Agents for regulating expression

Abstract

Agents that target nucleic acids (e.g., processed mRNA) can regulate the expression of the protein encoded by that nucleic acid, for example, through regulation of the level of that nucleic acid. In aspects, provided herein are compositions, methods, kits, and systems related to agents that regulate protein expression by targeting nucleic acid molecules that encode proteins.

Description

Technical Field

[0001] Cross-reference This application claims the benefit of U.S. Provisional Application No. 63 / 349,659, filed Jun. 7, 2022, which is hereby incorporated by reference in its entirety.

Summary of the Invention

[0002] In some aspects, provided herein is a method for reducing the expression of UBE3A protein in mammalian cells having a duplication, overexpression, or gain-of-function mutation of the UBE3A gene encoding the UBE3A protein, the method comprising contacting the mammalian cells with an agent or a vector encoding the agent, wherein the agent reduces the level of processed mRNA encoding the UBE3A protein in the mammalian cells.

[0003] In some cases, the agent comprises a polynucleotide sequence that is at least 80% complementary to at least 8 contiguous nucleic acids of the sequence set forth in any one of SEQ ID NOs: 93-120.

[0004] In some cases, the agent comprises a polynucleotide sequence that is at least 80% complementary to at least 8 contiguous nucleic acids of each mRNA transcript listed in Table 2.

[0005] In some cases, the agent comprises an antisense oligomer.

[0006] In some cases, the agent comprises an antisense oligomer having at least 80% identity to the sequence set forth in any one of SEQ ID NOs: 1-92.

[0007] In some embodiments, methods are disclosed herein for regulating the expression of the UBE3A gene encoding the UBE3A protein in mammalian cells, the method comprising contacting a mammalian cell with an agent or a vector encoding the agent, the agent comprising a polynucleotide sequence comprising an antisense oligomer having at least 80% identity with the sequence set forth in any one of SEQ ID NOs: 1-92.

[0008] In some cases, the antisense oligomer comprises a backbone modification, a modified sugar moiety, or a combination thereof.

[0009] In some cases, the antisense oligomer comprises phosphorothioate linkages or phosphorodiamidate linkages.

[0010] In some cases, the antisense oligomer comprises phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl moiety, 2'-fluoro moiety, 2'-O-methoxyethyl moiety, or 2'-NMA moiety.

[0011] In some cases, the antisense oligomer comprises at least one modified sugar moiety.

[0012] In some cases, the antisense oligomer comprises at least 1, 2, 3, 4, 5, or 6 modified nucleosides at the 5' end of the antisense oligomer.

[0013] In some cases, the antisense oligomer comprises 1, 2, 3, 4, 5, or 6 modified nucleosides at the 5' end of the antisense oligomer.

[0014] In some cases, the antisense oligomer comprises 1, 2, 3, 4, 5, or 6 2'-O-methoxyethyl modified nucleosides at the 5' end of the antisense oligomer.

[0015] In some cases, the antisense oligomer contains at least 1, 2, 3, 4, 5, or 6 modified nucleosides at the 3'-end of the antisense oligomer.

[0016] In some cases, the antisense oligomer contains 1, 2, 3, 4, 5, or 6 modified nucleosides at the 3'-end of the antisense oligomer.

[0017] In some cases, the antisense oligomer contains 1, 2, 3, 4, 5, or 6 2'-O-methoxyethyl modified nucleosides at the 3'-end of the antisense oligomer.

[0018] In some cases, the antisense oligomer contains 3, 4, 5, or 6 2'-O-methoxyethyl modified nucleosides at the 5'-end of the antisense oligomer, contains 3, 4, 5, or 6 2'-O-methoxyethyl modified nucleosides at the 3'-end of the antisense oligomer, and contains phosphorothioate bonds between any two adjacent nucleosides of the antisense oligomer.

[0019] In some cases, the antisense oligomer contains a 5'-region consisting of 3, 4, 5, or 6 linked nucleosides, a central region consisting of 8, 9, 10, 11, or 12 linked nucleosides, and a 3'-region consisting of 3, 4, 5, or 6 linked nucleosides. Each of the 3, 4, 5, or 6 linked nucleosides in the 5'-region and each of the 3, 4, 5, or 6 linked nucleosides in the 3'-region contain a modified sugar moiety, and each of the 8, 9, 10, 11, or 12 linked nucleosides in the central region is a deoxyribonucleoside.

[0020] In some cases, the antisense oligomer consists of 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, 12 to 15 nucleobases, 15 to 50 nucleobases, 15 to 40 nucleobases, 15 to 35 nucleobases, 15 to 30 nucleobases, 15 to 25 nucleobases, 15 to 20 nucleobases, 15 to 19 nucleobases, 15 to 18 nucleobases, 15 to 16 nucleobases, 16 to 20 nucleobases, 16 to 19 nucleobases, 16 to 18 nucleobases, 17 to 20 nucleobases, 17 to 19 nucleobases, or 18 to 20 nucleobases.

[0021] In some cases, the antisense oligomer is a modified oligonucleotide comprising the sequence set forth in any one of SEQ ID NOs: 154 to 189 or 192 to 247.

[0022] In some cases, the vector comprises a viral vector encoding a drug.

[0023] In some cases, the viral vector comprises an adenoviral vector, an adeno-associated virus (AAV) vector, a lentiviral vector, a herpes simplex virus (HSV) viral vector, or a retroviral vector.

[0024] In some cases, the antisense oligomer comprises a sequence having at least 80% identity with the sequence set forth in any one of SEQ ID NOs: 1 to 92.

[0025] In some cases, the antisense oligomer comprises a sequence having at least 90% identity with the sequence set forth in any one of SEQ ID NOs: 1 to 92.

[0026] In some cases, the antisense oligomer comprises a sequence having 100% identity with the sequence set forth in any one of SEQ ID NOs: 1 to 92.

[0027] In some cases, the antisense oligomer consists of a sequence having at least 80% identity with the sequence set forth in any one of SEQ ID NOs: 1 to 92.

