Oligonucleotide-Based Delivery Vehicles for Oligonucleotide Agents and Methods of Use Thereof

Abstract

This application relates to nucleic acids, and in particular to oligonucleotide agents comprising double-stranded RNA (dsRNA, double stranded) and non-targeting auxiliary oligonucleotides (ACOs) covalently linked to the dsRNA, and their pharmaceutical uses.

Description

[Technical Field]

[0001] The present application relates to the technical field of nucleic acids, in particular to oligonucleotide agents comprising double-stranded RNA (dsRNA, double-stranded) and non-targeting auxiliary oligonucleotides (ACOs) covalently linked to the dsRNA, and their pharmaceutical uses.

[0002] This application claims priority to the filing date of provisional patent application serial number PCT / CN2021 / 105081, filed on July 7, 2021, and the filing date of provisional patent application serial number PCT / CN2022 / 091076, filed on May 6, 2022, the disclosures of which are incorporated herein by reference.

[0003] The instant application contains a Sequence Listing that has been submitted electronically in computer readable format and is hereby incorporated by reference in its entirety. [Background technology]

[0004] Oligonucleotides are an emerging class of therapeutics currently under active development for the treatment of a wide variety of diseases through numerous mechanisms of action (MOAs). Major categories of oligonucleotide therapeutics include single-stranded antisense oligonucleotides (ASOs) and double-stranded RNA (dsRNA). Single-stranded ASOs in the form of "gapmers" can be used to suppress gene expression by degrading target mRNAs via the RNase H mechanism. Gapmer ASOs contain a central DNA region necessary to support RNase H activity and two ribonucleotide wings to enhance the ASO's target binding affinity. Another category of ASOs is the steric blocker, which is generally composed entirely of ribonucleotides and alters mRNA splicing by binding to pre-mRNA in the nucleus and inhibiting the binding of certain splicing factors to the mRNA.

[0005] dsRNA can be further divided into two categories: small interfering RNA (siRNA) and small activating RNA (saRNA), both of which require Argonaute (AGO) proteins as protein partners to function. siRNAs primarily bind to target mRNAs in the cytoplasm and downregulate gene expression post-transcriptionally via the RNA interference (RNAi) mechanism. saRNAs target regulatory sequences in the nucleus, such as gene promoters, and upregulate gene expression at the transcriptional level via the RNAa (RNA activation) mechanism.

[0006] Nearly all monogenic and most polygenic disorders are caused by loss, rather than gain, of gene function. Loss of function can occur through epigenetic abnormalities or gene mutations, leading to transcriptional silencing or incorrect forms of the transcribed mRNA, respectively. One such error is missplicing of pre-mRNA, resulting in exon skipping or inclusion, which results in a nonfunctional protein when the mRNA is translated. In such cases, ASOs have been used to correct the incorrect splicing event and ultimately increase the protein production of the gene by sterically blocking the protein-RNA binding interaction between components of the splicing machinery and the pre-mRNA. Several such ASO drugs have been approved by the U.S. Food and Drug Administration (FDA), including the exon-enclosing ASOSPINRAZA® for the treatment of spinal muscular atrophy (SMA) and three exon-skipping ASOs for the treatment of Duchenne muscular dystrophy (DMD). Summary of the Invention [Problem to be solved by the invention]

[0007] However, the therapeutic potential of such oligonucleotides is limited by the delivery technologies that provide access to target tissues, organs, and cell types. Therefore, improved oligonucleotide-based delivery mechanisms are needed to address these challenges. [Means for solving the problem]

[0008] This application provides novel oligonucleotide agents comprising double-stranded RNA (dsRNA, double-stranded) and non-targeting auxiliary oligonucleotides (ACOs or single-stranded oligonucleotides) covalently linked to the dsRNA, which themselves constitute a system called an oligonucleotide-based delivery vehicle (ODV) with "self-delivery" properties. [Effects of the Invention]

[0009] The present inventors have surprisingly found that application of ODV to dsRNA (i.e., siRNA or saRNA) results in good biodistribution and activity upon local administration to selected tissues and upon systemic administration across several organs / tissues, including liver, muscle, lung, kidney, bladder, brain, spinal cord, heart, eye, spleen, etc. The agents possess unconventional nucleic acid chemistry and modification patterns that facilitate certain advantages associated with single-stranded oligonucleotide therapeutics, e.g., delivery, biodistribution, bioavailability, stability, cellular uptake, and other pharmacological properties, without the risk of compromising double-stranded activity.

[0010] ODV designs include RNA duplexes, such as siRNA and saRNA, consisting of two complementary or partially complementary strands, one of which is covalently linked to an ACO containing at least six nucleotides, with or without one or more linker moieties. The RNA duplex targets at least one nucleic acid sequence (e.g., mRNA) and is optionally chemically modified using oligonucleotide chemistry techniques (e.g., 2'-fluoro, 2'-O-methyl, phosphorothioate, mesylphosphoramidate or boranophosphate backbones, LNA, etc.) to promote in vivo activity, stability, and safety. The ACO component is not designed to specifically target the complementary nucleic acid sequence to be administered. ACO components can be chemically modified at their backbone, nucleoside, or other positions, such as phosphorothioate, mesyl phosphoramidate, or boranophosphate backbones, 2'-fluoro-2'-deoxynucleosides (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-(2-methoxyethyl) (2'-O-MOE), locked nucleic acids (LNA), bridged nucleic acids (BNA), peptide nucleic acids (PNA), 5'-(E)-vinylphosphonic acid moieties, and 5'-methylcytosine moieties, to confer physiochemical properties useful for improving drug bioavailability and delivery. The covalent linker moiety can be natural or unnatural nucleotides, ethylene glycol, carbohydrates, alkyl chains, or any other linker used to covalently link any two oligonucleotides located at the 3'- or 5'-end of one or both strands within an RNA duplex.

[0011] Embodiments of the present application are based, in part, on the surprising discovery that an oligonucleotide agent comprises: (a) a double-stranded oligonucleotide comprising a sense strand and an antisense strand, wherein the antisense strand has complementarity to a target nucleic acid; and (b) a non-targeting single-stranded oligonucleotide, wherein the single-stranded oligonucleotide is 6 to 22 nucleotides in length, wherein the double-stranded oligonucleotide and the single-stranded oligonucleotide are covalently linked, with or without one or more linking moieties, to form the oligonucleotide agent.

[0012] In certain embodiments of the present application, the double-stranded oligonucleotide is a small interfering RNA (siRNA) or a small activating RNA (saRNA).

[0013] In certain embodiments, the single-stranded oligonucleotide contains at least one phosphorothioate (PS) backbone substitution. In certain embodiments, the single-stranded oligonucleotide has at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the phosphodiester linkages on the backbone of the nucleotide sequence replaced with phosphorothioate (PS) linkages. In certain embodiments, the single-stranded oligonucleotide has 85-95% or 95-100% PS linkages.

[0014] In certain embodiments, the chemical modification in the double-stranded oligonucleotide or single-stranded oligonucleotide is the addition of a 5'-phosphonate moiety at the 5'-end of the nucleotide sequence.In certain embodiments, the chemical modification is the addition of a 5'-(E)-vinylphosphonate moiety.In certain embodiments, the chemical modification is the addition of a 5'-methylcytosine moiety at the 5'-end of the nucleotide sequence.

[0015] In certain embodiments, the single-stranded oligonucleotide is RNA, DNA, BNA, LNA, or PNA. In certain embodiments, the single-stranded oligonucleotide is 8 to 16 nucleotides in length. In certain embodiments, the single-stranded oligonucleotide is 10 to 14 nucleotides in length.

[0016] In certain embodiments of the present application, the sense strand of the double-stranded oligonucleotide in the oligonucleotide agent is at least 10 nucleotides in length. In certain embodiments of the present application, the sense strand has a length ranging from 10 to 60 nucleotides. In certain embodiments, the sense strand has a length ranging from 27 to 41 nucleotides.

[0017] In certain embodiments of the present application, the antisense strand has a nucleotide length ranging from 10 to 60 nucleotides, hi certain embodiments, the antisense strand has a nucleotide length ranging from 19 to 25 nucleotides.

[0018] In certain embodiments, the single-stranded oligonucleotide comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence selected from SEQ ID NOs: 1-22.

[0019] An ACO may also have a specific composition of nucleotides. In some embodiments, an ACO may have a specific percentage of adenines within the nucleotide sequence of the ACO. In some embodiments, the percent adenine composition is about 35% to about 65%. In some embodiments, the percent cytosine composition is about 35% to about 72%. In some embodiments, the percent guanosine composition is about 35% to about 65%. In some embodiments, the percent uracil composition is about 35% to about 72%. In some embodiments, the percent purine composition is about 64% to about 78%. In some embodiments, the percent pyrimidine composition is about 64% to about 86%. In some embodiments, the specific combination of purines and pyrimidines is about 42% to about 58% purines and about 42% to about 58% pyrimidines.

[0020] In some embodiments, the nucleotide sequence of the single-stranded oligonucleotide comprises at least about 14%, at least about 28%, at least about 42%, at least about 57%, at least about 71%, at least about 85%, at least about 92%, or about 70-100% of nucleotides having 2'Ome modifications.

[0021] In some embodiments, the nucleotide sequence of the single-stranded oligonucleotide is a palindromic sequence.

[0022] In some embodiments, the single-stranded oligonucleotide comprises a chemically modified nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% homologous, or 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1299-1379. In some embodiments, the single-stranded oligonucleotide comprises a chemically modified nucleotide sequence that has 0, 1, 2, or 3 different chemical modifications than the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1299-1379.

[0023] In some embodiments, the single-stranded oligonucleotide comprises a chemically modified nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% homologous, or 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1299-1379. In some embodiments, the single-stranded oligonucleotide comprises a chemically modified nucleotide sequence that has 0, 1, 2, or 3 different chemical modifications than the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1299-1379.

[0024] In some embodiments, the single-stranded oligonucleotide and the double-stranded oligonucleotide are linked without a linking moiety. In some embodiments, the single-stranded oligonucleotide and the double-stranded oligonucleotide are linked with one or more linking moieties. In some embodiments, the double-stranded oligonucleotide and the single-stranded oligonucleotide are covalently linked by a linking moiety. In some embodiments, the single-stranded oligonucleotide is linked with a linking moiety. In some embodiments, the 5'-end, 3'-end, or an internal nucleotide of the single-stranded oligonucleotide is linked to a linking moiety. In some embodiments, the double-stranded oligonucleotide comprises a sense strand and an antisense strand, and the single-stranded oligonucleotide is covalently linked to the sense strand, the antisense strand, or both the sense and antisense strands of the double-stranded oligonucleotide by a linking moiety. In some embodiments, the single-stranded oligonucleotide is covalently linked to the 3'-end, 5'-end, both the 3'-end and 5'-end, or an internal nucleotide of the sense strand of the double-stranded oligonucleotide. In some embodiments, the single-stranded oligonucleotide is covalently linked to the 3'-end, 5'-end, both the 3'-end and 5'-end, or an internal nucleotide of the antisense strand of the double-stranded oligonucleotide. In some embodiments, an internal nucleotide of the sense or antisense strand of the double-stranded oligonucleotide is replaced by a linking moiety, and the single-stranded oligonucleotide is covalently linked to the linking moiety.

[0025] In some embodiments, two or more single-stranded oligonucleotides are covalently linked to the double-stranded oligonucleotide, and in some embodiments, about 2-10 single-stranded oligonucleotides are covalently linked to the double-stranded oligonucleotide.

[0026] In some embodiments, two or more double-stranded oligonucleotides are covalently linked to the single-stranded oligonucleotide, and in some embodiments, about 2-10 double-stranded oligonucleotides are covalently linked to the single-stranded oligonucleotide.

[0027] In some embodiments, the linking moiety is linked to the nucleotides of the single-stranded oligonucleotide or the double-stranded oligonucleotide, or both the single-stranded oligonucleotide and the double-stranded oligonucleotide, via a phosphorothioate (PS) bond. In some embodiments, the substituted linking moiety is linked to each adjacent nucleotide or both adjacent nucleotides on the double-stranded oligonucleotide via a phosphorothioate (PS) bond.

[0028] In some embodiments, the linking moiety comprises a direct bond, or an oxygen or sulfur atom, or a unit selected from the group consisting of: NR, C(O), C(O)O, C(O)NR, SO, SO, and SONH; where R is hydrogen, acyl, aliphatic, or substituted aliphatic.

[0029] In some embodiments, the linking moiety is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkynyl and alkylaryl, alkenylheteroarylalkynylalkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynylalkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, where one or more methylenes are interrupted or terminated by O, S, S(O), SO, N(R'), C(O), a cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocycle.

[0030] In some embodiments, the linking moiety is selected from one or more of an ethylene glycol chain, an alkyl chain, an alkenyl chain, an alkynyl chain, a peptide, a carbohydrate, a thiol linkage, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, a tetrazole linkage, and a benzimidazole linkage.

[0031] In some embodiments, the connected component is selected from the group consisting of: a) L1 or S18 (spacer-18 linker) (1,1-bis(4-methoxyphenyl)-1-phenyl-2,5,8,11,14,17-hexaoxanonadecan-19-yl(2-cyanoethyl)diisopropylphosphoramidite); b) L4 or C6 (spacer-C6 linker) (6-(bis(4-methoxyphenyl)(phenyl)methoxy)hexyl(2-cyanoethyl)diisopropylphosphoramidite); c) L6 (1,1-bis(4-methoxyphenyl)-1-phenyl-2,5,8,11,14-pentaoxahexadecan-16-yl(2-cyanoethyl)diisopropylphosphoramidite); d) L6 (1,1-bis(4-methoxyphenyl)-1-phenyl-2,5,8,11,14-pentaoxahexadecan-16-yl(2-cyanoethyl)diisopropylphosphoramidite); e) L10 or C3 (spacer-C3 linker) (3-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl(2-cyanoethyl)diisopropylphosphoramidite); f) L12 (d spacer) ((2R,3S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuranpal-3-yl(2-cyanoethyl)diisopropylphosphoramidite); g) L13 or C12 (spacer-C12 linker) (12-(bis(4-methoxyphenyl)(phenyl)methoxy)dodecyl(2-cyanoethyl)diisopropylphosphoramidite); h) L14 (spacer-L14 linker) (((1r,4r)-4-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)cyclohexyl)methyl(2-cyanoethyl)diisopropylphosphoramidite); i) L15 (spacer-L15 linker) (4-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)phenethyl(2-cyanoethyl)diisopropylphosphoramidite); j) L16 (spacer-L16 linker) (2-(1-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)cyclohexyl)ethyl(2-cyanoethyl)diisopropylphosphoramidite); k) C6x1((2S,3S,4S,5S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-methoxy-4-(pent-4-yn-1-yloxy)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite); l) C6x2((2S,3S,4S,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2-methoxy-4-(pent-4-yn-1-yloxy)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite); m) C6x5(2-((2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(pent-4-yn-1-yl)amino)ethyl(2-cyanoethyl)diisopropylphosphoramidite); and n) C6x7 ((9H-Fluoren-9-yl)methyl (4-((2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((bis(diisopropylamino)phosphanyl)oxy)pyrrolidin-1-yl)-4-oxobutyl)carbamate).

[0032] In some embodiments, the oligonucleotide agent comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence selected from the group consisting of: a) siSOD1M2-AC2(N22)-S1V3v-Qu5 (SEQ ID NO: 58) and an antisense strand having the nucleotide sequence of SEQ ID NO: 57, which is partially complementary to the sense strand of siSOD1M2-AC2(N22)-S1V3v-Qu5 (SEQ ID NO: 58); b) siSOD1M2-AC2(N15)-S1V3v-Qu5 (SEQ ID NO: 60) and an antisense strand having the nucleotide sequence of SEQ ID NO: 57, which is partially complementary to the sense strand of siSOD1M2-AC2(N15)-S1V3v-Qu5 (SEQ ID NO: 60); c) siSOD1M2-AC2(N12)-S1V3v-Qu5 (SEQ ID NO: 62) and an antisense strand having the nucleotide sequence of SEQ ID NO: 57, which is partially complementary to the sense strand of siSOD1M2-AC2(N12)-S1V3v-Qu5 (SEQ ID NO: 62); and d) siSOD1M2-AC2(N6)-S1V3v-Qu5 (SEQ ID NO: 64) and an antisense strand having the nucleotide sequence of SEQ ID NO: 57 which is partially complementary to the sense strand of siSOD1M2-AC2(N6)-S1V3v-Qu5 (SEQ ID NO: 64): e) the oligonucleotide agent of any one of claims 1-27, wherein the oligonucleotide agent comprises a nucleotide sequence that is at least 90% identical to the following nucleotide sequence: f) siHTT-AC2-S1L1 (SEQ ID NO: 28) and an antisense strand having the nucleotide sequence of SEQ ID NO: 27, which has partial complementarity to the sense strand of siHTT-AC2-S1L1 (SEQ ID NO: 28).

[0033] In some embodiments, the oligonucleotide agent comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence selected from the group consisting of: a) siApp-8-AC2(N18)-S1L1V3v (SEQ ID NO: 32) and an antisense strand having the nucleotide sequence of SEQ ID NO: 31, which is partially complementary to the sense strand of siApp-8-AC2(N18)-S1L1V3v (SEQ ID NO: 32); b) siApp-8-AC2(N15)-S1L1V3v (SEQ ID NO: 34) and an antisense strand having the nucleotide sequence of SEQ ID NO: 31, which has partial complementarity to the sense strand of siApp-8-AC2(N15)-S1L1V3v (SEQ ID NO: 34); and c) siApp-8-AC2(N12)-S1L1V3v (SEQ ID NO: 36) and an antisense strand having the nucleotide sequence of SEQ ID NO: 31, which is partially complementary to the sense strand of siApp-8-AC2(N12)-S1L1V3v (SEQ ID NO: 36).

[0034] In some embodiments, the sense strand or the antisense strand of the double-stranded oligonucleotide has a nucleotide sequence that is at least 90% identical to a nucleotide sequence selected from the group consisting of: R6-04(20)-S1V1v(CM-4) (SEQ ID NO: 66) or R6-04(20)-S1V1v(CM-4) (SEQ ID NO: 67). In some embodiments, the oligonucleotide agent comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence selected from the group consisting of: a) R6-04M1-AC2(18)-S1L1V3v (SEQ ID NO: 68) and an antisense strand having the nucleotide sequence of SEQ ID NO: 67, which has partial complementarity to the sense strand of R6-04M1-AC2(18)-S1L1V3v (SEQ ID NO: 68); b) R6-04M1-AC2(16)-S1L1V3v (SEQ ID NO: 70) and an antisense strand having the nucleotide sequence of SEQ ID NO: 67, which has partial complementarity to the sense strand of R6-04M1-AC2(16)-S1L1V3v (SEQ ID NO: 70); c) R6-04M1-AC2(15)-S1L1V3v (SEQ ID NO: 72) and an antisense strand having the nucleotide sequence of SEQ ID NO: 67, which is partially complementary to the sense strand of R6-04M1-AC2(15)-S1L1V3v (SEQ ID NO: 72); d) R6-04M1-AC2(14)-S1L1V3v (SEQ ID NO: 74) and an antisense strand having the nucleotide sequence of SEQ ID NO: 67, which has partial complementarity to the sense strand of R6-04M1-AC2(14)-S1L1V3v (SEQ ID NO: 74); e) R6-04M1-AC2(13)-S1L1V3v (SEQ ID NO: 76) and an antisense strand having the nucleotide sequence of SEQ ID NO: 67, which is partially complementary to the sense strand of R6-04M1-AC2(13)-S1L1V3v (SEQ ID NO: 76); f) R6-04M1-AC2(12)-S1L1V3v (SEQ ID NO: 78) and an antisense strand having the nucleotide sequence of SEQ ID NO: 67, which is partially complementary to the sense strand of R6-04M1-AC2(12)-S1L1V3v (SEQ ID NO: 78); g) R6-04M1-AC2(11)-S1L1V3v (SEQ ID NO: 80) and an antisense strand having the nucleotide sequence of SEQ ID NO: 67, which is partially complementary to the sense strand of R6-04M1-AC2(12)-S1L1V3v (SEQ ID NO: 80); h) R6-04M1-AC2(10)-S1L1V3v (SEQ ID NO: 82) and an antisense strand having the nucleotide sequence of SEQ ID NO: 67, which is partially complementary to the sense strand of R6-04M1-AC2(12)-S1L1V3v (SEQ ID NO: 82); i) R6-04M1-AC2(9)-S1L1V3v (SEQ ID NO: 84) and an antisense strand having the nucleotide sequence of SEQ ID NO: 67, which has partial complementarity to the sense strand of R6-04M1-AC2(9)-S1L1V3v (SEQ ID NO: 84); and j) R6-04M1-AC2(8)-S1L1V3v (SEQ ID NO: 86) and an antisense strand having the nucleotide sequence of SEQ ID NO: 67, which is partially complementary to the sense strand of R6-04M1-AC2(12)-S1L1V3v (SEQ ID NO: 86).

[0035] In some embodiments, single-stranded oligonucleotide is bound to one or more conjugate groups.In some embodiments, double-stranded oligonucleotide is bound to one or more conjugate groups.In some embodiments, the sense strand or antisense strand of double-stranded oligonucleotide is bound to one or more conjugate groups.

[0036] In some embodiments, the conjugate group is selected from one or more of a lipid, a fatty acid, a fluorophore, a ligand, a sugar, a peptide, and an antibody. In some embodiments, the one or more conjugate groups are selected from a cell-penetrating peptide, polyethylene glycol, an alkaloid, a tryptamine, a benzimidazole, a quinolone, an amino acid, cholesterol, glucose, and N-acetylgalactosamine.

[0037] In some embodiments, each of the sense and antisense strands independently has a length of nucleotides in the range of 15 to 35 nucleotides.

[0038] In some embodiments, the sense strand or the antisense strand of the double-stranded oligonucleotide has a nucleotide sequence that is at least 90% identical to the nucleotide sequence of siApp-8-S1V1 (SEQ ID NO: 28) or siApp-8-S1V1 (SEQ ID NO: 27).

[0039] In some embodiments, the oligonucleotide agent comprises a small interfering RNA (siRNA), wherein the siRNA comprises a sense strand and an antisense strand and forms a double-stranded structure, wherein the antisense strand comprises a nucleotide sequence comprising at least 10 consecutive nucleotides, having 0, 1, 2 or 3 mismatches, and having at least 85% nucleotide sequence complementarity or homology to a portion of the nucleotide sequence of SEQ ID NO:895, wherein the oligonucleotide agent is capable of inhibiting expression of superoxide dismutase 1 (SOD1) in a cell.

[0040] In some embodiments, the sense strand of the double-stranded oligonucleotide has a nucleotide sequence that is at least 90% identical to a nucleotide sequence selected from the group consisting of: siSOD1-5 (SEQ ID NO: 357), siSOD1-8 (SEQ ID NO: 358), siSOD1-10 (SEQ ID NO: 359), siSOD1-11 (SEQ ID NO: 360), siSOD-17 (SEQ ID NO: 357), siSOD1-35 (SEQ ID NO: 362), and siSOD1-37 to siSOD1-447 (SEQ ID NOs: 363-624). In some embodiments, the antisense strand of the double-stranded oligonucleotide has a nucleotide sequence that is at least 90% identical to a nucleotide sequence selected from the group consisting of: siSOD1-5 (SEQ ID NO: 626), siSOD1-8 (SEQ ID NO: 627), siSOD1-10 (SEQ ID NO: 628), siSOD1-11 (SEQ ID NO: 629), siSOD-17 (SEQ ID NO: 630), siSOD1-35 (SEQ ID NO: 631), and siSOD1-37 to siSOD1-447 (SEQ ID NOs: 632 to 893).

[0041] In some embodiments, the sense strand of the double-stranded oligonucleotide has a nucleotide sequence that is at least 90% identical to a nucleotide sequence selected from the group consisting of: siSOD1-231-E (SEQ ID NO: 38); siSOD1-231-TT (SEQ ID NO: 40); siSOD1-231-M1 (SEQ ID NO: 42); siSOD1-231-S2 (SEQ ID NO: 44); siSOD1-388-E (SEQ ID NO: 46); siSOD1-388-TT (SEQ ID NO: 48); siSOD1-388-M1 (SEQ ID NO: 50); siSOD1-388-S2 (SEQ ID NO: 52); siSOD1M2-L1 (SEQ ID NO: 54); and siSOD1M2-S1V5 (SEQ ID NO: 56).

[0042] In some embodiments, the antisense strand of the double-stranded oligonucleotide has a nucleotide sequence that is at least 90% identical to a nucleotide sequence selected from the group consisting of: siSOD1-231-E (SEQ ID NO: 39); siSOD1-231-TT (SEQ ID NO: 41); siSOD1-231-M1 (SEQ ID NO: 43); siSOD1-231-S2 (SEQ ID NO: 45); siSOD1-388-E (SEQ ID NO: 47); siSOD1-388-TT (SEQ ID NO: 49); siSOD1-388-M1 (SEQ ID NO: 51); siSOD1-388-S2 (SEQ ID NO: 53); siSOD1M2-L1 (SEQ ID NO: 47); and siSOD1M2-S1V1v-Qu5 (SEQ ID NO: 57).

[0043] In some embodiments, the sense strand of the siRNA has a nucleotide sequence having at least 85% homology to a nucleotide sequence selected from the group consisting of: DS17-0001 (SEQ ID NO: 384), DS17-0002 (SEQ ID NO: 372), DS17-0003 (SEQ ID NO: 409), DS17-0004 (SEQ ID NO: 357), DS17-0005 (SEQ ID NO: 486), DS17-0029 (SEQ ID NO: 588), DS17-01N3 (SEQ ID NO: 912), DS17-02N3 (SEQ ID NO: 914), DS17-03N3 (SEQ ID NO: 916), DS17-04N3 (SEQ ID NO: 918), DS17-05N3 (SEQ ID NO: 920), and any of SEQ ID NOs: 976-1021.

[0044] In some embodiments, the antisense strand of the siRNA has a nucleotide sequence having at least 85% homology to a nucleotide sequence selected from the group consisting of: DS17-0001 (SEQ ID NO: 653), DS17-0002 (SEQ ID NO: 641), DS17-0003 (SEQ ID NO: 678), DS17-0004 (SEQ ID NO: 626), DS17-0005 (SEQ ID NO: 755), DS17-0029 (SEQ ID NO: 857), DS17-01N3 (SEQ ID NO: 913), DS17-02N3 (SEQ ID NO: 915), DS17-03N3 (SEQ ID NO: 917), DS17-04N3 (SEQ ID NO: 919), DS17-05N3 (SEQ ID NO: 921), and SEQ ID NOs: 1022-1067.

[0045] In some embodiments, the sense and antisense strands of the siRNA have nucleotide sequences that are independently at least 85% homologous to a pair of nucleotide sequences selected from the group consisting of: a) DS17-0001 (SEQ ID NO: 384 and SEQ ID NO: 653), b) DS17-0002 (SEQ ID NO: 372 and SEQ ID NO: 641); c) DS17-0003 (SEQ ID NO: 409 and SEQ ID NO: 678); d) DS17-0004 (SEQ ID NO: 357 and SEQ ID NO: 626); e) DS17-0005 (SEQ ID NO: 486 and SEQ ID NO: 755); f) DS17-0029 (SEQ ID NO: 588 and SEQ ID NO: 857); g) DS17-01N3 (SEQ ID NO: 912 and SEQ ID NO: 913); h) DS17-02N3 (SEQ ID NO: 914 and SEQ ID NO: 915); i) DS17-03N3 (SEQ ID NO: 916 and SEQ ID NO: 917); j) DS17-04N3 (SEQ ID NO: 918 and SEQ ID NO: 919), and k) DS17-05N3 (SEQ ID NO: 920 and SEQ ID NO: 921).

[0046] In some embodiments, the sense and antisense strands of the siRNA have nucleotide sequences that are independently at least 85% homologous to a pair of nucleotide sequences selected from the group consisting of: a) DS17-01M3 (SEQ ID NO: 922 and SEQ ID NO: 923); b) DS17-02M3 (SEQ ID NO: 924 and SEQ ID NO: 925); c) DS17-03M3 (SEQ ID NO: 926 and SEQ ID NO: 927); d) DS17-04M3 (SEQ ID NO: 928 and SEQ ID NO: 929), and e) DS17-05M3 (SEQ ID NO: 930 and SEQ ID NO: 931).

[0047] In some embodiments, the oligonucleotide agent comprises an siRNA and a non-targeted ACO, wherein the ACO comprises a nucleotide sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO:954, and the oligonucleotide agent is capable of inhibiting expression of superoxide dismutase 1 (SOD1) in a cell.

[0048] In some embodiments, the sense and antisense strands of the siRNA have nucleotide sequences that are independently at least 85% homologous to a pair of nucleotide sequences selected from the group consisting of: a) DS17-01M3 (SEQ ID NO: 922 and SEQ ID NO: 923); b) DS17-02M3 (SEQ ID NO: 924 and SEQ ID NO: 925); c) DS17-03M3 (SEQ ID NO: 926 and SEQ ID NO: 927); d) DS17-04M3 (SEQ ID NO: 928 and SEQ ID NO: 929); e) DS17-05M3 (SEQ ID NO: 930 and SEQ ID NO: 931); f) DS17-01M3-AC1(mel4)-L9V3 (SEQ ID NO: 932 and SEQ ID NO: 933); g) DS17-02M3-AC1(mel4)-L9V3 (SEQ ID NO: 934 and SEQ ID NO: 935); h) DS17-03M3-AC1(mel4)-L9V3 (SEQ ID NO: 936 and SEQ ID NO: 937); i) DS17-04M3-AC1(mel4)-L9V3 (SEQ ID NO: 938 and SEQ ID NO: 939); j) DS17-05M3-AC1(mel4)-L9V3 (SEQ ID NO: 940 and SEQ ID NO: 941); k) DS17-29M2-AC1(mel4)-L9V3 (SEQ ID NO: 942 and SEQ ID NO: 47) l) DS17-01M3v-AC1(mel4)-L9V3 (SEQ ID NO: 932 and SEQ ID NO: 47); m) DS17-02M3v-AC1(mel4)-L9V3 (SEQ ID NO: 934 and SEQ ID NO: 943); n) DS17-03M3v-AC1(mel4)-L9V3 (SEQ ID NO: 936 and SEQ ID NO: 944); o) DS17-04M3v-AC1(mel4)-L9V3 (SEQ ID NO: 938 and SEQ ID NO: 950); p) DS17-05M3v-AC1(mel4)-L9V3 (SEQ ID NO: 940 and SEQ ID NO: 951), and q) DS17-04M3-asSOD1-1-L9V3 (SEQ ID NO: 952 and SEQ ID NO: 939).

[0049] In some embodiments, the oligonucleotide agent comprises a non-targeted ACO-conjugated sense strand of an siRNA and an antisense strand of the siRNA, wherein the non-targeted ACO-conjugated sense strand comprises a linking moiety covalently linking the ACO and the sense strand, wherein the antisense strand comprises a nucleotide sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 57. In some embodiments, the non-targeted ACO-conjugated sense strand comprises a nucleotide sequence that is at least 90%, at least 95%, or 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1197-1288 and 1291-1298. In some embodiments, the linking moiety is selected from the group of linking moieties listed in SEQ ID NOs: 1197-1288 in Table 28 and SEQ ID NOs: 1291-1298 in Table 30.

[0050] In some embodiments, the single-stranded oligonucleotide of an oligonucleotide agent improves the stability, bioavailability, biodistribution, and / or cellular uptake of the double-stranded oligonucleotide compared to the oligonucleotide agent without the single-stranded oligonucleotide.

[0051] In some embodiments, the single-stranded oligonucleotide of the oligonucleotide agent increases the biodistribution of the double-stranded oligonucleotide in one or more target tissues compared with the oligonucleotide agent that does not contain the single-stranded oligonucleotide.In some embodiments, the one or more target tissues are selected from the tissues of the brain, spinal cord, muscle, spleen, lung, heart, liver, bladder, and kidney.In some embodiments, the one or more target tissues are selected from the group consisting of the prefrontal cortex, cerebellum, and the rest of the brain; the cervical, thoracic, and lumbar regions of the spinal cord; the heart, forelimbs, hind limbs, nape, and buttocks.

[0052] At least some of the present disclosure provides siRNAs comprising an oligonucleotide sequence having a length ranging from 16 to 35 contiguous nucleotides, wherein the oligonucleotide sequence comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% homology or complementarity to an equal length portion of SEQ ID NO:59, and wherein the siRNA inhibits mRNA transcription of the SOD1 gene by at least 80% compared to baseline Sod1 mRNA levels.

[0053] In certain embodiments, the nucleotide sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% homology or complementarity to an equal length portion of SEQ ID NO:61 or 63, and the oligonucleotide agent inhibits mRNA transcription of the SOD1 gene by at least 80% compared to baseline Sod1 mRNA levels.

[0054] A particular embodiment of the present application relates to a hotspot in the 3'UTR of the SOD1 gene, wherein the hotspot has a nucleic acid sequence selected from SEQ ID NO: 61 (H1) and SEQ ID NO: 63 (H2).

[0055] Certain embodiments of the present application relate to siRNA target sequences in mRNA transcription products from the 3'-UTR of the SOD1 gene, wherein the target sequence in the mRNA transcription product has at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% homology to a sequence selected from SEQ ID NOs: 1068-1113.

[0056] Certain embodiments of the present application also relate to siRNA target sequences in mRNA transcription products from the SOD1 gene, wherein the target sequence in the mRNA transcription product has at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% homology to a sequence selected from SEQ ID NOs: 88-355.

[0057] Certain embodiments of the present application also relate to siRNAs comprising a sense strand and an antisense strand, wherein the sense strand of the siRNA has a nucleotide sequence having at least 85%, at least 90%, at least 95%, or 100% homology to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 976-1021, and wherein the siRNA inhibits mRNA transcription of the SOD1 gene by at least 80% compared to baseline Sod1 mRNA levels.

[0058] The present disclosure further provides an siRNA comprising a sense strand and an antisense strand, wherein the antisense strand of the siRNA has a nucleotide sequence having at least 85%, at least 90%, at least 95%, or 100% homology to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1022-1067, and wherein the siRNA inhibits mRNA transcription of the SOD1 gene by at least 80% compared to baseline Sod1 mRNA levels.

[0059] In some embodiments, the sense strand and antisense strand of the siRNA have nucleotide sequences that are independently at least 85%, at least 90%, at least 95%, or 100% homologous to a pair of nucleotide sequences selected from the group consisting of siSOD1-547 to siSOD1-694 (sense strands of SEQ ID NOs: 976 to 1021 and antisense strands of SEQ ID NOs: 1022 to 1067, respectively, in Table 22).

[0060] At least a portion of the present disclosure also relates to an antisense oligonucleotide (ASO) comprising an oligonucleotide sequence having a length ranging from 12 to 30 contiguous nucleotides, wherein the oligonucleotide sequence comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% homology or complementarity to an equal length portion of SEQ ID NO:59, and wherein the ASO inhibits mRNA transcription of the SOD1 gene by at least 60% compared to baseline mRNA levels of the SOD1 gene.

