotide sequence 5'-VPusUfsucdAc(Tgn)gguccaUfuAfcuuucscsu-3' (SEQ ID NO: 18), where VP is 5'-vinylphosphonate. where (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate, (Tgn) is thymidine-glycol nucleic acid (GNA), S isomer; s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA is 2'-deoxy A, and Af, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, G, and U; e) the nucleotide sequence of the sense strand differs by no more than two bases from the nucleotide sequence 5'-asgsga(Uhd)gaaGfAfGfaggcaugususa-3' (SEQ ID NO: 19), and the nucleotide sequence of the antisense strand differs by no more than two bases from the nucleotide sequence 5'-VPusAfsacdAu(G2p)ccucucUfuCfauccususu-3' (SEQ ID NO: 20), wherein VP is 5'-vinylphosphonate. (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate, (G2p) is guanosine-2'-phosphate, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA is 2'-deoxy A, and Af, Cf, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, G, and U; f) the nucleotide sequence of the sense strand differs by no more than two bases from the nucleotide sequence 5'-asasgga(Ahd)agUfAfAfuggaccagsusa-3' (SEQ ID NO:21), and the nucleotide sequence of the antisense strand differs by no more than two bases from the nucleotide sequence 5'-VPusdAscudGg(Tgn)ccaudTaCfuuuccuuscsu-3' (SEQ ID NO:22), where VP is 5'-vinylphosphonate, and (Ahd ) is 2'-O-hexadecyl-adenosine-3'-phosphate, (Tgn) is thymidine-glycol nucleic acid (GNA), S isomer; s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA, dG, and dT are 2'-deoxy A, G, T, and Af, Cf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, and U; g) the nucleotide sequence of the sense strand differs by no more than two bases from the nucleotide sequence 5'-asuscaa(Uhd)uuCfGfAfgcagaaggsasa-3' (SEQ ID NO:23), and the nucleotide sequence of the antisense strand differs by no more than two bases from the nucleotide sequence 5'-VPusUfsccdTu(C2p)ugcucgAfaAfuugausgsg-3' (SEQ ID NO:24), wherein VP is 5'-vinylphospho nate, (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate, (C2p) is cytidine-2'-phosphate, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dT is 2'-deoxy T, and Af, Cf, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, G, and U; h) the nucleotide sequence of the sense strand differs by no more than two bases from the nucleotide sequence 5'-cscsuca(Chd)uuUfAfAfuccucuauscsa-3' (SEQ ID NO:25), and the nucleotide sequence of the antisense strand differs by no more than two bases from the nucleotide sequence 5'-VPusdGsaudAg(Agn)ggaudTaAfagugaggsasc-3' (SEQ ID NO:26), where VP is 5'-vinylphosphonate; (C (hd) is 2'-O-hexadecyl-cytidine-3'-phosphate, (Agn) is adenosine-glycol nucleic acid (GNA), S isomer, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA, dG, and dT are 2'-deoxy A, G, and T; Af and Uf are 2'-deoxy-2'-fluoro (2'-F) A and U; i) the nucleotide sequence of the sense strand differs by no more than two bases from the nucleotide sequence 5'-asasgga(Uhd)gaAfGfAfgaggcaugsusa-3' (SEQ ID NO:27), and the nucleotide sequence of the antisense strand differs by no more than two bases from the nucleotide sequence 5'-VPusAfscadTg(C2p)cucucuUfcAfuccuususg-3' (SEQ ID NO:28), wherein VP is 5'-vinylphosphoryl phosphate, (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate, (C2p) is cytidine-2'-phosphate, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dT is 2'-deoxy T, and Af, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, G, and U; or j) the nucleotide sequence of the sense strand differs by no more than two bases from the nucleotide sequence 5'-asasuuu(Chd)gaGfCfAfgaaggaaasgsa-3' (SEQ ID NO:29) and the nucleotide sequence of the antisense strand differs by no more than two bases from the nucleotide sequence 5'-VPusCfsuudTc(C2p)uucugcUfcGfaaauusgsg-3' (SEQ ID NO:30), wherein VP is 5'-vinylphosphonate. where (Chd) is 2'-O-hexadecyl-cytidine-3'-phosphate, (C2p) is cytidine-2'-phosphate; s is a phosphorothioate bond, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dT is 2'-deoxy T, and Af, Cf, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, G, and U.

[0011] In one embodiment, a) the nucleotide sequence of the sense strand differs by no more than one base from the nucleotide sequence 5'-csascuu(Uhd)aaUfCfCfucuauccasgsa-3' (SEQ ID NO: 11), and the nucleotide sequence of the antisense strand differs by no more than one base from the nucleotide sequence 5'-VPusdCsugdGadTagagdGaUfuaaagugsasg-3' (SEQ ID NO: 12), where VP is 5'-vinylphosphonate, (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dC, dG, and dT are 2'-deoxy C, G, and T; and Cf and Uf are 2'-deoxy-2'-fluoro (2'-F) C and U; b) the nucleotide sequence of the sense strand differs by no more than one base from the nucleotide sequence 5'-csasggu(Chd)cuCfAfCfuuuaauccsusa-3' (SEQ ID NO: 13), and the nucleotide sequence of the antisense strand differs by no more than one base from the nucleotide sequence 5'-VPusdAsggdAudTaaagdTgAfggaccugscsg-3' (SEQ ID NO: 14), where VP is 5'-vinylphosphonate, (Chd) is 2'-O-hexadecyl-cytidine-3'-phosphate, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA and dT are 2'-deoxy A and T; and Af and Cf are 2'-deoxy-2'-fluoro (2'-F) A and C; c) the nucleotide sequence of the sense strand differs by no more than one base from the nucleotide sequence 5'-ususcgag(Chd)aGfAfAfggaaaguasasa-3' (SEQ ID NO: 15) and the nucleotide sequence of the antisense strand differs by no more than one base from the nucleotide sequence 5'-VPusUfsuadCu(Tgn)uccuucUfgCfucgaasasu-3' (SEQ ID NO: 16), where VP is 5'-vinylphosphonate; (Chd) is 2'-O-hexadecyl-cytidine-3'-phosphate, (Tgn) is thymidine-glycol nucleic acid (GNA), S isomer; s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dC is 2'-deoxy C, and Af, Cf, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, G, and U; d) the nucleotide sequence of the sense strand differs by no more than one base from the nucleotide sequence 5'-gsasaag(Uhd)aaUfGfGfaccagugasasa-3' (SEQ ID NO: 17) and the nucleotide sequence of the antisense strand differs by no more than one base from the nucleotide sequence 5'-VPusUfsucdAc(Tgn)gguccaUfuAfcuuucscsu-3' (SEQ ID NO: 18), where VP is 5'-vinylphosphonate, (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate, and (Tgn) is thymidine glycol nucleic acid (GNA), S isomer; s is a phosphorothioate bond; a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA is 2'-deoxy A; Af, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, G, and U; e) the nucleotide sequence of the sense strand differs by no more than one base from the nucleotide sequence 5'-asgsga(Uhd)gaaGfAfGfaggcaugususa-3' (SEQ ID NO: 19), and the nucleotide sequence of the antisense strand differs by no more than one base from the nucleotide sequence 5'-VPusAfsacdAu(G2p)ccucucUfuCfauccususu-3' (SEQ ID NO: 20), wherein VP is 5'-vinylphosphonate. (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate, (G2p) is guanosine-2'-phosphate, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA is 2'-deoxy A, and Af, Cf, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, G, and U; f) the nucleotide sequence of the sense strand differs by no more than one base from the nucleotide sequence 5'-asasgga(Ahd)agUfAfAfuggaccagsusa-3' (SEQ ID NO:21), and the nucleotide sequence of the antisense strand differs by no more than one base from the nucleotide sequence 5'-VPusdAscudGg(Tgn)ccaudTaCfuuuccuuscsu-3' (SEQ ID NO:22), where VP is 5'-vinylphosphonate, and (Ahd ) is 2'-O-hexadecyl-adenosine-3'-phosphate, (Tgn) is thymidine-glycol nucleic acid (GNA), S isomer; s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA, dG, and dT are 2'-deoxy A, G, T, and Af, Cf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, and U; g) the nucleotide sequence of the sense strand differs by no more than one base from the nucleotide sequence 5'-asuscaa(Uhd)uuCfGfAfgcagaaggsasa-3' (SEQ ID NO:23), and the nucleotide sequence of the antisense strand differs by no more than one base from the nucleotide sequence 5'-VPusUfsccdTu(C2p)ugcucgAfaAfuugausgsg-3' (SEQ ID NO:24), wherein VP is 5'-vinylphospho nate, (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate, (C2p) is cytidine-2'-phosphate, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dT is 2'-deoxy T, and Af, Cf, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, G, and U; h) the nucleotide sequence of the sense strand differs by no more than one base from the nucleotide sequence 5'-cscsuca(Chd)uuUfAfAfuccucuauscsa-3' (SEQ ID NO:25), and the nucleotide sequence of the antisense strand differs by no more than one base from the nucleotide sequence 5'-VPusdGsaudAg(Agn)ggaudTaAfagugaggsasc-3' (SEQ ID NO:26), where VP is 5'-vinylphosphonate; (C (hd) is 2'-O-hexadecyl-cytidine-3'-phosphate, (Agn) is adenosine-glycol nucleic acid (GNA), S isomer, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA, dG, and dT are 2'-deoxy A, G, and T; Af and Uf are 2'-deoxy-2'-fluoro (2'-F) A and U; i) the nucleotide sequence of the sense strand differs by no more than one base from the nucleotide sequence 5'-asasgga(Uhd)gaAfGfAfgaggcaugsusa-3' (SEQ ID NO:27), and the nucleotide sequence of the antisense strand differs by no more than one base from the nucleotide sequence 5'-VPusAfscadTg(C2p)cucucuUfcAfuccuususg-3' (SEQ ID NO:28), where VP is 5'-vinylphosphoryl phosphate, (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate, (C2p) is cytidine-2'-phosphate, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dT is 2'-deoxy T, and Af, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, G, and U; or j) the nucleotide sequence of the sense strand differs by no more than one base from the nucleotide sequence 5'-asasuuu(Chd)gaGfCfAfgaaggaaasgsa-3' (SEQ ID NO:29) and the nucleotide sequence of the antisense strand differs by no more than one base from the nucleotide sequence 5'-VPusCfsuudTc(C2p)uucugcUfcGfaaauusgsg-3' (SEQ ID NO:30), wherein VP is 5'-vinylphosphonate. where (Chd) is 2'-O-hexadecyl-cytidine-3'-phosphate, (C2p) is cytidine-2'-phosphate; s is a phosphorothioate bond, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dT is 2'-deoxy T, and Af, Cf, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, G, and U.

[0012] In one embodiment, a) the nucleotide sequence of the sense strand comprises the nucleotide sequence 5'-csascuu(Uhd)aaUfCfCfucuauccasgsa-3' (SEQ ID NO: 11), and the nucleotide sequence of the antisense strand comprises the nucleotide sequence 5'-VPusdCsugdGadTagagdGaUfuaaagugsasg-3' (SEQ ID NO: 12), wherein: VP is 5'-vinylphosphonate, (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate, s is a phosphorothioate bond, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, dC, dG, and dT are 2'-deoxy C, G, and T, Cf and Uf are 2'-deoxy-2'-fluoro (2'-F) C and U, b) the nucleotide sequence of the sense strand comprises the nucleotide sequence 5'-csasggu(Chd)cuCfAfCfuuuaauccsusa-3' (SEQ ID NO: 13), and the nucleotide sequence of the antisense strand comprises the nucleotide sequence 5'-VPusdAsggdAudTaaagdTgAfggaccugscsg-3' (SEQ ID NO: 14), where VP is 5'-vinylphosphonate, (Chd) is 2'-O-hexadecyl-cytidine-3'-phosphate, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA and dT are 2'-deoxy A and T; and Af and Cf are 2'-deoxy-2'-fluoro (2'-F) A and C; c) the nucleotide sequence of the sense strand comprises the nucleotide sequence 5'-ususcgag(Chd)aGfAfAfggaaaguasasa-3' (SEQ ID NO: 15), and the nucleotide sequence of the antisense strand comprises the nucleotide sequence 5'-VPusUfsuadCu(Tgn)uccuucUfgCfucgaasasu-3' (SEQ ID NO: 16), where VP is 5'-vinylphosphonate and (Chd) is 2' -O-hexadecyl-cytidine-3'-phosphate, (Tgn) is thymidine-glycol nucleic acid (GNA), S isomer; s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dC is 2'-deoxy C, and Af, Cf, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, G, and U; d) the nucleotide sequence of the sense strand comprises the nucleotide sequence 5'-gsasaag(Uhd)aaUfGfGfaccagugasasa-3' (SEQ ID NO: 17) and the nucleotide sequence of the antisense strand comprises the nucleotide sequence 5'-VPusUfsucdAc(Tgn)gguccaUfuAfcuuucscsu-3' (SEQ ID NO: 18), where VP is 5'-vinylphosphonate and (Uhd) is , 2'-O-hexadecyl-uridine-3'-phosphate, (Tgn) is thymidine-glycol nucleic acid (GNA), S isomer; s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA is 2'-deoxy A, and Af, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, G, and U; e) the nucleotide sequence of the sense strand comprises the nucleotide sequence 5'-asgsga(Uhd)gaaGfAfGfaggcaugususa-3' (SEQ ID NO: 19), and the nucleotide sequence of the antisense strand comprises the nucleotide sequence 5'-VPusAfsacdAu(G2p)ccucucUfuCfauccususu-3' (SEQ ID NO: 20), where VP is 5'-vinylphosphonate, and (Uh d) is 2'-O-hexadecyl-uridine-3'-phosphate, (G2p) is guanosine-2'-phosphate, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA is 2'-deoxy A, and Af, Cf, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, G, and U; f) the nucleotide sequence of the sense strand comprises the nucleotide sequence 5'-asasgga(Ahd)agUfAfAfuggaccagsusa-3' (SEQ ID NO: 21), and the nucleotide sequence of the antisense strand comprises the nucleotide sequence 5'-VPusdAscudGg(Tgn)ccaudTaCfuuuccuuscsu-3' (SEQ ID NO: 22), where VP is 5'-vinylphosphonate and (Ahd) is 2'-O-heterocyclic amino acid. is hexadecyl-adenosine-3'-phosphate, (Tgn) is thymidine-glycol nucleic acid (GNA), S isomer; s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA, dG, and dT are 2'-deoxy A, G, T, and Af, Cf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, and U; g) the nucleotide sequence of the sense strand comprises the nucleotide sequence 5'-asuscaa(Uhd)uuCfGfAfgcagaaggsasa-3' (SEQ ID NO: 23), and the nucleotide sequence of the antisense strand comprises the nucleotide sequence 5'-VPusUfsccdTu(C2p)ugcucgAfaAfuugausgsg-3' (SEQ ID NO: 24), where VP is 5'-vinylphosphonate, and (U (hd) is 2'-O-hexadecyl-uridine-3'-phosphate, (C2p) is cytidine-2'-phosphate, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dT is 2'-deoxy T, and Af, Cf, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, G, and U; h) the nucleotide sequence of the sense strand comprises the nucleotide sequence 5'-cscsuca(Chd)uuUfAfAfuccucuauscsa-3' (SEQ ID NO: 25), and the nucleotide sequence of the antisense strand comprises the nucleotide sequence 5'-VPusdGsaudAg(Agn)ggaudTaAfagugaggsasc-3' (SEQ ID NO: 26), where VP is 5'-vinylphosphonate and (Chd) is 2'-O -hexadecyl-cytidine-3'-phosphate, (Agn) is adenosine-glycol nucleic acid (GNA), S isomer, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA, dG, and dT are 2'-deoxy A, G, and T; Af and Uf are 2'-deoxy-2'-fluoro (2'-F) A and U; i) the nucleotide sequence of the sense strand comprises the nucleotide sequence 5'-asasgga(Uhd)gaAfGfAfgaggcaugsusa-3' (SEQ ID NO:27), and the nucleotide sequence of the antisense strand comprises the nucleotide sequence 5'-VPusAfscadTg(C2p)cucucuUfcAfuccuususg-3' (SEQ ID NO:28), where VP is 5'-vinylphosphonate, (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate, (C2p) is cytidine-2'-phosphate, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dT is 2'-deoxy T, and Af, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, G, and U; or j) the nucleotide sequence of the sense strand comprises the nucleotide sequence 5'-asasuuu(Chd)gaGfCfAfgaaggaaasgsa-3' (SEQ ID NO: 29), and the nucleotide sequence of the antisense strand comprises the nucleotide sequence 5'-VPusCfsuudTc(C2p)uucugcUfcGfaaauusgsg-3' (SEQ ID NO: 30), where VP is 5'-vinylphosphonate; (Chd) is 2'-O-hexadecyl-cytidine-3'-phosphate, (C2p) is cytidine-2'-phosphate; s is a phosphorothioate bond, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dT is 2'-deoxy T, and Af, Cf, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, G, and U.