[0028] In some cases, the antisense oligomer consists of a sequence having at least 90% identity with the sequence set forth in any one of SEQ ID NOs: 1 to 92.

[0029] In some cases, the antisense oligomer consists of a sequence having 100% identity with the sequence set forth in any one of SEQ ID NOs: 1 to 92.

[0030] In some cases, the method reduces the level of processed mRNA encoding the UBE3A protein in mammalian cells.

[0031] In some cases, the level of processed mRNA encoding UBE3A protein in mammalian cells that have been contacted with an agent or vector is reduced by about 10% to about 99%, about 10% to about 95%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, about 50% to about 99%, about 50% to about 95%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 99%, about 60% to about 95%, about 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 70% to about 99%, about 70% to about 95%, about 70% to about 90%, about 70% to about 80%, about 80% to about 99%, about 80% to about 95%, about 80% to about 90%, about 90% to about 99%, about 90% to about 95%, or about 95% to about 99% compared to mammalian cells that have not been contacted with the agent or vector but are otherwise the same.

[0032] In some cases, the level of processed mRNA encoding UBE3A protein in mammalian cells that have been contacted with an agent or vector is reduced by at least about 10%, 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%, or at least about 99% compared to mammalian cells that have not been contacted with the agent or vector but are otherwise the same.

[0033] In some cases, the method reduces the level of UBE3A protein in mammalian cells.

[0034] In some cases, the level of UBE3A protein in mammalian cells that have been contacted with an agent or vector is about 10% to about 99%, about 10% to about 95%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, about 50% to about 99%, about 50% to about 95%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 99%, about 60% to about 95%, about 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 70% to about 99%, about 70% to about 95%, about 70% to about 90%, about 70% to about 80%, about 80% to about 99%, about 80% to about 95%, about 80% to about 90%, about 90% to about 99%, about 90% to about 95%, or about 95% to about 99% lower compared to cells that have not been contacted with the agent or vector but are otherwise the same.

[0035] In some cases, the level of UBE3A protein in mammalian cells that have been contacted with an agent or vector is at least about 10%, 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%, or at least about 99% lower compared to cells that have not been contacted with the agent or vector but are otherwise the same.

[0036] In some cases, the method comprises contacting an agent or vector with a population of mammalian cells.

[0037] In some cases, the agent reduces the level of processed mRNA encoding UBE3A protein in a population of mammalian cells by up to about 75%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, or up to about 20% compared to a population of the same mammalian cells that has not been contacted with the agent or vector.

[0038] In some cases, the agent reduces the level of UBE3A protein in a population of mammalian cells by up to about 75%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, or up to about 20% compared to a population of the same mammalian cells that has not been contacted with the agent or the vector.

[0039] In some cases, the mammalian cells are ex vivo.

[0040] In some cases, the mammalian cells are in vivo.

[0041] In some cases, the genome of the mammalian cells has a duplication of a genomic region containing the UBE3A gene encoding UBE3A protein.

[0042] In some cases, the mammalian cells are human cells and the genome of the cells has a duplication of chromosome 15q11.2 - q13.1.

[0043] In some cases, the mammalian cells are obtained from a human subject suffering from Dup15q syndrome or are descendants of sample cells obtained from a human subject.

[0044] In some embodiments, the present specification discloses an antisense oligomer comprising a sequence having at least 80% identity with the sequence set forth in any one of SEQ ID NOs: 1 - 92.

[0045] In some cases, the antisense oligomer comprises a backbone modification, a modified sugar moiety, or a combination thereof.

[0046] In some cases, the antisense oligomer contains phosphorothioate linkages or phosphorodiamidate linkages.

[0047] In some cases, the antisense oligomer contains phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl moieties, 2'-fluoro moieties, 2'-O-methoxyethyl moieties, or 2'-NMA moieties.

[0048] In some cases, the antisense oligomer contains at least one modified sugar moiety.

[0049] In some cases, the antisense oligomer contains at least 1, 2, 3, 4, 5, or 6 modified nucleosides at the 5'-end of the antisense oligomer.

[0050] In some cases, the antisense oligomer contains 1, 2, 3, 4, 5, or 6 modified nucleosides at the 5'-end of the antisense oligomer.

[0051] In some cases, the antisense oligomer contains 1, 2, 3, 4, 5, or 6 2'-O-methoxyethyl modified nucleosides at the 5'-end of the antisense oligomer.

[0052] In some cases, the antisense oligomer contains at least 1, 2, 3, 4, 5, or 6 modified nucleosides at the 3'-end of the antisense oligomer.

[0053] In some cases, the antisense oligomer contains 1, 2, 3, 4, 5, or 6 modified nucleosides at the 3'-end of the antisense oligomer.

[0054] In some cases, the antisense oligomer contains 1, 2, 3, 4, 5, or 6 2'-O-methoxyethyl modified nucleosides at the 3'-end of the antisense oligomer.

[0055] In some cases, the antisense oligomer comprises 3, 4, 5, or 6 2'-O-methoxyethyl modified nucleosides at the 5' end of the antisense oligomer, 3, 4, 5, or 6 2'-O-methoxyethyl modified nucleosides at the 3' end of the antisense oligomer, and phosphorothioate linkages between any two adjacent nucleosides of the antisense oligomer.

[0056] In some cases, the antisense oligomer comprises a 5' region consisting of 3, 4, 5, or 6 linked nucleosides, a central region consisting of 8, 9, 10, 11, or 12 linked nucleosides, and a 3' region consisting of 3, 4, 5, or 6 linked nucleosides, wherein each of the 3, 4, 5, or 6 linked nucleosides in the 5' region and each of the 3, 4, 5, or 6 linked nucleosides in the 3' region comprises a modified sugar moiety, and each of the 8, 9, 10, 11, or 12 linked nucleosides in the central region is a deoxyribonucleoside.