[0061] In some embodiments, the ASO has at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% complementarity to an equal length portion of SEQ ID NO:65, and the ASO inhibits mRNA transcription of the SOD1 gene by at least 60% compared to baseline Sod1 mRNA levels.

[0062] A particular embodiment of the present application relates to a hotspot in the 3'UTR of the SOD1 gene, wherein the hotspot has the nucleic acid sequence of SEQ ID NO: 65 (H3).

[0063] Certain embodiments of the present application relate to an ASO target sequence in an mRNA transcription product from the 3'-UTR of the SOD1 gene, wherein the target sequence in the mRNA transcription product has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% homology to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1114-1154.

[0064] Certain embodiments of the present application also relate to ASOs comprising a single-stranded oligonucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% homology or 100% identity to a nucleotide sequence selected from the group consisting of chemically modified SEQ ID NOs: 1155-1195, and their unmodified SEQ ID NOs: 1114-1154, wherein the ASO inhibits mRNA transcription of the SOD1 gene by at least 60% compared to baseline Sod1 mRNA levels.

[0065] Also provided herein are vectors and cells comprising the disclosed oligonucleotide agents. In some embodiments, the cells are mammalian cells, optionally human cells. In some embodiments, the cells are host cells. In some embodiments, the cells are in vitro. In some embodiments, the cells are present in a mammal.

[0066] Certain embodiments of the present application relate to pharmaceutical compositions containing an oligonucleotide agent, including: (a) a double-stranded oligonucleotide comprising a sense strand and an antisense strand, wherein the antisense strand is complementary to a target nucleic acid; and (b) a non-targeted single-stranded oligonucleotide, wherein the single-stranded oligonucleotide is 6 to 22 nucleotides in length, and the double-stranded oligonucleotide and the single-stranded oligonucleotide are covalently linked, with or without one or more linking moieties, to form the oligonucleotide agent. The target nucleic acid can be any target nucleic acid. Examples of target nucleic acids include, but are not limited to, the SOD1 gene, the HTT gene, the App gene, the SMN2 gene, and the like.

[0067] In certain embodiments, the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier selected from an aqueous carrier, a liposome or LNP, a polymer, a micelle, a colloid, a metallic nanoparticle, a non-metallic nanoparticle, a bioconjugate, and a polypeptide.

[0068] In certain embodiments, the pharmaceutical composition reduces or inactivates transcription of the SOD1 gene or SOD1 protein.

[0069] In certain embodiments, the pharmaceutical composition increases or activates expression of the HTT, App or SMN2 gene or HTT, App or SMN2 protein.

[0070] Also provided herein are kits that include an oligonucleotide agent described herein or a pharmaceutical composition of this disclosure.

[0071] Certain embodiments relate to kits comprising the pharmaceutical compositions of the present disclosure.

[0072] Certain embodiments relate to methods of reducing or silencing transcription of the SOD1 gene or protein, comprising administering to a subject a pharmaceutical composition of the present disclosure.

[0073] Certain embodiments relate to methods of treating or delaying the onset or progression of amyotrophic lateral sclerosis (ALS) in a subject, the method comprising: administering to the subject a pharmaceutical composition of the present disclosure. In certain embodiments, the subject has sporadic ALS (sALS). In certain embodiments, the subject has familial ALS (fALS).

[0074] Certain embodiments of the present application relate to methods for increasing or activating expression of the HTT gene or huntingtin protein, comprising administering to a subject a pharmaceutical composition.

[0075] Certain embodiments of the present application relate to methods for treating or delaying the onset or progression of Huntington's disease in a subject, the methods comprising: administering to the subject a pharmaceutical composition.

[0076] Certain embodiments of the present application relate to methods for treating or delaying the onset or progression of spinal muscular atrophy (SMA) in a subject, the methods comprising: administering to the subject a pharmaceutical composition.

[0077] Certain embodiments of the present application relate to methods for increasing or activating expression of the App gene or amyloid precursor protein (APP), comprising administering to a subject a pharmaceutical composition.

[0078] Certain embodiments of the present application relate to methods for treating or delaying the onset or progression of APP-related diseases, including cerebral amyloid angiopathy App-associated (CAA-APP) and Alzheimer's disease (AD), in a subject, the methods comprising administering to the subject a pharmaceutical composition.

[0079] Certain embodiments of the present application relate to methods for increasing or activating expression of the SMN2 gene, comprising administering to a subject a pharmaceutical composition.

[0080] Certain embodiments of the present application relate to methods for treating or delaying the onset or progression of spinal muscular atrophy (SMA) in a subject, the methods comprising: administering to the subject a pharmaceutical composition of the present disclosure.

[0081] In certain embodiments, the pharmaceutical composition reduces or inactivates the expression of the SOD1 gene or protein.

[0082] In certain embodiments, the single-stranded oligonucleotide of an oligonucleotide agent improves the stability, bioavailability, biodistribution, and / or cellular uptake of the double-stranded oligonucleotide compared to the oligonucleotide agent without the single-stranded oligonucleotide.

[0083] In certain embodiments, the single-stranded oligonucleotide of an oligonucleotide agent increases the biodistribution of the double-stranded oligonucleotide in one or more target tissues compared to an oligonucleotide agent that does not include the single-stranded oligonucleotide.

[0084] In certain embodiments, the single-stranded oligonucleotide of an oligonucleotide agent increases the biodistribution of the double-stranded oligonucleotide in two or more target cell types in tissues compared to an oligonucleotide agent that does not contain a single-stranded oligonucleotide.In certain embodiments, one or more target tissues are selected from tissues from the brain, spinal cord, muscle, spleen, lung, heart, liver, bladder, and kidney.In certain embodiments, one or more target tissues are selected from the group consisting of the prefrontal cortex, cerebellum, and the rest of the brain; the cervical, thoracic, and lumbar regions of the spinal cord; the heart, forelimbs, hind limbs, nape, and buttocks.

[0085] Certain embodiments of the present application relate to the use of an oligonucleotide agent of the present disclosure in the manufacture of a medicament for treating or delaying the onset or progression of amyotrophic lateral sclerosis (ALS).

[0086] Certain embodiments of the present application relate to the use of a pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating or delaying the onset or progression of amyotrophic lateral sclerosis (ALS). In certain embodiments, ALS includes sporadic ALS (sALS) and / or familial ALS (fALS).

[0087] Certain embodiments of the present application relate to an oligonucleotide agent of the present disclosure for use in treating or delaying the onset or progression of amyotrophic lateral sclerosis (ALS), optionally wherein ALS includes sporadic ALS (sALS) and / or familial ALS (fALS).

[0088] Certain embodiments of the present application also relate to pharmaceutical compositions of the present disclosure for use in treating or delaying the onset or progression of amyotrophic lateral sclerosis (ALS), optionally wherein ALS includes sporadic ALS (sALS) and / or familial ALS (fALS). [Brief explanation of the drawings]

[0089] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are employed, and the accompanying drawings (also referred to herein as "Figure" and "FIG"), in which: [Figures 1A-1C] Visual illustration of an exemplary ODV structure. A double-stranded RNA (dsRNA) duplex (siRNA or saRNA) is linked to a single-stranded accessory oligonucleotide (ACO) at the 3'- (Figure 1A), 5'- (Figure 1B), or internal position (Figure 1C) of its passenger (P) strand. Also labeled are the guide (G) strand and linker (L) that bridge the ACO to each dsRNA. [Figure 2]The ESI mass spectra and RP-HPLC profiles of purified ODV compounds siSOD1M2-AC2(N22)-S1V3v and siSOD1M2-AC2(N6)-S1V3v are shown, compared with siRNA duplexes with and without a linker (siSOD1M2-L1 and siSOD1-388-E). All duplexes contained Quasar 570 (Qu5) dye attached to the 5'-end of the passenger strand. RP-HPLC analysis by reversed-phase chromatography was performed using an acetonitrile gradient at a flow rate of 1.0 mL / min and a detection wavelength of 260 nm. [Figure 3A-3B] Figure 3 shows the in vivo knockdown activity of ODV-optimized siRNA (siHTT-AC2-S1L1) on Htt mRNA expression in the brain and spinal cord of C57BL / 6 pups (PND4). siRNA was injected intravenously at the indicated doses. Saline was injected as a negative control. siHTT-S1V1 lacks the ODV component and served as a comparison for siHTT-AC2-S1L1 activity. Mice were sacrificed 3 days after treatment. Brain (Figure 3A) and spinal cord (Figure 3B) tissue samples were collected for analysis by RT-qPCR. Htt mRNA levels are the average of two mice per group (n=2) after normalization to baseline Tbp levels relative to saline treatment. [Figure 4] Figure 1 shows the in vitro knockdown activity of App mRNA by ODV-siRNA with different lengths of ACO. NSC-34 cells were treated with 1 or 10 nM siRNA for 24 hours. Mock treatment was transfected in the absence of oligonucleotide. dsCon2 was used as a nonspecific control duplex. App mRNA levels were quantified by RT-qPCR using a gene-specific primer set. Tbp was amplified as an internal reference. The mean expression value of App mRNA relative to mock treatment after normalization with the Tbp reference level is shown. [Figure 5]The effect of ACO on the in vivo knockdown activity of ODV-siRNA in the CNS is shown. All siRNAs were injected ICV at a dose of 40 mg / kg into C57BL / 6 pups (PND4). Saline was injected as a negative control. Mice were sacrificed 3 days after administration. Brain and spinal cord tissue samples were collected for analysis by RT-qPCR. App mRNA levels are the average of three mice / group (n=3) relative to saline treatment after normalization to Tbp baseline levels. [Figures 6A-6B] This study demonstrates upregulation of SMN2 mRNA transcripts via ODV-optimized saRNA in human primary cells. The saRNA duplex, designated R6-04(20)-S1V1v(CM-4), is an activator of human SMN2 gene expression. Several ODV variants of R6-04(20)-S1V1v(CM-4) were synthesized with ACOs ranging in length from 8 to 18 nucleotides. Primary human fibroblasts derived from SMA Type-2 (GM03813 cells) and SMA Type-1 (GM09677 cells) patients were transfected with each ODV-saRNA variant at 25 nM for 3 days. Mock treatment was performed without oligonucleotide transfection. dsCon2 was used as a nonspecific control duplex. Both the full-length (SMN2FL) and Δ7 (SMN2Δ7) splice variants of SMN2 were quantified by RT-qPCR using isoform-specific primer sets. TBP was amplified as an internal reference. The mean expression values ​​of SMN2FL and SMN2Δ7 transcripts in GM03813 (FIG. 6A) and GM09677 (FIG. 6B) cells relative to mock treatment after normalization with TBP reference levels are shown. [Figures 7A-7B]This figure shows the associated upregulation of SMN2 protein by ODV-saRNA in human primary cells. R6-04(20)-S1V1v(CM-4) and its ODV-saRNA variants were transfected into patient-derived cells from GM03813 and GM09677 at 25 nM for 3 days. Mock treatment was performed without oligonucleotide transfection. dsCon2 was used as a nonspecific control duplex. Whole-cell protein extracts were harvested for immunoblot analysis. Total SMN protein levels were detected using a promiscuous monoclonal antibody that recognizes both the SMN1 and SMN2 gene products. Immunodetection of α / β-tubulin was used as protein loading. Scanning optical densitometry was used to quantify protein band intensity from immunoblot images (not shown). The relative changes in total SMN protein levels after normalization with α / β-tubulin band intensity in GM03813 (FIG. 7A) and GM09677 (FIG. 7B) cells are shown relative to mock treatment. [Figure 8] This figure shows ODV-saRNA-mediated upregulation of SMN2 mRNA transcripts in primary mouse hepatocytes (PMH) transfected with the human SMN2 gene. PMH cells derived from livers harvested from an SMA-like mouse model were transfected with R6-04(20)-S1V1v (CM-4) and its ODV-saRNA variants at 25 nM for 3 days. Mock treatment was performed without oligonucleotide transfection. dsCon2 was used as a nonspecific control duplex. The full-length (SMN2FL) and Δ7 (SMN2Δ7) splice variants of the human SMN2 gene were quantified by RT-qPCR using isoform-specific primer sets. Mouse Tbp (mTbp) was amplified as an internal reference. The mean expression values ​​of SMN2FL and SMN2Δ7 transcripts relative to mock treatment are shown after normalization to the mTbp reference level. [Figure 9]High-throughput screening data comparing the knockdown activity of 268 siRNAs against human Sod1 mRNA. HEK293A cells were transfected with 0.1 and 10 nM of each siRNA duplex for 24 hours. Mock treatment was transfected in the absence of oligonucleotides. SOD1 expression levels were quantified by RT-qPCR using a gene-specific primer set. TBP was amplified as an internal reference. Average Sod1 mRNA expression values ​​are shown for mock treatment after normalization to the TBP reference level at both treatment concentrations. [Figure 10] Figure 1 shows the lack of cytotoxicity of the top 30 SOD1 siRNAs in HEK293A cells. HEK293A cells were transfected with increasing dose concentrations representing approximate multiples of the IC50 (half-maximal inhibitory concentration) values. Mock treatments were transfected in the absence of oligonucleotide. mRNA expression and cytotoxicity were quantified for each siRNA at each dose by RT-qPCR and PI staining, respectively. Plotted is the optical density (OD) of PI staining compared to SOD1 knockdown relative to mock treatment.

[0090] [Figure 11] Figure 1 shows SOD1 knockdown by six lead siRNAs in human neuroblastoma cell lines. SH-SY5Y cells were treated with 1 and 10 nM SOD1 siRNAs (i.e., siSOD1-63, siSOD1-47, siSOD1-104, siSOD1-5, siSOD1-231, and siSOD1-388) for 24 hours. Mock treatments were transfected in the absence of oligonucleotides. dsCon2 was used as a nonspecific control duplex. Sod1 mRNA levels were quantified by RT-qPCR using a gene-specific primer set. TBP was amplified as an internal reference. Average SOD1 expression values ​​relative to mock treatment after normalization with TBP are shown. [Figures 12A-12B]Figure 12 shows the Sod1 knockdown of four lead siRNAs targeting conserved sequences in mouse motor neuron-like cell lines. NSC-34 and N-2a cells were treated with 1 and 10 nM SOD1 siRNAs (i.e., siSOD1-231, siSOD1-229, siSOD1-388, and siSOD1-387) for 24 hours. Mock treatments were transfected in the absence of oligonucleotides. dsCon2 was used as a nonspecific control duplex. Mouse Sod1 levels were quantified by RT-qPCR using a gene-specific primer set. Mouse Tbp was amplified as an internal reference. The mean expression values ​​of Sod1 transcripts relative to mock treatment are shown in NSC-34 (Figure 12A) and N-2a (Figure 12B) cells after normalization to the mTbp reference level. [Figures 13A-13C] Figure 13 shows the effect of medicinal chemistry on the knockdown activity of siSOD1-231 and siSOD1-388. The chemical modification patterns and duplex structures used to test knockdown activity are shown in Figure 13A using siSOD1-388 as a model sequence. Modification symbols: bold uppercase letters are 2'Ome, lowercase letters are 2'F, chevron (^) is PS, and T is deoxythymidine. Human 239A and mouse NE-4C (neuroepithelial) cells were treated with chemically modified versions of siSOD1-231 (i.e., siSOD1-231-E, siSOD1-231-TT, siSOD1-231-M1, or siSOD1-231-S2) and siSOD1-388 (i.e., siSOD1-388-E, siSOD1-388-TT, siSOD1-388-M1, or siSOD1-388-S2) at 1 and 10 nM for 24 h. Mock treatment was performed by transfection in the absence of oligonucleotides. dsCon2 was used as a nonspecific control duplex. SOD1 / SOD1 expression levels were quantified by RT-qPCR using species-specific gene primer sets. TBP / Tbp was amplified as an internal reference. The mean expression values ​​of SOD1 / Sod1 transcripts in HEK293A cells (FIG. 13A) and NE-2C cells (FIG. 13B) are shown relative to mock treatment after normalization with internal reference levels. [Figure 14]Figure 1 shows the in vitro knockdown activity of Sod1 mRNA by ODV-optimized siSOD1-388-E [siSOD1M2-AC2(N15)-S1V3v-Qu5]. NSC-34 cells were treated with 0.1 or 1 nM of the indicated siRNA for 3 days. Mock treatments were transfected in the absence of oligonucleotides. dsCon2 was used as a nonspecific control duplex. Sod1 mRNA levels were quantified by RT-qPCR using a gene-specific primer set. Tbp was amplified as an internal reference. Average Sod1 mRNA expression values ​​relative to mock treatment are shown after normalization to the Tbp reference level. [Figures 15A-15C] Biodistribution and in vivo knockdown activity of siSOD1M2-AC2(N15)-S1V3v-Qu5 in organs of pups (PND 4) are shown. Qu5-labeled ODV-siRNA (siSOD1M2-AC2(N15)-S1V3v-Qu5) was injected ICV into C57BL / 6 pups (PND 4) at a dose of 40 mg / kg. The Qu5-labeled siRNA variant siSOD1M2-S1V1v-Qu5, which lacks any auxiliary oligonucleotides, served as a control. Mice were sacrificed three days after treatment, and whole-organ fluorescence was quantified using an IVIS Imaging System with an excitation filter of 520 nm and an emission filter of 570 nm. Figure 15A shows example IVIS images showing the biodistribution of siSOD1M2-AC2(N15)-S1V3v-Qu5 via Qu5 signal in all major organs compared to siSOD1M2-AC2(N15)-S1V3v-Qu5 after ICV injection. Figure 15B shows quantification of the fluorescence intensity of siSOD1M2-AC2(N15)-S1V3v-Qu5 released from each organ. Sod1 mRNA knockdown was quantified in organ tissues by RT-qPCR using gene-specific primer sets. Tbp was amplified as an internal reference. Figure 15C shows Sod1 knockdown in each organ relative to mRNA levels from untreated animals after normalization with Tbp. [Figures 16A-16B]Figure 16A shows the biodistribution and in vivo knockdown activity of siSOD1M2-AC2(N12)-S1V3v-Qu5 in organs of adult mice after ICV injection. Qu5-labeled ODV-siRNA (siSOD1M2-AC2(N12)-S1V3v-Qu5) was administered to adult C57BL / 6 mice via bilateral ICV injection at a total dose of 10 mg / kg. The Qu5-labeled siRNA variant siSOD1M2-S1V1v-Qu5, which lacks any auxiliary oligonucleotides, served as a control. Five days after injection, mice were sacrificed, and whole-organ fluorescence was quantified using an IVIS imaging system with 520 nm excitation and 570 nm emission filters. Figure 16A quantifies the Qu5 signal intensity of siSOD1M2-AC2(N12)-S1V3v-Qu5 released from each major organ compared to siSOD1M2-S1V1v-Qu5 after ICV injection. Sod1 mRNA knockdown was quantified in tissues from the central nervous system and selected peripheral tissues (i.e., muscle and kidney) by RT-qPCR using gene-specific primer sets. Tbp was amplified as an internal reference. Figure 16B shows the mean Sod1 knockdown levels in each of the indicated adult tissues relative to mRNA levels in saline-treated animals after normalization to Tbp. [Figure 17] We demonstrate that the length of the ACO affects the distribution of ODV-siRNA knockdown activity in CNS tissues in vivo. Sod1 ODV-siRNA mutants carrying ACOs of either 22 nucleotides (siSOD1M2-AC2(N22)-S1V3v-Qu5) or 6 nucleotides (siSOD1M2-AC2(N6)-S1V3v-Qu5) were administered to adult C57BL / 6 mice via bilateral ICV injection at a total dose of 10 mg / kg. Ten days after administration, mice were sacrificed, and knockdown of Sod1 mRNA in various tissues of the central nervous system and liver was quantified by RT-qPCR using gene-specific primer sets. Tbp was amplified as an internal reference. The average expression levels from two animals (n=2) are shown relative to the mRNA levels in untreated animals in each of the indicated adult tissues. [Figure 18] Figure 1 shows the inhibitory potency of six lead siRNAs on Sod1 mRNA levels in SH-SY5Y human neuroblastoma cells. SH-SY5Y cells were treated with 0.1 and 1 nM SOD1 siRNAs (i.e., DS17-0001, DS17-0002, DS17-0003, DS17-0004, DS17-0005, and DS17-0029) for 24 hours. Mock treatments were transfected in the absence of oligonucleotides. dsCon2 was used as a nonspecific duplex control. Sod1 mRNA levels were quantified by RT-qPCR using a gene-specific primer set. TBP was amplified as an internal reference. Average values ​​of Sod1 mRNA relative to mock treatment after normalization with TBP are shown. [Figure 19] The inhibitory efficacy of seven siRNAs on Sod1 mRNA levels in HEK293A cells is shown. HEK293A cells were treated with DS17-04N3 siRNA and six prior art siRNAs (DS17-Vol49, DS17-Vol49(c), DS17-Vol53, and DS17-Vol53(c) from US10570395B2; DS17-Al289 and DS17-Al102 from WO2006066203A2) at 0.004, 0.016, 0.063, 0.250, 1.000, and 4.000 nM, respectively, for 24 hours. Mock treatment was performed by transfection without oligonucleotide. dsCon2 was used as a nonspecific duplex control. Data for Mock and dsCon2 are not shown. Sod1 mRNA levels were quantified by RT-qPCR using gene-specific primer sets. TBP was amplified as an internal reference. Mean values ​​of Sod1 mRNA levels relative to mock treatment after normalization with TBP are shown. [Figures 20A-20B]Figure 20A shows the inhibitory efficacy of nine siRNAs on Sod1 mRNA levels in HeLa and HEK293A cells. HeLa and HEK293A cells were treated with DS17-02N3 siRNA and eight prior art siRNAs (DS17-Vo195&60 and DS17-Vo195 (D-2763) in US20170314028A1, and DS17-Al148, DS17-Al194, DS17-Al290, DS17-Al405, DS17-Al447, and DS17-Al600 in WO2006066203A2) at 0.004, 0.016, 0.063, 0.250, 1.000, and 4.000 nM, respectively, for 24 hours. Figure 20B shows the Sod1 mRNA levels in HeLa cells. Figure 20B shows Sod1 mRNA levels in HEK293A cells. Mock treatment was transfected in the absence of oligonucleotide. dsCon2 was used as a nonspecific duplex control. Mock and dsCon2 data are not shown. Sod1 mRNA levels were quantified by RT-qPCR using a gene-specific primer set. TBP was amplified as an internal reference. Average values ​​of SOD1 relative to mock treatment after normalization with TBP are shown.

[0091] [Figures 21A-21B]Figure 21 shows the inhibitory efficacy of five chemically modified lead siRNAs on Sod1 mRNA levels in T98G and HEK293A cells. T98G and HEK293A cells were treated with 0.002, 0.005, 0.015, 0.046, 0.137, 0.412, 1.235, 3.704, 11.111, 33.333, and 100 nM SOD1 siRNAs (i.e., DS17-01M3, DS17-02M3, DS17-03M3, DS17-04M3, and DS17-05M3) for 24 hours, respectively. Figure 21A shows Sod1 mRNA levels in T98G cells. Figure 21B shows Sod1 mRNA levels in HEK293A cells. Mock treatment was performed without oligonucleotide transfection. dsCon2 was used as a nonspecific duplex control. Data for Mock and dsCon2 are not shown. Sod1 mRNA levels were quantified by RT-qPCR using a gene-specific primer set. TBP was amplified as an internal reference. Mean values ​​of SOD1 relative to Mock treatment after normalization with TBP are shown. [Figures 22A-22B]Figure 22A shows the inhibitory efficacy of four chemically modified lead ODV-siRNAs on Sod1 mRNA levels in HEK293A cells. Cells were treated with four SOD1 ODV-siRNAs (i.e., DS17-02M3-AC1(mel4)-L9V3, DS17-03M3-AC1(mel4)-L9V3, DS17-04M3-AC1(mel4)-L9V3, and DS17-29M2-AC1(mel4)-L9V3) at 0.00002, 0.00009, 0.00037, 0.0015, 0.0059, 0.023, 0.094, 0.375, 1.5, and 6 nM, respectively, for 24 hours. Figure 22B shows Sod1 mRNA levels in HEK293A cells. Figure 22B shows caspase 3 / 7 activity in HEK293A cells. Mock treatment was transfected in the absence of oligonucleotide. dsCon2 was used as a nonspecific duplex control. Data for Mock and dsCon2 are not shown. Sod1 mRNA levels were quantified by RT-qPCR using a gene-specific primer set. TBP was amplified as an internal reference. Average values ​​of SOD1 relative to mock treatment after normalization with TBP are shown. [Figures 23A-23B]This figure shows the in vivo inhibitory efficacy of ODV-siRNA leads on Sod1 mRNA levels via ICV injection in the central nervous system of adult SOD1G93A mice. Three ODV-siRNAs (i.e., DS17-01M3-AC1(me14)-L9V3, DS17-04M3-AC1(me14)-L9V3, and DS17-05M3-AC1(me14)-L9V3) were administered at 0.4 mg via unilateral ICV injection to adult SOD1G93A mice on postnatal day 46. The ODV duplex dsCon2M3-AC1(me14)-L9V3 was used as a nonspecific duplex control. As a positive control, asSOD1-1 (Tofersen) was also injected at the same dose. Seven days after administration, mice were sacrificed, and Sod1 mRNA knockdown was quantified in various CNS tissues by RT-qPCR using gene-specific primer sets. Figure 23A shows Sod1 mRNA levels in different brain tissues. Figure 23B shows Sod1 mRNA levels in different spinal cord tissues. Tbp was amplified as an internal reference. The mean mRNA levels of four animals (n=4) in each treatment group are shown in the indicated tissue relative to the untreated group after normalization to Tbp. [Figures 24A-24B] Figure 24 shows the latency and weight change of adult SOD1G93A mice after ICV injection of siRNA. ODV-siRNA (DS17-04M3-AC1(mel4)-L9V3) was administered to adult SOD1G93A mice on PND 46 via unilateral ICV injection at a total dose of 20 nmole. Figure 24A shows efficacy using the rotarod behavioral test after PND 68. Figure 24B shows weight changes after injection on PND 46 (normalized to day 0). Tofersen was injected at a dose of 20 nmole as a positive control. Artificial CSF (aCSF) was injected as a non-treated negative control. The average Sod1 mRNA level of four mice (n=4) for each treatment is shown. [Figure 25]Figure 1 shows siRNA concentrations in the adult SOD1G93A mouse brain after ICV injection. ODV-siRNA (DS17-04M3-AC1(me14)-L9V3) was unilaterally injected ICV into adult SOD1G93A mice at PND46 at a total dose of 0.4 mg. Mice were sacrificed 1, 7, 28, and 56 days after administration, and the concentration of DS17-04M3-AC1(me14)-L9V3 in different brain tissues was quantified by stem-loop RT-qPCR using gene-specific primer sets. The average siRNA concentration from three animals (n = 3) for each treatment is shown. [Figure 26] This figure shows the in vivo inhibitory efficacy of ODV-siRNA lead on Sod1 mRNA levels in different central nervous system tissues via ICV injection in adult SOD1G93A mice. ODV-siRNA (DS17-04M3-AC1(me14)-L9V3) was administered at 0.1, 0.4, 1, or 1.6 mg via unilateral ICV injection on PND 46. ACSF was injected as a non-treated negative control. Mice were sacrificed 14 days after administration, and knockdown of Sod1 mRNA in different central nervous system and liver tissues was quantified by RT-qPCR using gene-specific primer sets. Tbp was amplified as an internal reference. The mean Sod1 mRNA levels from 1 to 3 animals (n = 1–3) are shown relative to the mRNA levels in the untreated group. [Figure 27]Figure 1 shows the in vitro inhibitory potency of ODV-siRNA leads on Sod1 mRNA levels in T98G cells. T98G cells were treated with five SOD1 ODV-siRNAs (i.e., DS17-01M3v-AC1(me14)-L9V3, DS17-01M3v-AC1(me14)-L9V3, DS17-02M3v-AC1(me14)-L9V3, DS17-03M3v-AC1(me14)-L9V3, DS17-04M3v-AC1(me14)-L9V3, and DS17-05M3v-AC1(me14)-L9V3) at concentrations of 0.0003, 0.0011, 0.0044, 0.0176, 0.0703, 0.2813, 1.1250, 4.5, and 18 nM, respectively, for 24 hours. Mock treatment was transfected in the absence of oligonucleotide. dsCon2 was used as a nonspecific duplex control. Data for Mock and dsCon2 are not shown. Sod1 mRNA levels were quantified by RT-qPCR using a gene-specific primer set. TBP was amplified as an internal reference. Average values ​​of Sod1 mRNA relative to mock treatment after normalization with TBP are shown. [Figure 28]Figure 1 shows the in vivo inhibitory efficacy of ODV-siRNA leads on Sod1 mRNA levels via ICV injection in adult SOD1G93A mice. Five ODV-siRNAs (i.e., DS17-01M3v-AC1(me14)-L9V3, DS17-02M3v-AC1(me14)-L9V3, DS17-03M3v-AC1(me14)-L9V3, DS17-04M3v-AC1(me14)-L9V3, and DS17-05M3v-AC1(me14)-L9V3) were administered at a total dose of 0.2 mg via unilateral ICV injection into adult SOD1G93A mice on PND 46. ACSF was injected as a non-treated negative control. DS17-04M3(Scr)-AC1(me14)-L9V3 was used as a non-specific duplex control. Mice were sacrificed 14 days after treatment, and Sod1 mRNA knockdown was quantified in different central nervous system and liver tissues by RT-qPCR using gene-specific primer sets. Tbp was amplified as an internal reference. Average Sod1 mRNA levels from 2–4 animals (n = 2–4) were normalized to Tbp and shown relative to the mRNA levels in the untreated group. [Figures 29A-29C]Figure 29 shows the in vivo inhibitory efficacy of different ODV-siRNA leads on Sod1 mRNA levels by ICV injection in adult SOD1G93A mice. Two types of ODV-siRNA (DS17-04M3-AC1(mel4)-L9V3 and DS17-04M3v-AC1(mel4)-L9V3) were administered by unilateral ICV injection on PND46. aCSF was injected as a non-treated negative control. Figure 29A shows the Sod1 mRNA transcript levels in each tissue at a low dose (0.2 mg) after 14 days of treatment. Figure 29B shows the amount of Sod1 mRNA transcripts in each tissue at a high dose (0.4 mg) after 14 days of treatment. Figure 29C shows the Sod1 mRNA transcript levels in different tissues at a high dose (0.4 mg) after 56 days of treatment. Mice were sacrificed on days 14 and 56 post-treatment, and Sod1 mRNA knockdown was quantified in different central nervous system and liver tissues by RT-qPCR using gene-specific primer sets. Tbp was amplified as an internal reference. Average Sod1 mRNA levels from 2–4 animals (n = 2–4) are shown relative to the mRNA levels in the untreated group after normalization with Tbp. [Figures 30A-30G]Figure 30 shows the in vivo inhibitory efficacy of ODV-siRNA leads on Sod1 mRNA transcription levels via ICV injection in adult SOD1G93A mice. ODV-siRNA (DS17-04M3-AC1(mel4)-L9V3 and DS17-04M3v-AC1(mel4)-L9V3) were administered via unilateral ICV injection at a total dose of 20 nmoles on PND46. Tofersen was injected at the same dose as a positive control. aCSF was injected as an untreated negative control. Figures 30A, 30B, 30C, 30D, 30E, 30F, and 30G show Sod1 mRNA levels in the brain-frontal cortex, brain-cerebellum, rest of the brain, spinal cord-cervical, spinal cord-thoracic, spinal cord-lumbar, and liver tissues, respectively. Mice were sacrificed at weeks 2 and 8 post-treatment, and Sod1 mRNA knockdown was quantified in different central nervous system and liver tissues by RT-qPCR using gene-specific primer sets. Tbp was amplified as an internal reference. Average Sod1 mRNA levels from four animals (n=4) are shown relative to the mRNA levels in the untreated group after normalization with Tbp. [Figures 31A-31C] Figure 3 shows the immunostimulatory effect of SOD1ODV-siRNA in ICR mice. ICR mice were SC-injected with DS17-04M3, AC1-L9V3, and DS17-04M3-AC1(me14)-L9V3 at 10.18 nmoles (low dose) and 40.71 nmoles (high dose), respectively. Saline treatment alone served as a vehicle control to establish baseline values. Mice were sacrificed 8 hours after treatment to detect IL-1β (Figure 31A), IFN-γ (Figure 31B), and TNF-α (Figure 31C) protein levels in serum by ELISA assay, as described in the Materials and Methods section. [Figures 32A-32C]Figure 32 shows the levels of ALT (32A), AST (32B), and CREA (32C) in ICR mice after SC injection of DS17-04M3, AC1-L9V3, and DS17-04M3-AC1(mel4)-L9v3. ICR mice were SC injected with 10.18 nmoles and 40.71 nmoles of the title test compounds, respectively. Serum levels of ALT (Figure 32A), AST (Figure 32B), and CREA (Figure 32C) were detected using their specific detection kits as described in Materials and Methods. [Figure 33A-33B] Screening data comparing the knockdown activity of 46 siRNAs targeting the human SOD1 3'UTR. HEK293A cells were transfected with 0.1 and 1 nM of each siRNA duplex for 24 hours. Figure 33A shows the Sod1 mRNA expression levels of 46 siRNAs sorted by position in HEK293A cells. Figure 33B shows the Sod1 mRNA levels of 46 siRNAs sorted by activity on the 3'UTR in HEK293A cells. Mock treatment was transfected in the absence of oligonucleotide. dsCon2 was used as a nonspecific duplex control. Mock and dsCon2 data are not shown. Sod1 mRNA levels were quantified by RT-qPCR using gene-specific primer sets. TBP and HPRT1 were amplified as internal references. Average Sod1 mRNA expression values ​​for mock treatment are shown after normalization to TBP and HPRT1 reference levels at both treatment concentrations. Values ​​(y-axis) indicate the fold change in Sod1 mRNA expression levels induced by each siRNA relative to mock treatment, after normalization to TBP and HPRT1. siRNAs are sorted on the x-axis by their position in the 3'UTR, 540 bp and 700 bp downstream of the SOD1 transcription start site (TSS). The locations of the two siRNA hotspot regions are marked H1 and H2 within the dotted rectangular boxes. [Figure 34]Dose-dependent characteristics of SOD1 candidates in HEK293A cells are shown. siRNAs were transfected into HEK293A cells at increasing dose concentrations representing approximate multiples of the IC50 (half-maximal inhibitory concentration) values. Mock treatments were transfected in the absence of oligonucleotides (not shown). Sod1 mRNA expression levels were quantified by RT-qPCR using gene-specific primer sets. TBP and HPRT1 were amplified as internal references. The mean Sod1 mRNA expression values ​​for mock treatment are shown after normalization to TBP and HPRT1 reference levels at both treatment concentrations. [Figure 35A-35B] Screening data comparing the knockdown activity of ASOs targeting the human SOD13'UTR. HEK293A cells were transfected with 16 ASOs at 5 nM and 50 nM and 33 ASOs at 3.125, 12.5, 50, and 200 nM for 24 hours. Figure 35A shows the Sod1 mRNA levels of the 16 ASOs in HEK293A cells. Figure 35B shows the Sod1 mRNA levels of the 33 ASOs in HEK293A cells. Mock treatment was transfected in the absence of oligonucleotide. dsCon2 was used as a nonspecific duplex control. Mock and dsCon2 data are not shown. Sod1 mRNA levels were quantified by RT-qPCR using gene-specific primer sets. TBP and HPRT1 were amplified as internal references. Average Sod1 mRNA expression values ​​for mock treatment are shown after normalization to TBP and HPRT1 reference levels at both treatment concentrations. Values ​​(y-axis, log2) represent the fold change in Sod1 mRNA expression levels by each ASO relative to mock treatment, after normalization to TBP and HPRT1. ASOs are sorted on the x-axis by their position in the 3'UTR, 544 and 576 bp downstream of the SOD1 transcription start site (TSS). The location of the ASO hotspot region, H3, is indicated by a dotted rectangular box. [Figures 36A-36C]Figure 36 shows the dose-dependence of SOD1 ASO candidates in HEK293A cells. ASOs were transfected into HEK293A cells at increasing dose concentrations representing approximate multiples of the IC50 (half-maximal inhibitory concentration) value. Mock treatments were transfected in the absence of oligonucleotide (not shown). Figure 36A shows mRNA expression levels, Figure 36B shows apoptosis, and Figure 36C shows cytotoxicity quantified for each dose of each ASO by RT-qPCR in HEK293A cells using a caspase 3 / 7 kit (G8092, Promega) and a CCK8 kit (CK04, Dojindo, Japan). [Figure 37A-37B]Figure 37 shows the screening data for knockdown activity and cytotoxicity of 90 ODV-siRNA designs by free uptake in PMH cells. The indicated ODV-siRNAs were added to PMH cell culture media at final concentrations of 0.1 μM and 1 μM. Cells were then incubated for 3 days. RD-12556 and RD-12559 were used as duplex and ODV controls, respectively. Mock treatment was performed in the absence of oligonucleotides and is not shown. Figure 37A shows the Sod1 mRNA levels induced by each ODV-siRNA in PMH cells. Sod1 mRNA levels were quantified by RT-qPCR using gene-specific primer sets. Tbp and Hprt1 were amplified as internal references. Values ​​(y-axis) indicate the mean expression values ​​of Sod1 mRNA relative to mock treatment after normalization to Tbp and Hprt1 reference levels at both treatment concentrations. Figure 37B shows the cytotoxicity levels of 90 ODV-siRNAs in PMH cells by PI staining. The optical density (OD) of PI staining was measured using an Infinite M2000 Pro microplate reader system at an excitation wavelength of 535 nm and an emission wavelength of 615 nm. The values ​​(y-axis) represent the average PI staining of each ODV-siRNA at both concentrations relative to the mock. A total of 90 ODV-siRNAs are shown in 12 dotted rectangles (A) through (L). The dotted rectangle boxes represent various design groups with different linkers (A), palindromic AC1 sequence variants (B), various numbers of PS modifications (C), various numbers of 2' omegas (D), various ACO sizes (E), adenine-rich sequence composition (F), cytosine-rich sequence composition (G), guanine-rich sequence composition (H), uracil-rich sequence composition (I), purines-rich sequence composition (J), pyrimidine-rich sequence composition (K), and purines / pyrimidine equivalent composition (L). [Figure 38]Figure 37 shows the effect of various ODV linkers used in ODV-siRNA on in vitro knockdown activity in PMH cells, as shown in group (A). Group A (linker group) included 10 compounds (i.e., RD-12941, RD-12942, RD-12943, RD-12944, RD-12945, RD-12947, RD-12948, RD-12949, RD-12950, ​​and RD-12951). RD-12556 and RD-12559 served as duplex and ODV controls, respectively. Figure 38 shows Sod1 mRNA levels in PMH cells after 3 days of free uptake treatment with ODV-siRNA at 0.1 μM and 1 μM. Mock treatment was performed without oligonucleotides and is not shown. Sod1 mRNA levels were quantified by RT-qPCR using gene-specific primer sets. Tbp and Hprt1 were amplified as internal references. Values ​​(y-axis) show the mean expression values ​​of Sod1 mRNA relative to mock treatment after normalization to Tbp and Hprt1 reference levels at both treatment concentrations. [Figure 39]As shown in group (B) of Figure 37, the effect of different palindromic AC1 sequences used in ODV-siRNA on in vitro knockdown activity in PMH cells is shown. Group B (palindromic AC1 sequence group) included 15 compounds containing sequence derivatives based on an exemplary ACO called AC1 (i.e., RD-12952, RD-12953, RD-12954, RD-12955, RD-12956, RD-12957, RD-12958, RD-12959, RD-12960, RD-12961, RD-12962, RD-12963, RD-12964, RD-12965, and RD-12966). RD-12556 and RD-12559 were used as duplex and ODV controls, respectively. Figure 39 shows Sod1 mRNA levels in PMH cells after 3 days of free uptake treatment with ODV-siRNA at 0.1 μM and 1 μM. Mock treatment was performed in the absence of oligonucleotides and is not shown. Sod1 mRNA levels were quantified by RT-qPCR using gene-specific primer sets. Tbp and Hprt1 were amplified as internal references. Values ​​(y-axis) represent the mean expression values ​​of Sod1 mRNA relative to mock treatment after normalization to Tbp and Hprt1 reference levels at both treatment concentrations. [Figure 40]As shown in group (C) of Figure 37, the effect of PS modifications used in ODV-siRNA on in vitro knockdown activity in PMH cells is shown. Group C (PS modification group) includes six compounds (i.e., RD-12967, RD-12968, RD-12969, RD-12970, RD-12971, and RD-12972), each with the same 14-nt ACO containing either 2, 4, 6, 8, 10, or 12 PS modifications. RD-12556 and RD-12559 were used as duplex and ODV controls, respectively. Mock treatment was performed in the absence of oligonucleotides and is not shown. Sod1 mRNA levels were quantified by RT-qPCR using gene-specific primer sets. Tbp and Hprt1 were amplified as internal references. Values ​​(y-axis) indicate the mean expression values ​​of Sod1 mRNA relative to mock treatment after normalization to the Tbp and Hprt1 reference levels at both treatment concentrations. [Figure 41] As shown in Figure 37, group (D) shows the effect of 2'ome modifications used in ODV-siRNA on in vitro knockdown activity in PMH cells. Group D (2'ome modification group) includes seven compounds (i.e., RD-12973, RD-12974, RD-12975, RD-12976, RD-12977, RD-12978, and RD-12979), each with the same 14-nt ACO, containing either 2, 4, 6, 8, 10, or 12 substitutions of the 2'MOE chemistry for the 2'ome. RD-12556 and RD-12559 served as duplex and ODV controls, respectively. Mock treatment was performed in the absence of oligonucleotides and is not shown. Sod1 mRNA levels were quantified by RT-qPCR using gene-specific primer sets. Tbp and Hprt1 were amplified as internal controls. Values ​​(y-axis) show the mean expression values ​​of Sod1 mRNA relative to mock treatment after normalization to Tbp and Hprt1 reference levels at both treatment concentrations. [Figure 42]As shown in group (E) of Figure 37, the effect of various ACO sizes used in ODV-siRNA on in vitro knockdown activity in PMH cells is shown. Group E (ACO size group) includes eight compounds (i.e., RD-12980, RD-12981, RD-12982, RD-12983, RD-12984, RD-12985, RD-12986, and RD-12987), which were derived by truncating the 14-nt ACO of RD-12559 to lengths of 13, 12, 11, 10, 9, 8, 7, or 6 nucleotides, respectively. RD-12556 and RD-12559 served as duplex and ODV controls, respectively. Mock treatment was performed in the absence of oligonucleotides and is not shown. Sod1 mRNA levels were quantified by RT-qPCR using gene-specific primer sets. Tbp and Hprt1 were amplified as internal controls. Values ​​(y-axis) show the mean expression values ​​of Sod1 mRNA relative to mock treatment after normalization to Tbp and Hprt1 reference levels at both treatment concentrations. [Figure 43]Figure 37 shows the effect of the adenine-rich content of ODV-siRNA on in vitro knockdown activity in PMH cells, as shown in group (F). Group F (the adenine-rich group) includes five compounds (i.e., RD-12988, RD-12989, RD-12990, RD-12991, and RD-12992), each with a different 14-nt ACO sequence containing either 5, 6, 7, 8, or 9 total adenine nucleotides. RD-12556 and RD-12559 served as duplex and ODV controls, respectively. Figure 43 shows Sod1 mRNA levels in PMH cells after 3 days of free uptake treatment with 0.1 μM and 1 μM ODV-siRNA. Mock treatment was performed without oligonucleotides and is not shown. Sod1 mRNA levels were quantified by RT-qPCR using gene-specific primer sets. TBP and Hprt1 were amplified as internal controls. Values ​​(y-axis) show the mean expression values ​​of Sod1 mRNA relative to mock treatment after normalization to Tbp and Hprt1 reference levels at both treatment concentrations.