[0013] In one embodiment, a) the nucleotide sequence of the sense strand consists of the nucleotide sequence 5'-csascuu(Uhd)aaUfCfCfucuauccasgsa-3' (SEQ ID NO: 11), and the nucleotide sequence of the antisense strand consists of the nucleotide sequence 5'-VPusdCsugdGadTagagdGaUfuaaagugsasg-3' (SEQ ID NO: 12), where VP is 5'-vinylphosphonate, (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dC, dG, and dT are 2'-deoxy C, G, and T; and Cf and Uf are 2'-deoxy-2'-fluoro (2'-F) C and U; b) the nucleotide sequence of the sense strand consists of the nucleotide sequence 5'-csasggu(Chd)cuCfAfCfuuuaauccsusa-3' (SEQ ID NO: 13) and the nucleotide sequence of the antisense strand consists of the nucleotide sequence 5'-VPusdAsggdAudTaaagdTgAfggaccugscsg-3' (SEQ ID NO: 14), where VP is 5'-vinylphosphonate, (Chd) is 2'-O-hexadecyl-cytidine-3'-phosphate, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA and dT are 2'-deoxy A and T; and Af and Cf are 2'-deoxy-2'-fluoro (2'-F) A and C; c) the nucleotide sequence of the sense strand consists of the nucleotide sequence 5'-ususcgag(Chd)aGfAfAfggaaaguasasa-3' (SEQ ID NO: 15), and the nucleotide sequence of the antisense strand consists of the nucleotide sequence 5'-VPusUfsuadCu(Tgn)uccuucUfgCfucgaasasu-3' (SEQ ID NO: 16), where VP is 5'-vinylphosphonate and (Chd) is 2 '-O-hexadecyl-cytidine-3'-phosphate, (Tgn) is thymidine-glycol nucleic acid (GNA), S isomer; s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dC is 2'-deoxy C, and Af, Cf, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, G, and U; d) the nucleotide sequence of the sense strand consists of the nucleotide sequence 5'-gsasaag(Uhd)aaUfGfGfaccagugasasa-3' (SEQ ID NO: 17) and the nucleotide sequence of the antisense strand consists of the nucleotide sequence 5'-VPusUfsucdAc(Tgn)gguccaUfuAfcuuucscsu-3' (SEQ ID NO: 18), where VP is 5'-vinylphosphonate, and (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate, (Tgn) is thymidine-glycol nucleic acid (GNA), S isomer; s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA is 2'-deoxy A, and Af, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, G, and U; e) the nucleotide sequence of the sense strand consists of the nucleotide sequence 5'-asgsga(Uhd)gaaGfAfGfaggcaugususa-3' (SEQ ID NO: 19), and the nucleotide sequence of the antisense strand consists of the nucleotide sequence 5'-VPusAfsacdAu(G2p)ccucucUfuCfauccususu-3' (SEQ ID NO: 20), where VP is 5'-vinylphosphonate, and (U (hd) is 2'-O-hexadecyl-uridine-3'-phosphate, (G2p) is guanosine-2'-phosphate, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA is 2'-deoxy A, and Af, Cf, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, G, and U; f) the nucleotide sequence of the sense strand consists of the nucleotide sequence 5'-asasgga(Ahd)agUfAfAfuggaccagsusa-3' (SEQ ID NO: 21), and the nucleotide sequence of the antisense strand consists of the nucleotide sequence 5'-VPusdAscudGg(Tgn)ccaudTaCfuuuccuuscsu-3' (SEQ ID NO: 22), where VP is 5'-vinylphosphonate and (Ahd) is 2'-O- is hexadecyl-adenosine-3'-phosphate, (Tgn) is thymidine-glycol nucleic acid (GNA), S isomer; s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA, dG, and dT are 2'-deoxy A, G, T, and Af, Cf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, and U; g) the nucleotide sequence of the sense strand consists of the nucleotide sequence 5'-asuscaa(Uhd)uuCfGfAfgcagaaggsasa-3' (SEQ ID NO: 23), and the nucleotide sequence of the antisense strand consists of the nucleotide sequence 5'-VPusUfsccdTu(C2p)ugcucgAfaAfuugausgsg-3' (SEQ ID NO: 24), where VP is 5'-vinylphosphonate; (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate, (C2p) is cytidine-2'-phosphate, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dT is 2'-deoxy T, Af, Cf, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, G, and U; h) the nucleotide sequence of the sense strand consists of the nucleotide sequence 5'-cscsuca(Chd)uuUfAfAfuccucuauscsa-3' (SEQ ID NO: 25) and the nucleotide sequence of the antisense strand consists of the nucleotide sequence 5'-VPusdGsaudAg(Agn)ggaudTaAfagugaggsasc-3' (SEQ ID NO: 26), where VP is 5'-vinylphosphonate and (Chd) is 2'- is O-hexadecyl-cytidine-3'-phosphate, (Agn) is adenosine-glycol nucleic acid (GNA), S isomer, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA, dG, and dT are 2'-deoxy A, G, and T; Af and Uf are 2'-deoxy-2'-fluoro (2'-F) A and U; i) the nucleotide sequence of the sense strand consists of the nucleotide sequence 5'-asasgga(Uhd)gaAfGfAfgaggcaugsusa-3' (SEQ ID NO:27), and the nucleotide sequence of the antisense strand consists of the nucleotide sequence 5'-VPusAfscadTg(C2p)cucucuUfcAfuccuususg-3' (SEQ ID NO:28), wherein VP is 5'-vinylphosphonate, (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate, (C2p) is cytidine-2'-phosphate, s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dT is 2'-deoxy T, and Af, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, G, and U; or j) the nucleotide sequence of the sense strand consists of the nucleotide sequence 5'-asasuuu(Chd)gaGfCfAfgaaggaaasgsa-3' (SEQ ID NO: 29), and the nucleotide sequence of the antisense strand consists of the nucleotide sequence 5'-VPusCfsuudTc(C2p)uucugcUfcGfaaauusgsg-3' (SEQ ID NO: 30), where VP is 5'-vinylphosphonate, and (Chd ) is 2'-O-hexadecyl-cytidine-3'-phosphate, (C2p) is cytidine-2'-phosphate; s is a phosphorothioate linkage, a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dT is 2'-deoxy T, and Af, Cf, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, G, and U.

[0014] In one embodiment, the dsRNA agent is in sodium salt form.

[0015] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of superoxide dismutase 1 (SOD1), wherein the dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, and the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, , 17, 18, 19, 20, or 21 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO:2, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO:2, wherein (i) the dsRNA agent comprises at least one modified nucleotide, (ii) the double-stranded region is 15 to 30 nucleotide pairs in length, and (iii) the sense strand or the antisense strand is conjugated to one or more lipophilic moieties.

[0016] In one embodiment, the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides from any one of the nucleotide sequences 207-229, 219-241, 328-350, or 336-358 of SEQ ID NO:1, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO:2.

[0017] In another embodiment, the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides from any one of the nucleotide sequences 207-229, 328-350, or 336-358 of SEQ ID NO:1, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO:2.

[0018] In one embodiment, the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides from nucleotides 336-358 of SEQ ID NO:1, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO:2.

[0019] In another embodiment, the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides from any one of the antisense strand nucleotide sequences of a duplex selected from the group consisting of AD-1395762, AD-1395756, AD-1395731, AD-1395743, AD-1395771, AD-1395738, AD-1395718, AD-1395760, AD-1395764, or AD-1395724.

[0020] In one embodiment, the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides from any one of the antisense strand nucleotide sequences of a duplex selected from the group consisting of AD-1395762, AD-1395756, AD-1395731, and AD-1395743.

[0021] In one embodiment, the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides from any one of the antisense strand nucleotide sequences of a duplex selected from the group consisting of AD-1395762, AD-1395756, and AD-1395731.

[0022] In another embodiment, the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides from the antisense strand nucleotide sequence of duplex AD-1395762.

[0023] In one embodiment, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand comprise a modification.

[0024] In one embodiment, at least one of the modified nucleotides is 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 2'-5'-linked ribonucleotide (3'-RNA), 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 nucleotide containing a non-natural base, a tetrahydropyran-modified nucleotide, a The nucleotide is selected from the group consisting of a nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-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 vinylphosphonate, a glycol nucleic acid (GNA), a 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.

[0025] In one embodiment, 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.

[0026] In another embodiment, the modifications on the nucleotide are independently selected from the group consisting of a 2'-deoxy modification, a 2'-O-methyl modification, a 3'-RNA modification, a GNA modification, an S-GNA modification, and a 2'-deoxy-2'-fluoro modification.

[0027] In one embodiment, the dsRNA agent further comprises at least one phosphorothioate internucleotide linkage.

[0028] In one embodiment, the dsRNA agent includes 6 to 8 phosphorothioate internucleotide linkages.

[0029] In one embodiment, at least one strand comprises a 3' overhang of at least 1 nucleotide.

[0030] In another embodiment, at least one strand comprises a 2-nucleotide 3' overhang.

[0031] In one embodiment, the double-stranded region is 17 to 23 nucleotide pairs in length.

[0032] In another embodiment, the double-stranded region is 19-21 nucleotide pairs in length.

[0033] In one embodiment, the double-stranded region is 21-23 nucleotide pairs in length.

[0034] In one embodiment, each strand has between 19 and 30 nucleotides.

[0035] In one embodiment, one or more lipophilic moieties are conjugated to one or more interior positions on at least one strand.

[0036] In one embodiment, one lipophilic moiety is conjugated to an internal position 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.

[0037] In one embodiment, the internal positions are selected from the group consisting of positions 5, 6, 7, 15, and 17 on the sense strand, counting from the 5' end of the strand.

[0038] In another embodiment, the internal positions are selected from the group consisting of positions 15 and 17 on the antisense strand, counting from the 5' end of the strand.

[0039] In one embodiment, the internal positions are selected from the group consisting of positions 6 and 7 of the sense strand.

[0040] In one embodiment, the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound.

[0041] In one embodiment, 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.

[0042] In one embodiment, the lipophilic moiety contains a saturated or unsaturated C6 to C18 hydrocarbon chain.

[0043] In one embodiment, the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain.

[0044] In one embodiment, the saturated or unsaturated C16 hydrocarbon chain is conjugated to position 6 or 7 of the sense strand, counting from the 5' end of the sense strand.

[0045] In one embodiment, the dsRNA agent further comprises a phosphate or a phosphate mimic at the 5' end of the antisense strand.

[0046] In one embodiment, the phosphate mimic is a 5'-vinylphosphonate (VP).

[0047] In another embodiment, the phosphate mimetic is a 5'-E-vinylphosphonate (VP).

[0048] In one embodiment, the base pair at position 1 of the 5'-end of the antisense strand of the duplex is an AU base pair.

[0049] In one embodiment, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

[0050] The invention also provides pharmaceutical compositions comprising cells and a pharmaceutically acceptable diluent comprising a dsRNA agent disclosed herein.

[0051] In one aspect, the present invention provides a method for inhibiting expression of the SOD1 gene in a cell, the method comprising contacting the cell with a dsRNA agent or a pharmaceutical composition of the present invention, and maintaining the cell produced in step (a) for a time sufficient to obtain degradation of mRNA transcripts of the SOD1 gene, thereby inhibiting expression of the SOD1 gene in the cell.

[0052] In one embodiment, the cell is in a human subject.

[0053] In one embodiment, the subject meets at least one diagnostic criterion for or has been diagnosed with an SOD1-associated neurodegenerative disease.

[0054] In one embodiment, the SOD1-associated neurodegenerative disease is selected from the group consisting of amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), and Down's syndrome (DS).

[0055] In one aspect, the invention provides a method of treating a subject diagnosed with an SOD1-associated neurodegenerative disease, the method comprising administering to the subject a therapeutically effective amount of a dsRNA agent or a pharmaceutical composition of the invention, thereby treating the subject.

[0056] In one embodiment, treating includes ameliorating at least one sign or symptom of the disease.

[0057] In another embodiment, treating includes preventing the progression of the disease.

[0058] In one embodiment, the SOD1-associated neurodegenerative disease is selected from the group consisting of amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), and Down's syndrome (DS).

[0059] In another aspect, the present invention provides a method for preventing the onset of an SOD1-associated neurodegenerative disease in a subject who meets at least one diagnostic criterion for an SOD1-associated neurodegenerative disease, the method comprising administering to the subject a therapeutically effective amount of a dsRNA agent or a pharmaceutical composition of the invention, thereby preventing the onset of an SOD1-associated neurodegenerative disease in the subject who meets at least one diagnostic criterion for an SOD1-associated neurodegenerative disease.

[0060] In one embodiment, the subject is a human.

[0061] In one embodiment, the subject has been diagnosed with an SOD1-associated neurodegenerative disease.

[0062] In one embodiment, the SOD1-associated neurodegenerative disease is selected from the group consisting of amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), and Down's syndrome (DS).

[0063] In one embodiment, the dsRNA agent is administered to the subject intrathecally or intracerebroventricularly.

[0064] In one aspect, the disclosure provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting expression of the superoxide dismutase 1 (SOD1) gene, wherein the RNAi agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity comprising at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 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 2-7, 12, 13, and 18-20. In certain embodiments, the antisense strand comprises a region of complementarity comprising at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides, to any one of the antisense sequences listed in any one of Tables 2-7, 12, 13, and 18-20. In certain embodiments, the antisense strand comprises a region of complementarity comprising at least 19, e.g., 19, 20, 21, 22, or 23, contiguous nucleotides that differ by no more than 3 nucleotides (i.e., differ by 3, 2, 1, or 0 nucleotides) from any one of the antisense sequences listed in any one of Tables 2-7, 12, 13, and 18-20. In certain embodiments, the antisense strand comprises a region of complementarity comprising at least 19, e.g., 19, 20, 21, 22, or 23, contiguous nucleotides from any one of the antisense sequences listed in any one of Tables 2-7, 12, 13, and 18-20. 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.