[0057] In some cases, the antisense oligomer consists of 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, 12 to 15 nucleobases, 15 to 50 nucleobases, 15 to 40 nucleobases, 15 to 35 nucleobases, 15 to 30 nucleobases, 15 to 25 nucleobases, 15 to 20 nucleobases, 15 to 19 nucleobases, 15 to 18 nucleobases, 15 to 16 nucleobases, 16 to 20 nucleobases, 16 to 19 nucleobases, 16 to 18 nucleobases, 17 to 20 nucleobases, 17 to 19 nucleobases, or 18 to 20 nucleobases.

[0058] In some cases, the antisense oligomer includes a sequence having at least 80% identity with the sequence set forth in any one of SEQ ID NOs: 1 to 92.

[0059] In some cases, the antisense oligomer includes a sequence having at least 90% identity with the sequence set forth in any one of SEQ ID NOs: 1 to 92.

[0060] In some cases, the antisense oligomer includes a sequence having 100% identity with the sequence set forth in any one of SEQ ID NOs: 1 to 92.

[0061] In some cases, the antisense oligomer consists of a sequence having at least 80% identity with the sequence set forth in any one of SEQ ID NOs: 1 to 92.

[0062] In some cases, the antisense oligomer consists of a sequence having at least 90% identity with the sequence set forth in any one of SEQ ID NOs: 1 to 92.

[0063] In some cases, the antisense oligomer consists of a sequence having 100% identity with the sequence set forth in any one of SEQ ID NOs: 1 to 92.

[0064] In some cases, the antisense oligomer is a modified oligonucleotide comprising the sequence set forth in any one of SEQ ID NOs: 154 to 189 or 192 to 247.

[0065] In some cases, the antisense oligomer is a modified oligonucleotide consisting of the sequence set forth in any one of SEQ ID NOs: 154 to 189 or 192 to 247.

[0066] In some cases, the antisense oligomer is configured to reduce the level of processed mRNA transcripts encoding the UBE3A protein in a population of mammalian cells when contacted with the population.

[0067] In some cases, the antisense oligomer is configured to reduce the level of processed mRNA encoding UBE3A protein in the population by about 10% to about 99%, about 10% to about 95%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, about 50% to about 99%, about 50% to about 95%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 99%, about 60% to about 95%, about 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 70% to about 99%, about 70% to about 95%, about 70% to about 90%, about 70% to about 80%, about 80% to about 99%, about 80% to about 95%, about 80% to about 90%, about 90% to about 99%, about 90% to about 95%, or about 95% to about 99% compared to a population of mammalian cells that are otherwise the same but not in contact with the antisense oligomer.

[0068] In some cases, the antisense oligomer is configured to reduce the level of processed mRNA encoding UBE3A protein in the population by at least about 10%, 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%, or at least about 99% compared to cells that are otherwise the same but not in contact with the antisense oligomer.

[0069] In some cases, the antisense oligomer is configured to reduce the level of processed mRNA encoding the UBE3A protein in a population of mammalian cells by up to about 75%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, or up to about 20% compared to a population of mammalian cells that is otherwise the same but not in contact with the antisense oligomer.

[0070] In some cases, the antisense oligomer is configured to reduce the level of UBE3A protein in a population of mammalian cells.

[0071] In some cases, the antisense oligomer is configured to reduce the level of UBE3A protein in a population of mammalian cells by about 10% to about 99%, about 10% to about 95%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, about 50% to about 99%, about 50% to about 95%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 99%, about 60% to about 95%, about 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 70% to about 99%, about 70% to about 95%, about 70% to about 90%, about 70% to about 80%, about 80% to about 99%, about 80% to about 95%, about 80% to about 90%, about 90% to about 99%, about 90% to about 95%, or about 95% to about 99% compared to a population of mammalian cells that is otherwise the same but not in contact with the antisense oligomer.

[0072] In some cases, the antisense oligomer is configured to reduce the level of UBE3A protein in the population by at least about 10%, 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%, or at least about 99% compared to a population of mammalian cells that are otherwise the same but not in contact with the antisense oligomer.

[0073] In some cases, the antisense oligomer is configured to reduce the level of UBE3A protein in the population by up to about 75%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, or up to about 20% compared to a population of mammalian cells that are otherwise the same but not in contact with the antisense oligomer.

[0074] In some cases, the mammalian cells are ex vivo.

[0075] In some cases, the mammalian cells are in vivo.

[0076] In some cases, the genome of the mammalian cells has a duplication of a genomic region containing the UBE3A gene that encodes the UBE3A protein.

[0077] In some cases, the mammalian cells are human cells.

[0078] In some cases, the genome of the mammalian cells has a duplication of chromosome 15q11.2 - q13.1.

[0079] In some cases, the mammalian cells are obtained from a human subject suffering from Dup15q syndrome or are descendants of sample cells obtained from a human subject.

[0080] In some embodiments, the present specification discloses a pharmaceutical composition comprising (a) a pharmaceutically acceptable excipient or carrier and (b) an antisense oligomer disclosed herein.

[0081] In some embodiments, the present specification discloses a pharmaceutical composition comprising (a) a pharmaceutically acceptable excipient or carrier and (b) an agent or a vector encoding the agent configured to reduce the level of processed mRNA transcripts encoding the UBE3A protein in mammalian cells when contacted with mammalian cells.

[0082] In some cases, the agent comprises a polynucleotide sequence that is at least 80% complementary to at least 8 consecutive nucleic acids of the sequence set forth in any one of SEQ ID NOs: 93 - 120.

[0083] In some cases, the agent comprises a polynucleotide sequence that is at least 80% complementary to at least 8 consecutive nucleic acids of each mRNA transcript listed in Table 2.

[0084] In some cases, the agent comprises an antisense oligomer.

[0085] In some cases, the antisense oligomer has at least 80% identity with the sequence set forth in any one of SEQ ID NOs: 1 - 92.

[0086] In some cases, the pharmaceutical composition comprises a vector, and the vector comprises a viral vector encoding the agent.