[0092] [Figure 44]As shown in group (G) of Figure 37, the effect of cytosine enrichment used in ODV-siRNA on in vitro knockdown activity in PMH cells is shown. Group G (cytosine-rich group) includes six compounds (i.e., RD-12993, RD-12994, RD-12995, RD-12996, RD-12997, and RD-12998), each with a different 14-nt ACO sequence containing either 5, 6, 7, 8, 9, or 10 total cytosine nucleotides. RD-12556 and RD-12559 were used as duplex and ODV controls, respectively. Mock treatment was performed in the absence of oligonucleotides and is not shown. Sod1 mRNA levels were quantified by RT-qPCR using gene-specific primer sets. Tbp and Hprt1 were amplified as internal controls. Values ​​(y-axis) show the mean expression values ​​of Sod1 mRNA relative to mock treatment after normalization to Tbp and Hprt1 reference levels at both treatment concentrations.

[0093] [Figure 45] As shown in Group (H) of Figure 37, the effect of guanine-rich compounds used in ODV-siRNA on in vitro knockdown activity in PMH cells is shown. Group H (Guanine-rich group) includes five compounds (i.e., RD-12999, RD-13000, RD-13001, RD-13002, and RD-13003), each with a different 14-nt ACO sequence containing either 5, 6, 7, 8, or 9 total guanine nucleotides. RD-12556 and RD-12559 were used as duplex and ODV controls, respectively. Mock treatment was performed in the absence of oligonucleotides and is not shown. Sod1 mRNA levels were quantified by RT-qPCR using gene-specific primer sets. Tbp and Hprt1 were amplified as internal references. Values ​​(y-axis) indicate the mean expression values ​​of Sod1 mRNA relative to mock treatment after normalization to the Tbp and Hprt1 reference levels at both treatment concentrations. [Figure 46]Group (I) in Figure 37 shows the effect of uracil-rich used in ODV-siRNA on in vitro knockdown activity in PMH cells. Group G (uracil-rich group) includes six compounds (i.e., RD-13004, RD-13005, RD-13006, RD-13007, RD-13008, and RD-13009), each with a different 14-nt ACO sequence containing either 5, 6, 7, 8, 9, or 10 total uracil nucleotides. RD-12556 and RD-12559 served as duplex and ODV controls, respectively. Mock treatment was performed in the absence of oligonucleotides and is not shown. Sod1 mRNA levels were quantified by RT-qPCR using gene-specific primer sets. Tbp and Hprt1 were amplified as internal controls. Values ​​(y-axis) show the mean expression values ​​of Sod1 mRNA relative to mock treatment after normalization to Tbp and Hprt1 reference levels at both treatment concentrations. [Figure 47]As shown in group (J) of Figure 37, the effect of Purinerich used in ODV-siRNA on in vitro knockdown activity in PMH cells is shown. Group J (Purinerich group) contains eight compounds, each with a 14-nt ACO sequence containing nine purines (i.e., RD-13010, RD-13011, and RD-13012), ten purines (i.e., RD-13013, RD-13014, and RD-13015), or eleven purines (i.e., RD-13016 and RD-13017) consisting of different amounts of adenosine and guanine. RD-12556 and RD-12559 were used as duplex and ODV controls, respectively. Figure 47 shows Sod1 mRNA levels in PMH cells after 3 days of free-uptake treatment with 0.1 μM and 1 μM ODV-siRNA. Mock treatment was performed without the presence of oligonucleotides and is not shown. Sod1 mRNA levels were quantified by RT-qPCR using gene-specific primer sets. Tbp and Hprt1 were amplified as internal controls. Values ​​(y-axis) represent the mean expression values ​​of Sod1 mRNA relative to mock treatment after normalization to Tbp and Hprt1 reference levels at both treatment concentrations. [Figure 48]Figure 37 shows the effect of pyrimidine-rich ODV-siRNA (group (K)) on in vitro knockdown activity in PMH cells. Group K (pyrimidine-rich group) contained eight compounds, each with a different 14-nt ACO sequence, containing nine pyrimidines (i.e., RD-13018, RD-13019, RD-13020), ten pyrimidines (i.e., RD-13021, RD-13022), eleven pyrimidines (i.e., RD-13023, RD-13024), or twelve pyrimidines (i.e., RD-13025), with varying amounts of cytosine and uracil. Figure 48 shows Sod1 mRNA levels in PMH cells after 3 days of free uptake treatment with 0.1 μM and 1 μM ODV-siRNA. Mock treatment was performed without oligonucleotides and is not shown. Sod1 mRNA levels were quantified by RT-qPCR using gene-specific primer sets. Tbp and Hprt1 were amplified as internal controls. Values ​​(y-axis) represent the mean expression values ​​of Sod1 mRNA relative to mock treatment after normalization to Tbp and Hprt1 reference levels at both treatment concentrations. [Figure 49]Figure 37 shows the effect of the balanced pur:pyr ratio used in ODV-siRNA (group (L)) on in vitro knockdown activity in PMH cells. Group L (the pur:pyr balanced group) includes six compounds (i.e., RD-13026, RD-13027, RD-13028, RD-13029, RD-13030, and RD-13031), each with a different 14-nt ACO sequence containing a fixed 1:1 ratio of purine to pyrimidine. Figure 49 shows Sod1 mRNA levels in PMH cells after 3 days of free uptake treatment with 0.1 μM and 1 μM ODV-siRNA. Mock treatment was performed without oligonucleotides and is not shown. Sod1 mRNA levels were quantified by RT-qPCR using gene-specific primer sets. Tbp and Hprt1 were amplified as internal controls. Values ​​(y-axis) show the mean expression values ​​of Sod1 mRNA relative to mock treatment after normalization to Tbp and Hprt1 reference levels at both treatment concentrations. [Figures 50A-50C] Figure 50A shows the in vivo efficacy of ODV-siRNA in knocking down Sod1 mRNA levels in lung tissue by intratracheal administration (ITI) in adult C57BL / 6J mice. In Figure 50A, the indicated oligonucleotides (i.e., RD-12401, RD-12402, RD-12403, RD-12557, RD-12929, and RD-12559) were administered ITI at a dose of 0.6 mg. The indicated oligonucleotides (RD-12556, RD-12929, and RD-12559) were administered ITI at doses of 0.1 mg (Figure 50B) and 0.6 mg (Figure 50C). Saline was administered as a non-treated negative control. RD-12404 was a nonspecific duplex used as a negative control. Mice were sacrificed on days 7 and 21 post-injection, and Sod1 mRNA knockdown was quantified in lung tissue by RT-qPCR using gene-specific primer sets after RNA isolation and RT reaction. Tbp was amplified as an internal reference. The mean Sod1 mRNA levels in lung tissue from 2–7 animals (n = 2–7) are shown relative to the mRNA levels in the untreated group after normalization with Tbp. [Figure 51A-51B] This figure shows the in vivo efficacy of ODV-siRNAs injected intravenously and subcutaneously into adult SOD1G93A mice for knocking down Sod1 mRNA levels in muscle tissue. The indicated siRNA, RD-12293, was administered intravenously and subcutaneously at 20 mg / kg and 50 mg / kg, respectively. Saline was injected as a non-treated negative control. Mice were sacrificed 14 days after administration, and Sod1 mRNA levels in muscle and liver tissue were quantified by RT-qPCR using gene-specific primer sets after RNA isolation and RT reaction. Tbp was amplified as an internal reference. The mean Sod1 mRNA levels in muscle tissue from 2–4 animals (n=2–4) are shown relative to the mRNA levels in the untreated group after normalization with Tbp.

[0094] [Figure 52] Figure 1 shows the in vivo efficacy of different ODV-siRNA leads for knocking down Sod1 mRNA expression by intravenous injection in adult C57BL / 6J mice. The indicated ODV-siRNAs (i.e., RD-12559, RD-12556, RD-12967, RD-13180, RD-12941, RD-12942, RD-12952, RD-12982, RD-12983, RD-12979, RD-13015, RD-13006, and RD-12998) were administered intravenously at 20 mg / kg. Saline injections served as untreated negative controls. RD-12556 served as a non-ODV duplex control. Mice were sacrificed 7 days after administration, and Sod1 mRNA levels were quantified in different types of muscle tissue (i.e., forelimbs, hindlimbs, nape, and rump) by RT-qPCR using gene-specific primer sets after RNA isolation and RT reaction. Tbp was amplified as an internal reference. The mean Sod1 mRNA levels in muscle tissue from 3–6 animals (n = 3–6) are shown relative to the mRNA levels in the untreated group after normalization with Tbp. [Figures 53A-53C]This figure shows the in vivo efficacy of different ODV-siRNA leads for knockdown of Sod1 mRNA expression by ICV injection in adult C57BL / 6J mice. The indicated siRNAs or ODV-siRNAs (i.e., RD-12559, RD-12556, RD-13334, RD-12967, RD-13180, RD-12941, RD-12942, RD-12952, RD-12982, RD-12983, RD-12979, RD-13015, RD-13006, and RD-12998) were administered by unilateral ICV injection. Saline was injected as an untreated negative control. RD-12556 and RD-13334 served as non-ODV duplex controls. Figure 53A shows Sod1 mRNA transcription levels in the brain-frontal cortex, the rest of the brain (excluding the frontal cortex and cerebellum), and the brain-cerebellum tissues at 0.2 mg. Figure 53B shows Sod1 mRNA transcription levels in the spinal cord-cervical tissue, spinal cord-thoracic tissue, and spinal cord-lumbar tissue at 0.2 mg. Figure 53C shows Sod1 mRNA transcription levels in the liver tissue at 0.2 mg. Mice were sacrificed 7 days after administration. After RNA isolation and RT reactions, Sod1 mRNA knockdown was quantified in the CNS (i.e., the brain-frontal cortex, the rest of the brain, the brain-cerebellum, the spinal cord-cervical, the spinal cord-thoracic, and the spinal cord-lumbar) and selected peripheral tissues (i.e., the liver) by RT-qPCR using gene-specific primer sets. Tbp was amplified as an internal reference. The mean Sod1 mRNA levels in each designated tissue from 3 to 6 animals (n = 3 to 6) are shown relative to the mRNA levels in the untreated group after normalization with Tbp. [Figure 54A-54B]Figure 54 shows the in vivo efficacy of different ODV-siRNA leads to knockdown Sod1 mRNA expression after intravenous injection into adult C57BL / 6J mice. The indicated siRNAs or ODV-siRNAs (i.e., RD-12559, RD-12556, RD-13334, RD-12967, RD-13180, RD-12941, RD-12942, RD-12952, RD-12982, RD-12983, RD-12979, RD-13015, RD-13006, and RD-12998) were administered intravenously at 20 mg / kg. Saline injections served as untreated negative controls. RD-12556 and RD-13334 served as non-ODV duplex controls. Figure 54A shows Sod1 mRNA transcription levels in heart, liver, and spleen tissues at 20 mg / kg. Figure 54B shows Sod1 mRNA transcription levels in lung, kidney, and bladder tissues at 20 mg / kg. Mice were sacrificed 7 days after administration, and Sod1 mRNA knockdown in selected peripheral tissues (i.e., heart, liver, spleen, lung, kidney, and bladder) was quantified by RT-qPCR using gene-specific primer sets after RNA isolation and RT reactions. Tbp was amplified as an internal reference. The mean Sod1 mRNA levels in each designated tissue from 2 to 6 animals (n = 2 to 6) are shown relative to the mRNA levels in the untreated group after normalization with Tbp. [Figures 55A-55D]Figure 55 shows the dose-dependence of intracellularly conjugated ODV (iODV)-siRNA in PMH cells. PMH cells were transfected with the indicated siRNA or ODV-siRNA (i.e., RD-12559, RD-12556, and RD-13351) at increasing dose concentrations that represent approximate multiples of the IC50 value using RNAiMAX (Figures 55A and 55B). The indicated siRNA (i.e., RD-12559, RD-13180, and RD-13351) was added to PMH cell cultures at increasing dose concentrations that represent approximate multiples of the IC50 value (Figures 55C and 55D). RD-12556 was used as a non-ODV duplex control. RD-12559 was used as an ODV-siRNA positive control. Mock treatments were transfected in the absence of oligonucleotide (not shown). Figures 55A and 55C show Sod1 mRNA levels, and Figures 55B and 55D show cell viability quantified for each oligonucleotide at each dose by RT-qPCR and CCK8 assay, respectively. [Figure 56] This is an interpretation of some of the in vivo data for siSOD1 using ACO mutants in Example 28. The average mean knockdown data (mean % reduction) in CNS tissues (brain and spinal cord compared to liver) are shown in the table. ODV-SiSOD1 mutants capable of better and worse retention of knockdown activity in local and surrounding tissues are illustrated. [Figure 57] The design of exemplary AC1-derived ACO sequence variants is shown. The variants include 15 sequences in which the siRNA, linker (L9), 2'MOE modification, and PS backbone are fixed, but the ACO sequence and length are altered relative to the AC1 sequence. Examples of palindromes, deletions, and purine / pyrimidine substitutions that may affect delivery efficiency are shown. DETAILED DESCRIPTION OF THE INVENTION

[0095] While various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0096] Before the present invention is described, it is to be understood that the invention is not limited to the particular embodiments described. Further, the scope of the present invention will be limited only by the appended claims, and it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0097] Where a range of values ​​is provided, unless the context clearly dictates otherwise, each intervening value between the upper and lower limits of that range, to the tenth of the unit of the lower limit, is understood to be specifically disclosed. Each subrange between any stated or intervening value in a stated range and any other stated or intervening value within that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may each independently be included or excluded, and each range in which either, either, or both upper limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded upper limits in the stated range. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included within the invention.

[0098] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Any method and material similar or equivalent to those described herein can be used to carry out or test the present invention, but here, exemplary method and material are described.All publications mentioned in this specification are incorporated herein by reference to disclose and describe the method and / or material related to the publication cited.It is understood that this disclosure shall prevail over the disclosure of the incorporated publication if there is any discrepancy.

[0099] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. Thus, for example, reference to a "sample" includes a plurality of such samples, reference to a "molecule" includes a reference to one or more molecules and equivalents thereof known to those skilled in the art, and so forth.

[0100] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0101] The term "amyotrophic lateral sclerosis" or "ALS" includes, but is not limited to, familial ALS (FALS), sporadic ALS (sALS), Lou Gehrig's disease, diseases associated with mutant genes chromosome 9 open reading frame 72 gene (C9orf72; 40%), superoxide dismutase 1 (SOD1; 20%), transactive response DNA binding protein 43 (TDP43; 4%), and fusion in sarcoma / translocation in sarcoma (FUS / TLS; 4%).

[0102] The terms "oligonucleotide agent" and "oligonucleotide" can be used interchangeably and refer to a polymer of nucleotides, including, but not limited to, single- or double-stranded nucleic acid molecules of DNA, RNA, or DNA / RNA hybrids, oligonucleotide strands containing alternating deoxyribosyl and ribosyl moieties in regular and irregular sequences, and modified naturally or non-naturally occurring frameworks for such oligonucleotides. Specifically, oligonucleotide agents for inhibiting the amount of mRNA transcription of target genes described herein are small inhibitory nucleic acid molecules (siRNAs), antisense oligonucleotide molecules (ASOs), or oligonucleotide delivery vehicle (ODV)-linked siRNA molecules (siRNA-ACOs). Also specifically, oligonucleotide agents for activating transcription of target genes described herein are small activating nucleic acid molecules (saRNAs) or oligonucleotide delivery vehicle (ODV)-linked saRNA molecules (saRNA-ACOs).

[0103] As used herein, the terms "subject" and "individual" are used interchangeably herein to mean any living organism that can be treated with the agents of the present application. The term "patient" refers to a human subject or individual, including infants, children, and adults, as disclosed herein.

[0104] A "therapeutically effective amount" of a composition is an amount sufficient to achieve the desired therapeutic effect, and therefore does not require a cure or complete remission. In embodiments of the present application, the therapeutic effect is an improvement in any of the disease indicators, and a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition / symptom in the treated individual. The terms "therapeutically effective amount" and "effective amount" are used herein to mean an amount sufficient to reduce a clinically significant deficit in the activity, function, and response of the treated individual by at least about 15%, preferably at least 50%, more preferably at least 90%, or to increase by at least about 50%, at least about 100%, at least about 200%, more preferably at least about 500%, and most preferably prevent.

[0105] An effective amount may vary depending on factors such as the subject's size and weight, the type of illness, or the particular drug being administered. For example, the choice of drug administered may affect what constitutes an "effective amount." One of ordinary skill in the art would be able to study the factors contained herein and make the determination regarding the effective amount of the drug of the present application without undue experimentation.

[0106] The administration regimen can affect what constitutes an effective amount. The agents of the present application can be administered to a subject either before or after disease diagnosis or pathology. Furthermore, several divided doses and staggered doses can be administered daily or continuously, or the dose can be continuously infused or bolus injected. Furthermore, the dosage of the agent(s) of the present application can be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0107] As used herein, the terms "treat," "treating," "treating," or "treatment" have the meaning commonly understood in the medical arts, and thus include any beneficial or desired clinical result, not requiring a cure or complete remission. Non-limiting examples of such beneficial or desired clinical results include increased survival compared to expected survival without treatment, alleviation of symptoms, including one or more of the following: proximal skeletal muscle weakness and atrophy, inability to sit or walk independently, difficulty swallowing, difficulty breathing, etc.

[0108] As used herein, "preventing" or "delaying" a disease refers to inhibiting the complete onset of the disease.

[0109] The term "biological sample" refers to any tissue, cell, fluid, or other substance derived from an organism (e.g., a human subject). In certain embodiments, the biological sample is serum or blood.

[0110] As used herein, the term "sequence identity" or "sequence homology" refers to, for example, one oligonucleotide strand (sense or antisense) of a saRNA or siRNA having at least 80% similarity to a region on the coding or template strand of a promoter, or the sequence of a target gene.

[0111] In the embodiment of the present application, one target gene is SOD1. " Target sequence " refers to the sequence fragment that siRNA or saRNA sense strand or antisense oligonucleotide is homologous or complementary to.For example, in certain embodiments, SOD1 siRNA is homologous or complementary to the target selection sequence in human SOD1 transcription product.

[0112] As used herein, the term "non-target" refers to a referenced accessory oligonucleotide (ACO) (e.g., siRNA, saRNA, etc.) that binds to a target oligonucleotide not being specifically complementary to the target sequence for which the target oligonucleotide functions, and / or a referenced oligonucleotide (i.e., ACO) not sharing the same target sequence as the target oligonucleotide (e.g., siRNA, saRNA, etc.) specifically functions. Target oligonucleotides disclosed herein are nucleic acid sequences that are specifically complementary to a target sequence or a region thereof. In some embodiments, the term "non-target oligonucleotide" can include any referenced oligonucleotide other than the "target sequence." In some cases, "specifically complementary" may refer to at least about 95% complementarity between the target oligonucleotide and the target sequence or a region thereof. Non-targeted oligonucleotides (i.e., auxiliary oligonucleotides, or "ACOs," used interchangeably) do not induce biological activity through a known mechanism, nor do they induce activity indicative of ASO (i.e., "mixer" or "gapmer") function on complementary nucleic acid sequences (i.e., mRNA) in a particular subject, subject's organ, subject's tissue, or subject's cell when the oligonucleotide is administered. The purpose of non-targeted oligonucleotides (i.e., ACOs) is to facilitate the introduction of the target oligonucleotide (e.g., siRNA, saRNA, etc.) to which they are attached into a particular subject, subject's organ, subject's tissue, subject's cell, or subject's cell nucleus when the oligonucleotide conjugate is administered.

[0113] As used herein, the term "gapmer" refers to a short DNA antisense oligonucleotide (ASO) structure with modified RNA segments on either side of a central DNA structure. In some embodiments, at least one of the modified RNA segments contains one or more modified nucleotides selected from locked nucleic acid (LNA) and 2'-OMe or 2'-F modified nucleotides to increase affinity for the target, increase nuclease resistance, reduce immunogenicity, and / or reduce toxicity. In some embodiments, the gapmer contains at least one nucleotide modified with a phosphorothioate (PS) group. In some embodiments, the gapmer is designed to hybridize to a target portion of RNA and silence a gene transcript through induction of RNase H cleavage. As an example, the ASO drug "Toferson" is a gapmer that knocks down Sod1 mRNA for the treatment of ALS. A possible example of a dual-acting oligonucleotide (DAO) with the gapmer ASO disclosed in this application could be "siSOD1-Toferson."

[0114] As used herein, the term "mixer" refers to an antisense oligonucleotide (ASO) characterized by a mixed structure of DNA and chemically modified nucleic acid analogs. Optionally, the mixer is composed of fully modified nucleotides or nucleic acid analogs. In some embodiments, the mixer is designed to bind to and mask complementary RNA sequences to sterically inhibit proteins, factors, or other RNAs from interacting with the target RNA. In some embodiments, the mixer is designed to alter pre-mRNA splicing by displacing the spliceosome. In some embodiments, the mixer is designed to bind to and sequester microRNAs (miRNAs), and the mixer is alternatively referred to as an "antagomir" or "anti-miR."

[0115] As used herein, the terms "sense strand" and "passenger strand" are interchangeable. The sense strand of a dsRNA (e.g., siRNA, saRNA) molecule may comprise, for example, the first nucleic acid strand of the siRNA, which is composed of a fragment of the mRNA sequence of a target gene.

[0116] As used herein, the terms "antisense strand" and "guide strand" are interchangeable. The antisense strand of a dsRNA molecule can include, for example, the second nucleic acid strand in a saRNA or siRNA duplex that is complementary to the sense strand.

[0117] As used herein, the term "first oligonucleotide strand" can be a sense strand or an antisense strand.For example, the sense strand of saRNA refers to the oligonucleotide strand that has homology with the coding strand of the promoter DNA sequence of the target gene of saRNA.The sense strand of siRNA refers to the oligonucleotide strand that has homology with the mRNA sequence of the target gene of siRNA.Antisense strand refers to the oligonucleotide strand that is complementary to the sense strand of dsRNA.

[0118] As used herein, the term " second oligonucleotide strand " can also be sense strand or antisense strand.When the first oligonucleotide strand is sense strand, the second oligonucleotide strand is antisense strand, and when the first oligonucleotide strand is antisense strand, the second oligonucleotide strand is sense strand.

[0119] As used herein, the term "promoter" refers to a nucleic acid sequence that does not encode a protein but plays a regulatory role in transcription by spatially associating with a protein-coding or RNA-coding nucleic acid sequence. Generally, eukaryotic promoters contain 100 to 5,000 base pairs, although this length range is not intended to limit the term "promoter" as used herein. Although promoter sequences are generally located at the 5' end of a protein-coding or RNA-coding sequence, they can also be found in exon and intron sequences.

[0120] As used herein, the term "coding strand" refers to the DNA strand of a target gene that is not transcribed, the nucleotide sequence of which is identical to the sequence of the RNA produced by transcription (in which the DNA Ts are replaced by Us). The coding strand of the double-stranded DNA sequence of a target gene promoter described in this disclosure refers to the promoter sequence on the same DNA strand as the DNA coding strand of the target gene.

[0121] As used herein, the term "template strand" refers to the other strand of double-stranded DNA of a target gene that is complementary to the coding strand and can be transcribed as a template into RNA complementary to the transcribed RNA bases (AU, GC). During transcription, RNA polymerase binds to the template strand, moves along the template strand in the 3'→5' direction, and catalyzes RNA synthesis in the 5'→3' direction. The template strand of the double-stranded DNA sequence of a target gene promoter described in this disclosure refers to the promoter sequence on the same DNA strand as the DNA template strand of the target gene.

[0122] As used herein, the term "transcription start site" or TSS refers to the nucleotide that marks the start of transcription on the template strand of a gene. A transcription start site may be present on the template strand in the promoter region. A gene may have more than one transcription start site.

[0123] As used herein, the term "overhang" refers to an oligonucleotide having non-base-paired nucleotide(s) at the end (5' or 3') of an oligonucleotide strand that extends beyond one of the strands in a double-stranded oligonucleotide. The single-stranded region that extends beyond the 3' and / or 5' end of the duplex is called an overhang. In certain embodiments, the overhang is 0 to 6 nucleotides in length. An overhang of 0 nucleotides is understood to mean no overhang.

[0124] As used herein, the terms "gene activation," "activation of gene expression," "gene upregulation," and "upregulation of gene expression" can be used interchangeably and refer to an increase or upregulation of the transcription, translation, expression, or activity of a specific nucleic acid sequence, as determined by measuring the gene's transcription level, mRNA level, protein level, enzyme activity, methylation status, chromatin state or configuration, translation level, or activity or state in a cell or biological system. These activities or states can be determined directly or indirectly. Furthermore, "gene activation" or "activation of gene expression" refers to an increase in activity associated with a nucleic acid sequence, regardless of the mechanism of such activation. For example, gene activation occurs at the transcription level, increasing transcription into RNA, which is translated into protein, thereby increasing protein expression.

[0125] As used herein, the terms "gene silencing," "knockdown of gene expression," "gene downregulation," and "downregulation of gene expression" can be used interchangeably and refer to the reduction or downregulation of the transcription, translation, expression, or activity of a specific nucleic acid sequence, as determined by measuring the transcription level, mRNA level, protein level, enzyme activity, methylation state, chromatin state or configuration, translation level, or activity or state of the gene in a cell or biological system. These activities or states can be determined directly or indirectly. Furthermore, "gene downregulation" or "downregulation of gene expression" refers to a decrease in activity associated with a nucleic acid sequence, regardless of the mechanism of such downregulation. For example, gene downregulation occurs at the transcription level, reducing or silencing transcription into RNA, which is not translated into protein, thereby reducing or silencing protein expression.