[0065] In some embodiments, the sense strand or the antisense strand is conjugated to one or more lipophilic moieties.

[0066] In some embodiments, the agent comprises one or more lipophilic moieties conjugated, optionally via a linker or carrier, to one or more interior nucleotide positions.

[0067] In other embodiments, the agent further comprises a targeting ligand that targets liver tissue, e.g., one or more GalNAc derivatives, optionally conjugated to the double-stranded RNAi agent via a linker or carrier.

[0068] In yet other embodiments, the agent further comprises one or more lipophilic moieties conjugated to one or more internal nucleotide positions, optionally via a linker or carrier, and a targeting ligand that targets liver tissue, e.g., one or more GalNAc derivatives, optionally conjugated to the double-stranded RNAi agent via a linker or carrier.

[0069] Another embodiment of the present disclosure provides a double-stranded RNAi agent for inhibiting expression of the superoxide dismutase 1 (SOD1) gene, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand 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 sense strand sequences presented in Tables 2-7, 12, 13, and 18-20, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by no more than 3 nucleotides, e.g., 3, 2, 1, or 1, from any one of the antisense strand nucleotide sequences presented in Tables 2-7, 12, 13, and 18-20. In certain embodiments, the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides of any one of the sense strand sequences presented in Tables 2-7, 12, 13, and 18-20, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of any one of the antisense strand nucleotide sequences presented in Tables 2-7, 12, 13, and 18-20. In certain embodiments, the sense strand comprises at least 19, e.g., 19, 20, or 21 contiguous nucleotides of any one of the sense strand sequences presented in Tables 2-7, 12, 13, and 18-20, and the antisense strand comprises at least 19, e.g., 19, 20, 21, 22, or 23 contiguous nucleotides of any one of the antisense strand nucleotide sequences presented in Tables 2-7, 12, 13, and 18-20.

[0070] In some embodiments, the agent comprises one or more lipophilic moieties conjugated, optionally via a linker or carrier, to one or more interior nucleotide positions.

[0071] In other embodiments, the agent further comprises a targeting ligand that targets liver tissue, e.g., one or more GalNAc derivatives, optionally conjugated to the double-stranded RNAi agent via a linker or carrier.

[0072] In yet other embodiments, the agent further comprises one or more lipophilic moieties conjugated to one or more internal nucleotide positions, optionally via a linker or carrier, and a targeting ligand that targets liver tissue, e.g., one or more GalNAc derivatives, optionally conjugated to the double-stranded RNAi agent via a linker or carrier.

[0073] Additional embodiments of the present disclosure provide double-stranded RNAi agents for inhibiting expression of the superoxide dismutase 1 (SOD1) gene, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand has at least 15 contiguous nucleotides that differ by no more than 3 nucleotides (i.e., differ by 3, 2, 1, or 0 nucleotides) from any one of the nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, or 9, or at least 90% nucleotide sequence identity to the entire nucleotide sequence of any one of SEQ ID NOs: 1, 3, 5, 7, or 9. 99, 910, 920, 930, 940, 950, 960, 970, 980, 99, or 100% identity, where the substitution of uracil for thymine in any of SEQ ID NOs: 1, 3, 5, 7, and 9 (when comparing aligned sequences) results in no more than 3 nucleotide differences (i.e., 3, 2, 1, or 0 nucleotide differences) from any one of the nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, or 9, or at least 90% nucleotide differences relative to the entire nucleotide sequence of any one of SEQ ID NOs: 1, 3, 5, 7, or 9. nucleotide sequence identity, e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity, is not counted as a difference contributing to a nucleotide sequence having at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NOs: 2, 4, 6, 8, or 10, or at least 90% nucleotide sequence identity, e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity, to the entire nucleotide sequence of any one of SEQ ID NOs: 2, 4, 6, 8, or 10. 95, 96, 97, 98, 99, or 100% identity, where the substitution of uracil for thymine in any of SEQ ID NOs: 2, 4, 6, 8, or 10 (when comparing aligned sequences) results in no more than 3 nucleotide differences from the nucleotide sequence of any one of SEQ ID NOs: 2, 4, 6, 8, or 10, or at least 90% nucleotide sequence identity to the entire nucleotide sequence of any one of SEQ ID NOs: 2, 4, 6, 8, or 10, e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,or differences that do not count as contributing to a nucleotide sequence having 100% identity, wherein at least one of the sense strand and the antisense strand contains one or more lipophilic moieties conjugated, optionally via a linker or carrier, to one or more internal nucleotide positions.

[0074] In one embodiment, a double-stranded RNAi agent targeting SOD1 comprises a sense strand that 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., differ by 3, 2, 1, or 0 nucleotides) from the nucleotide sequence of the sense strand nucleotide sequence of the duplex of Tables 2-7, 12, 13, and 18-20.

[0075] In one embodiment, a double-stranded RNAi agent targeting SOD1 comprises an antisense strand that comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by no more than 3 nucleotides (i.e., differ by 3, 2, 1, or 0 nucleotides) from the antisense strand nucleotide sequence of the duplex in one of Tables 2-7, 12, 13, and 18-20.

[0076] In some embodiments, the agent further comprises a targeting ligand that targets liver tissue, e.g., one or more GalNAc derivatives, optionally conjugated to the double-stranded RNAi agent via a linker or carrier.

[0077] Optionally, the double-stranded RNAi agent includes at least one modified nucleotide.

[0078] In certain embodiments, the lipophilicity of the lipophilic moiety is determined by the log K ow is measured and exceeds zero.

[0079] In some embodiments, the hydrophobicity of the double-stranded RNAi agent is greater than 0.2, as measured by the unbound fraction in a plasma protein binding assay of the double-stranded RNAi agent. In a related embodiment, the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.

[0080] In certain embodiments, substantially all of the nucleotides in the sense strand are modified nucleotides. Optionally, all of the nucleotides in the sense strand are modified nucleotides.

[0081] In some embodiments, substantially all of the nucleotides in the antisense strand are modified nucleotides. Optionally, all of the nucleotides in the antisense strand are modified nucleotides.

[0082] Optionally, every nucleotide in the sense strand and every nucleotide in the antisense strand is a modified nucleotide.

[0083] In one embodiment, at least one of the modified nucleotides is 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'-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 nucleotides, cyclohexenyl modified nucleotides, nucleotides containing a 5'-phosphorothioate group, nucleotides containing a 5'-methylphosphonate group, nucleotides containing a 5' phosphate or a 5' phosphate mimic, nucleotides containing vinyl phosphonate, nucleotides containing adenosine glycol nucleic acid (GNA), nucleotides containing thymidine glycol nucleic acid (GNA) S isomers, nucleotides containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides containing 2'-deoxythymidine-3' phosphate, nucleotides containing 2'-deoxyguanosine-3'-phosphate, or terminal nucleotides linked to cholesteryl derivatives, or dodecanoic acid bisdecylamide groups.

[0084] In related embodiments, the modified nucleotide is a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a 3'-terminal deoxythymidine nucleotide (dT), a locked nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, or a nucleotide containing a non-natural base.

[0085] In one embodiment, the modified nucleotides include a short sequence of 3'-terminal deoxythymidine nucleotides (dT).

[0086] In another embodiment, the modifications in the nucleotide are 2'-O-methyl, 2' fluoro, and GNA modifications.

[0087] In additional embodiments, the double-stranded RNAi agent includes at least one phosphorothioate internucleotide linkage. Optionally, the double-stranded RNAi agent includes 6 to 8 (e.g., 6, 7, or 8) phosphorothioate internucleotide linkages.

[0088] In certain embodiments, the region of complementarity is at least 17 nucleotides in length. Optionally, the region of complementarity is 19-23 nucleotides in length. Optionally, the region of complementarity is 19 nucleotides in length.

[0089] In one embodiment, each strand is 30 nucleotides or less in length.

[0090] In another embodiment, at least one strand comprises a 3' overhang of at least 1 nucleotide. Optionally, at least one strand comprises a 3' overhang of at least 2 nucleotides.

[0091] In certain embodiments, the double-stranded RNAi agent further comprises a lipophilic ligand, such as a C16 ligand, conjugated to the 3' end of the sense strand via a monovalent or branched bivalent or trivalent linker.In certain embodiments, the double-stranded RNAi agent further comprises a lipophilic ligand, such as a C16 ligand, conjugated to an internal nucleotide position via a monovalent or branched bivalent or trivalent linker.

[0092] In certain embodiments, the ligand is a C16 ligand. In one embodiment, the ligand is conjugated at the 2'-position of the nucleotide or modified nucleotide in the sense strand or antisense strand. For example, the C16 ligand may 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.

[0093] In other embodiments, the agent further comprises a targeting ligand that targets liver tissue, for example, one or more GalNAc derivatives, optionally conjugated to the double-stranded RNAi agent via a linker or carrier.In certain embodiments, one of the modified strands in Tables 3, 5, 7, 13, 18, and 20 is conjugated to the targeting ligand that targets liver tissue.In certain embodiments, the targeting ligand is an L96 ligand, for example, one or more GalNAc derivatives, optionally conjugated to the double-stranded RNAi agent via a linker or carrier.In certain embodiments, the L96 ligand is conjugated to one end of the strand.In certain embodiments, the L96 ligand is conjugated to the 3'-end of the sense strand.

[0094] In yet other embodiments, the agent further comprises a lipophilic ligand, e.g., a C16 ligand, conjugated to an internal nucleotide position, e.g., via a monovalent or branched bivalent or trivalent linker, and a targeting ligand that targets liver tissue, such as one or more GalNAc derivatives conjugated to the 3' end of the sense strand, e.g., via a monovalent or branched bivalent or trivalent linker.

[0095] In yet other embodiments, the agent further comprises a lipophilic ligand, e.g., a C16 ligand, conjugated to the 3' end of the sense strand, e.g., via a monovalent or branched bivalent or trivalent linker, and a targeting ligand that targets liver tissue, such as one or more GalNAc derivatives, conjugated to the 3' end of the sense strand, e.g., via a monovalent or branched bivalent or trivalent linker.

[0096] In another embodiment, the region of complementarity to SOD1 comprises any one of the antisense sequences in any one of Tables 2-7, 12, 13, and 18-20.

[0097] In additional embodiments, the region of complementarity to SOD1 is a region of any one of the antisense sequences in any one of Tables 2-7, 12, 13, and 18-20. In some embodiments, the internal nucleotide positions include all but the two terminal positions from each end of the strand.

[0098] In a related embodiment, the internal positions include all but the three most distal positions on each strand. Optionally, the internal positions exclude the cleavage site region of the sense strand.

[0099] In some embodiments, the internal positions exclude positions 9 to 12, counting from the 5' end of the sense strand. In certain embodiments, the sense strand is 21 nucleotides in length.

[0100] In other embodiments, the internal positions exclude positions 11-13 from the 3' end of the sense strand. Optionally, the internal positions exclude the cleavage site region of the antisense strand. In certain embodiments, the sense strand is 21 nucleotides in length.

[0101] In some embodiments, internal positions exclude positions 12 to 14, counting from the 5' end of the antisense strand. In certain embodiments, the antisense strand is 23 nucleotides in length.

[0102] In another embodiment, the internal positions exclude 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. In a particular embodiment, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

[0103] In additional embodiments, the one or more lipophilic moieties are conjugated to one or more of the following interior positions, counting from the 5' end of each strand: positions 4-8 and 13-18 on the sense strand and positions 6-10 and 15-18 on the antisense strand. Optionally, the one or more lipophilic moieties are conjugated to one or more of the following interior positions, counting from the 5' end of each strand: positions 5, 6, 7, 15, and 17 on the sense strand and positions 15 and 17 on the antisense strand. In certain embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

[0104] In certain embodiments, the lipophilic moiety is an aliphatic compound, an alicyclic compound, or a polyalicyclic compound. Optionally, the lipophilic moiety is a lipid, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, a heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

[0105] In some embodiments, the lipophilic moiety is a saturated or unsaturated C-C 30 It contains a hydrocarbon chain and an optional functional group selected from hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, or alkyne.

[0106] In certain embodiments, the lipophilic moiety is a saturated or unsaturated C-C 18 Optionally, the lipophilic moiety contains a saturated or unsaturated C 16It contains a hydrocarbon chain.In a related embodiment, the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotides at the internal position.In certain embodiments, the carrier is a cyclic group that is pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, or decalinyl, or is an acyclic moiety of a serinol skeleton or a diethanolamine skeleton system.

[0107] In some embodiments, the lipophilic moiety is conjugated to the double-stranded RNAi 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.

[0108] In one embodiment, the lipophilic moiety is conjugated to a nucleobase, a sugar moiety, or an internucleoside linkage.

[0109] In another embodiment, the double-stranded RNAi agent further comprises a phosphate or a phosphate mimic at the 5'-end of the antisense strand. Optionally, the phosphate mimic is 5'-vinylphosphonate (VP). When the phosphate mimic is 5'-vinylphosphonate (VP), the 5'-terminal nucleotide may have 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 double bond between the C5' carbon and R5' is in the E or Z conformation (e.g., E conformation), and B is a nucleobase or modified nucleobase, optionally B is adenine, guanine, cytosine, thymine, or uracil.

[0110] In certain embodiments, the double-stranded RNAi agent further comprises a targeting ligand, for example, a hydrophilic ligand, that targets a receptor that mediates delivery to CNS tissue. In certain embodiments, the targeting ligand is a C16 ligand.

[0111] In some embodiments, the double-stranded RNAi agent further comprises a targeting ligand that targets brain tissue, eg, the striatum.

[0112] In some embodiments, the double-stranded RNAi agent further comprises a targeting ligand that targets a liver tissue or cell type, eg, a hepatocyte.

[0113] In one embodiment, the lipophilic moiety or targeting ligand is conjugated via a bio-cleavable linker that is DNA, RNA, disulfide, amide, and functionalized mono- or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, or combinations thereof.

[0114] In a related embodiment, the 3' end of the sense strand is protected via an end cap that is an amine-bearing cyclic group, wherein the cyclic group is pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, or decalinyl.

[0115] In one embodiment, the RNAi agent comprises at least one modified nucleotide that is a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a nucleotide comprising a glycol nucleic acid (GNA), or a nucleotide comprising a vinyl phosphonate. Optionally, the RNAi agent comprises at least one of each of the following modifications: a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a nucleotide comprising a glycol nucleic acid (GNA), and a nucleotide comprising a vinyl phosphonate.

[0116] In another embodiment, the RNAi agent comprises a pattern of modified nucleotides such that the positions of 2'-C16, 2'-O-methyl, GNA, phosphorothioate, and 2'-fluoro modifications are provided in Tables 2-7, 12, 13, and 18-20, regardless of the individual nucleotide base sequence of the displayed RNAi agent.