[0087] In some cases, the viral vector comprises an adenoviral vector, an adeno - associated virus (AAV) vector, a lentiviral vector, a herpes simplex virus (HSV) viral vector, or a retroviral vector.

[0088] In some cases, the antisense oligomer comprises a sequence having at least 80% identity with the sequence set forth in any one of SEQ ID NOs: 1 to 92.

[0089] In some cases, the antisense oligomer comprises a sequence having at least 90% identity with the sequence set forth in any one of SEQ ID NOs: 1 to 92.

[0090] In some cases, the antisense oligomer comprises a sequence having 100% identity with the sequence set forth in any one of SEQ ID NOs: 1 to 92.

[0091] In some cases, the antisense oligomer consists of a sequence having at least 80% identity with the sequence set forth in any one of SEQ ID NOs: 1 to 92.

[0092] In some cases, the antisense oligomer consists of a sequence having at least 90% identity with the sequence set forth in any one of SEQ ID NOs: 1 to 92.

[0093] In some cases, the antisense oligomer consists of a sequence having 100% identity with the sequence set forth in any one of SEQ ID NOs: 1 to 92.

[0094] In some cases, the antisense oligomer is a modified oligonucleotide comprising the sequence set forth in any one of SEQ ID NOs: 154 to 189 or 192 to 247.

[0095] In some cases, the antisense oligomer is a modified oligonucleotide consisting of the sequence set forth in any one of SEQ ID NOs: 154 to 189 or 192 to 247.

[0096] In some cases, the agent is configured to reduce the level of processed mRNA encoding the UBE3A protein in a population of mammalian cells when contacted with the population.

[0097] In some cases, the antisense oligomer is configured to reduce the level of processed mRNA encoding the UBE3A protein in a population of mammalian cells that is not in contact with the agent or vector, but is otherwise the same, by about 10% to about 99%, about 10% to about 95%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, about 50% to about 99%, about 50% to about 95%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 99%, about 60% to about 95%, about 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 70% to about 99%, about 70% to about 95%, about 70% to about 90%, about 70% to about 80%, about 80% to about 99%, about 80% to about 95%, about 80% to about 90%, about 90% to about 99%, about 90% to about 95%, or about 95% to about 99%.

[0098] In some cases, the agent is configured to reduce the level of processed mRNA encoding the UBE3A protein in a population of mammalian cells that is not in contact with the agent or vector, but is otherwise the same, by at least about 10%, 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%, or at least about 99%.

[0099] In some cases, the agent is configured to reduce the level of processed mRNA encoding the UBE3A protein in the population by up to about 75%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, or up to about 20% compared to a population of mammalian cells that is otherwise the same but has not been contacted with the antisense oligomer.

[0100] In some cases, the agent is configured to reduce the level of UBE3A protein in the population.

[0101] In some cases, the agent is configured to reduce the level of UBE3A protein in the population by about 10% to about 99%, about 10% to about 95%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, about 50% to about 99%, about 50% to about 95%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 99%, about 60% to about 95%, about 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 70% to about 99%, about 70% to about 95%, about 70% to about 90%, about 70% to about 80%, about 80% to about 99%, about 80% to about 95%, about 80% to about 90%, about 90% to about 99%, about 90% to about 95%, or about 95% to about 99% compared to a population of mammalian cells that is otherwise the same but has not been contacted with the agent or vector.

[0102] In some cases, the agent is configured to reduce the level of UBE3A protein in the population by at least about 10%, 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%, or at least about 99% compared to a population of mammalian cells that is otherwise the same but has not been contacted with the agent or vector.

[0103] In some cases, the agent is configured to reduce the level of UBE3A protein in the population by up to about 75%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, or up to about 20% compared to a population of mammalian cells that is otherwise the same but has not been contacted with the antisense oligomer.

[0104] In some cases, the mammalian cells are ex vivo. In some cases, the mammalian cells are in vivo. In some cases, the genome of the mammalian cells has a duplication of a genomic region containing the UBE3A gene that encodes the UBE3A protein.

[0105] In some cases, the mammalian cells are human cells. In some cases, the genome of the mammalian cells has a duplication of chromosome 15q11.2 - q13.1.

[0106] In some cases, the mammalian cells are obtained from a human subject suffering from Dup15q syndrome or are descendants of sample cells obtained from a human subject.

[0107] In some cases, the pharmaceutical composition is formulated for intracerebroventricular injection, intraperitoneal injection, intramuscular injection, intrathecal injection, intracisternal injection, subcutaneous injection, oral administration, synovial injection, intravitreal administration, subretinal injection, topical application, transplantation, or intravenous injection.

[0108] In some cases, the pharmaceutical composition is formulated for intrathecal injection.

[0109] In some cases, the pharmaceutically acceptable excipient or carrier includes artificial cerebrospinal fluid.

[0110] In some cases, the pharmaceutical composition further includes a second therapeutic agent.

[0111] In some cases, the second therapeutic agent includes a small molecule, an antisense oligomer, or a gene editing molecule.

[0112] In some embodiments, disclosed herein is a method of treating a disease or condition of a subject or reducing the likelihood of onset thereof by reducing the expression of UBE3A protein in the cells of the subject in need of treatment of the disease or condition, the method comprising contacting the cells of the subject with a pharmaceutical composition disclosed herein.

[0113] In some cases, the disease or condition is related to overexpression of the UBE3A gene encoding the UBE3A protein or a gain-of-function mutation.

[0114] In some cases, the genome of the cells of the subject has at least one extra copy of the UBE3A gene encoding the UBE3A protein.

[0115] In some cases, the genome of the cells of the subject has a duplication of a genomic region containing the UBE3A gene encoding the UBE3A protein.

[0116] In some cases, the genome of the cells of the subject has a duplication of chromosome 15q11.2-q13.1.

[0117] In some cases, the disease or condition includes Dup15q syndrome, autism spectrum disorder, epilepsy, or intellectual disability.