[0126] As used herein, the terms "short interfering RNA," "siRNA," and "silencing RNA" can be used interchangeably and refer to ribonucleic acid molecules that can downregulate, knockdown, or silence target gene expression. They can be double-stranded nucleic acid molecules. siRNAs primarily bind to target mRNAs in the cytoplasm and post-transcriptionally downregulate gene expression via the RNA interference (RNAi) mechanism. siRNAs are sometimes designed to target the mRNA sequence of genes, such as SOD1, to silence their expression via the RNAi mechanism, for example, to maximize therapeutic efficacy in ALS patients. This modification does not eliminate cellular activity, but rather increases stability or activity. Examples of chemical modifications include phosphorothioate groups, 2'-deoxynucleotides, 2'-OCH3-containing ribonucleotides, 2'-F-ribonucleotides, 2'-methoxyethyl ribonucleotides, and combinations thereof. siRNAs can have a variety of lengths (e.g., 10-200 bps) and structures (e.g., hairpin, single-stranded / double-stranded, bulge, nick / gap, mismatch) and are processed intracellularly to provide active gene silencing. Double-stranded siRNAs can have the same number of nucleotides on each strand (blunt ends) or asymmetric ends (overhangs). For example, 1-2 nucleotide overhangs can be present on the sense and / or antisense strands, and on the 5'- and / or 3'-ends of a given strand. The length of an siRNA molecule is typically about 10 to about 60, about 10 to about 50, about 15 to about 30, about 17 to about 29, about 18 to about 28, about 19 to about 27, about 20 to about 26, about 21 to about 25, and about 22 to about 24 base pairs, and typically about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 23, about 25, about 30, about 40, or about 50 base pairs. Furthermore, the terms "small interfering RNA," "silencing RNA," and "siRNA" also encompass nucleic acids other than ribonucleotides, including, but not limited to, modified nucleotides or analogs.

[0127] As used herein, the terms "inhibition of gene expression" or "inhibiting gene expression" and "gene downregulation" or "downregulating gene expression" can be used interchangeably and refer to a decrease in the transcription, translation, expression, or activity of a specific nucleic acid, as determined by measuring the transcription level, mRNA level, protein level, enzyme activity, methylation status, chromatin state or configuration, translation level, or the activity or status of a gene in a cell or biological system. These activities or status can be determined directly or indirectly. Furthermore, "inhibition of gene expression," "suppression of gene expression," "gene downregulation," or "downregulation of gene expression" refers to a decrease in activity associated with a nucleic acid sequence, regardless of the mechanism of such activation. For example, inhibition of gene expression occurs at the transcription level, reducing transcription into RNA, which is translated into protein, thereby reducing protein expression.

[0128] As used herein, the terms "small activator RNA," "saRNA," and "small activator ribonucleic acid" can be used interchangeably to refer to ribonucleic acid molecules capable of upregulating the expression of a target gene. A saRNA can be a double-stranded nucleic acid molecule consisting of a first nucleic acid strand containing a ribonucleotide sequence with sequence homology to a non-coding nucleic acid sequence (such as a promoter or enhancer) of a target gene and a second nucleic acid strand containing a nucleotide sequence complementary to the first nucleic acid strand. saRNA can also be composed of synthetic or vector-expressed single-stranded RNA molecules that are prone to forming hairpin structures due to two complementary regions within the molecule, where the first region contains a ribonucleotide sequence with sequence homology to the target sequence in the gene's promoter and the second region contains a ribonucleotide sequence complementary to the first region. The length of the duplex region of the saRNA molecule is typically about 10 to about 60, about 10 to about 50, about 10 to about 40, about 12 to about 30, about 14 to about 28, about 16 to about 26, about 18 to about 24, or about 20 to about 22 base pairs, and typically about 10, about 13, about 15, about 17, about 18, about 19, about 20, about 21, about 22, about 25, about 30, about 40, about 50, or about 60 base pairs. Furthermore, the terms "small activator RNA," "saRNA," and "small activator ribonucleic acid" also include nucleic acids other than ribonucleotides, including, but not limited to, modified nucleotides or analogs.

[0129] As used herein, the terms "ASO" and "antisense oligonucleotide" can be used interchangeably and refer to a single-stranded oligonucleotide that binds to complementary mRNA and induces RNase H-dependent knockdown or alters mRNA protein binding through steric hindrance.

[0130] As used herein, the term "hotspot" of an siRNA or ASO refers to a nucleic acid region of at least 12 bp in length that is enriched for functional siRNAs / ASOs, i.e., at least 80%, for example, about 85%, about 90%, about 95%, or about 100% of the siRNAs / ASOs designed to target this region are functional and have the ability to inhibit the mRNA transcription level of a target gene. As used herein, a "hotspot" is defined by a nucleic acid region on the target sequence of an siRNA / ASO where the 5'-end of the functional siRNA or ASO is located. In a non-limiting example, the siRNA / ASO is designed according to the following criteria: (1) having a GC content between 35% and 70%; (2) having five or fewer consecutive identical nucleotides; (3) having three or fewer dinucleotide repeats; and (4) having three or fewer trinucleotide repeats. In a non-limiting example, each of the hotspot sequences disclosed herein contains at least four, at least five, or at least six (5'-end) functional siRNAs or ASOs.

[0131] As used herein, the term "functional siRNA" refers to an siRNA that inhibits the mRNA transcription level of an intended target gene by at least 80% compared to the baseline level of Sod1 mRNA at a treatment concentration of 1 nM, where free uptake by cells occurs. The term "non-functional siRNA" refers to an siRNA that is unable to inhibit the mRNA transcription level by 80% compared to the baseline level of Sod1 mRNA at a treatment concentration of 1 nM, where free uptake by cells occurs.

[0132] As used herein, the term "functional ASO" refers to an ASO that inhibits the mRNA transcription level of its intended target gene by at least 60% compared to the baseline level of Sod1 mRNA at a concentration of 200 nM free uptake by cells. The term "non-functional siRNA" refers to an ASO that is unable to inhibit the mRNA transcription level by 60% compared to the baseline level of Sod1 mRNA at a concentration of 200 nM free uptake by cells.

[0133] As used herein, the terms "isolated target site," "target site," and "isolated polynucleotide" can be used interchangeably, and refer herein to the nucleic acid target site to which siRNA has complementary or hybridization.For example, the isolated nucleic acid sequence of the target site can include the nucleic acid sequence to which the region of siRNA has complementary or hybridization.

[0134] As used herein, the term "complementarity" refers to the ability to form base pairs between two oligonucleotide strands. Base pairs are generally formed by hydrogen bonds between the nucleotides of antiparallel oligonucleotide strands. The bases of complementary oligonucleotide strands can be paired by Watson-Crick method (AT, AU, CG, etc.) or other methods (such as Hoogsteen or reverse Hoogsteen base pairing) that allow duplex formation.

[0135] Complementarity can be either perfect or imperfect. "Perfect complementarity" or "100% complementarity" refers to the fact that each nucleotide in a first oligonucleotide strand can form a hydrogen bond with the corresponding nucleotide in a second oligonucleotide strand in the double-stranded region of an siRNA molecule, without resulting in "mispaired" base pairs. "Incomplete complementarity," "partial complementarity," or "mismatch" refers to the fact that not all nucleotide units in the two strands are bound to each other by hydrogen bonds. For example, in two oligonucleotide strands, each 20 nucleotides long in the double-stranded region, if only two base pairs can be formed by hydrogen bonds in the double-stranded region, the complementarity of the oligonucleotide strands is 10%. In the same example, if 18 base pairs in the double-stranded region can be formed by hydrogen bonds, the oligonucleotide strands have 90% complementarity. Substantial complementarity refers to at least about 75%, about 79%, about 80%, about 85%, about 90%, about 95%, or 99% complementarity.

[0136] As used herein, the terms "3' untranslated region" and "3' UTR" can be used interchangeably and refer to a fragment of the 3' region of a messenger RNA (mRNA) that controls mRNA-based processes such as mRNA localization, mRNA stability, and translation. Furthermore, the 3' UTR can establish 3' UTR-mediated protein-protein interactions (PPIs), thus transmitting the genetic information encoded in the 3' UTR to a protein.

[0137] As used herein, "ODV" and "oligonucleotide delivery vehicle" are used interchangeably and refer to an oligonucleotide molecule comprising a duplex or double-stranded RNA (e.g., siRNA or saRNA) and an ACO covalently linked to the double-stranded RNA via a linker, as described in more detail below.

[0138] As used herein, the terms "covalent linker," "linker," and "linking moiety" are used interchangeably and refer to a molecule for covalently linking two molecules, such as a single-stranded oligonucleotide (e.g., ACO) and a dsRNA (e.g., siRNA or saRNA), two dsRNAs, etc. As described in more detail below, this term can include, for example, a nucleic acid linker, a peptide linker, etc., and also includes a disulfide linker.

[0139] As used herein, the term "synthetic" refers to the method by which an oligonucleotide is synthesized and includes any means by which RNA can be synthesized or chemically modified, such as chemical synthesis, in vitro transcription, vector expression, etc.

[0140] As used herein, the term "LNA" refers to a locked nucleic acid in which the 2'-oxygen atom and the 4'-carbon atom are linked by an extra bridge. As used herein, the term "BNA" refers to a 2'-O and 4'-aminoethylene bridged nucleic acid, which may contain a 5- or 6-membered bridge structure with an NO bond. As used herein, the term "PNA" refers to a nucleic acid mimic with a pseudopeptide backbone consisting of N-(2-aminoethyl)glycine units with the nucleobase linked to the glycine nitrogen via a carbonyl methylene linker.

[0141] As used herein, capitalized "SOD1" or "SOD1 gene" refers to the gene.

[0142] As used herein, the term "Sod1 mRNA" refers to the expression of the SOD1 gene or the message RNA (mRNA) produced from transcription of the SOD1 gene.

[0143] As used herein, the term "SOD1 protein" refers to the protein produced from expression of the SOD1 gene or translation of Sod1 mRNA.

[0144] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application is directed.

[0145] overview Aspects of the present application include oligonucleotide agents comprising oligonucleotide-based delivery vehicles (ODVs) to efficiently target one or more genes associated with a disease or condition without compromising oligonucleotide activity, and to provide improved delivery, chemical, biodistribution, bioavailability, and other pharmacological properties.

[0146] This application is based on a discussion of compositions and methods that combine targeted oligonucleotides (e.g., siRNA, saRNA) with ACOs to activate / upregulate gene expression, increasing the amount of full-length gene or protein expression, or knockout / silence gene expression, decreasing the amount of full-length gene or protein expression, thereby improving the therapeutic effect against genetic diseases. The term "oligonucleotide delivery vehicle (ODV)" refers to a structure that facilitates the introduction or uptake of a molecule into a cell, tissue, or organ of an individual by attaching an "auxiliary oligonucleotide (ACO)" to a chemical entity or moiety, such as a double-stranded oligonucleotide.

[0147] We found that ODV did not inhibit siRNA knockdown activity (Example 3) or dsRNA-induced gene activation (Example 5). We also found that the length, nucleotide composition, modifications (e.g., 2'-ome), linkage components, sequence palindromes, and number of phosphorothioate (PS) backbone substitutions of ACOs affected in vivo dsRNA activity. Compared to dsRNA without ODV, ODV-dsRNA showed improved in vivo activity in the CNS when administered to brain or spinal cord tissues by local injection.

[0148] A further aspect of the present application includes a method for treating amyotrophic lateral sclerosis (ALS) by administering an effective amount of an oligonucleotide agent comprising an SOD1-targeting siRNA. The siRNA inhibits expression of the SOD1 gene via the RNAi silencing mechanism. The present inventors have developed SOD1 siRNA with potent inhibitory effects for use in treating ALS.

[0149] Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is a fatal, adult-onset paralytic disease caused by motor neuron degeneration. ALS is characterized by adult-onset progressive degeneration of cranial, brainstem, and spinal motor neurons, leading to death due to respiratory failure within 3–5 years of diagnosis. ALS can be divided into familial and sporadic forms, depending on the family history. Sporadic ALS (sALS) accounts for 90% of ALS cases. The most commonly mutated genes account for approximately 75% of ALS cases in the United States: chromosome 9 open reading frame 72 (C9orf72; 40%), superoxide dismutase 1 (SOD1; 20%), transactivation response DNA-binding protein 43 (TDP43; 4%), and fusion-associated sarcoma / liposarcoma metastasis type (FUS / TLS; 4%). Mutations in the C9orf72 and SOD1 genes also account for approximately 5–8% and 2–3% of sALS cases, respectively. To date, most gene silencing studies have been performed using the SOD1 gene model, which involves high copy numbers of SOD1 G93A Transgenic mouse models remain the cornerstone of ALS research.

[0150] To date, only two drugs—riluzole and edaravone—have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of ALS. Riluzole inhibits glutamate release from presynaptic terminals and blocks postsynaptic N-methyl-D-aspartate (NMDA) receptors, prolonging survival by an average of 3 to 6 months. Edaravone is a free radical scavenger, reducing neuronal damage. It ameliorates oxidative damage by scavenging lipid peroxide hydroxyl radicals and transferring electrons to edaravone (the radical). These two drugs only slightly improve survival and disease progression, but they cannot cure ALS. Currently, there is no effective treatment for ALS, and new therapies are needed.

[0151] Among the known genes underlying ALS, the SOD1 gene remains the primary cause of fALS and has been considered an important target for ALS therapeutics. The human SOD1 gene is located on chromosome 21q22.11, from base pair 33,031,935 to base pair 33,041,241, with a genomic size of 9307 bp. The SOD1 gene encodes the monomeric SOD1 protein (153 amino acids, molecular weight 16 kDa) and also encodes the detoxifying copper / zinc-binding SOD1 enzyme, which has been found to be primarily localized in the cytoplasm, nucleus, peroxisomes, and mitochondria (Tafuri et al. 2015). Although the first description of ALS dates back to at least 1824 by Charles Bell, SOD1 as the first risk gene for ALS was discovered in 1993. In 1994, the first SOD1 transgenic mouse model (SOD1 G93A The establishment of SOD1 signaling pathways has ushered in a new era in ALS research. All of this evidence indicates that SOD1 mutants likely cause disease via gain-of-function, and that reducing their levels is beneficial. Excessive oxidation of wild-type SOD1 leads to toxic structural changes. Silencing SOD1 significantly attenuated astrocyte-mediated toxicity to motor neurons. Therefore, suppressing SOD1 expression is an important strategy for the treatment of ALS.

[0152] Oligonucleotide Agents Embodiments of the present application include an oligonucleotide agent comprising a single-stranded oligonucleotide (e.g., ACO) having a length of at least six nucleotides and a targeted double-stranded oligonucleotide covalently linked thereto, wherein the single-stranded oligonucleotide is a non-targeted oligonucleotide.

[0153] Embodiments of the present application include an oligonucleotide agent comprising a single-stranded oligonucleotide (e.g., ACO) having a length of at least six nucleotides and a target double-stranded oligonucleotide covalently linked thereto, wherein at least one phosphodiester bond between two adjacent nucleotides in the single-stranded oligonucleotide sequence is replaced with a phosphorothioate (PS), mesyl phosphoramidate, or boranophosphate bond.

[0154] Embodiments of the present application include an oligonucleotide agent comprising a single-stranded oligonucleotide having a length of at least six nucleotides and a targeted double-stranded oligonucleotide covalently linked thereto, wherein the single-stranded oligonucleotide comprises a palindromic sequence.

[0155] Embodiments of the present application include an oligonucleotide agent comprising a single-stranded oligonucleotide having a length of at least six nucleotides and a targeted double-stranded oligonucleotide covalently linked thereto, wherein at least about 14%, at least about 28%, at least about 42%, at least about 57%, at least about 71%, at least about 85%, at least about 92%, or about 100% of the nucleotides of the single-stranded oligonucleotide have a 2'-Ome modification.

[0156] Embodiments of the present application include an oligonucleotide agent comprising a single-stranded oligonucleotide having a length of at least six nucleotides and a target double-stranded oligonucleotide covalently linked thereto, wherein the single-stranded oligonucleotide contains no more than 72% or no more than 64% cytosines.

[0157] Embodiments of the present application include an oligonucleotide agent comprising a single-stranded oligonucleotide of 6 to 22 nucleotides in length and a targeted double-stranded oligonucleotide covalently linked thereto, wherein the single-stranded oligonucleotide can facilitate delivery of the double-stranded oligonucleotide in the central nervous system (CNS).

[0158] In some embodiments, the oligonucleotide agent comprises a double-stranded oligonucleotide, wherein the double-stranded oligonucleotide comprises a sense strand and an antisense strand, wherein the antisense strand has complementarity to a target nucleic acid; and a non-target single-stranded oligonucleotide, wherein the single-stranded oligonucleotide is 6 to 22 nucleotides in length. The double-stranded oligonucleotide and the single-stranded oligonucleotide are covalently linked, with or without one or more linking moieties, to form the oligonucleotide agent.

[0159] In some embodiments, the sense strand of the double-stranded target oligonucleotide is covalently linked to a non-target single-stranded oligonucleotide (NTO). In some embodiments, the antisense strand of the double-stranded target oligonucleotide is covalently linked to a non-target single-stranded oligonucleotide. In certain embodiments, the NTO does not have complementarity to the target nucleic acid of the double-stranded oligonucleotide. In certain embodiments, the NTO does not have complementarity to the target gene or target mRNA transcription product of the double-stranded oligonucleotide. In certain embodiments, the NTO does not have complementarity to the nucleic acid of the subject that has the target nucleic acid. In some embodiments, the target nucleic acid is a mammalian nucleic acid, for example, a nucleic acid derived from a human.

[0160] In some embodiments, an oligonucleotide agent has the following compound formula: JPEG2024523702000001.jpg17170O1 is a double-stranded oligonucleotide consisting of a sense strand and an antisense strand, where the antisense strand has complementarity to a target nucleic acid (e.g., a mammalian target nucleic acid); O2 is a non-target single-stranded oligonucleotide. In some embodiments, the single-stranded oligonucleotide is at least 6 nucleotides in length. L is a linker for covalently linking the double-stranded oligonucleotide and the single-stranded oligonucleotide.

[0161] In some embodiments, an oligonucleotide agent has the following compound formula: JPEG2024523702000002.jpg17170O1 is a double-stranded oligonucleotide consisting of a sense strand and an antisense strand, where the antisense strand has complementarity to a target nucleic acid; O2 is a non-target single-stranded oligonucleotide. In some embodiments, the single-stranded oligonucleotide is 6 to 22 nucleotides in length; L is a linker for covalently linking the double-stranded oligonucleotide to the single-stranded oligonucleotide; and optional components Cx, Cy, and Cz, where Cx, Cy, and Cz are independently absent or are conjugation groups selected from one or more of lipids, fatty acids, fluorophores, ligands, saccharides, peptides, antibodies, and any other commonly used conjugation groups. In some embodiments, the compound of Formula II contains one conjugation group. In some embodiments, the compound of Formula II contains two conjugation groups. In some embodiments, the compound of Formula II contains three conjugation groups.

[0162] In some embodiments, the double-stranded oligonucleotide is an siRNA. In some embodiments, the double-stranded oligonucleotide is an saRNA.

[0163] In some embodiments, the 5'-end, 3'-end, or internal nucleotide of the single-stranded oligonucleotide is linked to a linking moiety.In some embodiments, the internal nucleotide of the sense strand or antisense strand of the double-stranded oligonucleotide is replaced by a linking moiety, and the single-stranded oligonucleotide is covalently linked to the linking moiety.In some embodiments, the single-stranded oligonucleotide is covalently linked to the sense strand, antisense strand, or both the sense strand and antisense strand of the second oligonucleotide by the linking moiety.

[0164] In some embodiments, the single-stranded oligonucleotide is covalently linked to the 3'-end, or 5'-end, or both the 3'-end and 5'-end, or an internal nucleotide of the sense strand of the double-stranded oligonucleotide, as shown in Figures 1A, 1B, or 1C. In some embodiments, the single-stranded oligonucleotide is covalently linked to the 3'-end, or 5'-end, or both the 3'-end and 5'-end, or an internal nucleotide of the antisense strand of the double-stranded oligonucleotide.

[0165] In some embodiments, the covalently linked double-stranded target oligonucleotide and single-stranded oligonucleotide have a total nucleotide length of from 10 nucleotides to 500 nucleotides (e.g., from 10 nucleotides to 100 nucleotides, from 50 nucleotides to 100 nucleotides, from 50 nucleotides to 100 nucleotides, from 50 nucleotides to 200 nucleotides, from 20 nucleotides to 100 nucleotides, from 20 nucleotides to 200 nucleotides, from 20 nucleotides to 300 nucleotides, from 50 nucleotides to 300 nucleotides, from 20 nucleotides to 80 nucleotides, from 100 nucleotides to 300 nucleotides, from 300 nucleotides to 500 nucleotides).

[0166] Auxiliary Oligonucleotides (ACOs) Although several previous studies have demonstrated that siRNAs capable of inhibiting OD1 mRNA and reducing SOD1 protein expression can be used to treat patients with SOD1 protein-related diseases, such as amyotrophic lateral sclerosis (ALS), the present inventors have found that there are two unresolved problems: 1) the lack of efficacy of SOD1 siRNA molecules, and 2) the lack of an efficient delivery method for delivering siRNA molecules to cells in target organs or tissues. Surprisingly, the present invention has found that when a dsRNA agent, such as an siRNA, is linked to a non-targeting single-stranded auxiliary oligonucleotide (ACO) as disclosed, the bioavailability, biodistribution, and / or cellular uptake, and in vivo efficacy of the dsRNA are significantly improved compared to an oligonucleotide agent that does not contain an ACO. In particular, in some in vivo examples in the present application, the ACO of the oligonucleotide agent increases the biodistribution of the dsRNA in one, two, or more target tissues compared to an oligonucleotide agent that does not contain an ACO.

[0167] "Delivering into a cell," when referring to a targeted double-stranded oligonucleotide, e.g., a double-stranded RNA agent (dsRNA), such as siRNA or saRNA, refers to efficient uptake or absorption by the cell, as understood by those skilled in the art. Absorption or uptake of dsRNA can occur through unassisted diffusive or active cellular processes, or through the use of auxiliary agents or devices. This term is not limited to cells in vitro; dsRNA can also be "introduced into a cell," in which case the cell is part of a living organism. In such cases, introduction into a cell also includes delivery to an organism. For example, in vivo introduction can involve injection of dsRNA into a tissue site or systemic administration. In vitro introduction into a cell includes methods known in the art, such as electroporation, free uptake, and lipofection. Additional approaches not known in the art are described below.

[0168] ACO of oligonucleotide agent is a single-stranded oligonucleotide, and its delivery characteristics make it advantageous for oligonucleotide agent.Therefore, ACO does not target the nucleic acid in the subject that dsRNA targets, or the "natural" nucleic acid from the subject, such as the target nucleic acid of dsRNA.In some embodiments, ACO does not target the nucleic acid in the subject that dsRNA targets.In some embodiments, ACO does not have complementarity with the nucleic acid that dsRNA targets.In some embodiments, ACO does not have complementarity with the gene sequence or its mRNA transcript that dsRNA targets.

[0169] In some embodiments, the length of the ACO ranges from 6 to 19 nucleotides, such as 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 22 or more, 20 or more, 21 or more, or 22 or more nucleotides. In some embodiments, the length of the ACO is 6 to 18 contiguous oligonucleotides. In some embodiments, the length of the ACO is 10 to 14 nucleotides.

[0170] In some embodiments, the length of the ACO can regulate the activity and / or biodistribution of the oligonucleotide agent within a target tissue or cell of interest. For example, the inventors have found that shorter ACOs exhibit oligonucleotide agent activity throughout the central nervous system, while longer ACOs exhibit oligonucleotide agent activity only in specific regions of the brain, such as the cerebellum. In some embodiments, activity of the oligonucleotide agent throughout the central nervous system is desired. In some embodiments, activity of the oligonucleotide agent in specific regions of the brain is desired.

[0171] The ACO may contain a modified sequence to further enhance the ability of the oligonucleotide agent to deliver a second oligonucleotide. In some embodiments, the sequence of the ACO contains one or more of chemically modified nucleotides, or at least one phosphodiester bond between two adjacent nucleotides in the oligonucleotide sequence is replaced with a phosphorothioate bond or a boranophosphate bond. Chemical modifications of the ACO include, but are not limited to, modification of the 2'-OH of the ribose in the nucleotide, modification or absence of a base in the nucleotide, locking or bridging of the nucleic acid, peptide nucleic acid as the nucleotide, deoxyribonucleotide (DNA) as the nucleotide, nucleotides with a 5'-phosphate moiety, nucleotides with a 5'-(E)-vinylphosphonate moiety, nucleotides with a 5'-methylcytosine moiety, etc.

[0172] An ACO can contain at least one phosphodiester bond substituted with a phosphorothioate (PS) bond on the backbone of the nucleotide sequence. In some embodiments, an ACO contains multiple PS backbone modifications, such as at least two PS, at least three PS, at least four PS, at least five PS, at least six PS, or more than six PS backbone modifications. In some embodiments, an ACO contains at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% phosphodiester bonds substituted with phosphorothioate (PS) bonds on the backbone of the nucleotide sequence. As a non-limiting example, a 14 nt ACO can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 PS backbone modifications. In some embodiments, an ACO having a length of N nucleotides comprises N-1 PS modifications.

[0173] ACOs may also have a specific composition of nucleotides. ACOs may have any percent adenine composition. ACOs may have any percent adenine composition. Percent adenine compositions that find use in the present disclosure include, without limitation, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70% or more, etc., and in preferred embodiments, the percent adenine composition is about 35% to about 65%.

[0174] In some embodiments, the ACO may have a certain percentage of cytosines within the nucleotide sequence of the ACO. The ACO may have any percentage cytosine composition. Percent cytosine compositions that find use in the present disclosure include, without limitation, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80% or more, etc., and in preferred embodiments, the percent cytosine composition is from about 35% to about 72%.

[0175] In some embodiments, the ACO may have a certain percentage of cytosines within the nucleotide sequence of the ACO. The ACO may have any percentage guanosine composition. Percent adenine compositions that find use in the present disclosure include, without limitation, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70% or more, etc., and in preferred embodiments, the percent adenine composition is about 35% to about 65%.

[0176] ACOs may have any percent uracil composition. Percent uracil compositions that find use in the present disclosure include, without limitation, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80% or more, etc., and in preferred embodiments, the percent uracil composition is from about 35% to about 72%.

[0177] In some embodiments, the ACO can have a certain percentage of purines within the nucleotide sequence of the ACO. The ACO can have any percentage purine composition. Percent purine compositions that find use in the present disclosure include, without limitation, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80% or more, etc., and in preferred embodiments, the percent purine composition is about 65% to about 72%.

[0178] In some embodiments, the ACO can have a certain percentage of pyrimidines within the nucleotide sequence of the ACO. The ACO can have any percentage pyrimidine composition. Percent pyrimidine compositions that find use in the present disclosure include, without limitation, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-86%, 80% or more, etc., and in preferred embodiments, the percent pyrimidine composition is about 42% to about 58%.

[0179] In some embodiments, the ACO may have a specific combination of purines and pyrimidines. The specific combination of purines and pyrimidines may be any combination desired. Specific combinations of purines and pyrimidines that find use in the present disclosure include, but are not limited to, about 30% purines and about 70% pyrimidines, about 40% purines and about 60% pyrimidines, about 50% purines and about 50% pyrimidines, about 60% purines and about 40% pyrimidines, about 70% purines and about 30% pyrimidines, etc. In a preferred embodiment, the specific combination of purines and pyrimidines is about 42% purines and about 58% pyrimidines.

[0180] In some embodiments, the nucleotide sequence of the single-stranded oligonucleotide has at least 40%, at least 50%, at least 60%, or at least 70% of the nucleotides having a 2'-Ome modification, and in some embodiments, 70-100% of the nucleotides in the ACO have a 2'-Ome modification.

[0181] In some embodiments, the nucleotide sequence of the single-stranded oligonucleotide is a palindromic sequence. As used herein, the term "palindromic sequence" refers to a nucleic acid sequence in a double-stranded DNA or RNA molecule where the reading in one direction (e.g., 5' to 3') on one strand is identical to the sequence in the same direction (e.g., 5' to 3') on the complementary strand. In some embodiments, a single-stranded oligonucleotide having a palindromic sequence enhances the activity of a double-stranded oligonucleotide or the delivery of an oligonucleotide agent. In some embodiments, a palindromic ACO enhances the protein binding ability and knockdown activity of ODV-siRNA against Sod1 mRNA. In some embodiments, a single-stranded oligonucleotide having a palindromic sequence is selected from the group of SEQ ID NOs: 1300-1314.

[0182] Accordingly, aspects of the present application relate to oligonucleotide agents capable of inhibiting the expression of superoxide dismutase 1 (SOD1), including small interfering RNAs (siRNAs), and ACOs.

[0183] In some embodiments, the oligonucleotide agent comprises one or more conjugated ACOs to enhance biodistribution of the oligonucleotide agent in particular tissues and to increase the permeability and crossability of the oligonucleotide agent across membranes, such as the blood-brain barrier.

[0184] In some embodiments, the ACO is an oligonucleotide comprising a 5' end and a 3' end.

[0185] In some embodiments, the dsRNA and ACO are covalently linked to form an oligonucleotide agent, with or without one or more linking moieties.

[0186] In another aspect of the present application, an oligonucleotide agent is provided, comprising an siRNA and a non-targeting ACO. The ACO comprises a single-stranded oligonucleotide sequence consisting of a nucleotide sequence having at least 60% homology (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% homology, or 100% identity) to a nucleotide sequence selected from SEQ ID NOs: 953-954. In some embodiments, the ACO comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence selected from SEQ ID NOs: 953-954.

[0187] In some embodiments, the oligonucleotide agent comprises a single-stranded oligonucleotide (ACO) linked to a dsRNA. In some embodiments, the dsRNA is a naturally occurring nucleic acid. In some embodiments, the naturally occurring nucleic acid is a target nucleic acid. In certain embodiments, the naturally occurring nucleic acid is an intracellular nucleic acid.

[0188] In some embodiments, the ACO is a non-targeted oligonucleotide (NTO). In some embodiments, the ACO is a synthetic non-targeted oligonucleotide. In some embodiments, the ACO is a random non-targeted oligonucleotide. In some embodiments, the ACO comprises RNA, DNA, BNA, LNAPNA, or a combination thereof.

[0189] In some embodiments, ACOs interact with one or more of proteins in the cell membrane, cytoplasmic proteins, peptides, ligands, lipids, fatty acids, saccharides, proteoglycans, and zwitterionic phosphocholines. Such interactions of ACOs provide increased biodistribution and concentration of targeted double-stranded oligonucleotides for various targeting tasks and localized delivery to cells of interest. Furthermore, such interactions can reduce or eliminate the cytotoxicity of oligonucleotide agents, ensuring potent "on-target" activity without overtly affecting cell viability.

[0190] In certain embodiments, the protein that interacts with ACO is selected from one or more of the following: serum albumin, IgG, apolipoprotein AI, apolipoprotein A-II, complement factor C3, transferrin, alpha-1 antitrypsin, haptoglobin, hemopexin, fibrinogen, alpha-2-macroglobulin, prealbumin / TTR, antithrombin III, alpha-1-antichymotrypsin, beta-2-glycoprotein, ceruloplasmin, alpha-1 acid glycoprotein, complement component C1q, complement factor C4, histidine-rich glycoprotein, plasminogen, fibronectin, ApoB100, factor H, apolipoprotein E, and factor V.

[0191] In still other embodiments, the protein that interacts with ACO is selected from one or more of the following: ASGPR, EGFR, LDLR, M6PR, TLR, stabilin, SRB, nucleolin, AP2M1, EEA1, Rab5C, Rab7a, STX5, P115, COPII, M6PR, GCC2, ANXA2, TCP1, ALIX, TSG101, VPS28, GLP-1, and HSP-90.

[0192] In certain embodiments, the interaction of the ACO is mediated by direct binding or by one or more conjugated ligands covalently attached to the ACO, the double-stranded oligonucleotide, or both. In some embodiments, the one or more conjugated ligands include lipids, fatty acids, fluorophores, saccharides, peptides, antibodies, and any other commonly used conjugated ligands.

[0193] In certain embodiments, the conjugated ligand is selected from one or more of a cell-penetrating peptide, polyethylene glycol, an alkaloid, a tryptamine, a benzimidazole, a quinolone, an amino acid, cholesterol, glucose, and N-acetylgalactosamine. In certain embodiments, the one or more conjugated ligands is a fatty acid.

[0194] In some embodiments, an oligonucleotide agent comprising one or more conjugated ligands enhances the biodistribution of the oligonucleotide agent in particular tissues, reduces or eliminates the cytotoxicity of the oligonucleotide agent, and increases the permeability and crossability of the oligonucleotide agent across membranes such as the blood-brain barrier.

[0195] In some embodiments, the ACO comprises a single-stranded oligonucleotide sequence comprising a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to a nucleotide sequence selected from the sequences of SEQ ID NOs: 1-22.

[0196] In some embodiments, the oligonucleotide agent comprises an ACO. In certain embodiments, the ACO comprises a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to a nucleotide sequence selected from the group consisting of AC2(N22) (SEQ ID NO: 1), AC2(N20) (SEQ ID NO: 2), AC2(N18) (SEQ ID NO: 3), AC2(N16) (SEQ ID NO: 4), AC2(N15) (SEQ ID NO: 5), AC2(N14) (SEQ ID NO: 6), AC2(N12) (SEQ ID NO: 7), AC2(N12) (SEQ ID NO: 7), AC2(N10) (SEQ ID NO: 8), AC2(N8) (SEQ ID NO: 9), AC2(N6) (SEQ ID NO: 10), AC2(22) ( SEQ ID NO: 11), AC2(18) (SEQ ID NO: 12), AC2(16) (SEQ ID NO: 13), AC2(15) (SEQ ID NO: 14), AC2(14) (SEQ ID NO: 15), AC2(13) (SEQ ID NO: 16), AC2(12) (SEQ ID NO: 17), AC2(11) (SEQ ID NO: 18), AC2(10) (SEQ ID NO: 19), AC2(9) (SEQ ID NO: 20), AC2(8) (SEQ ID NO: 21), and AC2(6) (SEQ ID NO: 22).

[0197] In certain embodiments, the ACO comprises a chemically modified nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to a nucleotide sequence selected from the group of SEQ ID NOs: 1299-1379. In certain embodiments, the ACO has a chemically modified nucleotide sequence selected from the group of SEQ ID NOs: 1299-1379. In certain embodiments, the ACO has a chemically modified nucleotide sequence and a linker selected from the group of SEQ ID NOs: 1299-1379. In certain embodiments, the ACO has a chemically modified nucleotide sequence with 0, 1, 2, or 3 chemical modification differences relative to a nucleotide sequence selected from the group of SEQ ID NOs: 1299-1379.

[0198] In some embodiments, an oligonucleotide agent of the present application includes one or more ACOs, e.g., 2, 3, 4, 5, 6, 7, 9, or 10 ACOs, covalently linked to a dsRNA, with or without one or more linkers between the ACOs and the dsRNA. The amount of ACOs can vary from 2 to 10, 2 to 100, 2 to 1,000, or 2 to 10,000, as needed, and are linked to the dsRNA via a branched or linear multivalent linker, e.g., a polymer linker. In some embodiments, multiple ACOs are covalently linked to two or more dsRNAs in a single agent, e.g., 2, 3, 4, 5, 6, 7, 9, 10, or more dsRNAs, including saRNA and / or siRNA.