[0117] Another aspect of the present disclosure provides a double-stranded RNAi agent for inhibiting expression of the SOD1 gene, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding SOD1, each strand being about 14 to about 30 nucleotides in length, the double-stranded RNAi agent being represented by formula (III): Sense:5'n p -N a -(XXX) i -N b -YYY-N 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) 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 an oligonucleotide sequence containing 0 to 25 nucleotides that are either modified or unmodified, or a combination thereof, each sequence containing at least two differently modified nucleotides; each N b and N b ' independently represents an oligonucleotide sequence containing 0 to 10 nucleotides, either modified or unmodified, or a combination thereof; each n p , n p ',n q and n q ', each of which may or may not be present, independently represent 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; N b The modification to N is different from the modification to Y. b The modification to ' is different from the modification to Y', The sense strand is conjugated to at least one ligand.

[0118] In one embodiment, i is 0, j is 0, i is 1, j is 1, i and j are both 0, or i and j are both 1.

[0119] In another embodiment, k is 0, l is 0, k is 1, l is 1, k and l are both 0, or k and l are both 1.

[0120] In certain embodiments, XXX is complementary to X'X'X', YYY is complementary to Y'Y'Y', and ZZZ is complementary to Z'Z'Z'.

[0121] In another embodiment, the YYY motif occurs at or near the cleavage site of the sense strand.

[0122] In additional embodiments, the Y'Y'Y' motif occurs at positions 11, 12, and 13 of the antisense strand from the 5' end. Optionally, Y' is 2'-O-methyl.

[0123] In some embodiments, Formula (III) is represented by Formula (IIIa): Sense:5'n p -N a -YYY-N a -n q 3' Antisense: 3'n p’ -N a’ -Y'Y'Y'-N a’ -n q’ 5'(IIIa)

[0124] In another embodiment, formula (III) is represented by formula (IIIb): Sense:5'n p -N a -YYY-N b -ZZZ-N a -n q 3' Antisense: 3'n p’ -N a’ -Y'Y'Y'-N b’ -Z'Z'Z'-N a’ -n q’ 5'(IIIb) In the formula, each N b and N b ' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides.

[0125] In additional embodiments, formula (III) is represented by formula (IIIc): Sense:5'n p -N a -XXX-N b -YYY-N a -n q 3' Antisense: 3'n p’ -Na’ -X'X'X'-N b’ -Y'Y'Y'-N a’ -n q’ 5'(IIIc) In the formula, each N b and N b ' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides.

[0126] In certain embodiments, formula (III) is represented by formula (IIId): Sense:5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' Antisense: 3'n p’ -N a’ -X'X'X'-N b’ -Y'Y'Y'-N b’ -Z'Z'Z'-N a’ -n q’ 5'(IIId) In the formula, each N b and N b ' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides, and each N a and N a ' independently represents an oligonucleotide sequence containing 2 to 10 modified nucleotides.

[0127] In another embodiment, the double-stranded region is 15 to 30 nucleotide pairs in length. Optionally, the double-stranded region is 17 to 23 nucleotide pairs in length.

[0128] In certain embodiments, the double-stranded region is 17 to 25 nucleotide pairs in length. Optionally, the double-stranded region is 23 to 27 nucleotide pairs in length.

[0129] In some embodiments, the double-stranded region is 19-21 nucleotide pairs in length. Optionally, the double-stranded region is 21-23 nucleotide pairs in length.

[0130] In certain embodiments, each strand independently has between 15 and 30 nucleotides. Optionally, each strand independently has between 19 and 30 nucleotides. Optionally, each strand independently has between 19 and 23 nucleotides.

[0131] In certain embodiments, the double-stranded region is 19-21 nucleotide pairs in length, with each strand having 19-23 nucleotides.

[0132] In another embodiment, the modification in the nucleotide of RNAi agent is LNA, glycol nucleic acid (GNA), hexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy or 2'-hydroxyl, and combinations thereof.Optionally, the modification in nucleotide comprises 2'-O-methyl, 2'-fluoro, or GNA, and combinations thereof.In related embodiments, the modification in nucleotide is 2'-O-methyl modification or 2'-fluoro modification.

[0133] In one embodiment, the RNAi agent comprises a ligand that is or includes one or more lipophilic moieties, eg, C16, attached via a bivalent or trivalent branched linker.

[0134] In other embodiments, the agent further comprises a targeting ligand, eg, one or more GalNAc derivatives, that targets liver tissue.

[0135] In yet other embodiments, the agent further comprises a lipophilic ligand, e.g., a C16 ligand, conjugated to the 3' end of the sense strand, e.g., via a monovalent or branched bivalent or trivalent linker, and a targeting ligand that targets liver tissue, such as one or more GalNAc derivatives, conjugated to the 3' end of the sense strand, e.g., via a monovalent or branched bivalent or trivalent linker.

[0136] In certain embodiments, the ligand is attached to the 3' end of the sense strand.

[0137] In some embodiments, the RNAi agent further comprises at least one phosphorothioate internucleotide linkage or methylphosphonate internucleotide linkage. In related embodiments, 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 related embodiments, 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.

[0138] 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.

[0139] In additional embodiments, the base pair at one position at the 5'-end of the antisense strand of the RNAi agent duplex is an A:U base pair.

[0140] In certain embodiments, the Y nucleotide contains a 2-'fluoro modification.

[0141] In some embodiments, the Y' nucleotide contains a 2'-O-methyl modification.

[0142] In certain embodiments, p'>0. Optionally, p'=2.

[0143] In some embodiments, q'=0, p=0, q=0, and the p' overhanging nucleotides are complementary to the target mRNA.

[0144] In certain embodiments, q'=0, p=0, q=0, and the p' overhanging nucleotides are non-complementary to the target mRNA.

[0145] In one embodiment, the sense strand of the RNAi agent has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

[0146] In another embodiment, at least one of n p ' are linked to adjacent nucleotides via phosphorothioate bonds. p ' is linked to the adjacent nucleotide via a phosphorothioate bond.

[0147] In certain embodiments, the SOD1 RNAi agent of the disclosure is one of those listed in Tables 2-7, 12, 13, and 18-20. In some embodiments, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand comprise a modification.

[0148] Another aspect of the present disclosure provides a double-stranded RNAi agent for inhibiting expression of an SOD1 gene in a cell, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding SOD1 in the cell, each strand being about 14 to about 30 nucleotides in length, the double-stranded RNAi agent being represented by formula (III): Sense:5'n p -N a -(XXX) i -N b -YYY-N 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) 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 an oligonucleotide sequence containing 0 to 25 nucleotides that are either modified or unmodified, or a combination thereof, each sequence containing at least two differently modified nucleotides; each N b and N b ' independently represents an oligonucleotide sequence containing 0 to 10 nucleotides, either modified or unmodified, or a combination thereof; each n p , n p ',n q and n q ', each of which may or may not be present, independently represent 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, the modifications being 2'-O-methyl or 2'-fluoro modifications; N b The modification to N is different from the modification to Y. b The modification to ' is different from the modification to Y', The sense strand is conjugated to at least one ligand, optionally the ligand being one or more lipophilic, eg, C16, ligand, or one or more GalNAc derivatives.

[0149] An additional aspect of the present disclosure provides a double-stranded RNAi agent for inhibiting expression of an SOD1 gene in a cell, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding SOD1, each strand being about 14 to about 30 nucleotides in length, the double-stranded RNAi agent being represented by formula (III): Sense:5'n p -N a -(XXX) i -N b-YYY-N 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) During the ceremony, i, j, k, and l each independently represent 0 or 1; each n p , n q and n q ', each of which may or may not be present, independently represent an overhanging nucleotide; p, q, and q' each independently represent 0 to 6; n p '>0 and at least one n p ' is linked to the adjacent nucleotide via a phosphorothioate bond, each N a and N a ' independently represent an oligonucleotide sequence containing 0 to 25 nucleotides that are either modified or unmodified, or a combination thereof, each sequence containing at least two differently modified nucleotides; each N b and N b ' independently represents an oligonucleotide sequence containing 0 to 10 nucleotides, either modified or unmodified, or a combination thereof; 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, the modifications being 2'-O-methyl modifications, glycol nucleic acid (GNA) modifications, or 2'-fluoro modifications; N b The modification to N is different from the modification to Y. bThe modification to ' is different from the modification to Y', The sense strand is conjugated to at least one ligand, optionally the ligand being one or more lipophilic, eg, C16, ligand, or one or more GalNAc derivatives.

[0150] Another aspect of the present disclosure provides a double-stranded RNAi agent for inhibiting expression of the SOD1 gene in a cell, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding SOD1 (SEQ ID NO: 1, or a nucleotide sequence having at least 90% nucleotide sequence identity, e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the entire nucleotide sequence of SEQ ID NO: 1), each strand being about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent is represented by formula (III): Sense:5'n p -N a -(XXX) i -N b -YYY-N 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) During the ceremony, i, j, k, and l each independently represent 0 or 1; each n p , n q and n q ', each of which may or may not be present, independently represent an overhanging nucleotide; p, q, and q' each independently represent 0 to 6; n p '>0 and at least one np ' is linked to the adjacent nucleotide via a phosphorothioate bond, each N a and N a ' independently represent an oligonucleotide sequence containing 0 to 25 nucleotides that are either modified or unmodified, or a combination thereof, each sequence containing at least two differently modified nucleotides; each N b and N b ' independently represents an oligonucleotide sequence containing 0 to 10 nucleotides, either modified or unmodified, or a combination thereof; 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, the modifications being 2'-O-methyl or 2'-fluoro modifications; N b The modification to N is different from the modification to Y. b The modification to ' is different from the modification to Y', The sense strand is conjugated to at least one ligand, optionally the ligand being one or more lipophilic, eg, C16, ligand, or one or more GalNAc derivatives.

[0151] An additional aspect of the present disclosure provides a double-stranded RNAi agent for inhibiting expression of the SOD1 gene in a cell, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding SOD1 (SEQ ID NO: 1, or a nucleotide sequence having at least 90% nucleotide sequence identity, e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the entire nucleotide sequence of SEQ ID NO: 1), each strand being about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent is represented by formula (III): Sense:5'n p -N a -(XXX) i -N b -YYY-Nb -(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) During the ceremony, i, j, k, and l each independently represent 0 or 1; each n p , n q and n q ', each of which may or may not be present, independently represent an overhanging nucleotide; p, q, and q' each independently represent 0 to 6; n p '>0 and at least one n p ' is linked to the adjacent nucleotide via a phosphorothioate bond, each N a and N a ' independently represent an oligonucleotide sequence containing 0 to 25 nucleotides that are either modified or unmodified, or a combination thereof, each sequence containing at least two differently modified nucleotides; each N b and N b ' independently represents an oligonucleotide sequence containing 0 to 10 nucleotides, either modified or unmodified, or a combination thereof; 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, the modifications being 2'-O-methyl or 2'-fluoro modifications; N b The modification to N is different from the modification to Y. b The modification to ' is different from the modification to Y', the sense strand contains at least one phosphorothioate bond; The sense strand is conjugated to at least one ligand, optionally the ligand being one or more lipophilic, eg, C16, ligand, or one or more GalNAc derivatives.

[0152] Another aspect of the present disclosure provides a double-stranded RNAi agent for inhibiting expression of the SOD1 gene in a cell, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding SOD1 (SEQ ID NO: 1, or a nucleotide sequence having at least 90% nucleotide sequence identity, e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the entire nucleotide sequence of SEQ ID NO: 1), each strand being about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent is represented by formula (III): Sense:5'n p -N a -YYY-N a -n q 3' Antisense: 3'n p '-N a '-Y'Y'Y'-N a '-n q '5'(IIIa) During the ceremony, each n p , n q and n q ', each of which may or may not be present, independently represent an overhanging nucleotide; p, q, and q' each independently represent 0 to 6; n p '>0 and at least one n p ' is linked to the adjacent nucleotide via a phosphorothioate bond, each N a and N a' independently represent an oligonucleotide sequence containing 0 to 25 nucleotides that are either modified or unmodified, or a combination thereof, each sequence containing at least two differently modified nucleotides; YYY and Y'Y'Y' each independently represent a motif of three identical modifications on three consecutive nucleotides, the modifications being 2'-O-methyl or 2'-fluoro modifications; the sense strand contains at least one phosphorothioate bond; The sense strand is conjugated to at least one ligand, optionally the ligand is one or more lipophilic, eg, C16 ligand, or one or more GalNAc derivatives.

[0153] An additional embodiment of the present disclosure provides a double-stranded RNAi agent for inhibiting expression of the SOD1 gene, wherein the double-stranded RNAi agent targeting SOD1 comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand differs by 3 nucleotides or less (i.e., differs by 3, 2, 1, or 0 nucleotides) from any one of the nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, and 9, or at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides from any one of the nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, or 9. The antisense strand comprises a nucleotide sequence having at least 90% nucleotide sequence identity, e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity, to the entire nucleotide sequence of SEQ ID NOs: 2, 4, 6, 8, and 10, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 3 nucleotides or less (i.e., differ by 3, 2, 1, or 0 nucleotides) from any one of the nucleotide sequences of SEQ ID NOs: 2, 4, 6, 8, and 10, or at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by 3 nucleotides or less (i.e., differ by 3, 2, 1, or 0 nucleotides) from any one of the nucleotide sequences of SEQ ID NOs: 2, 4, 6, 8, and 10. , 96, 97, 98, 99, or 100% identity to the entire nucleotide sequence of any one of SEQ ID NOs: 1-6, wherein the substitution of uracil for any thymine in the sequences provided in SEQ ID NOs: 1-6 (when comparing aligned sequences) is not counted as a difference contributing to a difference of 3 nucleotides or less from any one of the nucleotide sequences provided in SEQ ID NOs: 1-6, and wherein substantially all nucleotides of the sense strand are the sense strand comprises two phosphorothioate internucleotide linkages at the 5'-terminus, and substantially all nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5'-terminus and two phosphorothioate internucleotide linkages at the 3'-terminus, and the sense strand is conjugated to one or more lipophilic ligands, for example, a C16 ligand;Optionally, the composition further comprises a liver-targeting ligand, e.g., a ligand comprising one or more GalNAc derivatives.

[0154] Another embodiment of the present disclosure provides a double-stranded RNAi agent for inhibiting expression of the SOD1 gene, wherein the double-stranded RNAi agent targeting SOD1 comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a nucleotide sequence having 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., differ by 3, 2, 1, or 0 nucleotides) from any one of the nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, and 9, or at least 90% nucleotide sequence identity, e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity, to the entire nucleotide sequence of any one of SEQ ID NOs: 1, 3, 5, 7, or 9, and the antisense strand differs by no more than 3 nucleotides (i.e., differs by 0 nucleotides) from any one of the nucleotide sequences of SEQ ID NOs: 2, 4, 6, 8, and 10. a nucleotide sequence having at least 90% nucleotide sequence identity, e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity, to at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, contiguous nucleotides (i.e., differing by 3, 2, 1, or 0 nucleotides), or the entire nucleotide sequence of any one of SEQ ID NOs: 2, 4, 6, 8, and 10; a substitution of uracil for any thymine in the sequence provided in SEQ ID NOs: 1-10 (when comparing aligned sequences) is not counted as a difference contributing to a difference of 3 nucleotides or less from any one of the nucleotide sequences provided in SEQ ID NOs: 1-10; and wherein the sense strand comprises at least one 3'-terminal deoxythymidine nucleotide (dT) and the antisense strand comprises at least one 3'-terminal deoxythymidine nucleotide (dT).

[0155] In one embodiment, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand are modified nucleotides.

[0156] In another embodiment, each strand has between 19 and 30 nucleotides.