[0118] In some cases, the subject is a human. In some cases, the subject is a fetus, embryo, or pediatric patient. In some cases, the cells are ex vivo.

[0119] In some cases, the method includes administering a pharmaceutical composition to the subject by intracerebroventricular injection, intraperitoneal injection, intramuscular injection, intrathecal injection, intracisternal injection, subcutaneous injection, oral administration, synovial injection, intravitreal administration, subretinal injection, topical application, transplantation, or intravenous injection.

[0120] In some cases, the method includes administering a pharmaceutical composition to the subject by intrathecal injection.

[0121] In some cases, the method treats a disease or condition.

[0122] Incorporation by reference All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0123] The novel features of the present disclosure are set forth in detail in the appended claims. The features and advantages of the present disclosure will be better understood by reference to the following detailed description of the exemplary embodiments in which the principles of the present disclosure are utilized, and the accompanying drawings.

Brief Description of the Drawings

[0124]

Figure 1A

Figure 1B

Figure 1C

Figure 1D

Figure 2A

Figure 2B

Figure 3A

Figure 3B

Figure 3C

Figure 3D

Figure 4

Figure 5A

Figure 5B

Figure 6

Modes for Carrying Out the Invention

[0125] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0126] Specific Terms Unless specific definitions are provided, the nomenclature, as well as the procedures and techniques used in connection with analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein, are well known and commonly used in the art. Standard techniques can be used for chemical synthesis and chemical analysis.

[0127] Unless otherwise specified, the following terms have the following meanings.

[0128] "Administering" can mean providing a pharmaceutical to an animal and includes, but is not limited to, administration by a medical professional and self - administration. "Amelioration" refers to reducing, delaying, halting, or reversing at least one measure of the severity of a syndrome or condition. The severity of the measure can be determined by subjective or objective scales known to those of ordinary skill in the art.

[0129] "Animal" can refer to human or non - human animals including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non - human primates (including but not limited to monkeys and chimpanzees).

[0130] "Antisense oligomer" can mean an oligomeric compound that can hybridize to a target nucleic acid via hydrogen bonding. Examples of antisense oligomers include single - stranded and double - stranded compounds such as antisense oligonucleotides, siRNA, shRNA, and ssRNA.

[0131] "Antisense inhibition" or "inhibition" can mean that the level of a target nucleic acid is reduced in the presence of an antisense oligomer complementary to the target nucleic acid as compared to the level of the target nucleic acid in the absence of the antisense oligomer.

[0132] The "antisense mechanism" can refer to all those mechanisms involved in the hybridization of a compound with a target nucleic acid, where the result or effect of the hybridization is the degradation of the target or the occupancy of the target, and at the same time, cell mechanisms involved in, for example, transcription or splicing are halted. The antisense oligomers provided herein may be "antisense" to a target nucleic acid, which means that the antisense oligomer can hybridize with the target nucleic acid via hydrogen bonds.

[0133] "Antisense oligonucleotide" can mean a single-stranded oligonucleotide having a nucleobase sequence that enables hybridization with a corresponding segment of a target nucleic acid.

[0134] "Base complementarity" can refer to the ability of the nucleobases of an antisense oligonucleotide to accurately base pair (i.e., hybridize) with the corresponding nucleobases of a target nucleic acid, mediated by Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonds between the corresponding nucleobases.

[0135] "Bicyclic sugar" can mean a furanose ring modified by a bridge between two atoms. A bicyclic sugar is a modified sugar.

[0136] "Bicyclic nucleoside" (also referred to as "BNA") can mean a nucleoside having a sugar moiety that includes a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4'-carbon and the 2'-carbon of the sugar ring.

[0137] "Cap structure" or "terminal cap portion" can mean a chemical modification incorporated at either end of an antisense oligomer. "cEt" or "constrained ethyl" can mean a bicyclic nucleoside having a sugar moiety containing a crosslink that links the 4'-carbon and the 2'-carbon, and the crosslink has the following formula: 4'-CH(CH3)-0-2'

[0138] "Constrained ethyl nucleoside" (also called cEt nucleoside) can mean a nucleoside containing a bicyclic sugar moiety containing a 4'-CH(CH3)-0-2' crosslink.

[0139] "Chimeric antisense oligomer" can mean an antisense oligomer having at least two chemically distinct regions, with each position having multiple subunits.

[0140] "Complementarity" can mean the pairing ability between the nucleobases of a first nucleic acid and a second nucleic acid.

[0141] "Contiguous nucleobases" can mean nucleobases that are directly adjacent to each other.

[0142] "Diluent" can mean a component in a composition that lacks pharmacological activity but is pharmaceutically necessary or desirable. For example, in an injectable drug, the diluent can be a liquid, such as physiological saline.

[0143] "Effective amount" in the context of modulating activity, or treating or preventing a condition, can mean administering to an individual in need of such modulation, treatment, or prevention, that amount of a pharmaceutical that is effective for modulating its effect, or treating or preventing or ameliorating its condition, as a single dose or as part of a series of doses. The effective amount may vary from individual to individual depending on the health and physical condition of the individual to be treated, the taxonomic group of the individual to be treated, the formulation of the composition, the assessment of the individual's medical condition, and other relevant factors.

[0144] As used herein in the same sense, "efficacy" or "effectiveness" can mean the ability to produce a desired effect.

[0145] "Expression" can include all processes by which the encoded information of a gene is converted into a structure that exists and functions within a cell. Such structures include, but are not limited to, the products of transcription and translation.

[0146] "Gapmer" can mean a chimeric antisense oligomer in which an internal region having a plurality of nucleosides that support RNase H cleavage is located between external regions having one or more nucleosides, where the nucleosides including the internal region are chemically different from the nucleoside(s) including the external region. The internal region can be referred to as the "gap" and the external region can be referred to as the "wing".

[0147] "Hybridization" can mean the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, antisense oligomers and target nucleic acids. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, antisense oligonucleotides and nucleic acid targets.