[0199] In some embodiments, an oligonucleotide agent of the present application comprises one ACO and multiple dsRNAs, e.g., 2, 3, 4, 5, 6, 7, 9, or 10 dsRNAs, which may or may not be linked by one or more linkers between the ACO and the dsRNA. Optionally, the amount of dsRNA, including saRNA and / or siRNA, can vary from 2 to 10, 2 to 100, 2 to 1,000, or 2 to 10,000, and is linked to the ACO via a branched or linear multivalent linker, e.g., a polymer linker.

[0200] Targeted Oligonucleotides In some embodiments, target oligonucleotide comprises double-stranded oligonucleotide.In some embodiments, double-stranded oligonucleotide is double-stranded RNA (dsRNA).DsRNA can be any dsRNA that is considered useful, but the dsRNA that can be used in the present disclosure includes but is not limited to siRNA, saRNA, etc.

[0201] In some embodiments, the double-stranded oligonucleotide comprises a sense strand and an antisense strand, and the antisense strand is complementary to the target nucleic acid.In some embodiments, the antisense strand that is complementary to the target nucleic acid is located in a promoter sequence.In some embodiments, the antisense strand that is complementary to the target nucleic acid is located in the coding sequence or template sequence of a gene.In some embodiments, one of the sense strand or the antisense strand is complementary to the target nucleic acid that is a gene transcript, for example, mRNA or pre-mRNA.

[0202] In some embodiments, dsRNA comprises a sense strand that is at least 17 consecutive nucleotides.In some embodiments, dsRNA comprises a sense strand that is at least 18 consecutive nucleotides.In some embodiments, dsRNA comprises a sense strand that is at most 60 consecutive nucleotides.

[0203] In some embodiments, the sense strand has a length ranging from about 10 nucleotides or more, about 15 nucleotides or more, about 20 nucleotides or more, about 25 nucleotides or more, about 30 nucleotides or more, about 35 nucleotides or more, about 40 nucleotides or more, about 45 nucleotides or more, about 50 nucleotides or more, about 55 nucleotides or more, or about 60 nucleotides or more. In some embodiments, the sense strand has a length of 10 to 100 nucleotides (e.g., 10 to 20 nucleotides, 10 to 50 nucleotides, 10 to 90 nucleotides, 20 to 95 nucleotides, 30 to 70 nucleotides, 40 to 80 nucleotides, 50 to 100 nucleotides, 10 to 40 nucleotides, or 10 to 30 nucleotides). In some embodiments, the sense strand has a length ranging from 10 to 60 nucleotides (e.g., 10 to 20 nucleotides, 10 to 50 nucleotides, 10 to 40 nucleotides, or 10 to 30 nucleotides). In some embodiments, the sense strand has a length ranging from 27 to 41 nucleotides.

[0204] In some embodiments, the antisense strand has a length ranging from about 10 nucleotides or more, about 15 nucleotides or more, about 20 nucleotides or more, about 25 nucleotides or more, about 30 nucleotides or more, about 35 nucleotides or more, about 40 nucleotides or more, about 45 nucleotides or more, about 50 nucleotides or more, about 55 nucleotides or more, or about 60 nucleotides or more. In some embodiments, the antisense strand is 10 to 100 nucleotides in length (e.g., 10 to 20 nucleotides, 10 to 50 nucleotides, 10 to 90 nucleotides, 20 to 95 nucleotides, 30 to 70 nucleotides, 40 to 80 nucleotides, 50 to 100 nucleotides, 10 to 40 nucleotides, or 10 to 30 nucleotides). In some embodiments, the antisense strand is 19 to 30 nucleotides in length. In some embodiments, the antisense strand is 18 to 26 nucleotides in length.

[0205] The double-stranded oligonucleotide may contain a modified sequence to further increase the stability and / or ability of the double-stranded oligonucleotide to regulate gene expression. In some embodiments, the sequence of the double-stranded oligonucleotide contains one or more of chemically modified nucleotides, or at least one phosphodiester bond between two adjacent nucleotides in the oligonucleotide sequence is replaced with a phosphorothioate bond or a boranophosphate bond. Chemical modifications of the double-stranded oligonucleotide include, but are not limited to, modification of the 2'-OH of the ribose in the nucleotide, modification or absence of a base in the nucleotide, locked nucleic acid or crosslinked nucleus, nucleotides that are peptide nucleic acids, nucleotides that are deoxyribonucleotides (DNA), nucleotides with a 5'-phosphate moiety, nucleotides with a 5'-(E)-vinylphosphonate moiety, nucleotides with a 5'-methylcytosine moiety, etc.

[0206] Short interfering RNA (siRNA) Embodiments of the present application are based, in part, on the surprising discovery that oligonucleotide agents (e.g., siRNAs, also referred to herein as "SOD1 gene siRNAs," "SOD1 siRNAs," or "siSOD1") can inhibit or down-regulate expression of the SOD1 gene in cells. Reduction of functional SOD1 gene transcripts following administration of an oligonucleotide agent of the present invention can achieve a significant reduction or down-regulation of Sod1 mRNA and SOD1 protein levels in a cell or mammal.

[0207] In particular, the present inventors have discovered a functional oligonucleotide agent capable of inhibiting the expression of superoxide dismutase 1 (SOD1) that consists of an siRNA, wherein the siRNA comprises a sense strand and an antisense strand that form a duplex, and the antisense strand comprises a nucleotide sequence of at least 10 consecutive nucleotides that has at least 85% nucleotide sequence complementarity or homology to a portion of the nucleotide sequence of Sod1 mRNA, with 0, 1, 2, or 3 mismatches.

[0208] As a beneficial result, the target sequence (e.g., an isolated nucleic acid sequence comprising the target sequence) can, upon interaction with the siRNA, inhibit / downregulate Sod1 mRNA transcripts by at least 10% compared to baseline levels of Sod1 mRNA. Based at least in part on these discoveries, the present application features siRNAs, compositions, and pharmaceutical compositions for inhibiting / downregulating Sod1 mRNA transcripts by at least 10% compared to baseline levels of Sod1 mRNA. In some embodiments, the siRNA inhibits or downregulates Sod1 mRNA by 10% or more. For example, the siRNA inhibits or downregulates Sod1 mRNA by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, or more than 100% compared to baseline levels of Sod1 mRNA.

[0209] Also provided herein are methods for preventing or treating diseases or conditions in an individual induced by overexpression of SOD1 protein, mutations in the SOD1 gene, and / or high or abnormal SOD1 levels, comprising administering to the individual any of the siRNAs, compositions, and / or pharmaceutical compositions described herein.

[0210] Embodiments of the present application are also based in part on the discovery that a Sod1 mRNA inhibitory oligonucleotide agent includes an siRNA having a sense strand having at least 85%, at least 90%, or at least 95% homology to a nucleotide sequence selected from the group consisting of DS17-0001 (SEQ ID NO: 384), DS17-0002 (SEQ ID NO: 372), DS17-0003 (SEQ ID NO: 409), DS17-0004 (SEQ ID NO: 357), DS17-0005 (SEQ ID NO: 486), DS17-0029 (SEQ ID NO: 588), DS17-01N3 (SEQ ID NO: 912), DS17-02N3 (SEQ ID NO: 914), DS17-03N3 (SEQ ID NO: 916), DS17-04N3 (SEQ ID NO: 918), and DS17-05N3 (SEQ ID NO: 920).

[0211] In some other embodiments, the Sod1 mRNA inhibitory oligonucleotide agent comprises an siRNA having an antisense strand having at least 85%, at least 90%, or at least 95% homology to a nucleotide sequence selected from the group consisting of DS17-0001 (SEQ ID NO: 653), DS17-0002 (SEQ ID NO: 641), DS17-0003 (SEQ ID NO: 678), DS17-0004 (SEQ ID NO: 626), DS17-0005 (SEQ ID NO: 755), DS17-0029 (SEQ ID NO: 857), DS17-01N3 (SEQ ID NO: 913), DS17-02N3 (SEQ ID NO: 915), DS17-03N3 (SEQ ID NO: 917), DS17-04N3 (SEQ ID NO: 919), and DS17-05N3 (SEQ ID NO: 921).

[0212] In some embodiments, the Sod1 mRNA inhibitory oligonucleotide agent comprises an siRNA, wherein the sense strand and the antisense strand of the siRNA have nucleotide sequences that are at least 85%, at least 90%, or at least 95% identical, independently, to a pair of nucleotide sequences selected from the group consisting of DS17-0001 (SEQ ID NO:384 and SEQ ID NO:653), DS17-0002 (SEQ ID NO:372 and SEQ ID NO:641), DS17-0003 (SEQ ID NO:409 and SEQ ID NO:678), DS17-0004 (SEQ ID NO:35 7 and SEQ ID NO: 626), DS17-0005 (SEQ ID NO: 486 and SEQ ID NO: 755), DS17-0029 (SEQ ID NO: 588) and (SEQ ID NO: 857), AC2 (DS17-01N3) (SEQ ID NO: 912 and SEQ ID NO: 913), DS17-02N3 (SEQ ID NO: 914 and (SEQ ID NO: 915), DS17-03N3 (SEQ ID NO: 916 and SEQ ID NO: 917), DS17-04N3 (SEQ ID NO: 918 and SEQ ID NO: 919), AC2 (DS17-05N3) (SEQ ID NO: 920 and SEQ ID NO: 921).

[0213] The present disclosure also provides an siRNA targeting the 3'UTR of the SOD1 gene to inhibit intracellular Sod1 mRNA transcript levels. The siRNA comprises an oligonucleotide sequence having a length ranging from 16 to 35 contiguous nucleotides, the contiguous oligonucleotide sequence comprising a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% homology or complementarity to an equal-length portion of SEQ ID NO: 59, and the siRNA inhibits SOD1 gene mRNA transcripts by at least 80% compared to baseline Sod1 mRNA levels.

[0214] Surprisingly, the present inventors discovered that functional siRNAs capable of inhibiting Sod1 mRNA transcription levels are not randomly distributed on the SOD1 gene, particularly on the 3'-UTR of the SOD1 gene, but are concentrated in specific hotspot regions. Only certain regions on the 3'-UTR of the SOD1 gene are favorable for the inhibitory function of siRNAs, such as the regions 549 to 562 (H1; SEQ ID NO: 61) and 568 to 580 (H2; SEQ ID NO: 63) of the SOD1 gene. Similarly, only one region on the 3'-UTR of the SOD1 gene is favorable for the inhibitory function of ASOs, namely, the region 552 to 566 (H3; SEQ ID NO: 65). The regions disclosed herein are so-called "hotspots."

[0215] The inventors have also discovered that the optimal target sequence / sense strand of an siRNA within the SOD1 gene, or particularly the 3'-UTR of the SOD1 gene, contains the following sequences: (1) a GC content of between 35% and 65%; (2) no more than five consecutive identical nucleotides; (3) no more than three dinucleotide repeats; and (4) no more than three trinucleotide repeats. As a beneficial result, upon interaction with an siRNA, the target sequence (e.g., an isolated nucleic acid sequence comprising the target sequence) can inhibit Sod1 mRNA transcript levels by at least 80% compared to baseline levels of Sod1 mRNA. Based at least in part on these discoveries, the present disclosure features siRNAs, compositions, and pharmaceutical compositions for inhibiting Sod1 mRNA transcription levels by at least 80% compared to baseline levels of Sod1 mRNA. Also provided herein are methods for preventing or treating a disease or condition induced by elevated intracellular SOD1 protein levels in an individual, comprising administering any of the siRNAs, compositions, and / or pharmaceutical compositions described herein.

[0216] Thus, the present application discloses siRNA hotspots in the 3'-UTR of the SOD1 gene, and the oligonucleotide agents disclosed in the present application have at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% homology or complementarity to equal length portions of the hotspot, and the oligonucleotide agents inhibit mRNA transcription of the SOD1 gene by at least 80% compared to baseline Sod1 mRNA levels.

[0217] The present application further discloses an isolated target site for siRNA in the 3'-UTR of the SOD1 gene, wherein the isolated target site has a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% homology to a sequence selected from SEQ ID NOs: 1068-1113.

[0218] In some embodiments, the sense strand of the siRNA has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a nucleotide sequence selected from SEQ ID NOs: 976-1021. In some embodiments, the antisense strand of the siRNA has a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a nucleotide sequence selected from SEQ ID NOs: 1022-1067. In certain embodiments, the selected target region of the SOD1 gene comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1068-1113.

[0219] In some embodiments, the Sod1 mRNA inhibitory oligonucleotide agent comprises an siRNA, wherein the siRNA comprises a sense strand and an antisense strand having a nucleotide sequence independently having at least 85%, at least 90%, or at least 95% homology to a pair of nucleotide sequences selected from the following groups: DS17-01M3 (SEQ ID NO: 922 and SEQ ID NO: 923), DS17-02M3 (SEQ ID NO: 924 and SEQ ID NO: 925), DS17-03M3 (SEQ ID NO: 926 and SEQ ID NO: 927), DS17-04M3 (SEQ ID NO: 928 and SEQ ID NO: 929), and DS17-05M3 (SEQ ID NO: 930 and SEQ ID NO: 931).

[0220] Furthermore, the siRNAs provided in Table 3 and Table 22 identify sites in the SOD1 transcription product that are susceptible to RISC-mediated cleavage. Thus, the present invention further features siRNAs that target one of these sequences. As used herein, an siRNA is said to target a specific site within an RNA transcript if the siRNA promotes cleavage of the transcript anywhere within that specific site. Such siRNAs generally comprise at least 15 consecutive nucleotides from one of the sequences provided in Table 3 or 22 combined with an additional nucleotide sequence taken from a region contiguous to the selected sequence of the SOD1 gene.

[0221] The siRNA oligonucleotide agents described herein comprise an RNA strand (antisense strand) having a region of 60 nucleotides or less in length, i.e., 15 to 40 nucleotides, typically 19 to 25 nucleotides in length, that is substantially complementary to at least a portion of the mRNA transcript of the SOD1 gene. The use of these siRNAs enables targeted degradation of mRNA of genes involved in pathologies associated with SOD1 expression in mammals. Low doses of SOD1 siRNA, in particular, can specifically and efficiently mediate RNAi and significantly inhibit SOD1 gene expression. Using cell-based assays, the inventors demonstrated that siRNAs targeting SOD1 specifically and efficiently mediate RNAi, resulting in significant inhibition of SOD1 gene expression. Therefore, methods and oligonucleotide agents comprising these siRNAs are useful for treating pathological processes that may be mediated by SOD1 downregulation, such as the treatment of diseases that cause elevated SOD1 levels, such as amyotrophic lateral sclerosis (ALS). The following detailed description discloses methods for preparing and using oligonucleotide agents, including siRNA, to inhibit expression of the SOD1 gene, as well as oligonucleotide agents and methods for treating diseases and disorders caused by expression of this gene.

[0222] In some embodiments, the contiguous oligonucleotide sequence of the siRNA has 5 or fewer nucleotide differences or mismatches with the equal-length portion of Sod1 mRNA, i.e., 5, 4, 3, 2, 1, or 0. In some embodiments, the contiguous oligonucleotide sequence of the sense strand of the siRNA has 3 or fewer nucleotide differences or mismatches with the equal-length portion of Sod1 mRNA, i.e., 3, 2, 1, or 0. In some embodiments, the contiguous oligonucleotide sequence of the antisense strand of the siRNA has 3 or fewer nucleotide differences or mismatches with the equal-length portion of Sod1 mRNA.

[0223] In some embodiments, the Sod1 mRNA disclosed herein does not contain a nucleotide mutation. In some embodiments, the Sod1 mRNA disclosed herein contains at least one nucleotide mutation. In some embodiments, the Sod1 mRNA disclosed herein contains at least one nucleotide mutation in the siRNA targeting site. In some embodiments, the Sod1 mRNA disclosed herein contains at least one nucleotide mutation upstream and / or downstream of the siRNA targeting site.

[0224] In some embodiments, the difference or mismatch is located in the middle or 3' end of the oligonucleotide sequence of siRNA.The method and principle of siRNA molecule design are well known to those skilled in the art, for example, Place et al., Molecular Therapy-Nucleic Acids (2012) 1, e15; and Liet. al., PNAS, 2006, vol.103, no.46, 17337-17342, are incorporated herein by reference in their entirety.

[0225] In one embodiment, an RNA interference agent comprises a single-stranded RNA that interacts with a target RNA sequence and induces cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNA introduced into plant or invertebrate cells is degraded into siRNAs by a type III endonuclease known as Dicer (Sharp et al., Genes Dev. 2001, 15:485). Dicer, an enzyme similar to RNase III, processes dsRNA into 19-23 base pair short 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, allowing the complementary antisense strand to recognize the target (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect, the present invention relates to a single-stranded RNA that promotes the formation of a RISC complex, resulting in the silencing of a target gene.

[0226] In some embodiments, the siRNA disclosed herein comprises a sense strand and an antisense strand. The sense strand and the antisense strand comprise complementary regions capable of forming a double-stranded nucleic acid structure that inhibits SOD1 transcription in cells via the RNAi mechanism. As used herein, the RNAi mechanism (also referred to as RNA interference) refers to a mechanism by which a double-stranded nucleic acid structure downregulates a target gene in a sequence-specific manner at the transcriptional level. The sense strand and the antisense strand of the siRNA may be present on two different nucleic acid strands or on a single nucleic acid strand (e.g., a contiguous nucleic acid sequence). When the sense strand and the antisense strand are present on two different strands, at least one strand of the siRNA has a 3' overhang of 0 to 6 nucleotides in length, for example, 0, 1, 2, 3, 4, 5, or 6 nucleotides in length, and in some cases, both strands have 3' overhangs of 2 or 3 nucleotides in length.

[0227] The overhanging nucleotide is optionally a thymine deoxyribonucleotide (dT), or optionally a natural overhang, which is a nucleotide selected from or complementary to the corresponding position on the DNA target. When the sense strand and antisense strand are located on a single nucleic acid strand, the siRNA is optionally a hairpin-type single-stranded nucleic acid molecule, in which the complementary regions of the sense strand and antisense strand form a double-stranded nucleic acid structure with each other. In the siRNAs disclosed herein, in some embodiments, the sense strand has a length ranging from 10 to 60 nucleotides. For example, in some embodiments, the sense strand and the antisense strand independently comprise a length of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides. In some embodiments, the antisense strand has a length ranging from 10 to 60 nucleotides. For example, in some embodiments, the sense and antisense strands are independently 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length.

[0228] In some embodiments, one strand of the siRNA has at least 75% (e.g., at least about 79%, about 80%, about 85%, about 90%, about 95%, or about 99%) sequence homology or complementarity to a nucleotide sequence fragment of an SOD1 gene transcript. Specifically, the sense strand of the siRNA disclosed herein has at least 75% (e.g., at least about 79%, about 80%, about 85%, about 90%, about 95%, or about 99%) sequence homology to a nucleotide sequence fragment of an SOD1 gene transcript, and the antisense strand of the siRNA disclosed herein has at least 75% (e.g., at least about 79%, about 80%, about 85%, about 90%, about 95%, or about 99%) sequence homology to a nucleotide sequence fragment of an SOD1 gene transcript.

[0229] In some embodiments, the sense strand of the siRNA disclosed herein has at least 75% (e.g., at least about 79%, about 80%, about 85%, about 90%, about 95%, or about 99%) sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 976-1021. In some embodiments, the antisense strand of the siRNA disclosed herein has at least 75% (e.g., at least about 79%, about 80%, about 85%, about 90%, about 95%, or about 99%) sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1022-1067.

[0230] In certain embodiments, one strand of the siRNA may have five or fewer nucleotide differences or mismatches, i.e., 5, 4, 3, 2, 1, or 0, with respect to the nucleotide sequence of any portion of SEQ ID NO: 59. Specifically, the sense strand of the siRNA disclosed herein may have three or fewer nucleotide differences, i.e., 3, 2, 1, or 0, with respect to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 976-1021, and the antisense strand of the siRNA disclosed herein may have three or fewer nucleotide differences, i.e., 3, 2, 1, or 0, with respect to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1022-1067. In some embodiments, the differences or mismatches are located in the middle or at the 3' end of the sense or antisense strand of the siRNA.

[0231] In some embodiments, the antisense strand disclosed herein can interact with the target nucleic acid sequence of the mRNA of SOD1 gene in a sequence-specific manner, which means that the antisense strand can undergo hybridization with the target nucleic acid through hydrogen bonds.In some embodiments, when written in 5' to 3' direction, the antisense strand has a nucleotide sequence that constitutes the reverse complement of the target portion of the target nucleic acid that it is targeted to.In certain such embodiments, when written in 5' to 3' direction, the antisense strand has a nucleotide sequence that constitutes the reverse complement of the target portion in the fragment of SOD1 gene transcript.

[0232] Antisense oligonucleotides (ASOs) The present disclosure also provides antisense oligonucleotides (ASOs) capable of inhibiting intracellular Sod1 mRNA levels. These ASOs regulate the RNAi-Sod1 mRNA-SOD1 protein pathway via an RNase H-dependent mechanism of action, and can therefore be used to treat patients with SOD1 protein-related diseases, such as amyotrophic lateral sclerosis (ALS).

[0233] Disclosed herein are oligonucleotide agents, including antisense oligonucleotides (ASOs), comprising an oligonucleotide sequence having a length ranging from 12 to 30 contiguous nucleotides, the contiguous oligonucleotide sequence comprising a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% homology or complementarity to an equal-length portion of SEQ ID NO:59, wherein the ASO inhibits mRNA transcription of the SOD1 gene by at least 60% compared to baseline Sod1 mRNA levels. In some embodiments, the ASO inhibits mRNA transcription of the SOD1 gene by 60% or more compared to baseline Sod1 mRNA levels. For example, the ASO inhibits mRNA transcription of the SOD1 gene by at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, or more than 100% compared to baseline Sod1 mRNA levels.

[0234] The present application further discloses that ASOs capable of inhibiting the Sod1 mRNA transcription level present in the 3'-UTR of the SOD1 gene are not randomly distributed on the SOD1 gene, or particularly on the 3'-UTR of the SOD1 gene, but are concentrated in specific hotspot regions. Only certain regions on the 3'-UTR of the SOD1 gene are advantageous for the ASO's inhibitory function, such as the SEQ ID NO: 65 (H3) region of the SOD1 gene. In some embodiments, the ASOs disclosed herein have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementarity to an isometric portion of the hotspot SEQ ID NO: 65 (H3). In some embodiments, the ASOs inhibit the mRNA transcription of the SOD1 gene by at least 60% compared to baseline Sod1 mRNA levels. In some embodiments, the ASO comprises a single-stranded oligonucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a chemically modified nucleotide sequence selected from SEQ ID NOs: 1155-1195. In some embodiments, the ASO comprises a single-stranded oligonucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a nucleotide sequence selected from the unmodified naked sequences of SEQ ID NOs: 1155-1195.

[0235] In some embodiments, the ASO is a single-stranded oligonucleotide comprising a 5' end and a 3' end.

[0236] In some embodiments, the length of the antisense oligonucleotide ranges from 12 to 30 nucleotides, including, for example, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, or 25 or more nucleotides. In some embodiments, the length of the ACO is 15 to 25 contiguous oligonucleotides.

[0237] In some embodiments, the ASO comprises a nucleotide sequence selected from the group of nucleotide sequences set forth in SEQ ID NOs: 1155-1195.

[0238] chemical modification All nucleotides of the oligonucleotide described herein can be natural nucleotides, i.e., non-chemically modified nucleotides, or at least one nucleotide can be chemically modified nucleotides.Non-limiting examples of chemical modification include one or more of the following combinations: a) modification of the phosphodiester bond of the nucleotide in the oligonucleotide sequence; b) modification of the 2'-OH of ribose in the nucleotide; c) modification of the base in the nucleotide; d) at least one nucleotide in the oligonucleotide sequence is a locked nucleic acid, and e) at least one nucleotide in the oligonucleotide sequence is deoxyribonucleotide (DNA).

[0239] In some embodiments, the nucleotides or oligonucleotides of the present application are chemically modified to enhance stability or other beneficial properties. The nucleic acids described herein may be prepared by conventional methods, such as those described in "Current Protocols in Nucleic Acid Chemistry," Beaucage, S. Lett. et al. (Eds.), John Wiley & Sons, Inc., incorporated herein by reference. Modifications include, for example, (a) terminal modifications, such as 5'-terminal modifications (phosphorylation, conjugation, reverse linkage, etc.) and 3'-terminal modifications (conjugation, DNA nucleotides, reverse linkage, etc.); (b) base modifications, such as substitution with stabilizing bases, destabilizing bases, or bases that base-pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions; and (d) backbone modifications, including modification or substitution of phosphodiester bonds. Specific examples of siRNA molecules that can be used in the present application include, but are not limited to, RNAs containing modified backbones or RNAs that do not contain natural internucleoside linkages. In some embodiments, the RNA with modified backbone includes, among others, those that do not have a phosphorus atom in the backbone.In some embodiments, the modified RNA that does not have a phosphorus atom in the internucleoside backbone can also be an oligonucleoside.In some embodiments, the modified oligonucleotide will have a phosphorus atom in its internucleoside backbone.

[0240] Chemical modifications of nucleotides or oligonucleotides in the present disclosure are well known to those skilled in the art, and modifications of phosphodiester bonds refer to modifications of the oxygen in the phosphodiester bond, including phosphorothioate modifications and boronated phosphate modifications.

[0241] The modifications disclosed herein stabilize the oligonucleotide structure, maintaining high specificity and high affinity for base pairing. The modifications disclosed herein also stabilize the ACO structure, maintaining delivery-aiding properties, including bioavailability, biodistribution, and / or cellular uptake of oligonucleotide drugs in various tissues, including the prefrontal cortex, cerebellum, spinal cord (e.g., cervical, thoracic, lumbar), muscle, liver, and kidney.

[0242] In some embodiments, the chemical modification is the replacement of phosphodiester linkages on the backbone of the nucleotide sequence of an oligonucleotide agent disclosed herein with phosphorothioate (PS) linkages. In some embodiments, an oligonucleotide agent disclosed herein includes at least one PS backbone modification. In some embodiments, an ACO includes at least one PS backbone modification. In some embodiments, an oligonucleotide agent includes at least two PS, at least three PS, at least four PS, at least five PS, at least six PS, or more than six PS backbone modifications. In some embodiments, about 90% to about 95% of the phosphodiester backbone linkages of the ACO are replaced with phosphorothioate (PS) linkages. In some embodiments, an oligonucleotide agent includes at least one PS backbone modification at the 5'-end, 3'-end, or an internal site of the sense strand of the dsRNA. In some embodiments, an oligonucleotide agent includes at least one PS backbone modification on the 5'-end, 3'-end, or an internal site of the antisense strand of the dsRNA. In some embodiments, an oligonucleotide agent includes at least one PS backbone modification at the 5'-end, 3'-end, or an internal site of one strand of the ACO.

[0243] In some embodiments, the nucleotide or oligonucleotide in the present application comprises at least one chemically modified nucleotide modified at the 2'-OH of the pentose of the nucleotide, for example, a 2'-fluoro modification, a 2'-oxymethyl modification, a 2'-oxyethylidenemethoxy modification, a 2,4'-dinitrophenol modification, a locked nucleic acid (LNA), a 2'-amino modification, or a 2'-deoxy modification, for example, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide.

[0244] In some embodiments, the nucleotides or oligonucleotides in the present application comprise at least one chemically modified nucleotide modified at the base of the nucleotide, for example, a 5'-bromouracil modification, a 5'-iodouracil modification, an N-methyluracil modification, or a 2,6-diaminopurine modification.

[0245] In some embodiments, the chemical modification of the nucleotide or oligonucleotide in the present application is the addition of an (E)-vinylphosphonate moiety at the 5'-end of the sense or antisense sequence. In some embodiments, the chemical modification of at least one chemically modified nucleotide is the addition of a 5'-methylcytosine moiety at the 5'-end of the sense or antisense sequence.

[0246] In some embodiments, the nucleotide or oligonucleotide in the present application is modified at the base of the nucleotide, for example, 5'-bromouracil modification, 5'-iodouracil modification, N-methyluracil modification, or 2,6-diaminopurine modification.In some embodiments, at least one oligonucleotide in the oligonucleotide agent contains at least one modified nucleotide, such as a 2'-O-methyl modified nucleotide, a nucleotide containing a 5'-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a basic nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, and an unnatural base consisting of a nucleotide.In some embodiments, the first and second dsRNAs contain "endo-light" modifications using 2'-O-methyl modified nucleotides and nucleotides containing a 5'-phosphorothioate group.

[0247] Modified oligonucleotide 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, thioalkylphosphonates, thioalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and those with reverse polarity, in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.Various salts, mixed salts, and free acid forms are also included.

[0248] Non-limiting examples of the preparation of phosphorus-containing conjugates include, but are not limited to, U.S. Patents 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,40 5,939;5,453,496;5,455,233;5,466,677;5,476,925;5,519,126;5,536,821;5,541,316;5,550,111;5,563,253;5,571,799;5,587,361;5,625,050;6,02 6,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590, 6,534,639, 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 RE39464, each of which is specifically incorporated by reference herein in its entirety.

[0249] In some embodiments, the nucleotides or oligonucleotides comprise one or more of RNA, DNA, BNA, LNA, or peptide nucleic acid (PNA).

[0250] The RNA of siRNA or saRNA can be modified to contain one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide with a modified ribose moiety consisting of an extra bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in the 3'-endo conformation. The addition of locked nucleic acids to siRNA has been shown to increase the stability of siRNA 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). Representative United States patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, U.S. Patent Nos. 6,268,490, 6,670,461, 6,794,499, 6,998,484, 7,053,207, 7,084,125, and 7,399,845, each of which is incorporated herein by reference in its entirety.

[0251] Other RNA mimics suitable or contemplated for use in siRNA involve the replacement of both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units with novel groups. The base units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an RNA mimic that has been shown 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, specifically an aminoethylglycine backbone. The nucleobases are retained and are linked directly or indirectly to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is incorporated herein by reference. Further teaching of PNA compounds can be found, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0252] In some embodiments, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or about 100% of the nucleotides of the single-stranded oligonucleotide are chemically modified nucleotides.

[0253] In some embodiments, the sense strand and antisense strand of an oligonucleotide agent independently comprise at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of nucleotides that are chemically modified nucleotides.

[0254] These modifications can increase the bioavailability of oligonucleotides, their affinity for target sequences, and their resistance to intracellular nuclease hydrolysis.

[0255] Furthermore, to facilitate entry of the oligonucleotide into cells, based on the above modifications, a lipophilic group such as cholesterol can be introduced at the end of the sense strand or antisense strand of the oligonucleotide to facilitate its action through the cell membrane and nuclear membrane, which are made up of lipid bilayer membranes, and gene promoter regions in the nucleus.

[0256] In some embodiments, oligonucleotides of the present invention that are effective, upon contact with a cell, to inactivate or downregulate expression of one or more genes in the cell, preferably by at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%).

[0257] In some embodiments, oligonucleotides of the present invention that are effective, upon contact with a cell, to activate or upregulate expression of one or more genes in the cell preferably by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 500%, at least 800%, at least 1000%, at least 2000%, or at least 5000%).

[0258] One aspect of the present application provides a cell comprising an oligonucleotide agent of the present application or a nucleic acid encoding the oligonucleotide agent of the present application. In one embodiment, the cell is a mammalian cell, preferably a human cell. Such cells may be ex vivo cells, such as cell lines or cell lines, or may be present in a mammal, such as a human, including an infant, child, or adult.

[0259] In some embodiments, at least one chemically modified oligonucleotide is a non-target single-stranded oligonucleotide.In some embodiments, at least one chemically modified oligonucleotide is a target double-stranded oligonucleotide.In certain embodiments, at least one chemically modified oligonucleotide is a non-target single-stranded oligonucleotide and a target double-stranded oligonucleotide.

[0260] covalent bond Embodiments of the present application include oligonucleotide drugs that include a covalently linked double-stranded targeting oligonucleotide and a non-targeting single-stranded oligonucleotide, such as ACO.

[0261] In some embodiments, the double-stranded target oligonucleotide and the non-target single-stranded oligonucleotide are covalently linked by a linking moiety.

[0262] In some embodiments, double-stranded oligonucleotide and non-target single-stranded oligonucleotide are linked by covalent linker.In some embodiments, linker is disulfide linker.Various combinations of chains can be linked, for example, the first and second dsRNA sense strands are linked by covalent bond, or for example, the first and second dsRNA antisense strands are linked by covalent bond.

[0263] In some embodiments, the sense strand of the double-stranded target oligonucleotide is covalently linked to the single-stranded oligonucleotide, hi some embodiments, the antisense strand of the double-stranded target oligonucleotide is covalently linked to the single-stranded oligonucleotide.

[0264] In some embodiments, any of the oligonucleotides in an oligonucleotide agent of the present application includes a linking moiety.

[0265] Linkers are typically a direct bond, or an atom such as oxygen or sulfur, a unit such as NR, C(O), C(O)O, C(O)NR, SO, SO, SONH, or a unit such as substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkynyl, heteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, where one or more methylene is O, S, S(O), SO, N(R'), C(O), a cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle; where R is hydrogen, acyl, aliphatic, or substituted aliphatic.

[0266] Various types of linker functionalities can be included in the subject conjugates, including, but not limited to, cleavable and non-cleavable linkers, and reversible and irreversible linkers.

[0267] In some embodiments, the linker is a cleavable linker. A cleavable linker is one that releases the two moieties to which it is attached, e.g., ACO and dsRNA, depending on a process within the target cell, such as reduction in the cytoplasm, exposure to acidic conditions in lysosomes or endosomes, or cleavage by a specific enzyme (e.g., protease) within the cell. Thus, the cleavable linker allows the dsRNA to be released in its original form after the conjugate is internalized and processed within the target cell. Cleavable linkers include, but are not limited to, those whose bond is cleavable by an enzyme (e.g., a peptide linker), those whose bond is cleavable under reducing conditions (e.g., a disulfide linker), and those whose bond is cleavable under acidic conditions (e.g., a hydrazone and a carbonate).