[0157] In certain embodiments, the antisense strand of the RNAi agent comprises at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions of the 5' region or its precursor. Optionally, the thermally destabilizing modification of the duplex is one or more of the following: [ka] wherein B is a nucleobase.

[0158] Another aspect of the present disclosure provides a cell containing a double-stranded RNAi agent of the present disclosure.

[0159] An additional aspect of the present disclosure provides a pharmaceutical composition for inhibiting expression of the SOD1 gene, comprising a double-stranded RNAi agent of the present disclosure.

[0160] In one embodiment, the double-stranded RNAi agent is administered in an unbuffered solution. Optionally, the unbuffered solution is saline or water.

[0161] In another embodiment, double-stranded RNAi agent is administered with a buffer solution.Optionally, the buffer solution comprises acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof.In another embodiment, the buffer solution is phosphate buffered saline (PBS).

[0162] Another aspect of the present disclosure provides a pharmaceutical composition comprising a double-stranded RNAi agent of the present disclosure and a lipid formulation.

[0163] In one embodiment, the lipid formulation comprises a lipid nanoparticle (LNP).

[0164] A further aspect of the present disclosure provides a method for inhibiting expression of the SOD1 gene in a cell, the method comprising: (a) contacting the cell with a double-stranded RNAi agent of the present disclosure or a pharmaceutical composition of the present disclosure; and (b) maintaining the cell resulting from step (a) for a time sufficient to obtain degradation of mRNA transcripts of the SOD1 gene, thereby inhibiting expression of the SOD1 gene in the cell.

[0165] In one embodiment, the cell is in a subject. Optionally, the subject is a human.

[0166] In certain embodiments, the subject is a rhesus monkey, a cynomolgus monkey (also known as a crab-eating macaque), a mouse, a dog, or a rat.

[0167] In certain embodiments, the human subject suffers from an SOD1-associated neurodegenerative disease, such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), and Down's syndrome (DS).

[0168] In certain embodiments, the method further comprises administering to the subject an additional therapeutic agent or therapy. Exemplary additional therapeutic agents and therapies 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), ...

[0169] In certain embodiments, the double-stranded RNAi agent is administered at a dose of about 0.01 mg / kg to about 50 mg / kg.

[0170] In some embodiments, the double-stranded RNAi agent is administered to the subject intrathecally.

[0171] In one embodiment, the method reduces expression of the SOD1 gene in brain (e.g., striatum) or spinal tissue. Optionally, the brain or spinal tissue is the striatum, frontal cortex, temporal cortex, cerebellum, hippocampus, cervical vertebrae, lumbar vertebrae, or thoracic vertebrae.

[0172] In one embodiment, the method reduces expression of the SOD1 gene in eye (with or without lens), heart, kidney, liver, lung and / or muscle tissue or cells.

[0173] In some embodiments, the double-stranded RNAi agent is administered to the subject subcutaneously.

[0174] In some embodiments, the double-stranded RNAi agent is administered to the subject intracerebroventricularly.

[0175] In some embodiments, the double-stranded RNAi agent is administered to the subject intrathecally.

[0176] In one embodiment, the method reduces expression of the SOD1 gene in the liver.

[0177] In other embodiments, the method reduces expression of the SOD1 gene in the liver and brain.

[0178] Another aspect of the present disclosure provides a method for inhibiting expression of SOD1 in a subject, the method involving administering to the subject a therapeutically effective amount of a double-stranded RNAi agent of the present disclosure or a pharmaceutical composition of the present disclosure, thereby inhibiting expression of SOD1 in the subject.

[0179] An additional aspect of the present disclosure provides a method for treating or preventing a disorder or SOD1-related neurodegenerative disease or disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent of the present disclosure or a pharmaceutical composition of the present disclosure, thereby treating or preventing the SOD1-related neurodegenerative disease or disorder in the subject.

[0180] In certain embodiments, the SOD1-associated neurodegenerative disease or disorder is selected from the group consisting of amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), and Down's syndrome (DS).

[0181] Another aspect of the present disclosure provides a kit for practicing the methods of the present disclosure, the kit including: a) a double-stranded RNAi agent of the present disclosure; and b) instructions for use; and c) optionally, a device for administering the double-stranded RNAi agent to a subject.

[0182] An additional embodiment of the present disclosure provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting expression of the SOD1 gene, wherein the RNAi agent has a sense strand and an antisense strand, and the antisense strand comprises a region of complementarity comprising at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides, e.g., at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides (i.e., differs by 3, 2, 1, or 0 nucleotides), at least 19, e.g., 19, 20, 21, 22, or 23 nucleotides (i.e., differs by 3, 2, 1, or 0 nucleotides), from any one of the antisense strand nucleobase sequences of Tables 2-7, 12, 13, and 18-20. In one embodiment, the RNAi agent comprises one or more of the following modifications: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-C-alkyl-modified nucleotides, nucleotides comprising glycol nucleic acids (GNAs), phosphorothioates (PS), and vinyl phosphonates (VP). Optionally, the RNAi agent comprises at least one of each of the following modifications: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-C-alkyl-modified nucleotides, nucleotides comprising glycol nucleic acids (GNAs), phosphorothioates, and vinyl phosphonates (VP).

[0183] In another embodiment, the RNAi agent comprises four or more PS modifications, optionally six to ten PS modifications, optionally eight PS modifications.

[0184] In additional embodiments, each of the sense and antisense strands of the RNAi agent has a 5'-end and a 3'-end, and the RNAi agent comprises eight PS modifications located at each of the penultimate and final internucleotide linkages from the 3'-end and 5'-end of each of the sense and antisense strands of the RNAi agent.

[0185] In another embodiment, the sense strand and antisense strand of the RNAi agent each comprise a 5'-end and a 3'-end, and the RNAi agent comprises only one nucleotide comprising a GNA. Optionally, the GNA-containing nucleotide is positioned on the antisense strand at the seventh nucleobase residue from the 5'-end of the antisense strand.

[0186] In additional embodiments, each of the sense strand and antisense strand of the RNAi agent comprises a 5'-end and a 3'-end, and the RNAi agent comprises one to four 2'-C-alkyl-modified nucleotides. Optionally, the 2'-C-alkyl-modified nucleotide is a 2'-C16-modified nucleotide. Optionally, the RNAi agent comprises a single 2'-C-alkyl, e.g., C16-modified nucleotide. Optionally, the single 2'-C-alkyl, e.g., C16-modified nucleotide is located on the sense strand at the sixth nucleobase residue from the 5'-end of the sense strand.

[0187] In another embodiment, each of the sense strand and antisense strand of the RNAi agent comprises a 5'-end and a 3'-end, and the RNAi agent comprises two or more 2'-fluoro modified nucleotides. Optionally, each of the sense strand and antisense strand of the RNAi agent comprises two or more 2'-fluoro modified nucleotides. Optionally, the 2'-fluoro modified nucleotides are located on the sense strand at nucleobase positions 7, 9, 10, and 11 from the 5'-end of the sense strand, and on the antisense strand at nucleobase positions 2, 14, and 16 from the 5'-end of the antisense strand.

[0188] In additional embodiments, each of the sense and antisense strands of the RNAi agent comprises a 5'-end and a 3'-end, and the RNAi agent comprises one or more VP modifications. Optionally, the RNAi agent comprises a single VP modification at the 5'-end of the antisense strand.

[0189] In another embodiment, each of the sense strand and antisense strand of the RNAi agent comprises a 5'-end and a 3'-end, and the RNAi agent comprises two or more 2'-O-methyl modified nucleotides.Optionally, the RNAi agent comprises 2'-O-methyl modified nucleotides at all nucleobase positions that are not modified by 2'-fluoro, 2'-C-alkyl, or glycol nucleic acid (GNA).Optionally, the two or more 2'-O-methyl modified nucleotides are located at positions 1, 2, 3, 4, 5, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 from the 5' end of the sense strand, and at positions 1, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 from the 5' end of the antisense strand.

[0190] 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).

[0191] In another embodiment, an antisense oligonucleotide, or a pharmaceutically acceptable salt thereof, is provided having any one of the antisense nucleotide sequences listed in any one of Tables 3, 5, 7, 13, 18, and 20, but lacking the 3'-terminal nucleotide (3'N-1 AS) (see, e.g., Figure 5). In another embodiment, a dsRNA duplex, or a pharmaceutically acceptable salt thereof, is provided, comprising an antisense strand having any one of the antisense nucleotide sequences listed in any one of Tables 3, 5, 7, 13, 18, and 20, but lacking the 3'-terminal nucleotide (3'N-1 AS), and a sense strand substantially complementary to the antisense oligonucleotide. In another embodiment, a dsRNA duplex, or a pharmaceutically acceptable salt thereof, is provided, comprising any one of the duplexes in any one of Tables 3, 5, 7, 13, 18, or 20, in which the antisense nucleotide sequence is replaced with an antisense oligonucleotide sequence lacking the 3'-terminal nucleotide (3'N-1 AS).

[0192] In one embodiment, an antisense oligonucleotide is provided having the nucleotide sequence of any one of SEQ ID NOs: 1369-1378 (see Table 23), or a pharmaceutically acceptable salt thereof. In one embodiment, an antisense oligonucleotide is provided having the nucleotide sequence of SEQ ID NO: 1369, or a pharmaceutically acceptable salt thereof. In one embodiment, an antisense oligonucleotide is provided having the nucleotide sequence of SEQ ID NO: 1370, or a pharmaceutically acceptable salt thereof. In one embodiment, an antisense oligonucleotide is provided having the nucleotide sequence of SEQ ID NO: 1371, or a pharmaceutically acceptable salt thereof.

[0193] In another embodiment, there is provided a dsRNA duplex comprising an antisense strand having the nucleotide sequence of SEQ ID NO: 1369 and a sense strand substantially complementary to the antisense oligonucleotide, or a pharmaceutically acceptable salt thereof. In another embodiment, there is provided a dsRNA duplex comprising an antisense strand having the nucleotide sequence of SEQ ID NO: 1370 and a sense strand substantially complementary to the antisense oligonucleotide, or a pharmaceutically acceptable salt thereof. In another embodiment, there is provided a dsRNA duplex comprising an antisense strand having the nucleotide sequence of SEQ ID NO: 1371 and a sense strand substantially complementary to the antisense oligonucleotide, or a pharmaceutically acceptable salt thereof.

[0194] In another embodiment, there is provided a dsRNA duplex, or a pharmaceutically acceptable salt thereof, comprising an antisense strand having the nucleotide sequence of SEQ ID NO: 1369 and a sense strand having the sequence of SEQ ID NO: 11. In another embodiment, there is provided a dsRNA duplex, or a pharmaceutically acceptable salt thereof, comprising an antisense strand having the nucleotide sequence of SEQ ID NO: 1370 and a sense strand having the nucleotide sequence of SEQ ID NO: 1371. In another embodiment, there is provided a dsRNA duplex, or a pharmaceutically acceptable salt thereof, comprising an antisense strand having the nucleotide sequence of SEQ ID NO: 1371 and a sense strand having the nucleotide sequence of SEQ ID NO: 15.

[0195] The present invention is further illustrated by the following detailed description and drawings. [Brief explanation of the drawings]

[0196] [Figure 1] 1 is a graph showing human SOD1 mRNA levels in mice subcutaneously administered a single 3 mg / kg dose of the indicated dsRNA duplexes. Human SOD1 mRNA levels are shown relative to control levels detected with PBS treatment. [Figure 2]Figure 2 is a graph showing human SOD1 mRNA levels in the indicated regions of the mouse brain and spinal cord after intracerebroventricular injection (ICV) of a single 25 μg, 50 μg, 100 μg, 150 μg, 200 μg, or 300 μg dose of AD-401824 or artificial CSF (aCSF). [Figure 3] Figure 3A is a graph showing the effect of a single dose of 50 nM, 10 nM, 1 nM, or 0.1 nM of the indicated duplex on hSOD1 mRNA expression in BE(2)c cells at 14 days post-administration. Figure 3B is a graph showing the effect of a single intrathecal 0.9 mg dose of the indicated duplex on hSOD1 mRNA expression in the lumbar, thoracic, and cervical spinal cord regions of G93A rats at 14 days post-administration. [Figure 4-1] Figure 4A is a graph showing the concentration of the indicated duplex in the cervical, thoracic, or lumbar spinal cord of rats administered a single dose of the indicated duplex. Figure 4B is a graph showing the concentration of the indicated duplex in the cerebral cortex or brainstem of rats administered a single dose of the indicated duplex. Figure 4C is a table showing the retention time of the indicated duplex in the cervical, thoracic, lumbar spinal cord, cerebral cortex, or brainstem of rats administered a single dose of the indicated duplex. [Figure 4-2] Same as above. [Figure 5] Figure 5 is a table showing metabolite profiles in the cerebral cortex and lumbar spinal cord of rats administered a single dose of the indicated duplexes. Figure 5 discloses SEQ ID NOs: 1366, 1366, 1366-1367, 1367, 1367-1368, and 1368, respectively, in order of appearance. [Figure 6] Figure 6 is a table summarizing the tissue exposure and metabolite profiling of duplexes AD-1395762, AD-1395756, and AD-1395731. [Figure 7] FIG. 7 is a table showing the study protocol for evaluating the effect of the indicated duplexes on SOD1 mRNA and protein expression in non-human primates. [Figure 8-1]Figure 8A is a table showing the number of non-human primates considered to have received "bad exposure" (duplex levels in CSF samples <1,000 ng / mL at 24 hours), "good exposure" (duplex levels in CSF samples >3,000 ng / mL at 24 hours), or "partial exposure" (duplex levels in CSF samples 1,000-3,000 ng / mL at 24 hours) to a single 70 mg intrathecal dose of AD-1395762, AD-1395756, or AD-1395731. Figure 8B is a graph showing SOD1 mRNA levels in lumbar spinal cord (L3), thoracic spinal cord (T1-T5), cervical spinal cord (C7), frontal cortex (FC), brainstem (BS), or pons samples from non-human primates after a single 70 mg intrathecal dose of the indicated duplexes on days 31 and 85 post-dose. Figure 8C is a graph showing SOD1 mRNA levels in lumbar spinal cord (L3), thoracic spinal cord (T1-T5), cervical spinal cord (C7), frontal cortex (FC), brainstem (BS), or pons samples in non-human primates after a single 70 mg intrathecal administration of the indicated duplexes on day 85. Figure 8C discloses SEQ ID NOs: 1281, 1284, 71, and 1366-1368, respectively, in order of appearance. [Figure 8-2] Same as above. [Figure 9-1]Figure 9A is a graph showing SOD1 protein levels in CSF samples from individual non-human primates after intrathecal administration of a single 70 mg dose of the indicated duplexes on days 0, 8, 14, 29, 57, and 85 post-administration. Figure 9A is a graph showing SOD1 protein levels in CSF samples from individual non-human primates after intrathecal administration of a single 70 mg dose of the indicated duplexes on days 0, 8, 14, 29, 57, and 85 post-administration. Figure 9B is a graph showing SOD1 protein levels in CFS samples from individual non-human primates after intrathecal administration of a single 70 mg dose of the indicated duplexes up to 85 days post-administration (top), and in CSF samples from three individual non-human primates after intrathecal administration of a single 70 mg dose of the indicated duplexes, where day 85 post-administration extended to day 141. Figure 9C is a graph showing SOD1 protein levels in the prefrontal cortex, thoracic spinal cord (T9-12), and CSF samples of non-human primates after intrathecal administration of a single 70 mg dose of the indicated duplexes at day 85 post-administration. [Figure 9-2] Same as above. [Figure 9-3] Same as above. [Figure 10] FIG. 10 is a graph showing that the reduction in mRNA and protein levels observed in prefrontal cortex and thoracic spinal cord (T9-12) samples in NHPs following intrathecal administration of a single 70 mg dose of the indicated duplexes is highly and significantly correlated. [Figure 11-1] Figure 11A is a graph showing mRNA remaining versus siRNA exposure in prefrontal cortex, brainstem, pons, cervical spinal cord (SC_C5-C6), cervical spinal cord (SC_C7), thoracic spinal cord (SC_T1-T5), thoracic spinal cord (SC_T9-T12), and lumbar spinal cord (SC_L3) tissues after intrathecal administration of a single 70 mg dose of the indicated duplex. Figure 11B is a graph showing a strong correlation between the reduction in both mRNA and protein versus siRNA exposure in both prefrontal cortex and thoracic spinal cord (SC_T0-T12) samples after intrathecal administration of a single 70 mg dose of the indicated duplex. [Figure 11-2] Same as above. [Figure 12-1] Figures 12A-12H are stacked bar graphs showing in vitro SOD1 knockdown for each of the duplexes in Tables 14 and 15 in PCH or Be(2)C cells mapped to NM_000454.4 for duplexes having target sequences starting at positions 199-225 (Figure 12A, PCH; Figure 12B, Be(2)C), 319-337 (Figure 12C, PCH; Figure 12D, Be(2)C), 364-382 (Figure 12E, PCH; Figure 12F, Be(2)C), and 516-540 (Figure 12G, PCH; Figure 12H, Be(2)C). [Figure 12-2] Same as above. [Figure 12-3] Same as above. [Figure 12-4] Same as above. [Figure 13] FIG. 13 is a graph showing the substantial lack of pharmacodynamic effect of the indicated dsRNA agents administered to the kidney and liver of NHPs at 31 and 85 days after intrathecal administration. DETAILED DESCRIPTION OF THE INVENTION