[0148] "Individual" can mean a human or non-human animal selected for treatment or therapy.

[0149] "Inhibiting UBE3A" or "inhibiting UBE3A" can mean reducing the level or expression of UBE3A mRNA and / or UBE3A protein. In certain embodiments, the UBE3A mRNA and / or UBE3A protein levels are inhibited in the presence of an antisense oligomer (including an antisense oligonucleotide) targeting UBE3A, as compared to the expression of UBE3A mRNA and / or UBE3A protein levels in the absence of the UBE3A antisense oligomer (such as an antisense oligonucleotide).

[0150] "Inhibiting expression or activity" can refer to a decrease or blockage of expression or activity, and does not necessarily indicate a complete elimination of expression or activity.

[0151] "Internucleoside linkage" can refer to a chemical bond between nucleosides.

[0152] "Intrathecal" or "ICM" injection or delivery can refer to injecting the agents or pharmaceutical compositions provided herein into the subarachnoid space filled with cerebrospinal fluid (CSF) between the cerebellum and the dorsal medulla.

[0153] "Linked nucleosides" can refer to adjacent nucleosides linked to each other by an internucleoside linkage.

[0154] "UBE3A antisense oligomer" can mean an antisense oligomer targeting UBE3A mRNA.

[0155] "Mismatch" or "non-complementary nucleobase" can refer to the case where the nucleobase of the first nucleic acid cannot pair with the corresponding nucleobase of the second nucleic acid or the target nucleic acid.

[0156] "Modified internucleoside linkage" can refer to a substitution or any change from a naturally occurring internucleoside linkage (i.e., a phosphodiester internucleoside linkage).

[0157] "Modified nucleobase" can refer to any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. "Unmodified nucleobase" means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0158] "Modified nucleoside" can independently refer to a nucleoside having a modified sugar moiety and / or a modified nucleobase.

[0159] "Modified nucleotide" can independently refer to a nucleotide having a modified sugar moiety, a modified internucleoside linkage, and / or a modified nucleobase.

[0160] "Modified antisense oligonucleotide" can refer to an oligonucleotide containing at least one modified internucleoside linkage, a modified sugar, and / or a modified nucleobase.

[0161] "Modified sugar" can refer to a substitution and / or any change from a natural sugar moiety.

[0162] "Monomer" can refer to a single unit of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides, whether naturally occurring or modified. "Motif" means the pattern of unmodified and modified nucleosides in an antisense oligomer.

[0163] "Natural sugar moiety" can refer to the sugar moiety contained in DNA (2'-H) or RNA (2'-OH).

[0164] "Naturally occurring internucleoside linkage" can refer to a 3'-5' phosphodiester linkage.

[0165] "Non-complementary nucleobases" can refer to a pair of nucleobases that do not form hydrogen bonds with each other or, otherwise, do not support hybridization.

[0166] "Nucleic acid" can refer to a molecule composed of monomeric nucleotides. Nucleic acids include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), single-stranded nucleic acids, double-stranded nucleic acids, small interfering ribonucleic acid (siRNA), and microRNA (miRNA).

[0167] "Nucleobase" can mean a heterocyclic moiety that can pair with the base of another nucleic acid. "Nucleobase complementarity" can refer to a nucleobase that can base pair with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In certain embodiments, complementary nucleobases refer to the nucleobases of an antisense oligomer that can base pair with the nucleobases of its target nucleic acid. For example, if the nucleobase at a particular position of an antisense oligomer can hydrogen bond with the nucleobase at a particular position of a target nucleic acid, the position of the hydrogen bond between the oligonucleotide and the target nucleic acid is considered to be complementary in that nucleobase pair.

[0168] "Nucleobase sequence" can refer to the order of consecutive nucleobases, independent of any sugar, linkage, and / or nucleobase modification.

[0169] "Nucleoside" can refer to a nucleobase linked to a sugar.

[0170] "Nucleoside mimics" do not necessarily replace the linkages at one or more positions of an oligomeric compound, and may include structures used to replace the sugar or both the sugar and the base. For example, there are nucleoside mimics having a morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo, or tricyclic sugar mimic (e.g., a non-furanose sugar unit). Nucleotide mimics include structures used to replace the nucleoside and the linkage at one or more positions of an oligomeric compound, such as peptide nucleic acids or morpholinos (morpholinos linked by -N(H)-C(=O)-O- or other non-phosphodiester linkages). Sugar substitutes overlap with the term nucleoside mimics which has a somewhat broader meaning, but are intended to indicate replacement of only the sugar unit (the furanose ring). The tetrahydropyranyl ring provided herein represents an example of a sugar substitute in which the furanosyl group is replaced by a tetrahydropyranyl ring system. "Mimic" can refer to a group used in place of a sugar, nucleic acid base, and / or internucleoside linkage. Generally, mimics can be used in place of a combination of a sugar or a sugar-nucleoside linkage, and the nucleic acid base is maintained for hybridization to a selected target.

[0171] "Nucleotide" can refer to a nucleoside in which a phosphate group is covalently attached to the sugar moiety of the nucleoside.

[0172] As used herein in the same sense, "oligomeric compound" or "oligomer" can refer to a polymer of linked monomer subunits that can hybridize to at least one region of a nucleic acid molecule.

[0173] "Oligonucleotide" can refer to a polymer of linked nucleosides, each of which may or may not be independently modified.

[0174] "Parenteral administration" can refer to administration by injection (e.g., bolus injection) or infusion. Parenteral administration can include subcutaneous administration, intravenous administration, intramuscular administration, intra-arterial administration, intraperitoneal administration, or intracranial administration (e.g., intrathecal, intraventricular, or intracisternal administration).

[0175] "Peptide" can refer to a molecule formed by linking at least two amino acids by an amide bond. Without limitation, as used herein, peptide refers to polypeptide and protein.

[0176] "Pharmaceutical" can refer to a substance that, when administered to an individual, produces a therapeutic effect. For example, in certain embodiments, an antisense oligonucleotide that targets UBE3A is a pharmaceutical.