[0268] In some embodiments, the linking moiety is selected from one or more of an ethylene glycol chain, an alkyl chain, a peptide, a nucleic acid, a carbohydrate, a thiol linkage, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, and a carbamate. In some embodiments, the linking moiety includes, but is not limited to, the following: a) L1 or S18 (spacer-18 linker) (1,1-bis(4-methoxyphenyl)-1-phenyl-2,5,8,11,14,17-hexaoxanonadecan-19-yl(2-cyanoethyl)diisopropylphosphoramidite); b) L4 or C6 (spacer-C6 linker) (6-(bis(4-methoxyphenyl)(phenyl)methoxy)hexyl(2-cyanoethyl)diisopropylphosphoramidite); c) L6 (1,1-bis(4-methoxyphenyl)-1-phenyl-2,5,8,11,14 pentoxahexadecan-16-yl(2-cyanoethyl)diisopropylphosphoramidite); d) L9 or S9 (spacer-9 linker) (2-(2-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethoxy)ethoxy)ethyl(2-cyanoethyl)diisopropylphosphoramidite); e) L10 or C3 (spacer-C3 linker) (3-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl(2-cyanoethyl)diisopropylphosphoramidite); f) L12 (d spacer) ((2R,3S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite); g) L13 or C12 (spacer-C12 linker) (12-(bis(4-methoxyphenyl)(phenyl)methoxy)dodecyl(2-cyanoethyl)diisopropylphosphoramidite); h) L14 (spacer-L14 linker) (((1r,4r)-4-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)cyclohexyl)methyl(di-cyanoethyl)diisopropylphosphoramidite); i) L15 (spacer-L15 linker) (4-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)phenethyl(2-cyanoethyl)diisopropylphosphoramidite); j) L16 (spacer-L16 linker) (2-(1-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)cyclohexyl)ethyl(2-cyanoethyl)diisopropylphosphoramidite) k) C6x1((2S,3S,4S,5S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-methoxy-4-(pent-4-yn-1-yloxy)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite); l) C6x2((2S,3S,4S,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2-methoxy-4-(pent-4-yn-1-yloxy)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite); m) C6x5(2-((2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(pent-4-yn-1-yl)amino)ethyl(2-cyanoethyl)diisopropylphosphoramidite); and n) C6x7 ((9H-Fluoren-9-yl)methyl (4-((2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((bis(diisopropylamino)phosphanyl)oxy)pyrrolidin-1-yl)-4-oxobutyl)carbamate).

[0269] In certain embodiments, the linking moiety comprises a compound structure shown in Table 1. In certain embodiments, the linking moiety is attached to a nucleotide in a single-stranded or double-stranded oligonucleotide. In certain embodiments, the linking moiety is attached to a nucleoside position selected from the 5'-phosphate, 3', base, and 2'-H / OH of a nucleotide in a single-stranded or double-stranded oligonucleotide. In certain embodiments, the linking moiety is spacer phosphoramidite 18 (phosphoramidic acid, N,N-bis(1-methylethyl)-,19,19-bis(4-methoxyphenyl)-19-phenyl-3,6,9,12,15,18-hexaoxanonadecyl-1-yl 2-cyanoethyl ester).

[0270] JPEG2024523702000003.jpg228170JPEG2024523702000004.jpg241170JPEG2024523702000005.jpg250170In some embodiments, the double-stranded targeting oligonucleotide and the single-stranded oligonucleotide are covalently linked by a phosphodiester bond. In some embodiments, the double-stranded targeting oligonucleotide and the single-stranded oligonucleotide are covalently linked by a phosphorothioate bond.

[0271] In some embodiments, the double-stranded target oligonucleotide comprises a sense strand covalently linked to a single-stranded oligonucleotide, hi some embodiments, the double-stranded target oligonucleotide comprises an antisense strand covalently linked to a single-stranded oligonucleotide.

[0272] In some embodiments, the double-stranded target oligonucleotide and the single-stranded oligonucleotide are covalently linked by one or more nucleotides.

[0273] Non-limiting examples of covalent linker are described in US Patent Application Publication No. 20200332292, which is incorporated herein by reference in its entirety.Covalent linker can link double-stranded target oligonucleotide and single-stranded oligonucleotide.In some embodiments, covalent linker can link two sense strands, two antisense strands, one sense strand and one antisense strand, two sense strands and one antisense strand, two antisense strands and one sense strand, two sense strands and two antisense strands, antisense strand and single-stranded oligonucleotide, sense strand and inactivation oligonucleotide, etc.

[0274] In certain embodiments, the covalent linker comprises a nucleic acid (e.g., RNA and / or DNA) and / or a peptide. The linker may be single-stranded, double-stranded, partially single-stranded, or partially double-stranded. In some embodiments, the linker comprises a disulfide bond. The linker may be cleavable or non-cleavable.

[0275] In certain embodiments, covalent linkers include, for example, dTsdTuu = (5'-2' deoxythymidyl-3'-thiophosphate-5'-2' deoxythymidyl-3'-phosphate-5'-uridyl-3'-phosphate); rUsrU (thiophosphate linker: 5'-uridyl-3'-thiophosphate-5'-uridyl-3'-phosphate); rUrU linker; dTsdTaa (aadTsdT, 5'-2' deoxythymidyl 5'-2'deoxythymidyl-3'-thiophosphate-5'-2'deoxythymidyl-3'-phosphate-5'-adenylated-3'-phosphate; dTsdT (5'-2'deoxythymidyl-3'-thiophosphate-5'-2'deoxythymidyl-3'-phosphate); or dTsdTuu = uudTsdT = 5'-2'deoxythymidyl-3'-thiophosphate-5'-2'deoxythymidyl-3'-phosphate-5'-uridyl-3'-phosphate When the covalent linker is RNA, the RNA linker can be composed of any combination of nucleotides. The combination of nucleotides can be adenine, uracil, guanosine, cytosine, or any combination thereof. The RNA linker can be of any length. In some embodiments, the RNA linker is 2 to 50 nucleotides in length. If the RNA linker is 2 to 50 nucleotides in length, the RNA linker can be any intervening length, such as 5 to 10, 10 to 15, or 15 to 20 nucleotides in length. In some embodiments, the covalent linker comprises a polyRNA such as poly(5'-adenylate-3'-phosphate-AAAAAAA) or poly(5'-cytidyl-3'-phosphate-5'-uridyl-3'-phosphate-CUCUCU)), e.g., an Xn single-stranded polyRNA linker, where n is an integer from 2 to 50, preferably an integer from 4 to 15, and most preferably an integer from 7 to 8. Modified nucleotides or mixtures of nucleotides can also be present in the polyRNA linker. When the covalent linker is DNA, the DNA linker can be composed of any combination of nucleotides. The combination of nucleotides can be adenine, thymine, guanosine, cytosine, or any combination thereof. The DNA linker can be any length. In some embodiments, the RNA linker is 2 to 50 nucleotides in length. When the DNA linker is 1 to 50 nucleotides in length, the DNA linker can be any intervening length, such as 5 to 10, 10 to 15, or 15 to 20 nucleotides in length. The covalent linker can be polyDNA, e.g., poly(5'-2'deoxythymidyl-3'-phosphate-TTTTTTTT), where n is an integer from 2 to 50, preferably an integer from 4 to 15, and most preferably an integer from 7 to 8. Modified nucleotides or mixtures of nucleotides can also be present in the polyDNA linker. A single-stranded polyDNA linker, where n is an integer from 2 to 50, preferably an integer from 4 to 15, and most preferably an integer from 7 to 8. Modified nucleotides or mixtures of nucleotides may also be present in the poly-DNA linker.

[0276] In some embodiments, the covalent bond linker comprises a disulfide bond, optionally a bis-hexyl-disulfide linker. In one embodiment, the disulfide linker is In some embodiments, the covalent linker comprises a peptide bond, e.g., an amino acid. In one embodiment, the covalent linker is a 1-10 amino acid long linker, preferably consisting of 4-5 amino acids, optionally X-Gly-Phe-Gly-Y, where X and Y represent any amino acid.

[0277] In some embodiments, the covalent linker comprises a hexaethylene glycol linker, HEG.

[0278] Aspects of the present application include covalently linking a double-stranded target oligonucleotide and a non-target single-stranded oligonucleotide, such as ACO, to form an oligonucleotide agent. In some embodiments, the orientation of the linkage and positioning of the double-stranded target oligonucleotide and the single-stranded oligonucleotide can enhance stability, oligonucleotide activity, or other beneficial properties, such as maximizing the output of the target gene, increasing or decreasing the activity or expression (e.g., mRNA expression, protein expression, etc.) of one or more target genes.

[0279] In some embodiments, the single-stranded oligonucleotide is covalently linked to the 3'-end of the sense strand or antisense strand of the double-stranded target oligonucleotide; b) the single-stranded oligonucleotide is covalently linked to the 5'-end of the sense strand or antisense strand of the double-stranded target oligonucleotide; or c) the single-stranded oligonucleotide is covalently linked to the internal nucleotide between the 5'-end and the 3'-end of the sense strand or antisense strand of the double-stranded target oligonucleotide.In some embodiments, the internal nucleotide of the sense strand or antisense strand of the double-stranded target oligonucleotide is located at the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th or 13th nucleotide position from the 5'-end of the sense strand or antisense strand; or located at the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th or 13th nucleotide position from the 3'-end of the sense strand or antisense strand.

[0280] In some embodiments, the internal nucleotides of the sense or antisense strand of the double-stranded target oligonucleotide are replaced by one or more linking moieties or spacers covalently linked to the single-stranded oligonucleotide at its 5'-end or 3'-end (i.e., internally conjugated ODVs). In some embodiments, the internally conjugated ODVs have enhanced efficacy compared to the 3'-end or 5'-end conjugated ODVs (i.e., ACOs conjugated to the 3'-end or 5'-end of the sense or antisense strand of the double-stranded oligonucleotide).

[0281] In certain embodiments, the 5' end of the single-stranded oligonucleotide is attached to the linking moiety. In some embodiments, the 3' end of the single-stranded oligonucleotide is attached to the linking moiety.

[0282] In certain embodiments, the linking moiety or spacer comprises a compound shown in Table 1.

[0283] Drugs that decrease SOD1 gene or protein expression In some embodiments, the oligonucleotide agent reduces expression of the SOD1 gene or protein. Administration of the oligonucleotide agent to a patient treats or delays the onset of ALS, such as familial ALS or sporadic ALS or Lew Lou Gehrig's disease. In certain embodiments, the described oligonucleotide agent reduces the amount of full-length SOD1 protein, e.g., by inactivating / downregulating SOD1 transcription to reduce the amount of full-length SOD1 mRNA. In certain embodiments, the full-length SOD1 protein is reduced by an amount sufficient to alleviate symptoms associated with ALS. In certain embodiments, the full-length SOD1 protein is reduced by at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%).

[0284] Administration can be by any route deemed useful. In some embodiments, the route of administration is local to a site in the central nervous system. In some embodiments, the route of administration is systemic.

[0285] In certain embodiments, the double-stranded target oligonucleotide of the oligonucleotide agent that reduces the expression of the SOD1 gene or protein is an siRNA. The SOD1 siRNA inactivates or downregulates the expression of the SOD1 gene, its mRNA transcript, or SOD1 protein in cells in which the SOD1 gene, its mRNA transcript, or SOD1 protein is normally or overexpressed.

[0286] In a typical embodiment, the first strand of the SOD1 siRNA consists of a segment having at least 75% sequence identity or sequence complementarity with a 6-60 nucleotide fragment of the selected target region of the Sod1 mRNA transcript, thereby inactivating or downregulating the expression of the SOD1 protein.

[0287] In the present application, SOD1 siRNA is composed of a sense nucleic acid fragment and an antisense nucleic acid fragment. The sense nucleic acid fragment and the antisense nucleic acid fragment have complementary regions that can form a double-stranded nucleic acid structure that knocks down the expression of the SOD1 gene in cells via the RNA interference mechanism. The sense nucleic acid fragment and the antisense nucleic acid fragment of the siRNA may exist on two different nucleic acid strands or on the same nucleic acid strand. When the sense nucleic acid fragment and the antisense nucleic acid fragment exist on two strands, at least one strand of the siRNA has a 3' overhang of 0 to 6 nucleotides in length, and preferably both strands have 3' overhangs of 2 or 3 nucleotides in length, and the nucleotides in the overhangs are preferably deoxythymine (dT). When the sense nucleic acid fragment and the antisense nucleic acid fragment of the siRNA exist on the same nucleic acid strand, the siRNA is preferably a single-stranded hairpin nucleic acid molecule, and the complementary regions of the sense nucleic acid fragment and the antisense nucleic acid fragment form a double-stranded nucleic acid structure. In such siRNAs, the length of the sense nucleic acid fragment and the antisense nucleic acid fragment are each 16 to 60 nucleotides, and may be 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, or 60 nucleotides.

[0288] In certain embodiments of the present application, the SOD1 siRNA comprises a sense nucleic acid strand and an antisense nucleic acid strand, wherein the sense nucleic acid strand comprises at least one region complementary to at least one region on the antisense nucleic acid strand, forming a double-stranded nucleic acid structure capable of inactivating the expression of SOD1 protein in a cell.

[0289] In certain embodiments of the present application, the sense nucleic acid strand and the antisense nucleic acid strand are located on two different nucleic acid strands.In certain embodiments of the present application, the sense nucleic acid fragment and the antisense nucleic acid fragment are located on the same nucleic acid strand to form a hairpin-type single-stranded nucleic acid molecule, and the complementary regions of the sense nucleic acid fragment and the antisense nucleic acid fragment form a double-stranded nucleic acid structure.

[0290] In certain embodiments of the present application, at least one of the nucleic acid strands has a 3' overhang with a length of 0 to 6 nucleotides. In certain embodiments of the present application, both of the nucleic acid strands have a 3' overhang with a length of 2 to 3 nucleotides. In certain embodiments of the present application, the sense nucleic acid strand and the antisense nucleic acid strand are each 16 to 35 nucleotides in length.

[0291] In some embodiments, the oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of DS17-01M3-AC1(me14)-L9V3, whose antisense strand has the nucleotide sequence of SEQ ID NO:933, which has complementarity to a fragment of the ODV-structured sense strand of SEQ ID NO:928.

[0292] In some embodiments, the oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of DS17-02M3-AC1(me14)-L9V3, which has the nucleotide sequence of SEQ ID NO:935, whose antisense strand has complementarity to a fragment of the ODV-structured sense strand of SEQ ID NO:928.

[0293] In some embodiments, the oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of DS17-03M3-AC1(me14)-L9V3, which has the nucleotide sequence of SEQ ID NO:937, whose antisense strand has complementarity to a fragment of the ODV-structured sense strand of SEQ ID NO:936.

[0294] In some embodiments, the oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of DS17-04M3-AC1(me14)-L9V3, which has the nucleotide sequence of SEQ ID NO:939, whose antisense strand has complementarity to a fragment of the ODV-structured sense strand of SEQ ID NO:938.

[0295] In some embodiments, the oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of 04M3v04M3v(me14)-L9V3, which has the nucleotide sequence of SEQ ID NO:950, whose antisense strand has complementarity to a fragment of the ODV-structured sense strand of SEQ ID NO:938.

[0296] In some embodiments, the oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of DS17-05M3-AC1(me14)-L9V3, which has the nucleotide sequence of SEQ ID NO:941, whose antisense strand has complementarity to a fragment of the ODV-structured sense strand of SEQ ID NO:940.

[0297] In some embodiments, the oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of DS17-29M2-AC1(me14)-L9V3, which has the nucleotide sequence of SEQ ID NO:47, whose antisense strand has complementarity to a fragment of the ODV-structured sense strand of SEQ ID NO:942.

[0298] In some embodiments, the oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of 04M3v04M3v(me14)-L9V3, which has the nucleotide sequence of SEQ ID NO:47, whose antisense strand has complementarity to a fragment of the ODV-structured sense strand of SEQ ID NO:932.

[0299] In some embodiments, the oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of 04M3v04M3v(me14)-L9V3, whose antisense strand has the nucleotide sequence of SEQ ID NO:943, which has complementarity to a fragment of the ODV-structured sense strand of SEQ ID NO:934.

[0300] In some embodiments, the oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of 04M3v04M3v(me14)-L9V3, which has the nucleotide sequence of SEQ ID NO:944, whose antisense strand has complementarity to a fragment of the ODV-structured sense strand of SEQ ID NO:936.

[0301] In some embodiments, the oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of 04M3v04M3v(me14)-L9V3, which has the nucleotide sequence of SEQ ID NO:951, whose antisense strand has complementarity to a fragment of the ODV-structured sense strand of SEQ ID NO:940.

[0302] In some embodiments, the oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of DS17-04M3-asSOD1-1-L9V3, which has the nucleotide sequence of SEQ ID NO:939, whose antisense strand has complementarity to a fragment of the ODV-structured sense strand of SEQ ID NO:952.

[0303] In some aspects, the present application provides isolated SOD1 gene siRNA targeting sites having any contiguous 16-35 nucleotide sequence on a selected target region of the SOD1 gene (the full-length SOD1 sequence of SEQ ID NO: 895). In certain embodiments, the selected target region of the SOD1 gene comprises a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to a nucleotide sequence selected from the following: at least the nucleotide sequence of SEQ ID NOs: 88-356.

[0304] In some embodiments, the siRNA comprises a nucleotide sequence of the sense strand that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to a nucleotide sequence selected from the following: SEQ ID NOs: 357-624. In some embodiments, the siRNA comprises a nucleotide sequence of the antisense strand that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to a nucleotide sequence selected from the following: SEQ ID NOs: 626-893.

[0305] In some embodiments, the siRNA comprises a nucleotide sequence of the sense strand that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to a nucleotide sequence selected from the following: SEQ ID NOs: 38, 40, 42, 44, 46, 50, 52, 54, 56, 58, 60, 62, and 64. In some embodiments, the siRNA comprises a nucleotide sequence of the antisense strand that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to a nucleotide sequence selected from the following: SEQ ID NOs: 39, 41, 43, 45, 47, 49, 51, 53, and 57.

[0306] In certain embodiments, the oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of siHTT-AC2-S1L1 (SEQ ID NO:28), and an antisense strand that has the nucleotide sequence of SEQ ID NO:27, which has partial complementarity to the sense strand of SEQ ID NO:28.

[0307] In certain embodiments, an oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of siHTT-S1V1 (SEQ ID NO:30), and an antisense strand that has the nucleotide sequence of SEQ ID NO:31, which has partial complementarity to the sense strand of SEQ ID NO:30.

[0308] Certain embodiments include an oligonucleotide agent that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of siSOD1-231-ESC (SEQ ID NO:38), and an antisense siRNA strand having the nucleotide sequence of SEQ ID NO:39, which has partial complementarity to the sense strand of SEQ ID NO:38.

[0309] In certain embodiments, the oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of siSOD1-231-TT (SEQ ID NO:40), and an antisense siRNA strand having the nucleotide sequence of SEQ ID NO:41, which has partial complementarity to the sense strand of SEQ ID NO:40.

[0310] In certain embodiments, an oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of (SEQ ID NO:42), and an antisense siRNA strand having the nucleotide sequence of SEQ ID NO:43, which has partial complementarity to the sense strand of SEQ ID NO:42.

[0311] In certain embodiments, an oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of (SEQ ID NO:44), and an antisense siRNA strand having the nucleotide sequence of SEQ ID NO:45, which has partial complementarity to the sense strand of SEQ ID NO:44.

[0312] In certain embodiments, an oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of (SEQ ID NO:46), and an antisense siRNA strand having the nucleotide sequence of SEQ ID NO:47, which has partial complementarity to the sense strand of SEQ ID NO:46.

[0313] In certain embodiments, an oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of (SEQ ID NO:46), and an antisense siRNA strand that has the nucleotide sequence of SEQ ID NO:49, which has partial complementarity to the sense strand of SEQ ID NO:46.

[0314] In certain embodiments, an oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of (SEQ ID NO:50), and an antisense siRNA strand having the nucleotide sequence of SEQ ID NO:51, which has partial complementarity to the sense strand of SEQ ID NO:50.

[0315] In certain embodiments, an oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of (SEQ ID NO:52), and an antisense siRNA strand having the nucleotide sequence of SEQ ID NO:53, which has partial complementarity to the sense strand of SEQ ID NO:52.

[0316] In certain embodiments, an oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of (SEQ ID NO:54), and an antisense siRNA strand having the nucleotide sequence of SEQ ID NO:47, which has partial complementarity to the sense strand of SEQ ID NO:54.

[0317] In certain embodiments, an oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of (SEQ ID NO:56), and an antisense siRNA strand having the nucleotide sequence of SEQ ID NO:56 that has partial complementarity to the sense strand of SEQ ID NO:57.

[0318] In certain embodiments, the oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of siSOD1M2-AC2(N22)-S1V3v-Qu5 (SEQ ID NO:58), and an antisense siRNA strand having the nucleotide sequence of SEQ ID NO:57, which has partial complementarity to the sense strand of SEQ ID NO:58.

[0319] In certain embodiments, the oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of siSOD1M2-AC2(N15)-S1V3v-Qu5 (SEQ ID NO:60), and an antisense siRNA strand having the nucleotide sequence of SEQ ID NO:57, which has partial complementarity to the sense strand of SEQ ID NO:60.

[0320] In certain embodiments, the oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of siSOD1M2-AC2(N12)-S1V3v-Qu5 (SEQ ID NO:62), and an antisense siRNA strand having the nucleotide sequence of SEQ ID NO:57, which has partial complementarity to the sense strand of SEQ ID NO:62.

[0321] In certain embodiments, the oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of siSOD1M2-AC2(N6)-S1V3v-Qu5 (SEQ ID NO:64), and an antisense siRNA strand having the nucleotide sequence of SEQ ID NO:57, which has partial complementarity to the sense strand of SEQ ID NO:64.

[0322] In certain embodiments, the oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of siSOD1M2-AC2(N6)-S1V3v-Qu5 (SEQ ID NO: 1196), and an antisense siRNA strand having the nucleotide sequence of SEQ ID NO: 57, which has partial complementarity to the sense strand of SEQ ID NO: 64.

[0323] Furthermore, to facilitate entry of siRNA into cells, chemical conjugation groups other than those disclosed in ACO can be introduced to the end of the sense or antisense strand of the siRNA based on the above modifications to facilitate its action through the nuclear membrane and the cell membrane consisting of the intranuclear lipid bilayer and mRNA region.

[0324] In some embodiments, the siRNA disclosed in the present application is covalently linked to one or more conjugate groups.In some embodiments, the conjugate group modifies one or more properties of the linked oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge and clearance.In some embodiments, the conjugate group gives the linked oligonucleotide a new property, for example, a fluorophore or reporter group that allows the oligonucleotide to be detected. Specific conjugated groups and moieties include, for example, cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manohara et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060), thioethers such as hexyl-S-tritylthiol (Manohara et al., Ann. NY Acad. Sci., 1992, 660, 306-309; Manohara et al., Bioorg. Med. Chem. Lett., 1993, 3, 2765-2770), thiocholesterols (Oberhaus ...thioesters such as hexyl-S-tritylthiol (Manohara et al., Ann. NY Acad. Sci., 1992, 660, 306-309; Manohara al., Nucl. Acids Res., 1992, 20, 533-538), aliphatic chains, e.g., dodecane-diol or undecyl residues (Saison-Behmoara et al., EMBO1, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), polyamine or polyethylene glycol chains (Manohara et al., Nucleosides & Nucleotides, 1995, 14, 969-973), adamantane acetic acid 、palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., 1996, 277, 923-937), tocopherol group (Nishina et al., Molecular Therapy Nucleic Acids, 2015, 4, e220; and Nishina et al., Molecular Therapy, 2008, 16, 734-740), or GalNAc cluster (e.g., WO2014 / 179620) In some embodiments, the siRNA of the present application relates to a sense or antisense strand of an siRNA conjugated to one or more conjugation groups selected from the following: an intercalator, a reporter molecule, a polyamine, a polyamide, a peptide, a carbohydrate, a vitamin moiety, a polyethylene glycol, a thioether, a polyether, a cholesterol, a thiocholesterol, a cholic acid moiety, a folic acid, a lipid, a phospholipid, a biotin, a phenazine, a phenanthridine, an anthraquinone, an adamantane, an acridine, a fluorescein, a rhodamine, a coumarin, a fluorophore, and a dye.

[0325] In some embodiments, the conjugate group is selected from, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, benzothiazide, chlorothiazide, diazepine, indomethicine, barbiturates, cephalosporins, sulfa drugs, antidiabetic drugs, antibacterial drugs, or antibiotics.

[0326] In some embodiments, the siRNA of the present application is conjugated to one or more conjugate groups selected from lipids, fatty acids, fluorophores, ligands, saccharides, peptides, and antibodies.

[0327] In some embodiments, the siRNA of the present application relates to a sense or antisense strand of siRNA conjugated to one or more conjugate groups selected from a cell-penetrating peptide, polyethylene glycol, an alkaloid, a tryptamine, a benzimidazole, a quinolone, an amino acid, cholesterol, glucose, and N-acetylgalactosamine.

[0328] In some embodiments, the siRNA attached to one or more conjugate groups disclosed in the embodiments is directly contacted, transferred, delivered or administered to a cell or subject.

[0329] Oligonucleotide agents that increase expression of the SMN2 gene or SMN2 protein In some embodiments, an oligonucleotide agent, e.g., ACO, comprising a double-stranded targeting oligonucleotide and a non-targeting single-stranded oligonucleotide, increases the expression of the SMN2 gene or protein. Administering the oligonucleotide agent to a patient treats or delays the onset of an SMN deficiency-associated disease, such as spinal muscular atrophy (SMA). In certain embodiments, the described oligonucleotide agent increases the amount of full-length SMN protein by activating / upregulating SMN2 transcription in conjunction with, for example, regulating the splicing of exon 7 inclusion to increase the amount of full-length SMN2 mRNA. In certain embodiments, the full-length SMN protein is increased by an amount sufficient to alleviate symptoms associated with SMN deficiency. In certain embodiments, full-length SMN protein is increased by at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%).

[0330] Administration can be by any route deemed useful. In some embodiments, the route of administration is local to a site in the central nervous system. In some embodiments, the route of administration is systemic.

[0331] In certain embodiments, the double-stranded targeting oligonucleotide of the oligonucleotide agent that increases SMN2 gene or protein expression is a saRNA. The SMN2 saRNA activates or upregulates SMN2 gene expression in cells in which the SMN2 gene is normally, insufficiently, or incorrectly expressed.

[0332] In typical embodiments, the first strand of the SMN2 saRNA comprises a segment having at least 75% sequence identity or sequence complementarity to a 16-35 nucleotide fragment of the promoter region of the SMN2 gene, thereby activating the gene or upregulating expression.

[0333] In certain embodiments of the present application, the SMN2 saRNA comprises a sense nucleic acid strand and an antisense nucleic acid strand, and the sense nucleic acid strand comprises at least one region that is complementary to at least one region on the antisense nucleic acid strand to form a double-stranded nucleic acid structure that can activate expression of the SMN2 gene in a cell.

[0334] In certain embodiments, an oligonucleotide agent has a nucleotide sequence at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to any of the saRNA sense strand sequences SEQ ID NOs: 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, and 86, and an antisense saRNA strand having a nucleotide sequence selected from SEQ ID NO: 67, which has partial complementarity to the sense strand SEQ ID NOs: 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, and 86, respectively.

[0335] In certain embodiments, the oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to any one of the saRNA sense strand sequences of SEQ ID NO:66, and the antisense saRNA strand having a nucleotide sequence selected from SEQ ID NO:67 has partial complementarity to a sense strand selected from SEQ ID NO:66.

[0336] In certain embodiments, an oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of R6-04M1-AC2(16)-S1L1V3v (SEQ ID NO:70), and an antisense siRNA strand having the nucleotide sequence of SEQ ID NO:67, which has partial complementarity to the sense strand of SEQ ID NO:70.

[0337] In certain embodiments, an oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of R6-04M1-AC2(15)-S1L1V3v (SEQ ID NO:72), and an antisense siRNA strand having the nucleotide sequence of SEQ ID NO:67, which has partial complementarity to the sense strand of SEQ ID NO:72.

[0338] In certain embodiments, an oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of R6-04M1-AC2(14)-S1L1V3v (SEQ ID NO:74), and an antisense siRNA strand having the nucleotide sequence of SEQ ID NO:67, which has partial complementarity to the sense strand of SEQ ID NO:74.

[0339] In certain embodiments, an oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of R6-04M1-AC2(13)-S1L1V3v (SEQ ID NO:76), and an antisense siRNA strand having the nucleotide sequence of SEQ ID NO:67, which has partial complementarity to the sense strand of SEQ ID NO:76.

[0340] In certain embodiments, an oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of R6-04M1-AC2(12)-S1L1V3v (SEQ ID NO:78), and an antisense siRNA strand having the nucleotide sequence of SEQ ID NO:67, which has partial complementarity to the sense strand of SEQ ID NO:78.

[0341] In certain embodiments, an oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of R6-04M1-AC2(11)-S1L1V3v (SEQ ID NO:80), and an antisense siRNA strand having the nucleotide sequence of SEQ ID NO:67, which has partial complementarity to the sense strand of SEQ ID NO:80.

[0342] In certain embodiments, an oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of R6-04M1-AC2(10)-S1L1V3v (SEQ ID NO:82), and an antisense siRNA strand having the nucleotide sequence of SEQ ID NO:67, which has partial complementarity to the sense strand of SEQ ID NO:82.

[0343] In certain embodiments, an oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of R6-04M1-AC2(9)-S1L1V3v (SEQ ID NO:84), and an antisense siRNA strand having the nucleotide sequence of SEQ ID NO:67, which has partial complementarity to the sense strand of SEQ ID NO:84.

[0344] In certain embodiments, an oligonucleotide agent has a nucleotide sequence that is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%) identical to the nucleotide sequence of R6-04M1-AC2(8)-S1L1V3v (SEQ ID NO:86), and an antisense siRNA strand having the nucleotide sequence of SEQ ID NO:67, which has partial complementarity to the sense strand of SEQ ID NO:86.

[0345] Methods for regulating gene expression In some embodiments, the oligonucleotide agents of the present application are useful in therapeutic approaches to treating diseases such as spinal muscular atrophy (SMA) or ALS.

[0346] By way of non-limiting embodiment, the present application provides a method for reducing or silencing the level of mRNA transcripts of the SOD1 gene or SOD1 protein in a cell or individual, comprising administering to a subject a pharmaceutical composition disclosed herein.

[0347] In some embodiments, the present application relates to a method for treating or delaying the onset or progression of amyotrophic lateral sclerosis (ALS) in a subject, the method comprising administering to the subject a pharmaceutical composition disclosed herein. In some embodiments, the subject has sporadic ALS (sALS). In some embodiments, the subject has familial ALS (fALS). In some embodiments, the pharmaceutical composition reduces or silences the level of mRNA transcripts of the SOD1 gene or SOD1 protein in a cell or individual.

[0348] In some embodiments, an ACO of an oligonucleotide agent improves the stability, bioavailability, biodistribution, and / or cellular uptake of the siRNA compared to the oligonucleotide agent without the ACO. In some embodiments, an ACO of an oligonucleotide agent increases the biodistribution of the siRNA in one or more target tissues compared to the oligonucleotide agent without the ACO. In some embodiments, an ACO of an oligonucleotide agent increases the biodistribution of the siRNA in two or more target tissues compared to the oligonucleotide agent without the ACO.

[0349] In some embodiments, one or more target tissues are selected from the group consisting of brain, spinal cord, muscle, spleen, lung, heart, liver, bladder, and kidney tissue. In some embodiments, one or more target tissues are selected from the group consisting of the prefrontal cortex, cerebellum, and the rest of the brain; the cervical, thoracic, and lumbar regions of the spinal cord; the heart, forelimbs, hind limbs, nape, and buttocks.

[0350] In some embodiments, the oligonucleotide agent of the present application achieves a reduction in full-length SOD1 protein that is less than the amount achieved by administration of the same amount of a double-stranded oligonucleotide, such as an siRNA agent without an ODV structure, used individually, with greater efficacy, reduced toxicity, or undesirable side effects. In some embodiments, the oligonucleotide agent of the present application achieves a reduction in full-length SOD1 protein that is less than the additive effect of treatment with the same amount of a double-stranded targeting oligonucleotide used individually.

[0351] Specifically, an oligonucleotide agent of the present application inhibits / downregulates Sod1 mRNA transcripts by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100%) compared to baseline Sod1 mRNA transcripts. In some embodiments, upon administration of an oligonucleotide agent disclosed in the embodiments to, for example, a cell or a subject, Sod1 mRNA transcripts are inhibited / downregulated in an in vitro cell line by at least 50%, 60%, 70%, 77%, 79%, 81%, 84%, 85%, and 88% at 10 nM treatment compared to baseline Sod1 mRNA transcripts in a control group. In some embodiments, the oligonucleotide agent inhibits or downregulates Sod1 mRNA transcripts by about 90%.

[0352] In some embodiments, expression of the SOD1 gene is at least 0.01 nM, e.g., 0.02 nM, 0.05 nM, 0.08 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.8 nM, 1 nM, 5 nM, 10 nM, 25 nM, 50 nM, 75 nM, 100 nM, or 150 nM, of an oligonucleotide agent disclosed in the embodiments. In some embodiments, the protein encoding the SOD1 gene (SOD1 protein) is inhibited / downregulated, for example, by administering an oligonucleotide agent disclosed in the embodiments to a cell or subject. Knockdown of SOD1 protein is at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100%) compared to baseline expression of SOD1 protein. In some embodiments, the oligonucleotide agent inhibits or downregulates expression of SOD1 protein by about 90%. In some embodiments, SOD1 protein is inhibited / downregulated by administering to a cell an oligonucleotide agent disclosed in the embodiments at a concentration of at least 0.01 nM, e.g., 0.02 nM, 0.05 nM, 0.08 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.8 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 10 nM, 25 nM, 50 nM, 75 nM, 100 nM, or 150 nM.

[0353] In some embodiments, the oligonucleotide agents disclosed in the embodiments have dose-dependent knockdown activity in cells. In some embodiments, the oligonucleotide agents knockdown Sod1 mRNA transcripts in cells with an IC50 of less than 10 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.8 nM, less than 0.6 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2 nM, or less than 0.1 nM, 0.08 nM, 0.06 nM, 0.04 nM, 0.02 nM, 0.01 nM, 0.008 nM, or 0.005 nM. Another aspect of the present application relates to a method for preventing or treating a disorder or condition induced by overexpression of SOD1 protein, SOD1 gene mutation, and / or high Sod1 mRNA levels in an individual, comprising the steps of: administering to the individual an effective amount of an siRNA, oligonucleotide agent, or composition comprising an oligonucleotide agent disclosed herein. In some embodiments, the effective amount of an siRNA disclosed herein is a concentration ranging from 0.01 nM to 50 nM, e.g., 0.01 nM, 0.02 nM, 0.05 nM, 0.08 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.8 nM, 1 nM, 5 nM, 10 nM, 25 nM, 50 nM, 75 nM, 100 nM, or 150 nM. In some embodiments, the disorder or condition is ALS. In some embodiments, the individual is a mammal. In some embodiments, the individual is human.

[0354] In any of the embodiments provided herein, such cells may be in vitro, such as a cell line, or may be present in a mammal, such as a human. In some embodiments, the human is a subject or individual suffering from an SOD1 protein-related disease or ALS.

[0355] Another aspect of the present application relates to the use of an oligonucleotide agent of the present application, a nucleic acid encoding two or more oligonucleotides of the oligonucleotide agent of the present application, or a composition comprising an oligonucleotide agent of the present application, or a nucleic acid encoding two or more oligonucleotides of the oligonucleotide agent of the present application, for preparing a medicament for treating or delaying the onset of an SMN deficiency-associated condition or ALS. The subject may be a mammal, such as a human. The subject may be an infant, a child, or an adult.