[0197] The present disclosure provides an RNAi composition that performs RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the SOD1 gene. The SOD1 gene can be present in a cell, for example, in a cell of a subject, such as a human. The present disclosure also provides a method of using the RNAi composition of the present disclosure to inhibit the expression of the SOD1 gene or to treat a subject with a disorder that would benefit from inhibiting or reducing the expression of the SOD1 gene, for example, an SOD1-related neurodegenerative disease, such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), and Down's syndrome (DS).

[0198] 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 agent of the present disclosure comprises 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 SOD1 gene.

[0199] In certain embodiments, RNAi agents of the present disclosure comprise an RNA strand (antisense strand) that can comprise 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 SOD1 gene. These RNAi agents with longer antisense strand lengths preferably comprise a second RNA strand (sense strand) 20-60 nucleotides in length, where the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.

[0200] The use of these RNAi agents allows for targeted degradation of the mRNA of the SOD1 gene in mammals. Thus, methods and compositions comprising these RNAi agents are useful for treating subjects who would benefit from a reduction in the level or activity of SOD1 protein, such as subjects with SOD1-related neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), and Down's syndrome (DS).

[0201] The following detailed description discloses methods for making and using compositions containing RNAi agents that inhibit expression of the SOD1 gene, as well as compositions and methods for treating subjects with diseases and disorders that would benefit from inhibiting or reducing expression of the gene.

[0202] 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.

[0203] 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 more than one element, e.g., a plurality of elements.

[0204] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to."

[0205] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless context clearly indicates otherwise.

[0206] 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.

[0207] 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 the context indicates otherwise. 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.

[0208] 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.

[0209] As used herein, a method of detection can include determining that the amount of analyte present is below the detection level of the method.

[0210] 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.

[0211] If the chemical structure and chemical name do not match, the chemical structure takes precedence.

[0212] As used herein, the term "superoxide dismutase 1," which is used interchangeably with the term "SOD1," refers to the well-known gene and polypeptide also known in the art as superoxide dismutase [Cu-Zn], Cu / Zn superoxide dismutase, epididymal secretory protein Li 44, EC 1.15.1.1, and indophenol oxidase A. The term "SOD1" includes human SOD1, the amino acid and nucleotide sequences of which can be found, for example, in GenBank Accession No. NM_000454.4 (GI:48762945; SEQ ID NO:1), mouse SOD1, the amino acid and nucleotide sequences of which can be found, for example, in GenBank Accession No. NM_011434.1 (GI:45597446; SEQ ID NO:3), and crab-eating macaque (cynomolgus monkey). monkey) SOD1, its amino acid and nucleotide sequence can be found, for example, in GenBank Accession No. NM_001285406.1 (GI:549432988; SEQ ID NO:5), canine SOD1, its amino acid and nucleotide sequence can be found, for example, in GenBank Accession No. NM_001003035.1 (GI:50978673; SEQ ID NO:7), and rat SOD1, its amino acid and nucleotide sequence can be found, for example, in GenBank Accession No. NM_017050.1 (GI:8394327; SEQ ID NO:9).

[0213] Further examples of SOD1 mRNA sequences are readily available using, for example, GenBank, UniProt, OMIM, and the Macaca Genome Project websites.

[0214] Exemplary SOD1 nucleotide sequences can also be found in SEQ ID NOs: 1 to 10. SEQ ID NOs: 2, 4, 6, 8, and 10 are the reverse complements of SEQ ID NOs: 1, 3, 5, 7, and 9, respectively.

[0215] Further information on SOD1 is provided in the NCBI gene database, e.g., www.ncbi.nlm.nih.gov / gene / 6647.

[0216] 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.

[0217] As used herein, the terms "superoxide dismutase 1" and "SOD1" also refer to naturally occurring DNA sequence variations of the SOD1 gene. Numerous sequence variations within the SOD1 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=6647, the entire contents of which are incorporated herein by reference as of the filing date of this application).

[0218] As used herein, "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during transcription of SOD1 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 will be 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 transcription of SOD1 gene.In one embodiment, the target sequence is located within the protein coding region of SOD1 gene.In another embodiment, the target sequence is located within the 3'UTR of SOD1 gene.

[0219] 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.

[0220] 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.

[0221] " G ", " C ", " Z ", " 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 a GU wobble base that pairs with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods featured in this disclosure.

[0222] As used herein interchangeably, the terms "iRNA," "RNAi agent," "iRNA agent," and "RNA interference agent" refer to agents that contain RNA as defined herein and mediate 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 SOD1 in cells, for example, in cells of a subject, such as a mammalian subject.

[0223] In one embodiment, the RNAi agent of the present disclosure comprises a single-stranded RNAi that interacts with a target RNA sequence, e.g., an SOD1 target mRNA sequence, to mediate cleavage of 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) comprising a sense strand and an antisense strand by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, an RNase III-like enzyme, 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 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 in the RISC cleave the target to 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 SOD1 gene. Therefore, the term "siRNA" is used herein to also refer to the RNAi described above.

[0224] 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.

[0225] 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., SOD1 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, herein referred to as RNA interference or RNAi.

[0226] 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, such as deoxyribonucleotide, modified nucleotide.In addition, as used herein, " RNAi agent " can comprise the ribonucleotide with chemical modification, and RNAi agent can comprise the substantial modification in multiple nucleotides.

[0227] 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 modifications 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.

[0228] 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.

[0229] 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.

[0230] 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.

[0231] 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.

[0232] 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.

[0233] 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."

[0234] 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.

[0235] 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., an SOD1 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).

[0236] 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 SOD1 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 SOD1 RNA sequence to induce cleavage of the target RNA.

[0237] 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.

[0238] 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.

[0239] 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.

[0240] 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.

[0241] 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.

[0242] 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.

[0243] 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'-fhioro-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 a preferred embodiment, 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.

[0244] Exemplary 2' adem modified nucleotides are shown below. [ka]

[0245] 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.

[0246] 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.

[0247] 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.

[0248] 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.

[0249] 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., SOD1 mRNA.

[0250] 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, SOD1 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.

[0251] 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.

[0252] 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 SOD1 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 SOD1 gene. It is important to consider the efficacy of mismatched RNAi agents in inhibiting SOD1 expression, particularly when specific regions of complementarity in the SOD1 gene are known to have polymorphic sequence variation within the population.

[0253] 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.

[0254] 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.

[0255] 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.

[0256] 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.

[0257] 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.

[0258] "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, including, but not limited to, G:U wobble base pairs or Hoogsteen base pairs, so long as they meet the above requirements regarding their ability to hybridize.

[0259] 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.

[0260] 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 SOD1). For example, a polynucleotide is complementary to at least a portion of an SOD1 mRNA if the sequence is substantially complementary to an uninterrupted portion of the mRNA encoding SOD1.

[0261] Thus, in some embodiments, the antisense strand polynucleotides disclosed herein are perfectly complementary to the target SOD1 sequence.

[0262] In other embodiments, the antisense strand polynucleotides disclosed herein are substantially complementary to the target SOD1 sequence and comprise a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 90%, or about 95% complementary, over its entire length to the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, or 9 for SOD1, or an equivalent region of a fragment of SEQ ID NO: 1, 3, 5, 7, or 9.

[0263] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a target SOD1 sequence and comprise a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 90%, or about 95% complementary over its entire length, to any one of the sense strand nucleotide sequences in any one of Tables 2-7, 12, 13, and 18-20, or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 2-7, 12, 13, and 18-20.

[0264] 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 SOD1 sequence, and the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 90%, or about 95% complementary over its entire length to an equivalent region of the nucleotide sequence of SEQ ID NO: 2, 4, 6, 8, or 10, or a fragment of any one of SEQ ID NO: 2, 4, 6, 8, or 10.

[0265] 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 SOD1 sequence, and the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 90%, or about 95% complementary over its entire length, to any one of the antisense strand nucleotide sequences in any one of Tables 2-7, 12, 13, and 18-20, or a fragment of any one of the antisense strand nucleotide sequences in any one of Tables 2-7, 12, 13, and 18-20.

[0266] 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.

[0267] 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.

[0268] 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.

[0269] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each independently 15 to 30 nucleotides in length.

[0270] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each independently 19 to 25, or 19 to 30 nucleotides in length.

[0271] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each independently 21 to 23 nucleotides in length.

[0272] In one embodiment, the sense strand of the iRNA agent is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, in which case the strands form a double-stranded region of 21 contiguous base pairs with a 2-nucleotide long single-stranded overhang at the 3' end.

[0273] 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 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.

[0274] In one embodiment, at least partial suppression of SOD1 gene expression is assessed by a decrease in the amount of SOD1 mRNA, which can be isolated from or detected in a first cell or group of cells in which the SOD1 gene is transcribed and which has been treated to inhibit expression of the SOD1 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

[0275] 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.

[0276] 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.

[0277] 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 by intrathecal injection, intracerebroventricular 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.

[0278] 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.

[0279] 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.

[0280] 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.

[0281] 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.

[0282] 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.

[0283] Thus, conjugating a lipophilic moiety to an internal position of a double-stranded RNAi agent provides optimal hydrophobicity for enhanced in vivo delivery in siRNA.

[0284] 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., an RNAi agent or a plasmid from which an 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.

[0285] As used herein, a "subject" is an animal, such as a mammal, including a primate (e.g., a human, a non-human primate, e.g., a monkey and a chimpanzee) or a non-primate (e.g., a rat or a mouse). In a preferred 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 SOD1 expression, a human being at risk for a disease, disorder, or condition that would benefit from reduced SOD1 expression, a human having a disease, disorder, or condition that would benefit from reduced SOD1 expression, or a human being treated for a disease, disorder, or condition that would benefit from reduced SOD1 expression as described herein.

[0286] As used herein, the term "treat" or "treatment" refers to a beneficial or desired result, including, but not limited to, reducing or ameliorating one or more signs or symptoms associated with SOD1 gene expression or SOD1 protein production, e.g., SOD1-associated neurodegenerative diseases, e.g., amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Down's syndrome (DS), reducing SOD1 expression, deposition, and / or activity in areas of increased neuronal death in subjects with such neurodegenerative diseases. "Treatment" can also mean increasing survival time compared to expected survival time in the absence of treatment.

[0287] The term "lower" in the context of SOD1 levels or disease markers or symptoms in a subject refers to a statistically significant decrease in such levels. 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 SOD1 levels in a subject preferably refers to lowering to a level that is accepted as being within the normal range for individuals without such a 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.

[0288] As used herein, "prevention" or "preventing," when used in reference to a disease, disorder, or condition that would benefit from a reduction in SOD1 gene expression or SOD1 protein production, refers to a reduction in the likelihood that a subject will develop symptoms associated with such a disease, disorder, or condition, such as symptoms of an SOD1-related neurodegenerative disease.Not developing a disease, disorder, or condition, or a reduction in the onset of symptoms associated with such a disease, disorder, or condition, such as neuroinflammation (e.g., a reduction of at least about 10% of the clinically acceptable magnitude for the disease or disorder), or a delay in the onset of symptoms (e.g., a delay of several days, weeks, months, or years), is considered effective prevention.

[0289] As used herein, the term "SOD1-related neurodegenerative disease" or "SOD1-related neurodegenerative disorder" refers to any disease or disorder that would benefit from a reduction in SOD1 expression or activity. Such SOD1-neurodegenerative diseases are characterized by misfolding of the SOD1 protein, such as increased SOD1 deposition in brain regions associated with neuronal death in diseases such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), and Down's syndrome (DS) (see, for example, Trist B, et al. (2020) Angew Chem Int Ed Engl. Accepted author's manuscript).

[0290] In one embodiment, the SOD1-associated neurodegenerative disease is "amyotrophic lateral sclerosis" ("ALS"), also known as Lou Gehrig's disease.

[0291] Amyotrophic lateral sclerosis (ALS) is a progressive disease that affects motor neurons in the spinal cord and brain. In ALS, motor neurons die (atrophy) over time, leading to muscle weakness, loss of muscle mass, and an inability to control movements.

[0292] Mutations in the SOD1 gene cause approximately 20% of hereditary familial amyotrophic lateral sclerosis (fALS) cases and up to approximately 5% of sporadic ALS (sALS) cases. The resulting mutations, such as amino acid substitutions, insertions, deletions, and / or genetic polymorphisms, destabilize the protein structure of SOD1, leading to its misfolding and self-assembly into neurotoxic oligomers and aggregates, a process that contributes to the characteristic motor neuron degeneration in affected individuals.

[0293] Patients with sporadic ALS usually first develop symptomatic symptoms in their late 50s or early 60s. Sporadic and familial ALS have similar pathological and clinical profiles.

[0294] The earliest symptoms of ALS include muscle spasms, cramps, stiffness, or weakness. Affected individuals may develop slurred speech (dysarthria) and later difficulty chewing and swallowing (dysphagia). Many ALS patients experience malnutrition due to reduced food intake caused by swallowing problems and increased body energy demands (metabolism) over time. As the disease progresses, muscles weaken, and the arms and legs begin to appear thinner as muscle tissue atrophies. ALS patients eventually lose muscle strength and the ability to walk. Affected individuals eventually become wheelchair dependent and require assistance with self-care and other activities of daily living. Over time, muscle strength declines, causing patients to lose use of their hands and arms. Breathing becomes difficult due to weakening of the respiratory muscles. Most ALS patients die from respiratory failure within 2 to 10 years of the onset of ALS signs and symptoms, but disease progression varies widely among individuals.