[0177] "Pharmaceutical composition" can refer to a mixture of substances suitable for administration to an individual. For example, a pharmaceutical composition can include an antisense oligonucleotide and a sterile aqueous solution.

[0178] "Pharmaceutically acceptable salt" can refer to a physiologically and pharmaceutically acceptable salt of a pharmaceutically active ingredient (e.g., an antisense oligomer provided herein), e.g., a salt that retains the desired biological activity of the active ingredient and does not impart undesired toxic effects.

[0179] "Phosphorothioate bond" can mean a bond between nucleosides in which a phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate bond is a modified internucleoside bond.

[0180] "Segment" can mean a defined number of consecutive (i.e., linked) nucleobases of a nucleic acid. In certain embodiments, a segment is a defined number of consecutive nucleobases of a target nucleic acid. In certain embodiments, a segment is a defined number of consecutive nucleobases of an antisense oligomer.

[0181] "Prevent" or "preventing" can mean delaying or preventing the onset or progression of a disorder or syndrome for minutes to days, weeks to months, or indefinitely.

[0182] "Preventive effective amount" can mean the amount of a pharmaceutical that produces a preventive or prophylactic effect in an animal.

[0183] "Ribonucleotide" can mean a nucleotide having a hydroxy group at the 2'-position of the sugar moiety of the nucleotide. Ribonucleotides may be modified with any of a variety of substituents.

[0184] "Segment" is defined as a smaller portion or sub-portion of a region within a target nucleic acid.

[0185] "Targeting" or "targeted" can mean the process of designing and selecting an antisense oligomer that specifically hybridizes to a target nucleic acid and induces a desired effect.

[0186] "Target nucleic acid", "target RNA", "target RNA transcript", and "nucleic acid target" can all mean a nucleic acid that can be targeted by an antisense oligomer. In certain embodiments, the target nucleic acid is a UBE2A nucleic acid.

[0187] "Target region" can mean a portion of a target nucleic acid that one or more antisense oligomers target.

[0188] "Target segment" can mean the nucleotide sequence of a target nucleic acid that an antisense oligomer targets. "5'-target site" refers to the most 5'-nucleotide of the target segment. "3'-target site" refers to the most 3'-nucleotide of the target segment.

[0189] "Therapeutically effective amount" can mean an amount of a pharmaceutical that produces a therapeutic effect in an individual. "Treat" or "treating" or "treatment" can refer to administering a composition to effect a change or improvement in a disorder or syndrome.

[0190] "Unmodified nucleobase" can mean the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0191] "Unmodified nucleotide" can mean a nucleotide composed of a naturally occurring nucleobase, sugar moiety, and internucleoside linkage. In certain embodiments, the unmodified nucleotide is an RNA nucleotide (i.e., β-D-ribonucleoside) or a DNA nucleotide (i.e., β-D-deoxyribonucleoside).

[0192] "Wing segment" can mean a plurality of nucleosides modified to impart properties such as enhanced inhibitory activity, improved binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases to an oligonucleotide.

[0193] In general, the present disclosure relates to agents (e.g., antisense oligomers, e.g., antisense oligonucleotides), compositions, kits, and methods related to the regulation of UBE3A levels in mammalian cells. In some cases, the agents provided herein regulate the level of processed mRNA transcripts encoding UBE3A protein in mammalian cells. In some cases, provided herein are agents, compositions, kits, and methods related to the reduction of the expression of UBE3A protein in mammalian cells in which UBE3A is overexpressed, e.g., mammalian cells having a duplication, overexpression, or gain-of-function mutation of the UBE3A gene encoding the UBE3A protein.

[0194] Target Nucleic Acid, Target Region, and Nucleotide Sequence Ubiquitin-protein ligase E3A (UBE3A), also known as E6AP ubiquitin-protein ligase (E6AP), is an enzyme involved in targeting and degrading proteins within cells. In humans, the UBE3A protein is encoded by the UBE3A gene. The UBE3A gene is located on the long arm (q) of chromosome 15 at 15q11.2, between positions 11 and 13. In cells, the UBE3A protein can bind ubiquitin to proteins that are degraded by the ubiquitin-proteasome degradation machinery. Through ubiquitin tagging, proteins can be recognized and digested by the proteasome. In humans, both copies of the UBE3A gene may be active in most tissues of the body. However, in most neurons, only the maternal copy of the UBE3A gene is normally active.

[0195] Examples of nucleotide sequences encoding UBE3A include, but are not limited to, the complementary sequence from position 25333728 to 25439056 of GENBANK accession number NC_000015.10, or the complementary sequence from position 556325 to 658147 of GENBANK accession number NG_002690.1. Examples of nucleotide sequences encoding UBE3A include, but are not limited to, the sequences listed in Table 1. Examples of nucleotide sequences encoding UBE3A include, but are not limited to, any of the mRNA transcripts listed in Table 2. It is understood that the sequences described in each SEQ ID NO in Table 1 and the Examples included herein are independent of any modifications to the sugar moiety, internucleoside linkage, or nucleobase. Thus, the antisense oligomers defined by SEQ ID NO can independently include one or more modifications to the sugar moiety, internucleoside linkage, or nucleobase.

[0196] In some cases, the agents provided herein (e.g., antisense oligomers) comprise a polynucleotide sequence that is at least 80% complementary to at least 8 contiguous nucleic acids of the sequence set forth in any one of SEQ ID NOs: 93 - 120. In some cases, the agents provided herein (e.g., antisense oligomers) comprise a polynucleotide sequence that is at least 85%, 90%, 95%, 98%, or 100% complementary to at least 8 contiguous nucleic acids of the sequence set forth in any one of SEQ ID NOs: 93 - 120. In some cases, the agents provided herein (e.g., antisense oligomers) comprise a polynucleotide sequence that is 100% complementary to at least 8 contiguous nucleic acids of the sequence set forth in any one of SEQ ID NOs: 93 - 120.