[0356] In certain embodiments, the oligonucleotide agents of the present application achieve a reduction in full-length SOD1 protein that is less than that achieved by administration of the same amount of double-stranded oligonucleotide agent used individually, while reducing toxicity or undesirable side effects. In some embodiments, the oligonucleotide agents of the present application achieve a reduction in full-length SOD1 protein that is less than the additive effect of treatment with the same amount of double-stranded targeting oligonucleotide used individually.

[0357] In certain embodiments, the effect of an oligonucleotide agent of the present invention achieves a greater clinical improvement than the effect of the same amount of either agent used individually, hi certain embodiments, the effect of an oligonucleotide agent achieves a clinical improvement that is greater than additive compared to the effect of the same amount of double-stranded oligonucleotide used individually.

[0358] In any of the embodiments provided herein, such an oligonucleotide agent, a nucleic acid encoding a subject oligonucleotide agent, or a composition comprising such an oligonucleotide agent or a nucleic acid encoding a subject oligonucleotide agent may be directly introduced into a cell or may be produced intracellularly upon introduction of a nucleotide sequence encoding the oligonucleotide agent into a cell, preferably a mammalian cell, more preferably a human cell. Such cells may be ex vivo, such as a cell line, or may be present in a mammal, such as a human. In some embodiments, the human is a patient or individual suffering from an SMN deficiency-associated disease or ALS. In certain embodiments, a composition comprises a sufficient amount of each of the aforementioned oligonucleotide agents or nucleic acids encoding a subject oligonucleotide agent to effectively treat ALS.

[0359] In certain embodiments, the baseline measurement is obtained from a biological sample, as defined herein, obtained from the individual prior to administration of a therapy described herein, hi certain embodiments, the biological sample is peripheral blood mononuclear cells, plasma, serum, skin tissue, or cerebrospinal fluid (CSF).

[0360] How to Treat ALS Embodiments of the methods include a method of treating ALS in a subject comprising administering to the subject a pharmaceutical composition comprising an oligonucleotide agent of the present application and a pharmaceutically acceptable carrier.

[0361] Among the known genes underlying ALS, the SOD1 gene remains the primary cause of fALS and has been considered an important target for ALS therapeutics. The human SOD1 gene is located on chromosome 21q22.11, from base pair 33,031,935 to base pair 33,041,241, with a genomic size of 9307 bp. The SOD1 gene encodes the monomeric SOD1 protein (153 amino acids, molecular weight 16 kDa) and also encodes the copper / zinc-binding SOD1 enzyme, which has detoxifying properties and has been found to be primarily localized in the cytoplasm, nucleus, peroxisomes, and mitochondria. Although the first description of ALS dates back to at least 1824 by Charles Bell, SOD1 as the first risk gene for ALS was discovered in 1993. In 1994, the first SOD1 transgenic mouse model (SOD1 G93A The establishment of SOD1 signaling pathways has ushered in a new era in ALS research. All of this evidence indicates that SOD1 mutants likely cause disease via gain-of-function, and that lowering their levels is beneficial. Excessive oxidation of wild-type SOD1 leads to toxic structural changes. Silencing SOD1 significantly attenuated astrocyte-mediated toxicity to motor neurons. Therefore, silencing SOD1 expression is an important strategy for the treatment of ALS.

[0362] The present application provides an oligonucleotide agent having an efficient and effective oligonucleotide delivery carrier. It has been observed that an agent containing a double-stranded siRNA-targeted oligonucleotide treats ALS by inhibiting the expression of the SOD1 gene through the RNAi silencing mechanism. The present application provides an SOD1 siRNA that has a strong inhibitory effect when covalently linked to ODV (ACO), which the present inventors have found to be useful for treating ALS.

[0363] In some embodiments, the subject has sporadic ALS (sALS). In some embodiments, the subject has familial ALS (fALS). In some embodiments, the subject with ALS has increased or abnormal SOD1 full-length protein expression. In some embodiments, the double-stranded oligonucleotide of the oligonucleotide agent reduces or silences the expression of the SOD1 gene or SOD1 protein.

[0364] In certain embodiments, an ACO of an oligonucleotide agent improves the stability, bioavailability, biodistribution, and / or cellular uptake of a double-stranded oligonucleotide compared to an oligonucleotide agent without an ACO. In some embodiments, an ACO of an oligonucleotide agent increases the biodistribution of a double-stranded oligonucleotide in one or more target tissues compared to an oligonucleotide agent without an ACO. In some embodiments, an ACO of an oligonucleotide agent increases the biodistribution of a double-stranded oligonucleotide in two or more target tissues compared to an oligonucleotide agent without an ACO.

[0365] In some embodiments, the one or more target tissues are selected from the prefrontal cortex, cerebellum, muscle, liver, and kidney.

[0366] Cells containing siRNA After contacting a cell, the oligonucleotide agents disclosed herein can effectively inhibit or downregulate expression of the SOD1 gene in the cell, for example, downregulating expression by at least 10% (e.g., compared to baseline SOD1 transcription).

[0367] In some embodiments, the present application relates to cells comprising the oligonucleotide agent disclosed herein. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells, for example, human cells of various tissues of organs, including the brain, spinal cord, muscle, spleen, lung, heart, liver, bladder, and kidney. In some embodiments, the cells of target tissues are selected from the group consisting of the prefrontal cortex, cerebellum, and the rest of the brain; the cervical, thoracic, and lumbar regions of the spinal cord; the heart, forelimbs, hind limbs, nape, and gluteal muscles.

[0368] The cells disclosed herein may be in vitro, such as cell lines or cell cultures, or ex vivo, and may be present in a mammalian body, such as a human body, which is a subject suffering from a disease or condition caused by a mutation in the SOD1 gene, abnormal Sod1 mRNA levels, and / or overexpression of SOD1 protein in the CNS.

[0369] In some embodiments, the cells are derived from CNS tissue of a subject suffering from ALS. In some embodiments, the cells are derived from a subject suffering from ALS.

[0370] Oligonucleotide Agent Compositions Another aspect of the present application provides a pharmaceutical composition comprising a double-stranded targeting oligonucleotide and a non-targeting single-stranded oligonucleotide described herein.

[0371] The present application provides compositions or pharmaceutical compositions that can downregulate the level of Sod1 mRNA transcripts by the mechanism of action (MoA) of RNA interference, comprising the oligonucleotide agents disclosed herein, for the treatment or prevention of SOD1-related diseases (particularly ALS).

[0372] In some embodiments, the present application relates to a composition or pharmaceutical composition comprising the siRNA of the present application. In some embodiments, the present application relates to a composition or pharmaceutical composition comprising the siRNA and ACO described herein. In some embodiments, the present application relates to a composition or pharmaceutical composition comprising the siRNA and ACO covalently linked by a linking moiety described herein.

[0373] In some embodiments, the pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier. In one embodiment, the pharmaceutically acceptable carrier comprises one or more of an aqueous carrier, a liposome or LNP, a polymer, a micelle, a colloid, a metal nanoparticle, a non-metal nanoparticle, a bioconjugate (e.g., GalNAc), and a polypeptide. In one embodiment, the aqueous carrier can be, for example, RNase-free water or an RNase-free buffer. The composition can comprise 1 to 150 nM, for example, 1 to 100 nM, for example, 1 to 50 nM, for example, 1 to 20 nM, for example, 10 to 100 nM, 10 to 50 nM, 20 to 50 nM, 20 to 100 nM, for example, 50 nM of the aforementioned oligonucleotide or nucleic acid encoding the oligonucleotide according to the present application.

[0374] In some embodiments, a composition comprises between 1 and 150 nM of an oligonucleotide agent of the invention.

[0375] Another aspect of the application relates to the use of an oligonucleotide agent described herein, a nucleic acid encoding an oligonucleotide agent described herein, or a composition comprising such an oligonucleotide agent or a nucleic acid encoding an oligonucleotide agent described herein, wherein the double-stranded target oligonucleotide and the single-stranded oligonucleotide are covalently linked, for the preparation of one or more compositions for modulating the expression of one or more genes or proteins expressed by a cell.

[0376] Another embodiment provides pharmaceutical compositions or medicaments comprising an agent of the present application and a therapeutically inert carrier, diluent or pharmaceutically acceptable excipient, as well as methods of using the agent of the present application to prepare such compositions and medicaments.

[0377] For oligonucleotide agent compositions of the present application, delivery can optionally be via parenteral injection, including intrathecal, intramuscular, intravenous, intraarterial, intraperitoneal, intravesical, intracerebroventricular, intravitreal, or subcutaneous administration; or oral, intranasal, inhalation, vaginal, or rectal administration.

[0378] A typical formulation is prepared by mixing the agent of the present application with a carrier or excipient. Suitable carriers and excipients are well known to those skilled in the art, and are described in, for example, Ansel HC et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems (2004) Lippincott, Williams & Wilkins, Philadelphia; Gennaro A.R. et al., Remington: The Science and Practice of Pharmacy (2000) Lippincott, Williams & Wilkins, Philadelphia; and Rowe R.C, Handbook of Pharmaceutical Excipients (2005) Pharmaceutical Press, Chicago. The formulation may also include one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifying agents, glidants, processing aids, colorants, sweeteners, flavoring agents, diluents, and other known additives to provide an elegant presentation of the agent (i.e., an agent of the present application or a pharmaceutical composition thereof) or to aid in the manufacture of a pharmaceutical product (i.e., a medicament).

[0379] The compositions of the present application are formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the administration schedule, and other factors known to medical practitioners.

[0380] In another aspect, the present application provides the use of an oligonucleotide agent according to any one of the embodiments described herein or a composition according to any one of the embodiments described herein in the manufacture of a medicament for treating a gene- or protein-related condition in an individual. In certain embodiments, the condition may include an SMN deficiency-associated condition, including ALS. In certain embodiments, the condition may include an SMN deficiency-associated condition that constitutes a genetic neuromuscular disease, preferably spinal muscular atrophy. In other embodiments, the condition may include an immune-related condition, such as cancer. Also provided are certain embodiments in which the individual is a mammal, preferably a human.

[0381] Dosage regimen and route of administration Aspects of the present application relate to pharmaceutical compositions comprising the oligonucleotide agent of the present application. In some embodiments, the present application relates to pharmaceutical compositions comprising the oligonucleotide agent of the present application and a pharmaceutically acceptable carrier, a therapeutically inert carrier, a diluent, or a pharmaceutically acceptable excipient. The pharmaceutical compositions disclosed herein are developed as medicaments for preventing or treating SOD1 protein-related diseases or ALS.

[0382] Aspects of the present application also relate to methods of using the oligonucleotide agents of the present application to prepare such compositions.

[0383] Another aspect of the present application pertains to the use of an oligonucleotide agent of the present application in the manufacture of a pharmaceutical composition disclosed herein.

[0384] Another aspect of the present application relates to the use of an oligonucleotide agent according to any one of the embodiments described herein or a composition according to any one of the embodiments described herein in the manufacture of a medicament for the prevention or treatment of a gene- or protein-related condition induced by overexpression of SOD1 protein, SOD1 gene mutation, and / or high levels of SOD1 protein in an individual. In the case of use according to certain embodiments, the condition may include a disorder or condition associated with SOD1 protein mutation that constitutes ALS. In the case of use according to certain embodiments, the condition induced by overexpression of abnormal SOD1 protein is ALS. Also relevant are uses according to certain embodiments in which the individual is a mammal, for example, a human.

[0385] The dose at which the oligonucleotide agent or composition of the present application can be administered can vary within a wide range and is adapted to the individual requirements in each case. In some embodiments, the first dose of the pharmaceutical composition of the present application is administered when the subject is less than 1 week old, less than 1 month old, less than 3 months old, less than 6 months old, less than 1 year old, less than 2 years old, less than 15 years old, or more than 15 years old.

[0386] A single dose of an oligonucleotide agent can be, for example, a single dose in the range of about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 25, 30, 40, 50, 75, 100, 120, 150, 200, 250, 300, 400, 500, 750, or 1000 mg / kg. The doses described herein can include two or more of any of the oligonucleotide agent sequences described herein.

[0387] In some embodiments, the proposed number of doses is approximate. For example, in some embodiments, if the proposed dosing frequency is a dose on day 1 and a second dose on day 29, the ALS patient can receive the second dose 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 days after receiving the first dose. In some embodiments, if the proposed dosing frequency is a dose on day 1 and a second dose on day 15, the ALS patient can receive the second dose 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after receiving the first dose. In some embodiments, if the proposed dosing frequency is a dose on day 1 and a second dose on day 85, the ALS patient can receive the second dose 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 days after receiving the first dose.

[0388] In some embodiments, the dose and / or volume of the injection is adjusted based on the age of the subject, the weight of the subject, and / or other factors that may require adjustment of the parameters of the injection.

[0389] In some embodiments, the pharmaceutical composition includes a cosolvent system. Some such cosolvent systems include, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In some embodiments, such cosolvent systems are used for hydrophobic compounds. A non-limiting example of such a cosolvent system is the VPD cosolvent system, which contains 3% w / v benzyl alcohol, 8% w / v of the nonpolar surfactant polysorbate 80, and TM and 65% w / v polyethylene glycol 300 in absolute ethanol. The proportions of such co-solvent systems can be varied considerably without significantly altering solubility or toxicity characteristics. Furthermore, the identity of the co-solvent components can be varied: for example, Polysorbate 80 TMOther surfactants may be substituted for the polyethylene glycol; the fraction size of the polyethylene glycol may be varied; other biocompatible polymers, e.g., polyvinylpyrrolidone, may be substituted for the polyethylene glycol; and other sugars or polysaccharides may be substituted for the dextrose.

[0390] Examples of other compositions or components related to the oligonucleotide agents, compositions, pharmaceutical compositions, and methods described herein include, but are not limited to, diluents, salts, buffers, chelating agents, preservatives, desiccants, antimicrobial agents, needles, syringes, packaging materials, tubes, bottles, flasks, beakers, etc., for example, using, modifying, assembling, storing, packaging, preparing, mixing, diluting, and / or preserving the components for a particular use. In embodiments in which a liquid form of any of the components is used, the liquid form may be concentrated or ready-to-use.

[0391] In some embodiments, lipid moieties used in nucleic acid therapy can be applied in the present application for delivery of the oligonucleotide agent molecules disclosed herein. In such methods, a nucleic acid (e.g., one or more oligonucleotide agents described herein) is introduced into a preformed liposome or lipoplex composed of a mixture of cationic lipids and neutral lipids. In certain methods, the complex between the oligonucleotide agent and the mono- or polycationic lipid is formed without the presence of neutral lipids. In some embodiments, the lipid moiety is selected to increase distribution of the pharmaceutical agent to specific cells or tissues. In some embodiments, the lipid moiety is selected to increase distribution of the pharmaceutical agent to adipose tissue. In some embodiments, the lipid moiety is selected to increase distribution of the pharmaceutical agent to muscle tissue.

[0392] In some embodiments, the pharmaceutical composition comprises a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions, including those containing hydrophobic compounds. In some embodiments, certain organic solvents, such as dimethyl sulfoxide, are used.

[0393] In some embodiments, the pharmaceutical composition comprises one or more tissue-specific delivery molecules designed to deliver one or more agents of the present application to a particular tissue or cell type. For example, in some embodiments, the pharmaceutical composition comprises a liposome coated with a tissue-specific antibody.

[0394] In some embodiments, oligonucleotide agents can be delivered or administered via vector.Any vector that can be used for gene delivery can be used.In some embodiments, viral vectors can be used.Non-limiting examples of viral vectors that can be used in the present application include but are not limited to: human immunodeficiency virus; HSV, herpes simplex virus; MMSV, Moloney murine sarcoma virus; MSCV, murine stem cell virus; SFV, Semliki Forest virus; SIN, Sindbis virus; VEE, Venezuelan equine encephalitis virus; VSV, vesicular stomatitis virus; VV, vaccinia virus; AAV, adeno-associated virus; adenovirus; lentivirus; and retrovirus.

[0395] In some embodiments, the vector is a recombinant AAV vector. AAV vectors are relatively small DNA viruses that can integrate into the genome of infected cells in a stable and site-specific manner. AAV can infect a variety of cells without affecting cell growth, morphology, or differentiation, and does not appear to be involved in human pathologies. The AAV genome has been cloned, sequenced, and characterized. The genome consists of approximately 4,700 bases and is flanked by inverted terminal repeat (ITR) regions of approximately 145 bases each, which serve as the origin of viral replication. The remainder of the genome is divided into two important regions responsible for encapsidation: the left part of the genome, which contains the rep gene, which is involved in viral replication and viral gene expression, and the right part, which contains the cap gene, which encodes the viral capsid protein.

[0396] AAV vectors can be prepared using standard methods in the art. Adeno-associated viruses of any serotype are suitable (e.g., Blacklow, pp. 165-174 of "Parvoviruses and Human Disease," J.R.P. Tattison, ed. (1988); Rose, Comprehensive Virology 3:1, 1974; P. Tattersall, "The Evolution of Parvovirus Taxonomy," In Parvovirus (J.R.K. Crull, S.F. Cotmore, M.E. Bloom, R.M. Linden, C.R. Parrish, Eds.), pp. 5-14, Hudder Arnold, London, UK (2006); D.E. Bowles, J.E. Rabinowitz, R.J. Samulski, "The Genus Dependovirus," (J.R.K. Crull, S.F. Cotmore, M.E. Bloom, R.M. Linden, C.R. Parrish, Eds.), pp. 15-23, Hudder Arnold, London, UK (2006), the disclosures of which are incorporated herein by reference in their entireties). Methods for purifying vectors can be found, for example, in U.S. Patent Nos. 6,566,118, 6,989,264, 6,995,006, and WO / 1999 / 011764, entitled "Methods for Generating High Titer Helper-free Preparation of Recombinant AAV Vectors," the disclosures of which are incorporated herein by reference in their entireties. The preparation of hybrid vectors is described, for example, in PCT Application No. PCT / US2005 / 027091, the disclosure of which is incorporated herein by reference in its entirety. The use of AAV-derived vectors for in vitro and in vivo gene transfer has been described (see, e.g., International Patent Application Publication Nos. 91 / 18088 and WO93 / 09239; U.S. Patent Nos. 4,797,368, 6,596,535, and 5,139,941; and European Patent No. 0488528, all of which are incorporated herein by reference in their entireties).These publications describe various AAV-derived constructs in which the rep and / or cap genes have been deleted and replaced with a gene of interest, and the use of these constructs to transfer the gene of interest in vitro (in cultured cells) or in vivo (directly into the organism). Replication-deficient recombinant AAVs according to the present application can be prepared by co-transfecting a plasmid containing a nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions with a plasmid carrying the AAV encapsulation genes (rep and cap genes) into a cell line infected with a human helper virus (e.g., adenovirus). The resulting AAV recombinants are then purified by standard techniques.

[0397] In some embodiments, the vector(s) used in the methods of the present application are encapsulated in a viral particle (e.g., including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16). Thus, the present application can include recombinant viral particles (recombinant, as they contain a recombinant polynucleotide) comprising any of the vectors described herein. Methods for producing such particles are known in the art and are described in U.S. Patent No. 6,596,535.

[0398] In certain embodiments, the oligonucleotide agent exhibits greater than additive or synergistic effects in treating, preventing, slowing the progression of, and / or ameliorating diseases caused by the SOD1 gene, and further in protecting cells involved in the pathophysiology of the disease, particularly in treating, preventing, slowing the progression of, and / or ameliorating ALS.

[0399] In some embodiments, delivery of a pharmaceutical composition comprising an oligonucleotide agent can be via parenteral injection, including intrathecal, intramuscular, intravenous, intraarterial, intraperitoneal, intravesical, intracerebroventricular, intravitreal, or subcutaneous administration, or oral, intranasal, inhalation, vaginal, or rectal administration.

[0400] In certain embodiments, when an oligonucleotide agent is administered as an intrathecal injection by lumbar puncture, the use of a smaller gauge needle can alleviate or improve one or more symptoms associated with the lumbar puncture procedure. In certain embodiments, symptoms associated with lumbar puncture include, but are not limited to, post-lumbar puncture syndrome, headache, back pain, fever, constipation, nausea, vomiting, and pain at the puncture site. In certain embodiments, the use of a 24-gauge or 25-gauge needle for lumbar puncture alleviates or improves one or more post-lumbar puncture symptoms. In certain embodiments, the use of a 21-gauge, 22-gauge, 23-gauge, 24-gauge, or 25-gauge needle for lumbar puncture alleviates or improves post-lumbar puncture syndrome, headache, back pain, fever, constipation, nausea, vomiting, and / or pain at the puncture site.

[0401] In certain embodiments, the dose and / or volume of the injection is adjusted based on the patient's age, the patient's CSF volume, or the patient's age and / or estimated CSF volume (e.g., Matsuzawa J, Matsui M, Konishi T, Noguchi K, Gur RC, Bilker W, Miyawaki T. Age-related volumetric changes of brain ray and white matter in healthy infants and children. Cereb Cortex 2001 April;11(4):335-342, which is incorporated herein by reference in its entirety).

[0402] kit In another aspect, any of the compositions described herein can be provided in one or more kits, optionally including instructions for using the composition. That is, the kit can include instructions for using the oligonucleotide agent or composition in any of the methods described herein. As used herein, a "kit" typically defines a package, assembly, or container (such as an insulated container) containing one or more components or embodiments of the present application and / or other components related to the present application, for example, as described above. Any agent or component of the kit can be provided in liquid form (e.g., in solution) or solid form (e.g., dry powder, frozen, etc.).

[0403] In some cases, the kit includes one or more components, which may be in the same container or in two or more receptacles, and / or any combination thereof. The containers may contain liquids, and non-limiting examples include bottles, vials, jars, tubes, flasks, beakers, etc. In some cases, the containers are leakproof (when closed, liquids do not leak from the container, regardless of the container's orientation).

[0404] Examples of other compositions or components related to the agents, compounds, and methods described herein include, but are not limited to, diluents, salts, buffers, chelating agents, preservatives, desiccants, antimicrobial agents, needles, syringes, packaging materials, tubes, bottles, flasks, beakers, etc., used to use, modify, assemble, store, package, prepare, mix, dilute, and / or preserve the components for a particular use. In embodiments in which a liquid form of any of the components is used, the liquid form may be concentrated or ready-to-use.

[0405] In further embodiments, the kits may include instructions in any form or on a website or other source provided for using the kit in connection with the components and / or methods described herein. For example, the instructions may include instructions for using, modifying, mixing, diluting, preserving, assembling, storing, packaging, and / or preparing the components and / or other components associated with the kit. In some cases, the instructions may also include instructions for shipping the components, e.g., for shipment or storage at room temperature, below freezing, cryogenic temperatures, etc. The instructions may be provided in any form and in any manner useful to a user of the kit, such as written or oral (e.g., telephone), digital, optical, visual (e.g., videotape, DVD, etc.), and / or electronic communication (including internet or web-based communication). Materials and Methods

[0406] General method Starting materials, reagents, and solvents for organic synthesis were purchased commercially and used as received unless otherwise noted. Purification of reaction products was performed by column chromatography using silica gel (200-300 mesh) eluting with hexane / ethyl acetate and DCM / MeOH. Thin-layer chromatography (TLC) was performed using precoated silica gel GF plates and visualized using KMnO stain. H-NMR spectra were recorded at 400 MHz or 500 MHz (Varian) using CDCl3 with TMS. Mass spectra (MS) were recorded using an LC / MS (Agilent Technologies 1260 Infinity II / 6120 Quadrupole) and a time-of-flight mass spectrometer with electrospray ionization (ESI) or matrix-assisted laser desorption / ionization (MALDI).

[0407] Oligonucleotide synthesis The oligonucleotides used were synthesized by solid-phase synthesis using a K&A DNA synthesizer (K&A LaborgeraeteGbR, Schaafheim, Germany). Briefly, during solid-phase synthesis, phosphoramidite monomers (0.1 M in acetonitrile or dichloromethane) containing various linkers and conjugations were sequentially added to the solid support to generate the desired full-length oligonucleotides. Each cycle of base addition consisted of four chemical reactions, including detritylation, coupling, oxidation / thiolation, and capping.

[0408] Detritylation was performed using 3% dichloroacetic acid (TCA) in DCM for 45 seconds, and capping was performed using 16% N-methylimidazole in THF (CAPA) and THF:acetic anhydride:2,6-lutidine (80:10:10, v / v / v) (CAPB) for 20 seconds. Sulfurization was performed using a 0.1 M solution of hydrogenated xanthan in pyridine / ACN (50:50, v / v) for 3 minutes. Oxidation was performed using 0.02 M iodine in THF:pyridine:water (70:20:10, v / v / v) for 60 seconds. Coupling times for phosphoramidites were 360 ​​seconds for all amidites.

[0409] Deprotection I (nucleobase deprotection): After synthesis, the solid support was transferred to a microcentrifuge tube with a screw cap. For a 1 μM synthesis scale, a mixture of 33% methylamine in ethanol and 1 ml of ammonium hydroxide was added. The tube containing the solid support was then heated in an oven at 60–65°C for 15 minutes and cooled to room temperature. The cleavage solution was collected and evaporated to dryness using a Speedvac.

[0410] Deprotection II (removal of 2'-TBDMS group): Crude RNA oligonucleotides bearing 2'-TBDMS groups were dissolved in 0.1 ml of DMSO. After adding 1 ml of triethylamine 3HF, the tube was capped and the mixture was vigorously shaken to ensure complete dissolution. The bottle was heated in an oven at 60-65°C for 3-3.5 hours. The tube was removed from the oven and allowed to cool to room temperature. The solution containing the fully desilylated oligonucleotides was chilled on dry ice. 2 ml of ice-cold n-butanol (-20°C) was carefully added in 0.5 ml increments to precipitate the oligonucleotides. The precipitates were filtered, washed with 1 ml of ice-cold n-butanol, and then dissolved in 2 M TEAA (triethylammonium acetate). The crude oligonucleotides were then purified by IEX HPLC using a source15Q column. The purity of the fractions was confirmed using Column DNAPac. TM The purified single-stranded oligonucleotides were analyzed by ion exchange (IEX) HPLC using PA100. After the desalted purified single-stranded solution was obtained, two complementary single-stranded oligonucleotides were annealed to form a double-stranded oligonucleotide, which was then lyophilized to a powder.

[0411] RP-HPLC and ESI-MS To confirm the purity of the oligonucleotides, they were analyzed using reverse-phase chromatography (i.e., RP-HPLC) (Waters XBridge oligonucleotide BEHC18130A) with acetonitrile loading and detection at 260 nm. Electrospray ionization mass spectrometry (ESI-MS) was performed in negative ion mode on desalted oligonucleotides suspended in water / acetonitrile (50:50) containing 1% (vol / vol) triethylamine.

[0412] Cell culture and treatments SMA patient-derived fibroblasts, including GM03813 (SMA type II with three copies of the SMN2 gene) and GM09677 (SMA type I with three copies of the SMN2 gene), were obtained from the Coriell Institute (Camden, NJ, USA). Both cultures were maintained at 37°C and 5% CO in modified MEM medium (Gibco, Thermo Fisher Scientific, Carlsbad, CA) supplemented with 15% calf serum (Sigma-Aldrich), 1% NEAA (Gibco), and 1% penicillin / streptomycin (Gibco). Mouse neural stem cell line NSC-34 (BNCC341122, Beijing, China), HEK293A (Cobioer / CBP60436, Nanjing, China), and NSC-34 (BNCC341122, Beijing, China) cells were cultured in DMEM (Gibco) medium supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% penicillin / streptomycin (Gibco) at 37°C and 5% CO. Primary mouse hepatocytes (PMH) were isolated from the livers of type Ш SMA (Smn1- / -, SMN2+ / +) mice. PMH cells were cultured in modified DMEM medium (Gibco, Thermo Fisher Scientific, Carlsbad, CA) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% penicillin / streptomycin (Gibco) at 37°C and 5% CO. SH-SY5Y cells (SCSP-5014, Chinese Academy of Sciences, Shanghai, China) were cultured in MEM medium (Gibco, Thermo Fisher Scientific, Carlsbad, CA) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% penicillin / streptomycin (Gibco) at 5% CO2 and 37°C. Neuro-2a (N-2a, BNCC338529, Beijing, China) were cultured in EMEM medium (Gibco, Thermo Fisher Scientific, Carlsbad, CA) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% penicillin / streptomycin (Gibco) at 5% CO2 and 37°C.Human glioma cell line T98G (ATCC) cells were cultured in modified MEM medium (Gibco, Thermo Fisher Scientific, Carlsbad, CA) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% penicillin / streptomycin (Gibco) at 37°C and 5% CO2. Human cervical cancer cell line HeLa (ATCC) cells were cultured in modified RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Carlsbad, CA) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% penicillin / streptomycin (Gibco) at 37°C and 5% CO2. T98G, HeLa, and HEK293A cells were seeded at 10 × 10^4 cells / well in 24-well plates. siRNA (Note: In the following Materials, Methods, and Examples, the oligonucleotide agent of the present invention will be abbreviated as "siRNA") was transfected into each well individually at the indicated or other concentrations using 0.3 μL of RNAiMAX (Invitrogen, Carlsbad, CA) according to the reverse transfection protocol. The transfection time was 24 hours. Other cells were seeded into 6-well and 96-well plates at final densities of 1–2 × 105 and 6,000 cells / well, respectively. Mock (blank control) was transfected in the absence of oligonucleotide. dsCon2 was transfected as a nonspecific duplex control. All oligonucleotide sequences, including RNA duplexes and ODV constructs used for cell therapy, are listed in Tables 2, 3, and 8. ACO sequences are listed in Table 7.

[0413] JPEG2024523702000007.jpg232170JPEG2024523702000008.jpg225170JPEG20245237020 00009.jpg231170JPEG2024523702000010.jpg240170JPEG2024523702000011.jpg235170

[0414] JPEG2024523702000012.jpg240170JPEG2024523702000013.jpg237170JPEG2024523702000014.jpg236170 JPEG2024523702000015.jpg237170JPEG2024523702000016.jpg238170JPEG2024523702000017.jpg242170 JPEG2024523702000018.jpg244170JPEG2024523702000019.jpg235170JPEG2024523702000020.jpg234170 JPEG2024523702000021.jpg237170JPEG2024523702000022.jpg241170JPEG2024523702000023.jpg241170

[0415] JPEG2024523702000024.jpg238170JPEG2024523702000025.jpg237170

[0416] JPEG2024523702000026.jpg238170JPEG2024523702000027.jpg246170JPEG2024523702000028.jpg247170 JPEG2024523702000029.jpg254170JPEG2024523702000030.jpg248170JPEG2024523702000031.jpg249170

[0417] RT-qPCR One-step reverse transcription-quantitative polymerase chain reaction (one-stepRT-qPCR) After transfection, the medium was discarded and the cells were washed once per well with 150 μL of PBS. After discarding the PBS, 100 μL of cell lysate was added to each well and incubated at room temperature for 5 minutes. 0.5 μL of cell lysate was collected from each well and analyzed by RT-qPCR using the OneStepTBGreen™ PrimeScript™ RT-PCR kit II (Takara, RR086A) on a Roche Lightcycler 480 real-time PCR instrument. PCR reactions were prepared using an Echo 525 Acoustic Liquid Handler (Beckman Coulter). Each transfection sample was amplified in triplicate. The PCR reaction conditions are shown in Table 9.

[0418] The reaction conditions were as follows: reverse transcription (step 1): 42°C for 5 minutes, 95°C for 10 seconds; PCR (step 2): 95°C for 5 seconds, 59°C for 20 seconds, 72°C for 10 seconds, 40 cycles of amplification, and a melting curve (step 3). Human or mouse SOD1 genes were amplified as target genes. TBP and HPRT1 were amplified as reference genes and internal controls for RNA loading. All primer sequences are listed in Tables 4 and 10.

[0419] JPEG2024523702000033.jpg124170JPEG2024523702000034.jpg167170 Two-step RT-qPCR To quantify intracellular mRNA expression, total cellular RNA was isolated from treated cells using the RNeasyPlus Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The resulting RNA (1 μg) was reverse transcribed into cDNA using the PrimeScript RT kit (Takara, Shulga, Japan) with gDNAEraser. The resulting cDNA was amplified using a Roche LightCycler 480 Multiwell Plate 384 (Roche, ref: 4729749001, US) with SYBR Premix ExTaq II (Takara, Shulga, Japan) reagent and primers specific for the target gene. The reaction conditions were as follows: reverse transcription (step 1): 42°C for 5 minutes, 95°C for 10 seconds; PCR (step 2): 95°C for 5 seconds, 60°C for 30 seconds, 72°C for 10 seconds, 40 amplification cycles, and a melting curve (step 3). The PCR reaction conditions are listed in Tables 11 and 12.

[0420] JPEG2024523702000035.jpg114170JPEG2024523702000036.jpg42170To calculate the expression level (Erel) of Sod1 mRNA in samples transfected with siRNA relative to the control treatment (Mock), the Ct values ​​of the target gene and two internal reference genes were substituted into Formula III. JPEG2024523702000037.jpg16170Here, CtTm is the Ct value of the target gene from the mock-treated sample, mCtTs is the Ct value of the target gene from the siRNA-treated sample, CtR1m is the Ct value of internal reference gene 1 from the mock-treated sample; CtR1s is the Ct value of internal reference gene 1 in the siRNA-treated sample, CtR2m is the Ct value of internal reference gene 2 in the mock-treated sample, and CtR2s is the Ct value of internal reference gene 2 in the siRNA-treated sample.

[0421] Isolation of primary mouse hepatocytes (PMH) and free uptake assay C57BL / 6J mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were anesthetized with isoflurane and perfused sequentially with initial flushing and digestion reagents. The livers were then placed in a 10 cm dish containing medium. The liver lobules were torn apart with two pairs of forceps and filtered through a 70-75 micron membrane to obtain a cell suspension. The cell suspension was then collected in a 50 mL conical tube. The cells were then centrifuged at 100 × g for 2 minutes at 4°C in a swing-arm centrifuge. The supernatant was removed, and the cells were washed twice with 20 mL of cold PBS. Cell viability was checked using 0.4% trypan blue. The cells were seeded onto collagen I4-coated cell culture plates at a confluence of 60% or more in medium 12 hours before seeding. Experiments were performed after confirming that all cells had fully grown and reached a final confluence of 90-95% after seeding.

[0422] ELISA assay To evaluate the immunostimulatory activity of the oligonucleotides, ICR mice (Code ID: 201, Beijing Vital River Laboratory Animal Technology Co., Ltd.) were treated with DS17-04M3, AC1-L9V3, and DS17-04M3-AC1(me14)-L9V3. IL-1β, TNF-α, and IFN-γ protein expression levels were measured in the serum of treated mice using IL-1β (70-EK201B / 3-96, MULTISCIENCES, China), TNF-α (1217202, Shanghai, China), and IFN-γ (70-EK280 / 3-96, 70-EK280 / 3-96, China) ELISA kits according to the manufacturer's instructions. To examine toxicity after oligonucleotide stimulation, serum from ICR mice was collected and the enzyme activities of alanine transaminase (ALT), aspartate transaminase (AST), and creatinine transaminase (CREA) were detected using an AU480 Chemistry Analyzer (Beckman Coulter, USA) according to the kit instructions. The enzyme activities were determined using an ALT kit (OSR6107, Beckman Coulter, USA), an AST kit (OSR6209, Beckman Coulter, USA), and a CREA kit (OSR6212, Beckman Coulter, USA), respectively.