[0295] SOD1 accumulation has been found in motor neurons, spinal cords, and associated cells in postmortem mutant SOD1 transgenic mice and human ALS patient tissue. Furthermore, SOD1 transgenic mice, an art-recognized model of ALS that express mutant SOD1 forms at levels similar to or elevated than the endogenous mouse protein, have been shown to recapitulate the ALS phenotype. These mice develop severe degeneration of motor neurons, as seen in human ALS patients, leading to progressive paralysis and death of the hind and forelimbs. These mice also exhibit pathology seen in postmortem tissue from human patients, including SOD1 cytoplasmic inclusions, gliosis, glutamate excitotoxicity, mitochondrial vacuolization, and impaired axonal transport (Mina M, et al. (2018) J Transl Neurosci. 3:9).

[0296] In one embodiment, the SOD1-associated neurodegenerative disease is "Alzheimer's disease" ("AD"). AD is a chronic neurodegenerative disease that usually begins gradually and worsens gradually over time. The most common early symptom is difficulty remembering recent events. As the disease progresses, symptoms include language problems, disorientation (e.g., getting lost easily), mood swings, loss of motivation, inability to care for oneself, and behavioral problems. As the condition worsens, individuals often withdraw from family and society. Gradual loss of physical function occurs, ultimately leading to death.

[0297] Neuropathologically, AD is characterized by the loss of neurons and synapses in the cerebral cortex and certain subcortical regions. This loss results in significant atrophy of affected areas, including degeneration of the temporal and parietal lobes, as well as parts of the frontal cortex and cingulate gyrus. Degeneration also occurs in brainstem nuclei such as the locus coeruleus. Studies using MRI and PET have demonstrated a decrease in the size of specific brain regions in AD patients compared with similar images from healthy elderly subjects as they progress from mild cognitive impairment to Alzheimer's disease.

[0298] In brains affected by AD, both amyloid plaques and neurofibrillary tangles are clearly visible under a microscope. Plaques are dense, mostly insoluble deposits of beta-amyloid peptides and cellular material outside and surrounding neurons. Tangles (neurofibrillary tangles) are aggregates of hyperphosphorylated microtubule-associated protein tau that accumulate intracellularly. While most older adults develop some plaques and tangles as a result of aging, the brains of AD patients are more prevalent in certain brain regions, such as the temporal lobe. Lewy bodies are not uncommon in the brains of AD patients.

[0299] Postmortem and in vivo studies of patients with AD have also shown the accumulation of free radical damage products in the central nervous system and peripheral tissues. Furthermore, SOD1 aggregates have been identified in AD brains and shown to be associated with amyloid plaques and neurofibrillary tangles (Choi J, et al. (2005) JBC. 280:11648-11655).

[0300] In one embodiment, the SOD1-associated neurodegenerative disease is "Parkinson's disease" ("PD"). Parkinson's disease is a progressive disorder of the nervous system. The disorder affects several areas of the brain, particularly the area called the substantia nigra, which controls balance and movement. PD results in the loss of dopaminergic neurons in the basal ganglia.

[0301] Often, the first symptom of Parkinson's disease is shaking or tremor in the hands and feet, especially when the body is at rest. The tremor typically begins on one side of the body, usually one hand. Tremor can also affect the arms, legs, feet, and face. Other characteristic symptoms of Parkinson's disease include stiffness or rigidity of the limbs and trunk, slowness of movement (bradykinesia) or inability to move (akinesia), and impaired balance and coordination (postural instability). These symptoms slowly worsen over time.

[0302] Parkinson's disease can also affect emotions and thinking skills (cognition). Some people with the disease develop psychiatric conditions such as depression and hallucinations. People with Parkinson's disease are also at increased risk of developing dementia, a decline in intellectual function, including judgment and memory.

[0303] Previous studies have shown that total levels of SOD1 protein are significantly increased in the brains of PD patients (Choi J, et al. (2005) JBC. 280:11648-11655). Furthermore, SOD1 deposits have been detected in Lewy bodies in the brains of PD patients (Nishiyama L, et al. (1995) Acta Neuropathologica, 89:471-474).

[0304] In one embodiment, the SOD1-associated neurodegenerative disease is "Down syndrome (DS)." Down syndrome ("DS") is caused by trisomy of human chromosome 21 (Hsa21) and increased expression of a subset of the encoded genes. DS patients exhibit distinct morphological features, such as short stature, obesity, and bilateral epicanthal folds. In addition, age-related hypotonia and neurodegeneration may occur throughout life. This syndrome is associated with mental retardation, congenital heart disease, immune system disorders, digestive system disorders, endocrine system disorders, and various biochemical disorders. Evidence from in vivo, in vitro, and animal model studies indicates that oxidative stress is involved in DS. Therefore, it has been proposed that the increased oxidative stress observed in these subjects is primarily due to excessive activity of SOD1, an enzyme encoded by HSA21 (21q22.1). DS patients also show elevated SOD1 levels, which appear to increase lipid peroxidation and oxidative damage to DNA, in addition to elevated glutathione peroxidase activity (Campos C and Casado A. (2015) Indian J Med Res. 142(2):113-119).

[0305] A "therapeutically effective amount," as used herein, is intended to include an amount of an RNAi agent that, when administered to a subject with an SOD1-associated neurodegenerative 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.

[0306] As used herein, a "prophylactically effective amount" is intended to include an amount of an RNAi agent that, when administered to a subject with an SOD1-associated neurodegenerative disease, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease includes slowing the course 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.

[0307] 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.

[0308] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds (including salts), materials, 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.

[0309] 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.

[0310] 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.

[0311] II. RNAi Agents of the Disclosure Described herein are RNAi agents that inhibit expression of the SOD1 gene. In one embodiment, the RNAi agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting expression of the SOD1 gene in a cell, e.g., a mammalian cell, of a subject, e.g., a human, with an SOD1-associated neurodegenerative disease, e.g., amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), and Down's syndrome (DS). 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 SOD1 gene. The region of complementarity is no more than about 15-30 nucleotides in length. When contacted with a cell expressing the SOD1 gene, the RNAi agent inhibits expression of the SOD1 gene (e.g., human gene, primate gene, 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 a preferred embodiment, the 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.

[0312] dsRNA comprises two RNA strands, which are complementary and hybridize to form a duplex structure under the conditions in which dsRNA is used. One strand of 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 mRNA formed during the expression of the SOD1 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.

[0313] 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 preferred 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.

[0314] 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.

[0315] In some embodiments, the dsRNA is 15-23 nucleotides long, 25-30 nucleotides long, 20-30 nucleotides long, or 19-30 nucleotides long. 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 function 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. Where relevant, a "portion" of an mRNA target is a contiguous sequence of the mRNA target long enough to allow it to serve as a substrate for RNAi-dependent cleavage (i.e., cleavage via the RISC pathway).

[0316] Those skilled in the art will appreciate that the duplex region is the 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, 18-26, 18-27, 18-24, 18-28, 18-26, 18-25, 18-24, 18-26 ... It will also be appreciated that the duplex region may be 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, an RNAi agent useful for targeting SOD1 expression is not generated in a target cell by cleavage of a larger dsRNA.

[0317] 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.

[0318] 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.

[0319] 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.

[0320] 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.

[0321] siRNAs can be produced by separately synthesizing a single-stranded RNA molecule, or each strand of a double-stranded RNA molecule, and then annealing the component strands.

[0322] 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.

[0323] Organic synthesis can be used to produce individual siRNA species.The complementarity of the species to SOD1 gene can be precisely specified.For example, the species can be complementary to the region containing polymorphism, for example, single nucleotide polymorphism.Furthermore, the position of polymorphism can be precisely defined.In some embodiments, the polymorphism is located in the internal region, for example, within at least 4, 5, 7 or 9 nucleotides from one or both ends.

[0324] 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 the 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.

[0325] 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 adjacent regions of the source dsiRNA molecule.

[0326] 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.

[0327] 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 SOD1 can be selected from the group of sequences provided in any one of Tables 2-7, 12, 13, and 18-20, and the corresponding nucleotide sequence of the antisense strand of the sense strand can be selected from the group of sequences provided in any one of Tables 2-7, 12, 13, and 18-20. 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 SOD1 gene. Thus, in this embodiment, the dsRNA will comprise two oligonucleotides, one of which is designated as the sense strand (passenger strand) in any one of Tables 2-7, 12, 13, and 18-20, and the second of which is designated as the corresponding antisense strand (guide strand) in any one of Tables 2-7, 12, 13, and 18-20 for SOD1.

[0328] 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.

[0329] 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 2-7, 12, 13, and 18-20, 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.

[0330] 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 SOD1 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 an RNA agent at a concentration of 10 nM and the PCR assays provided in the Examples herein are intended to be within the scope of this disclosure.

[0331] In addition, the RNA described herein identifies the site in SOD1 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 the specific sites, the RNAi agent is said to target within the specific site of the RNA transcript.This RNAi agent will generally comprise at least about 15 consecutive nucleotides, preferably 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 SOD1 gene.

[0332] 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 SOD1 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 SOD1 gene. It is important to consider the efficacy of mismatched RNAi agents in inhibiting SOD1 expression, particularly when specific regions of complementarity in the SOD1 gene are known to have polymorphic sequence variation within the population.

[0333] 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 a preferred embodiment, 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.

[0334] 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 ligation) or 3'-end modifications (conjugation, DNA nucleotides, inverted ligation, etc.), base modifications, such as substitution with stabilizing bases, destabilizing bases, or bases that base-pair with partners in the 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 modification or substitution of phosphodiester linkages. 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.

[0335] 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.

[0336] 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.

[0337] 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.

[0338] 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.

[0339] 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.

[0340] 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-.

[0341] 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(CH2)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).

[0342] 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.

[0343] 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.

[0344] 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.

[0345] 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 a 3'-endo conformation. The addition of locked nucleic acids 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 polynucleotides of the present disclosure include, but are not limited to, nucleosides containing a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agents of the present disclosure include one or more bicyclic nucleosides containing a 4' to 2' bridge.

[0346] 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, see, e.g., U.S. Pat. No. 7,399,845), 4'-C(CH3)(CH3)- 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-C12 alkyl, or a nitrogen protecting group (see, e.g., U.S. Pat. No. 7,427,672, Greene's Protective Groups in Organic Synthesis, Fourth Edition, 2006, eds. John Wiley & Sons, Inc.), 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.

[0347] 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, Nos. 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, U.S. Patent Nos. 2008 / 0039618, and 2009 / 0012281, the entire contents of each of which are incorporated herein by reference.

[0348] 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).

[0349] The RNAi agents of the present disclosure can also be modified to include 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 (i.e., 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."

[0350] 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 C3 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.

[0351] 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.

[0352] 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'-C3' bond of the sugar has been removed (i.e., a covalent carbon-carbon bond between the C2' and C3' 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).

[0353] 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.

[0354] 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'-O-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.

[0355] 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.

[0356] 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.

[0357] 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.

[0358] 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).

[0359] 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.

[0360] 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, excellent results can be obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides 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.

[0361] Thus, the present disclosure provides a double-stranded RNAi agent capable of inhibiting expression of a target gene (i.e., the SOD1 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.

[0362] 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 a preferred embodiment, the duplex region is 19 to 21 nucleotide pairs in length.

[0363] 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 a preferred embodiment, 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.

[0364] 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.

[0365] 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.

[0366] 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.

[0367] RNAi agent 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, which is located at the 5'-end of the antisense strand (or 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 its 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 guide strand into RISC process.

[0368] 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, 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.

[0369] 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, 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.

[0370] 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, 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.

[0371] In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand comprises 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 comprises 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 comprises a two-nucleotide overhang.Preferably, 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).

[0372] 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'-most 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'-most nucleotide, comprises at least 8 ribonucleotides at positions 1-23 of the sense strand, forming a duplex; wherein at least the 3'-most nucleotide of the antisense strand is unpaired with the sense strand, and up to 6 consecutive 3'-most 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 10-30 nucleotide single-stranded 5' overhang, 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.

[0373] 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 preferentially 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.

[0374] 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.

[0375] 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.

[0376] For RNAi agents with a duplex region 17-23 nucleotides in length, the cleavage sites of the antisense strand are typically approximately 10, 11, and 12 positions from the 5' end. Thus, three identical modification motifs can occur at positions 9, 10, 11, 10, 11, 12, 11, 12, 13, 12, 13, 14, or 13, 14, 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.

[0377] The sense strand of RNAi agent can contain at least one motif of three identical modifications on three consecutive nucleotides at the break 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 break 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.

[0378] 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.

[0379] 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.

[0380] 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.

[0381] 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 within the duplex region at the 3' end, 5' end, or both ends of the strand.

[0382] 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.

[0383] 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 an overlap of one, two, or three nucleotides, the two modifications from each single strand are at the other end of the duplex region with an overlap of one, two, or three nucleotides, and the two modifications from the single strand are on either side of a lead motif within the duplex region with an overlap of one, two, or three nucleotides.

[0384] 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.

[0385] 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.

[0386] 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.

[0387] 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.

[0388] In one embodiment, the sense strand sequence has formula (I): 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3'(I) It can be expressed as 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. Preferably, YYY are all 2'-F modified nucleotides.

[0389] In one embodiment, N a or N b includes alternating pattern modifications.

[0390] 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.

[0391] 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) It can be expressed as:

[0392] 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.

[0393] each N a can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0394] 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.

[0395] When the sense strand is represented by formula (Id), each N b independently represent an oligonucleotide sequence containing 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.

[0396] Each of X, Y and Z may be the same or different from one another.

[0397] 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 -nq 3'(Ia) It can be expressed as:

[0398] 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.

[0399] 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) It can be expressed as 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.

[0400] In one embodiment, N a ' or N b ' includes alternating pattern modifications.

[0401] Y'Y'Y' motif occurs at or near the cleavage site of antisense strand.For example, when RNAi agent has a double-stranded region of 17-23 nucleotides in length, Y'Y'Y' motif can occur at position 9, 10, 11, 10, 11, 12, 11, 12, 13, 12, 13, 14, or 13, 14, 15 of antisense strand, this number starting from the first nucleotide from the 5' end, or optionally this number starting from the first paired nucleotide in the double-stranded region from the 5' end.Preferably, Y'Y'Y' motif occurs at position 11, 12, 13.

[0402] In one embodiment, the Y'Y'Y' motif is all 2'-OMe modified nucleotides.

[0403] 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.

[0404] 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:

[0405] 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.

[0406] 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.

[0407] 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 ' 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.

[0408] 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:

[0409] 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.

[0410] Each of X', Y' and Z' may be the same as or different from one another.

[0411] 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.

[0412] 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.

[0413] 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.

[0414] 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.

[0415] 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 -YYY-N 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.

[0416] 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.

[0417] An exemplary combination of sense and antisense strands that form an RNAi duplex is of the following formula: 5'n p -N a -YYY-N a -n q 3' 3'n p ’ -N a ’ -Y'Y'Y'-N a ’ n q ’ 5' (IIIa) 5'n p -N a -YYY-N b -ZZZ-N 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 -XXX-N b -YYY-N a -nq 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 -XXX-N b -YYY-N b -ZZZ-N 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) Includes.

[0418] 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.

[0419] 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.

[0420] 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.

[0421] 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.

[0422] 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.

[0423] 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.

[0424] 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.

[0425] 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.

[0426] 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.

[0427] 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.

[0428] 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 double bond between the C5' carbon and R5' is in the E or Z conformation (e.g., E conformation), and B is a nucleobase or modified nucleobase, optionally B is adenine, guanine, cytosine, thymine, or uracil.

[0429] 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.

[0430] 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 preferred embodiments, the vinyl phosphonate of the present disclosure is attached to the antisense strand of the dsRNA at the 5' end of the antisense strand of the dsRNA, as appropriate.

[0431] 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 (e.g., E configuration).

[0432] E. 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.

[0433] The term "thermally destabilizing modification" includes modifications that result in a dsRNA having an overall melting temperature (Tm) that is lower than the Tm of a dsRNA that does not have such a modification. For example, a thermally destabilizing modification can decrease the Tm of a dsRNA by 1-4°C, e.g., 1, 2, 3, or 4 degrees Celsius. Additionally, the term "thermally destabilizing nucleotide" refers to a nucleotide that contains one or more thermally destabilizing modifications.

[0434] 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 thermally destabilizing modification of one or more duplexes is located at positions 2-9, or preferably 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.

[0435] 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").

[0436] 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.

[0437] Exemplary sugar modifications include, but are not limited to: [ka] wherein B is a modified or unmodified nucleobase.

[0438] 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.

[0439] 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).

[0440] 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'-C3', C3'-C4', C4'-O4', or C1'-O4') are absent, or at least one of the ribose carbons or oxygens (e.g., C1', C2', C3', 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'-C3' bond of the sugar (i.e., a carbon-carbon covalent bond between the C2' and C3' carbons) has been removed (see Mikhailov et al., Tetrahedron Letters, 26(17):2059 (1985), and Fluiter et al., Mol. Biosyst., 10:1039 (2009), which are incorporated herein 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.

[0441] 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]

[0442] 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 combinations 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.

[0443] 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]

[0444] 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.

[0445] Thermally destabilizing modifications can also include universal base and phosphate modifications that have reduced or eliminated ability to form hydrogen bonds with opposing bases.

[0446] 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]

[0447] 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.

[0448] Exemplary phosphate modifications that have been shown to decrease the thermal stability of dsRNA duplexes, relative to natural phosphodiester linkages, include the following: [ka]

[0449] 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.

[0450] 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.

[0451] 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 or antisense strand. For example, the stabilizing modifications may occur at any nucleotide in the sense or antisense strand, and each stabilizing modification may occur in an alternating pattern on the sense or antisense strand, or the sense or 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.

[0452] 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.

[0453] 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.

[0454] 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.

[0455] 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 two, three, or four blocks of stabilizing modifications.

[0456] 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.

[0457] Exemplary thermally stabilizing modifications include, but are not limited to, 2'-fluoro modifications. Other thermally stabilizing modifications include, but are not limited to, LNA.

[0458] 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.

[0459] 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.

[0460] 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 position and +1 position from the position of destabilizing modification.

[0461] 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.

[0462] 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.

[0463] 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.

[0464] 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. Preferably, a 2nT overhang is at the 3' end of the antisense strand.

[0465] 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), positions 1 to 23 of the sense strand comprise at least 8 ribonucleotides; 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 10 to 30 consecutive nucleotides that are not paired with the sense strand. wherein 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 2-9 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.

[0466] 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, the sense strand comprising 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 forming a blunt end, and the antisense strand is 1 to 4 nucleotides longer at its 3' end than the sense strand, the duplex region being at least 25 nucleotides in length, and the antisense strand being sufficiently complementary to a target mRNA along at least 19 nT 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 preferentially 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 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); (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.

[0467] 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.

[0468] 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.

[0469] 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.

[0470] 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.

[0471] At least two different modifications are typically present on the sense strand and the antisense strand. These modifications can 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) 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 should be understood that these modifications are in addition to at least one thermally destabilizing modification of the duplex present in the antisense strand.

[0472] 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 having 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 a similar pattern. 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.

[0473] 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.

[0474] 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.

[0475] 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.

[0476] 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.

[0477] 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.

[0478] 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.

[0479] 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.

[0480] 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.

[0481] 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 have phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides.Internucleotide linkage modification can also be made to link the overhang nucleotide with 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 with 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.Preferably, these terminal three nucleotides can be at the 3'-end of the antisense strand.

[0482] 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.

[0483] 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.

[0484] 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.

[0485] 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.

[0486] 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.

[0487] 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.

[0488] 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.

[0489] 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.

[0490] 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.

[0491] 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 on...

Claims

1. A double-stranded ribonucleic acid (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 nucleotide sequence of the antisense strand differs by no more than four bases from the nucleotide sequence 5'-VPusdCsugdGadTagagdGaUfuaaagugsa-3' (SEQ ID NO:1369); b) the nucleotide sequence of the antisense strand differs by no more than 3 bases from the nucleotide sequence 5'-VPusdCsugdGadTagagdGaUfuaaagugsasg-3' (SEQ ID NO:12); c) the nucleotide sequence of the sense strand differs by no more than four bases from the nucleotide sequence 5'-csascuu(Uhd)aaUfCfCfucuauccasgsa-3' (SEQ ID NO: 11) and the nucleotide sequence of the antisense strand differs by no more than four bases from the nucleotide sequence 5'-VPusdCsugdGadTagagdGaUfuaaagugsasg-3' (SEQ ID NO: 12); d) the nucleotide sequence of the sense strand differs from the nucleotide sequence 5'-csasggu(Chd)cuCfAfCfuuuaauccsusa-3' (SEQ ID NO: 13) by no more than 4 bases and the nucleotide sequence of the antisense strand differs from the nucleotide sequence 5'-VPusdAsggdAudTaaagdTgAfggaccugscsg-3' (SEQ ID NO: 14) by no more than 4 bases; e) the nucleotide sequence of the sense strand differs from the nucleotide sequence 5'-ususcgag(Chd)aGfAfAfggaaaguasasa-3' (SEQ ID NO: 15) by no more than 4 bases, and the nucleotide sequence of the antisense strand differs from the nucleotide sequence 5'-VPusUfsuadCu(Tgn)uccuucUfgCfucgaasasu-3' (SEQ ID NO: 16) by no more than 4 bases; f) the nucleotide sequence of the sense strand differs from the nucleotide sequence 5'-gsasaag(Uhd)aaUfGfGfaccagugasasa-3' (SEQ ID NO: 17) by no more than 4 bases and the nucleotide sequence of the antisense strand differs from the nucleotide sequence 5'-VPusUfsucdAc(Tgn)gguccaUfuAfcuuucscsu-3' (SEQ ID NO: 18) by no more than 4 bases; g) the nucleotide sequence of the sense strand differs from the nucleotide sequence 5'-asgsga(Uhd)gaaGfAfGfaggcaugususa-3' (SEQ ID NO:19) by no more than 4 bases and the nucleotide sequence of the antisense strand differs from the nucleotide sequence 5'-VPusAfsacdAu(G2p)ccucucUfuCfauccususu-3' (SEQ ID NO:20) by no more than 4 bases; h) the nucleotide sequence of the sense strand differs from the nucleotide sequence 5'-asasgga(Ahd)agUfAfAfuggaccagsusa-3' (SEQ ID NO:21) by no more than 4 bases and the nucleotide sequence of the antisense strand differs from the nucleotide sequence 5'-VPusdAscudGg(Tgn)ccaudTaCfuuuccuuscsu-3' (SEQ ID NO:22) by no more than 4 bases; i) the nucleotide sequence of the sense strand differs from the nucleotide sequence 5'-asuscaa(Uhd)uuCfGfAfgcagaaggsasa-3' (SEQ ID NO:23) by no more than 4 bases and the nucleotide sequence of the antisense strand differs from the nucleotide sequence 5'-VPusUfsccdTu(C2p)ugcucgAfaAfuugausgsg-3' (SEQ ID NO:24) by no more than 4 bases; j) the nucleotide sequence of the sense strand differs from the nucleotide sequence 5'-cscsuca(Chd)uuUfAfAfuccucuauscsa-3' (SEQ ID NO:25) by no more than 4 bases and the nucleotide sequence of the antisense strand differs from the nucleotide sequence 5'-VPusdGsaudAg(Agn)ggaudTaAfagugaggsasc-3' (SEQ ID NO:26) by no more than 4 bases; k) the nucleotide sequence of the sense strand differs from the nucleotide sequence 5'-asasgga(Uhd)gaAfGfAfgaggcaugsusa-3' (SEQ ID NO:27) by no more than 4 bases, and the nucleotide sequence of the antisense strand differs from the nucleotide sequence 5'-VPusAfscadTg(C2p)cucucuUfcAfuccuususg-3' (SEQ ID NO:28) by no more than 4 bases; l) the nucleotide sequence of the sense strand differs from the nucleotide sequence 5'-asasuuu(Chd)gaGfCfAfgaaggaaasgsa-3' (SEQ ID NO:29) by no more than 4 bases and the nucleotide sequence of the antisense strand differs from the nucleotide sequence 5'-VPusCfsuudTc(C2p)uucugcUfcGfaaauusgsg-3' (SEQ ID NO:30) by no more than 4 bases; During the ceremony, VP is 5'-vinylphosphonate; (Ahd) is 2'-O-hexadecyl-adenosine-3'-phosphate; (Chd) is 2'-O-hexadecyl-cytidine-3'-phosphate; (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate; (Agn) is adenosine glycol nucleic acid (GNA), S isomer; (Tgn) is thymidine glycol nucleic acid (GNA), S isomer; (C2p) is cytidine-2'-phosphate; (G2p) is guanosine-2'-phosphate; s is a phosphorothioate linkage; a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; dA, dC, dG, and dT are 2'-deoxy A, C, G, and T; Af, Cf, Gf, and Uf are 2'-deoxy-2'-fluoro (2'-F) A, C, G, and U; or m) the sense strand comprises at least 15 contiguous nucleotides from any one of the nucleotide sequences 201-223, 204-226, 207-229, 216-238, 219-241, 328-350, 333-355, 336-358, 372-394, or 373-395 of SEQ ID NO:1, and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO:2; (i) at least one nucleotide comprises a nucleotide modification; (ii) the double-stranded region is 15 to 30 nucleotide pairs in length; and (iii) a dsRNA agent, or a pharma- ceutically acceptable salt thereof, wherein said sense strand or said antisense strand is conjugated to one or more lipophilic moieties.

2. 2. The dsRNA agent of claim 1, or a pharma- ceutically acceptable salt thereof, which is a sodium salt.

3. 3. The dsRNA agent of claim 1m) or 2, or a pharma- ceutically acceptable salt thereof, wherein all of said nucleotides of said sense strand and all of said nucleotides of said antisense strand comprise a nucleotide modification.

4. At least one of the nucleotide modifications is a deoxy-nucleotide modification, a 3'-terminal deoxy-thymidine (dT) nucleotide modification, a 2'-O-methyl nucleotide modification, a 2'-fluoro nucleotide modification, a 2'-deoxy-nucleotide modification, a 2'-5'-linked ribonucleotide (3'-RNA) 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'-methoxyethyl nucleotide modification, a 2'-O-alkyl-nucleotide modification, a morpholino nucleotide modification, a phosphoramidate nucleotide modification, a nucleotide modification comprising a non-natural base, a tetrahydropyran nucleotide modification, a 1,5-anhydrohexyl ...amino-nucleotide modification, a dsRNA agent of any one of claims 1m), 2 and 3, or a pharmaceutically acceptable salt thereof, wherein the dsRNA agent is selected from the group consisting of a nucleotide having a 5'-phosphorothioate group modification, a cyclohexenyl nucleotide modification, a nucleotide having a 5'-methylphosphonate group modification, a nucleotide having a 5'phosphate or a 5'phosphate mimetic modification, a nucleotide having a vinylphosphonate modification, a glycol nucleic acid (GNA) modification, a glycol nucleic acid S isomer (S-GNA) modification, a nucleotide having a 2-hydroxymethyl-tetrahydrofuran-5-phosphate modification, a nucleotide having a 2'-deoxythymidine-3'phosphate modification, a nucleotide having a 2'-deoxyguanosine-3'-phosphate modification, and a terminal nucleotide linked to a cholesteryl derivative, and a dodecanoic acid bisdecylamide group modification, and combinations thereof.

5. 5. The dsRNA agent of any one of claims 1m) and 2-4, or a pharma- ceutically acceptable salt thereof, further comprising at least one phosphorothioate internucleotide linkage.

6. 6. The dsRNA agent of any one of claims 1m) and 2-5, 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.

7. 7. The dsRNA agent of any one of claims 1m) and 2-6, or a pharma- ceutically acceptable salt thereof, wherein said double-stranded region is 17-23 nucleotide pairs, 19-21 nucleotide pairs, or 21-23 nucleotide pairs in length.

8. The dsRNA agent of any one of claims 1m) and 2-7, or a pharma- ceutically acceptable salt thereof, wherein each strand is 19-30 nucleotides in length.

9. 9. The dsRNA agent of any one of claims 1m) and 2-8, or a pharma- ceutically acceptable salt thereof, wherein said one or more lipophilic moieties are conjugated to one or more interior positions in at least one strand.

10. 10. The dsRNA agent of claim 9, or a pharma- ceutically acceptable salt thereof, wherein one lipophilic moiety is conjugated to an internal position 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.

11. 11. The dsRNA agent of claim 9 or 10, or a pharma- ceutically acceptable salt thereof, wherein said lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound.

12. 12. The dsRNA agent of claim 11, 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.

13. 13. The dsRNA agent of any one of claims 1m) and 2-12, or a pharma- ceutically acceptable salt thereof, wherein said agent further comprises a phosphate or a phosphate mimetic at the 5'-end of the antisense strand.

14. A cell comprising the dsRNA agent or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 13.

15. A pharmaceutical composition comprising the dsRNA agent or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 13, and a pharma- ceutically acceptable excipient.

16. 1. An in vitro method for inhibiting expression of the SOD1 gene in a cell, comprising: (a) contacting said cell with a dsRNA agent according to any one of claims 1 to 13 or a pharma- ceutically acceptable salt thereof, or with a pharmaceutical composition according to claim 15; (b) maintaining the cells produced in step (a) for a period of time sufficient to obtain degradation of mRNA transcripts of the SOD1 gene, thereby inhibiting expression of the SOD1 gene in the cells.

17. 17. A pharmaceutical composition for treating a subject diagnosed with an SOD1-associated neurodegenerative disease, comprising a therapeutically effective amount of a dsRNA agent according to any one of claims 1 to 13, or a pharma- ceutical acceptable salt thereof, or the pharmaceutical composition according to claim 15.

18. 18. The pharmaceutical composition of claim 17, wherein the SOD1-associated neurodegenerative disease is selected from the group consisting of amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), and Down's syndrome (DS).

19. The pharmaceutical composition described in claim 18, wherein the SOD1-associated neurodegenerative disease is hereditary familial amyotrophic lateral sclerosis (fALS).

20. The pharmaceutical composition according to any one of claims 17 to 19, wherein the subject is a human.

21. The pharmaceutical composition according to any one of claims 17 to 20, wherein the pharmaceutical composition is administered to the subject intrathecally or intracerebroventricularly.

Images