[0423] Immunoblot analysis Whole-cell protein samples were harvested using RIPA buffer supplemented with protease inhibitors. Sample concentrations were measured using a BCA protein assay kit (Beyotime, P0010, Shanghai, China). Protein aliquots (10 μg / well) were separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and transferred to 0.45 μm polyvinylidene fluoride (PVDF) membranes for immunodetection. Diluted primary antibodies specific for SMN (CST, 19276, USA) or α / β-tubulin (CST, 2148s, USA) were incubated overnight at 4°C. Protein bands were visualized by chemiluminescence using the corresponding species-specific anti-IgG horseradish peroxidase secondary antibodies (CST, 7074s and 7076s, USA) on an ImageLab docking station (BIO-RAD, ChemistryDocMP Imaging System). Protein amounts were quantified by optical densitometry of the detected bands using ImageJ software.

[0424] Propidium iodide (PI) staining siRNA-treated HEK293A cells were cultured in 96-well plates for 24 hours. Cells were washed with cold PBS and lysed using 40 μL / well of Cell Lysis Buffer (0.25% Igeal CA-630, 140 mM NaCl, 2 mM DTT, 10 mM Tris, pH 7.4) containing 1.5 MPI. Plates were incubated on ice for 5 minutes before measuring the optical density (OD) using an Infinite M2000 Pro microplate reader system at an excitation wavelength of 535 nm and an emission wavelength of 615 nm.

[0425] Stem-loop RT-qPCR To quantify oligonucleotides in biological samples, animal tissues were harvested using heat-lysis, and the lysates were stored at -80°C. Duplex, antisense, and formulated siRNA (20 μM) were serially diluted 10-fold in 1x lysis buffer from tissue (100 mg / mL) or plasma (1:10 dilution) boiled at 95°C. Concentrations in nM were converted to ng / mL using the molecular weight of the corresponding siRNA. Two non-template controls were included in every experiment. The first control contained the water used to prepare the transcription master mix, and the second control contained the lysis buffer used as a diluent for samples and standards.

[0426] Reverse transcription reactions were performed using a Takara Reverse Transcription Kit (Takara, RR037A). A total of 4 μl of the cDNA obtained in the previous step (1:40 dilution) was added to a PCR amplification reaction mix (0.5 μM forward primer, 0.5 μM reverse primer, 2x TGGreen premix ExTaqII). qPCR reactions were performed on a Lightcycler 480 using the "Standard Curve" option. The stem-loop RT-qPCR reaction conditions are shown in Tables 13 and 14.

[0427] JPEG2024523702000038.jpg63170JPEG2024523702000039.jpg56170 Animal treatment All animal procedures were performed by certified laboratory personnel using protocols consistent with local and state regulations and approved by the Institutional Animal Care and Use Committee. Primary breeding pairs of SMA mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA). This model was created by incorporating the human SMN2 gene to complement a homozygous knockout of the mouse Smn gene (Hsieh-Lietal, 2000). Tail snips were collected on postnatal day 0 (PND0) and genotyped by PCR. Type 1 SMA mice (Smn - / - ,SMN2 + / - ), SMA type III mice (Smn- / - ,SMN2 + / + ), heterozygous (Het) control mice (Smn + / - ,SMN2 + / - C57BL / 6 mice were purchased from JOINNBiologics (Suzhou, Jiangsu, China). Bilateral intracerebroventricular (ICV) injections were performed under 2% isoflurane anesthesia in pups (2 μL per side) and adult mice (5 μL per side) using a 29-gauge needle at a depth of 1.5 mm or 3.6 mm, respectively. Subcutaneous (SC) injections were performed subcutaneously in the intrascapular region of pups and adult mice.

[0428] Intracerebroventricular (ICV) injection Animals were anesthetized with isoflurane inhalation. The eyes were treated with eye lubricant. The scalp and anterior dorsal hair were clipped and the animal was positioned in a stereotaxic apparatus. Buprenorphine was administered subcutaneously (0.1 mg / kg) prior to incision. A 1.5 cm, slightly off-center incision was made in the scalp. A 25-gauge needle attached to a Hamilton syringe was placed at the level of the bregma and moved to the appropriate anterior-posterior and medial / lateral coordinates (0.2 mm anterior-posterior, 1 mm medial / lateral right). The appropriate volume of injection solution was infused at a rate of approximately 1 μl / s, for a total of 10 μL. This flow rate has been shown to consistently deliver sufficient compound without causing adverse effects in the animals. The incision was closed with one horizontal mattress suture using 5-0 absorbable sutures.

[0429] Intratracheal instillation (ITI) injection Animals were anesthetized with Avertin and individually secured to an inclined wooden platform. A 1-cm incision was made in the ventral neck skin to expose the trachea. The test substance was dissolved in 50 μL of saline and injected directly into the lungs via endotracheal intubation with an 18-gauge plastic catheter. To ensure delivery and effective distribution of the test substance to the mouse's lungs, after injection of the test substance solution, 150 μL of pre-filled air was rapidly forced into the lungs using a 1 mL syringe with a blunt needle. A syringe-less catheter was placed in the mouse's trachea to assist breathing. The neck was sutured and wiped with povidone-iodine. Mice were anesthetized with Avertin and placed on an inclined wooden platform for 4 hours until the substance solution was completely absorbed into the lungs. A saline-treated group underwent the same surgery and served as the vehicle control.

[0430] Rotarod behavioral test SOD1 after administration of Tofersen and DS17-04M3-AC1(me14)-L9V3 on PND46 G93A SOD1 was used to assess motor coordination, muscle strength, and balance in mice. G93A Mice were tested on a rotarod apparatus (XR-6C, Shanghai XinRun Information Technology Co., Ltd., China) once or twice a week. G93A Mice were trained on the rotarod apparatus as follows: 1) the first speed was 5 revolutions per minute (rpm) and lasted for 5 seconds; 2) the second speed was 20 rpm and lasted for 100 seconds; 3) the third speed was 25 rpm and lasted for 100 seconds; 4) the fourth speed was 30 rpm and lasted for 100 seconds; and 5) the fifth speed was 20 rpm and lasted for 300 seconds. SOD1G93A mice were trained until they could stay on the apparatus for 300 seconds without falling. After training, SOD1 G93A Mice were placed on the rotarod in accelerating mode (5–30 rpm over 5 min) for up to 300 s. G93A Mice were given three courses with a 30-minute intertrial interval, and the average time spent on the rotarod was recorded.

[0431] In the present disclosure, it has been discovered that siRNA can effectively reduce the expression levels of Sod1 mRNA and SOD1 protein after being introduced into cells. The present invention will be further described below with reference to specific examples and drawings. It should be understood that these examples do not limit the scope of the present application, but are merely intended to illustrate the present application. In the following examples, test methods without specific conditions generally followed conventional conditions, such as those described in Sambrook, et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions recommended by manufacturers.

[0432] In vivo imaging of fluorescein-labeled oligonucleotides in animal organs Mice were terminally anesthetized on selected days after treatment and perfused with cold PBS to flush out residual Quasar 570 (Qu5) dye from the circulation. Major organs / tissues (i.e., muscle, liver, lung, heart, kidney, spinal cord, and specific regions of the brain) were harvested and imaged using an IVIS (in vivo imaging system) at excitation / emission wavelengths of 520 / 570 nm. Quasar 570 signal was quantified by encircling each tissue or organ within a standardized region of interest (ROI) and measuring the fluorescence intensity (p / s / cm2 / sr) via Living Image® software (Caliper Life Science, USA). [Example]

[0433] The following examples are set forth so as to provide those of skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent the following experiments as complete or exclusive. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviations should be accounted for. Unless otherwise specified, parts are parts by weight, molecular weight is weight average molecular weight, temperature is degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used. For example, bp is base pair, kb is kilobase, pl is picoliter, s or sec is seconds, min is minutes, h or hr is hours, aa is amino acid, nt is nucleotide, im is intramuscular, ip is intraperitoneal; sc is subcutaneous; icv or icv or ICV is intracerebroventricular, etc.

[0434] Example 1: Design and synthesis of ODV duplexes All single-stranded oligonucleotide sequences with chemical modifications, including nucleic acid analogs, backbone substitutions, linkers, and ACO conjugates, were synthesized as single molecules on a solid support. ODV duplexes were then created by annealing complementary single-stranded oligonucleotides. Figure 1 shows an example of the molecular structure of an annealed ODV duplex. Quality control of the synthesis was performed on purified duplexes using mass spectrometry and RP-HPLC. Figure 2 shows the mass spectrograms and elution profiles of example ODV duplexes with 6-nucleotide (siSOD1M2-AC2(N6)-S1V3v) or 22-nucleotide (siSOD1M2-AC2(N22)-S1V3v) ACOs linked with a Spacer18 linker (hexaethylene glycol). Spectroscopic data confirmed that on-support synthesis of both ODV-siRNAs was efficient and complete, with observed MWs of 9411.5 Da (calculated MW 9410.86 Da) and 14884.8 Da (calculated MW 14883.05 Da), respectively. In comparison, the same siRNA duplexes without the ACO exhibited observed MWs of 7065.1 Da (calculated 7065.08 Da) without the S18 linker (siSOD1-388-E) and 7409.3 Da (calculated 7409.38 Da) with the S18 linker (siSOD1M2-L1). RP-HPLC analysis also confirmed that post-synthesis ODV treatment yielded relatively pure duplexes (Figure 2).

[0435] Example 2: Inhibition of Htt mRNA expression in mouse brain and spinal cord by ODV-siRNA Using medicinal chemistry, we synthesized a fully modified siRNA duplex with knockdown activity targeting mouse Htt transcripts (siHTT-S1V1). While this compound provided the necessary advantages for in vivo function (e.g., extended stability and elimination of immune stimulation), it failed to demonstrate significant in vivo responses after local injection into the CNS. In an attempt to improve local diffusion and in vivo activity, an ODV mutant (siHTT-AC2-S1L1) was synthesized, which contained an 18-mer ACO-linked 2'MOE and PS at each position. Both siHTT-S1V1 and siHTT-AC2-S1L1 were injected into C57BL / 6 pups at PND4. Brains and spinal cords were harvested from the injected mice 72 hours later, and Htt expression was detected in the harvested tissues after RNA isolation and RT reaction. As shown in Figure 3A-B, ICV injection of siHTT-S1V1 did not significantly affect Htt expression in the brain or spinal cord 3 days after administration compared to saline controls. However, siHTT-AC2-S1L1 reduced target gene expression by approximately 37% and 30% in the brain and spinal cord, respectively. This data provides evidence that the ODV strategy can confer in vivo activity to siRNAs in the CNS via local injection.

[0436] Example 3: ODV does not inhibit siRNA knockdown activity in vitro To investigate the effect of ODV design on siRNA knockdown activity, we synthesized a series of ODV-siRNA mutants with ACOs of different sizes (i.e., 12, 15, or 18 nucleotides in ...

Claims

1. An oligonucleotide agent comprising a non-target single-stranded oligonucleotide, wherein the single-stranded oligonucleotide has a length of at least 6 nucleotides, the single-stranded oligonucleotide is capable of facilitating the delivery of a double-stranded oligonucleotide, and at least one phosphodiester bond between two adjacent nucleotides in the single-stranded oligonucleotide sequence is replaced by a phosphorothioate (PS), mesylphosphoramidate or boranophosphate bond, An oligonucleotide agent, wherein the double-stranded oligonucleotide comprises a sense strand and an antisense strand.

2. The oligonucleotide agent according to claim 1, wherein the single-stranded oligonucleotide and the double-stranded oligonucleotide are linked by 0, 1 or more linking components.

3. All nucleotides of the single-stranded oligonucleotide are unmodified nucleotides, or at least one nucleotide is a modified nucleotide, and / or All nucleotides of the double-stranded oligonucleotide are unmodified nucleotides, or at least one nucleotide is a modified nucleotide, or at least one phosphodiester bond between two adjacent nucleotides in the nucleotide sequence is replaced by a phosphorothioate, mesylphosphoramidate or boranophosphate bond, the oligonucleotide agent according to claim 1.

4. The oligonucleotide agent according to claim 3, wherein the chemically modified nucleotide comprises one or more of the following modifications: a) Modification of the 2'-OH of ribose in the nucleotide; b) Modification or non-modification of the base moiety on the nucleoside ring in the nucleotide; c) The nucleotide is a locked nucleic acid or a bridged nucleic acid, and d) The nucleotide is a deoxyribonucleotide (DNA), and / or The chemically modified nucleotide has a 2'-OH ribose modification selected from 2′-fluoro-2′-deoxynucleoside (2′-F) modification, 2′-O-methyl (2′-O-Me) modification, and 2′-O-(2-methoxyethyl) (2′-O-MOE) modification, and / or The single-stranded oligonucleotide is one or more nucleotides selected from the group consisting of RNA, DNA, bridged nucleic acid (BNA), locked nucleic acid (LNA), and peptide nucleic acid (PNA).

5. The oligonucleotide agent according to claim 1, wherein the single-stranded oligonucleotide contains at least one phosphorothioate (PS) backbone substitution.

6. The oligonucleotide agent according to claim 3, wherein at least one chemically modified nucleotide is a nucleotide having an addition of a 5'-phosphate moiety at the 5'-end of the nucleotide sequence or a nucleotide having an addition of a 5-methylcytosine moiety at the 5'-end of the nucleotide sequence.

7. The oligonucleotide agent according to claim 6, wherein at least one chemically modified nucleotide is a nucleotide having an addition of a 5'-(E)-vinylphosphonate moiety.

8. The oligonucleotide agent according to claim 1, wherein the single-stranded oligonucleotide has a length of 6 to 22 nucleotides.

9. The single-stranded oligonucleotide is 1) comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 22, 2) comprises a chemically modified nucleotide sequence that is at least 80% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1299 to 1379, or 3) comprises a chemically modified nucleotide sequence having 0, 1, 2, or 3 chemical modifications different from the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1299 - 1379, The oligonucleotide agent according to claim 1.

10. The nucleotide sequence of the single-stranded oligonucleotide contains 35 - 65% adenine and / or The nucleotide sequence of the single-stranded oligonucleotide contains 35 - 72% cytosine and / or The nucleotide sequence of the single-stranded oligonucleotide contains 35 - 65% guanosine and / or The nucleotide sequence of the single-stranded oligonucleotide contains 35 - 72% uracil and / or The nucleotide sequence of the single-stranded oligonucleotide contains 64 - 78% purine and / or The nucleotide sequence of the single-stranded oligonucleotide contains 64 - 86% pyrimidine and / or The nucleotide sequence of the single-stranded oligonucleotide contains 42 - 58% purine and 42 - 58% pyrimidine. The oligonucleotide agent according to claim 1.

11. The oligonucleotide agent according to claim 1, wherein the nucleotide sequence of the single-stranded oligonucleotide comprises at least about 14%, at least about 28%, at least about 42%, at least about 57%, at least about 71%, at least about 85%, at least about 92%, or about 100% of nucleotides having a 2'Ome modification.

12. The oligonucleotide agent according to claim 1, wherein the nucleotide sequence of the single-stranded oligonucleotide is a palindromic sequence.

13. The oligonucleotide agent according to claim 1, wherein one or more double-stranded oligonucleotides and one or more single-stranded oligonucleotides are covalently linked by a linking component.

14. The oligonucleotide agent according to claim 1, wherein the linking component is linked to the nucleotides of a single-stranded oligonucleotide or a double-stranded oligonucleotide, or both a single-stranded oligonucleotide and a double-stranded oligonucleotide, via a phosphorothioate (PS) bond.

15. The linking component comprises a unit selected from the group consisting of a direct bond, or an oxygen or sulfur atom, or NR1, C(O), C(O)O, C(O)NR1, SO, SO2, and SO2NH; (wherein R1 is hydrogen, acyl, aliphatic or substituted aliphatic), or The linking component is a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, wherein one or more methylenes are interrupted or terminated by O, S, S(O), SO2, N(R')2, C(O), a cleavable linking group, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, and a substituted or unsubstituted heterocycle, or is selected from the group consisting of, or The oligonucleotide agent according to claim 2, wherein the linking component is selected from one or more of an ethylene glycol chain, an alkyl chain, an alkenyl chain, an alkynyl chain, a peptide, a carbohydrate, a thiol bond, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, a tetrazole bond, and a benzimidazole bond.

16. The oligonucleotide agent according to claim 2, wherein the linking component is selected from the group consisting of: a) L1 or S18 (Spacer-18 linker) (1,1-bis(4-methoxyphenyl)-1-phenyl-2,5,8,11,14,17-hexaoxanonadecan-19-yl (2-cyanoethyl)diisopropylphosphoramidite); b) L4 or C6 (Spacer-C6 linker) (6-(bis(4-methoxyphenyl)(phenyl)methoxy)hexyl (2-cyanoethyl)diisopropylphosphoramidite); c) L6 (1,1-bis(4-methoxyphenyl)-1-phenyl-2,5,8,11,14-pentaoxahexadecane-16-yl (2-cyanoethyl)diisopropylphosphoramidite); d) L9 or S9 (Spacer-9 linker) (2-(2-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethoxy)ethoxy)ethyl (2-cyanoethyl)diisopropylphosphoramidite); e) L10 or C3 (Spacer-C3 linker) (3-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl (2-cyanoethyl)diisopropylphosphoramidite); f) L12 (d spacer) ((2R,3S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl (2-cyanoethyl)diisopropylphosphoramidite); g) L13 or C12 (Spacer-C12 linker) (12-(bis(4-methoxyphenyl)(phenyl)methoxy)dodecyl (2-cyanoethyl)diisopropylphosphoramidite); h) L14 (Spacer-L14 linker) (((1r,4r)-4-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)cyclohexyl)methyl (di-cyanoethyl)diisopropylphosphoramidite); i) L15 (Spacer-L15 linker) (4-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)phenethyl (2-cyanoethyl)diisopropylphosphoramidite); j) L16 (Spacer-L16 linker) (2-(1-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)cyclohexyl)ethyl (2-cyanoethyl)diisopropylphosphoramidite) k) C6x1((2S,3S,4S,5S)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-methoxy-4-(pent-4-yn-1-yloxy)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite); l) C6x2((2S,3S,4S,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2-methoxy-4-(pent-4-yn-1-yloxy)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite); m) C6x5(2-((2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(pent-4-yn-1-yl)amino)ethyl(2-cyanoethyl)diisopropylphosphoramidite); and n) C6x7((9H-fluoren-9-yl)methyl(4-((2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((bis(diisopropylamino)phosphanyl)oxy)pyrrolidin-1-yl)-4-oxobutyl)carbamate).

17. The oligonucleotide agent according to claim 2, comprising a nucleotide sequence that is at least 90% identical to a nucleotide sequence selected from the following group: 1) siSOD1M2-AC2(N22)-S1V3v-Qu5 (SEQ ID NO: 58) and an antisense strand having a nucleotide sequence of SEQ ID NO: 57 that has partial complementarity with the sense strand of siSOD1M2-AC2(N22)-S1V3v-Qu5 (SEQ ID NO: 58); 2) siSOD1M2-AC2(N15)-S1V3v-Qu5 (SEQ ID NO: 60) and an antisense strand having a nucleotide sequence of SEQ ID NO: 57 that has partial complementarity with the sense strand of siSOD1M2-AC2(N15)-S1V3v-Qu5 (SEQ ID NO: 60); 3) siSOD1M2-AC2(N12)-S1V3v-Qu5 (SEQ ID NO: 62) and an antisense strand having a nucleotide sequence of SEQ ID NO: 57 that has partial complementarity with the sense strand of siSOD1M2-AC2(N12)-S1V3v-Qu5 (SEQ ID NO: 62); 4) siSOD1M2-AC2(N6)-S1V3v-Qu5 (SEQ ID NO: 64) and an antisense strand having the nucleotide sequence of SEQ ID NO: 57 which has partial complementarity with the sense strand of siSOD1M2-AC2(N6)-S1V3v-Qu5 (SEQ ID NO: 64); 5) siHTT-AC2-S1L1 (SEQ ID NO: 28) and an antisense strand having the nucleotide sequence of SEQ ID NO: 27 which has partial complementarity with the sense strand of siHTT-AC2-S1L1 (SEQ ID NO: 28). 6) siApp-8-AC2(N18)-S1L1V3v (SEQ ID NO: 32) and an antisense strand having the nucleotide sequence of SEQ ID NO: 31 which has partial complementarity with the sense strand of siApp-8-AC2(N18)-S1L1V3v (SEQ ID NO: 32); 7) siApp-8-AC2(N15)-S1L1V3v (SEQ ID NO: 34) and an antisense strand having the nucleotide sequence of SEQ ID NO: 31 which has partial complementarity with the sense strand of siApp-8-AC2(N15)-S1L1V3v (SEQ ID NO: 34); 8) siApp-8-AC2(N12)-S1L1V3v (SEQ ID NO: 36) and an antisense strand having the nucleotide sequence of SEQ ID NO: 31 which has partial complementarity with the sense strand of siApp-8-AC2(N12)-S1L1V3v (SEQ ID NO: 36); 9) R6-04M1-AC2(18)-S1L1V3v (SEQ ID NO: 68) and an antisense strand having the nucleotide sequence of SEQ ID NO: 67 which has partial complementarity with the sense strand of R6-04M1-AC2(18)-S1L1V3v (SEQ ID NO: 68); 10) R6-04M1-AC2(16)-S1L1V3v (SEQ ID NO: 70) and an antisense strand having the nucleotide sequence of SEQ ID NO: 67 which has partial complementarity with the sense strand of R6-04M1-AC2(16)-S1L1V3v (SEQ ID NO: 70); 11) R6-04M1-AC2(15)-S1L1V3v (SEQ ID NO: 72) and an antisense strand having the nucleotide sequence of SEQ ID NO: 67 which has partial complementarity with the sense strand of R6-04M1-AC2(15)-S1L1V3v (SEQ ID NO: 72); 12) R6-04M1-AC2(14)-S1L1V3v (SEQ ID NO: 74) and an antisense strand having a nucleotide sequence of SEQ ID NO: 67 that is partially complementary to the sense strand of R6-04M1-AC2(14)-S1L1V3v (SEQ ID NO: 74); 13) R6-04M1-AC2(13)-S1L1V3v (SEQ ID NO: 76) and an antisense strand having a nucleotide sequence of SEQ ID NO: 67 that is partially complementary to the sense strand of R6-04M1-AC2(13)-S1L1V3v (SEQ ID NO: 76); 14) R6-04M1-AC2(12)-S1L1V3v (SEQ ID NO: 78) and an antisense strand having a nucleotide sequence of SEQ ID NO: 67 that is partially complementary to the sense strand of R6-04M1-AC2(12)-S1L1V3v (SEQ ID NO: 78);. 15) R6-04M1-AC2(11)-S1L1V3v (SEQ ID NO: 80) and an antisense strand having a nucleotide sequence of SEQ ID NO: 67 that is partially complementary to the sense strand of R6-04M1-AC2(11)-S1L1V3v (SEQ ID NO: 80); 16) R6-04M1-AC2(10)-S1L1V3v (SEQ ID NO: 82) and an antisense strand having a nucleotide sequence of SEQ ID NO: 67 that is partially complementary to the sense strand of R6-04M1-AC2(10)-S1L1V3v (SEQ ID NO: 82); 17) R6-04M1-AC2(9)-S1L1V3v (SEQ ID NO: 84) and an antisense strand having a nucleotide sequence of SEQ ID NO: 67 that is partially complementary to the sense strand of R6-04M1-AC2(9)-S1L1V3v (SEQ ID NO: 84); 18) R6-04M1-AC2(8)-S1L1V3v (SEQ ID NO: 86) and an antisense strand having a nucleotide sequence of SEQ ID NO: 67 that is partially complementary to the sense strand of R6-04M1-AC2(8)-S1L1V3v (SEQ ID NO: 86).

18. The oligonucleotide agent according to claim 1, wherein a single-stranded oligonucleotide or a double-stranded oligonucleotide is conjugated to one or more conjugate groups.

19. The oligonucleotide agent according to claim 1, wherein the double-stranded oligonucleotide is small interfering RNA (siRNA) or small activating RNA (saRNA).

20. The sense strand or antisense strand of the double-stranded oligonucleotide has a nucleotide sequence that is at least 90% identical to the nucleotide sequence siApp-8-S1V1 (SEQ ID NO: 28) or siApp-8-S1V1 (SEQ ID NO: 27), or The oligonucleotide agent according to claim 1, wherein the sense strand or antisense strand of the double-stranded oligonucleotide has a nucleotide sequence that is at least 90% identical to the nucleotide sequence R6-04(20)-S1V1v(CM-4) (SEQ ID NO: 66) or R6-04(20)-S1V1v(CM-4) (SEQ ID NO: 67).

21. The oligonucleotide agent according to claim 1, wherein the oligonucleotide agent contains small interfering RNA (siRNA), and the siRNA forms a double-stranded structure containing a sense strand and an antisense strand, and the antisense strand contains a nucleotide sequence consisting of at least 10 consecutive nucleotides having 0, 1, 2, or 3 mismatches, and has at least 85% nucleotide sequence complementarity or homology to a part of any one of the nucleotide sequences of SEQ ID NO: 895, SEQ ID NO: 59, SEQ ID NO: 61, or SEQ ID NOs: 1068 to 1113, wherein the oligonucleotide agent can inhibit the expression of superoxide dismutase 1 (SOD1) in cells.

22. The oligonucleotide agent according to claim 21, having a nucleotide sequence of a sense strand of a double-stranded oligonucleotide that is at least 90% identical to a nucleotide sequence selected from the group consisting of: siSOD1-5 (SEQ ID NO: 357), siSOD1-8 (SEQ ID NO: 358), siSOD1-10 (SEQ ID NO: 359), siSOD1-11 (SEQ ID NO: 360), siSOD-17 (SEQ ID NO: 357), siSOD1-35 (SEQ ID NO: 362), siSOD1-37 to siSOD1-447 (SEQ ID NOs: 363 to 624), siSOD1-547 to siSOD1-694 (SEQ ID NOs: 976-1021), siSOD1-231-E (SEQ ID NO: 38), siSOD1-231-TT (SEQ ID NO: 40), siSOD1-231-M1 (SEQ ID NO: 42), siSOD1-231-S2 (SEQ ID NO: 44), siSOD1-388-E (SEQ ID NO: 46), siSOD1-388-TT (SEQ ID NO: 48), siSOD1-388-M1 (SEQ ID NO: 50), siSOD1-388-S2 (SEQ ID NO: 52), siSOD1M2-L1 (SEQ ID NO: 54), and siSOD1M2-S1V5 (SEQ ID NO: 56).

23. The oligonucleotide agent according to claim 21, wherein the antisense strand of the double-stranded oligonucleotide has a nucleotide sequence that is at least 90% identical to a nucleotide sequence selected from the group consisting of: siSOD1-5 (SEQ ID NO: 626), siSOD1-8 (SEQ ID NO: 627), siSOD1-10 (SEQ ID NO: 628), siSOD1-11 (SEQ ID NO: 629), siSOD-17 (SEQ ID NO: 630), siSOD1-35 (SEQ ID NO: 631), and siSOD1-37 to siSOD1-447 (SEQ ID NO: 632 to 893), siSOD1-547 to siSOD1-694 (SEQ ID NO: 1022-1067), siSOD1-231-E (SEQ ID NO: 39), siSOD1-231-TT (SEQ ID NO: 41), siSOD1-231-M1 (SEQ ID NO: 43), siSOD1-231-S2 (SEQ ID NO: 45), siSOD1-388-E (SEQ ID NO: 47), siSOD1-388-TT (SEQ ID NO: 49), siSOD1-388-M1 (SEQ ID NO: 51), siSOD1-388-S2 (SEQ ID NO: 53), siSOD1M2-L1 (SEQ ID NO: 47) and siSOD1M2-S1V1v-Qu5 (SEQ ID NO: 57).

24. The oligonucleotide agent according to claim 21, wherein the sense strand of the siRNA has a nucleotide sequence that has at least 85% homology to a nucleotide sequence selected from the group consisting of: DS17-0001 (SEQ ID NO: 384), DS17-0002 (SEQ ID NO: 372), DS17-0003 (SEQ ID NO: 409), DS17-0004 (SEQ ID NO: 357), DS17-0005 (SEQ ID NO: 486), DS17-0029 (SEQ ID NO: 588), DS17-01N3 (SEQ ID NO: 912), DS17-02N3 (SEQ ID NO: 914), DS17-03N3 (SEQ ID NO: 916), DS17-04N3 (SEQ ID NO: 918), DS17-05N3 (SEQ ID NO: 920) and SEQ ID NO: 976-1021.

25. The oligonucleotide agent according to claim 21, wherein the antisense strand of the siRNA has a nucleotide sequence having at least 85% homology with a nucleotide sequence selected from the group consisting of: DS17-0001 (SEQ ID NO: 653), DS17-0002 (SEQ ID NO: 641), DS17-0003 (SEQ ID NO: 678), DS17-0004 (SEQ ID NO: 626), DS17-0005 (SEQ ID NO: 755), DS17-0029 (SEQ ID NO: 857), DS17-01N3 (SEQ ID NO: 913), DS17-02N3 (SEQ ID NO: 915), DS17-03N3 (SEQ ID NO: 917), DS17-04N3 (SEQ ID NO: 919), DS17-05N3 (SEQ ID NO: 921), and SEQ ID NOs: 1022 to 1067.

26. The oligonucleotide agent according to claim 21, wherein the sense strand and the antisense strand of the siRNA independently have a nucleotide sequence that is at least 85% homologous to a nucleotide sequence pair selected from the following group: a) DS17-0001 (SEQ ID NO: 384 and SEQ ID NO: 653), b) DS17-0002 (SEQ ID NO: 372 and SEQ ID NO: 641), c) DS17-0003 (SEQ ID NO: 409 and SEQ ID NO: 678), d) DS17-0004 (SEQ ID NO: 357 and SEQ ID NO: 626), e) DS17-0005 (SEQ ID NO: 486 and SEQ ID NO: 755), f) DS17-0029 (SEQ ID NO: 588 and SEQ ID NO: 857), g) DS17-01N3 (SEQ ID NO: 912 and SEQ ID NO: 913), h) DS17-02N3 (SEQ ID NO: 914 and SEQ ID NO: 915), i) DS17-03N3 (SEQ ID NO: 916 and SEQ ID NO: 917), j) DS17-04N3 (SEQ ID NO: 918 and SEQ ID NO: 919), k) DS17-05N3 (SEQ ID NO: 920 and SEQ ID NO: 921), l) DS17-01M3 (SEQ ID NO: 922 and SEQ ID NO: 923), m) DS17-02M3 (SEQ ID NO: 924 and SEQ ID NO: 925), n) DS17-03M3 (SEQ ID NO: 926 and SEQ ID NO: 927), o) DS17-04M3 (SEQ ID NO: 928 and SEQ ID NO: 929), and p) DS17-05M3 (SEQ ID NO: 930 and SEQ ID NO: 931).

27. The oligonucleotide agent contains siRNA and a non-target ACO, the siRNA contains a sense strand and an antisense strand, the ACO contains a nucleotide sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 954, and the oligonucleotide agent can inhibit the expression of superoxide dismutase 1 (SOD1) in cells. The oligonucleotide agent according to claim 1.

28. The sense strand and antisense strand of the siRNA independently have a nucleotide sequence that is at least 85% homologous to a nucleotide sequence pair selected from the following group: and a) DS17-01M3 (SEQ ID NO: 922 and SEQ ID NO: 923), b) DS17-02M3 (SEQ ID NO: 924 and SEQ ID NO: 925), c) DS17-03M3 (SEQ ID NO: 926 and SEQ ID NO: 927), d) DS17-04M3 (SEQ ID NO: 928 and SEQ ID NO: 929), e) DS17-05M3 (SEQ ID NO: 930 and SEQ ID NO: 931), f) DS17-01M3-AC1(me14)-L9V3 (SEQ ID NO: 932 and SEQ ID NO: 933), g) DS17-02M3-AC1(me14)-L9V3 (SEQ ID NO: 934 and SEQ ID NO: 935), h) DS17-03M3-AC1(me14)-L9V3 (SEQ ID NO: 936 and SEQ ID NO: 937), i) DS17-04M3-AC1(me14)-L9V3 (SEQ ID NO: 938 and SEQ ID NO: 939), j) DS17-05M3-AC1(me14)-L9V3 (SEQ ID NO: 940 and SEQ ID NO: 941), k) DS17-29M2-AC1(me14)-L9V3 (SEQ ID NO: 942 and SEQ ID NO: 47), l) DS17-01M3v-AC1(me14)-L9V3 (SEQ ID NO: 932 and SEQ ID NO: 47), m) DS17-02M3v-AC1(me14)-L9V3 (SEQ ID NO: 934 and SEQ ID NO: 943), n) DS17-03M3v-AC1(me14)-L9V3 (SEQ ID NO: 936 and SEQ ID NO: 944), o) DS17-04M3v-AC1(me14)-L9V3 (SEQ ID NO: 938 and SEQ ID NO: 950), p) DS17-05M3v-AC1(me14)-L9V3 (Accession No.: 940 and Accession No.: 951), and q) DS17-04M3-asSOD1-1-L9V3 (Accession No.: 952 and Accession No.: 939). **Claim 29** The oligonucleotide agent comprises a non-target ACO-conjugated sense strand of siRNA and an antisense strand of siRNA, the non-target ACO-conjugated sense strand comprises a linking component that covalently conjugates ACO and the sense strand, and the antisense strand comprises a nucleotide sequence that is at least 90%, at least 95% homologous, or 100% identical to SEQ ID NO:

57. The oligonucleotide agent according to claim 1. **Claim 30** The non-target ACO-conjugated sense strand comprises a nucleotide sequence that is at least 90%, at least 95%, or 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1197-1288 and SEQ ID NOs: 1291-1298. The oligonucleotide agent according to claim 29. **Claim 31** The linking component is selected from the linking component groups described in SEQ ID NOs: 1197-1288 in Table 28 and SEQ ID NOs: 1291-1298 in Table 30. The oligonucleotide agent according to claim 29. **Claim 32** The oligonucleotide agent according to claim 1, and optionally at least one pharmaceutically acceptable carrier selected from an aqueous carrier, liposome or LNP, polymer, micelle, colloid, metal nanoparticle, non-metal nanoparticle, bioconjugate, and polypeptide. The pharmaceutical composition according to claim 1. **Claim 33** A kit comprising the oligonucleotide agent according to claim 1. **Claim 34** Use of the oligonucleotide agent according to claim 1 in the manufacture of a medicament for treating or delaying the onset or progression of amyotrophic lateral sclerosis (ALS). **Claim 35** The use according to claim 34, wherein ALS comprises sporadic ALS (sALS) and / or familial ALS (fALS).