[0197] In some cases, the agents provided herein (e.g., antisense oligomers) comprise a polynucleotide sequence that is at least 80% complementary to at least 8 contiguous nucleic acids of the mRNA transcripts listed in Table 2. In some cases, the agents provided herein (e.g., antisense oligomers) comprise a polynucleotide sequence that is at least 85%, 90%, 95%, 98%, or 100% complementary to at least 8 contiguous nucleic acids of the mRNA transcripts listed in Table 2. In some cases, the agents provided herein (e.g., antisense oligomers) comprise a polynucleotide sequence that is 100% complementary to at least 8 contiguous nucleic acids of the mRNA transcripts listed in Table 2.

[0198] In certain embodiments, the target region is a structurally defined region of the target nucleic acid. For example, the target region can include a 3’UTR, 5’UTR, exon, intron, exon / intron junction, coding region, translation initiation region, translation termination region, or other defined nucleic acid regions. Structurally defined regions of UBE3A can be obtained by accession number from sequence databases such as NCBI, and such information is incorporated herein by reference. In certain embodiments, the target region includes the sequence from the 5’ target site of one target segment within the target region to the 3’ target site of another target segment within the same target region.

[0199] Targeting can include determining at least one target segment to which the antisense oligomer hybridizes such that a desired effect (e.g., degradation of an mRNA transcript comprising at least one target segment) occurs. In certain embodiments, the desired effect is a reduction in the mRNA target nucleic acid level. In certain embodiments, the desired effect is a reduction in the level of the protein encoded by the target nucleic acid, or a change in the phenotype associated with the target nucleic acid.

[0200] The target region can include one or more target segments. Multiple target segments within the target region may or may not overlap. In certain embodiments, the target segments within the target region are separated by about 300 nucleotides or fewer. In certain embodiments, the target segments within the target region are separated by 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides, about 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides or fewer, about 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides or fewer, or a number of nucleotides in a range defined by any two of the foregoing values on the target nucleic acid. In certain embodiments, the target segments within the target region are separated by 5 nucleotides or fewer or about 5 nucleotides or fewer on the target nucleic acid. In certain embodiments, the target segments are contiguous. Target regions defined by a range having a starting nucleic acid that is either a 5' target site or a 3' target site recited herein are contemplated.

[0201] In some embodiments, hybridization occurs between the antisense oligomers disclosed herein and the UBE3A nucleic acid. The most common mechanism of hybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reverse Hoogsteen hydrogen bonding) between complementary nucleobases of nucleic acid molecules.

[0202] Hybridization can occur under a variety of conditions. Stringent conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized.

[0203] Methods for determining whether an array can specifically hybridize to a target nucleic acid are well known in the art. In certain embodiments, the antisense oligomers provided herein can specifically hybridize to UBE3A nucleic acids.

[0204]

Table 1

[0205]

Table 2

[0206] Antisense oligomer In some embodiments, the agents provided herein are antisense oligomers. The antisense oligomers provided herein can include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, antisense oligomers, antisense oligonucleotides, and siRNA.

[0207] In certain embodiments, the antisense oligomer has a nucleobase sequence that, when written in the 5' to 3' direction, constitutes the reverse complement of the target segment of the target nucleic acid designed to be targeted by it. In certain such embodiments, the antisense oligonucleotide has a nucleobase sequence that, when written in the 5' to 3' direction, constitutes the reverse complement of the target segment of the target nucleic acid designed to be targeted by it.

[0208] In some cases, the agents provided herein include antisense oligomers, and the antisense oligomers include sequences having at least 80% identity with the sequences set forth in any one of SEQ ID NOs: 1-92. In some cases, the agents provided herein include antisense oligomers, and the antisense oligomers include sequences having at least 90% identity with the sequences set forth in any one of SEQ ID NOs: 1-92. In some cases, the antisense oligomers include sequences having 100% identity with the sequences set forth in any one of SEQ ID NOs: 1-92. In some cases, the antisense oligomers consist of sequences having at least 80% identity with the sequences set forth in any one of SEQ ID NOs: 1-92. In some cases, the antisense oligomers consist of sequences having at least 90% identity with the sequences set forth in any one of SEQ ID NOs: 1-92. In some cases, the antisense oligomers consist of sequences having 100% identity with the sequences set forth in any one of SEQ ID NOs: 1-92.

[0209] In some cases, the agents provided herein include antisense oligomers, and the antisense oligomers include the sequences set forth in any one of SEQ ID NOs: 1-92 having 0 to 4 nucleic acid substitutions. In some cases, the agents provided herein include antisense oligomers, and the antisense oligomers include the sequences set forth in any one of SEQ ID NOs: 1-92 having 0 to 3 nucleic acid substitutions. In some cases, the agents provided herein include antisense oligomers, and the antisense oligomers include the sequences set forth in any one of SEQ ID NOs: 1-92 having 0 to 2 nucleic acid substitutions. In some cases, the agents provided herein include antisense oligomers, and the antisense oligomers include the sequences set forth in any one of SEQ ID NOs: 1-92 having 0 to 1 nucleic acid substitutions.

[0210] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 1. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 1.

[0211] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 2. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 2.

[0212] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 3. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 3.

[0213] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 4. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 4.

[0214] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 5. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 5.

[0215] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 6. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 6.

[0216] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 7. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 7.

[0217] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 8. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 8.

[0218] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 9. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 9.

[0219] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 10. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 10.

[0220] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 11. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 11.

[0221] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 12. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 12.

[0222] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 13. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 13.

[0223] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 14. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 14.

[0224] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 15. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 15.

[0225] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 16. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 16.

[0226] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 17. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 17.

[0227] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 18. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 18.

[0228] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 19. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 19.

[0229] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 20. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 20.

[0230] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 21. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 21.

[0231] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 22. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 22.

[0232] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 23. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 23.

[0233] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 24. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 24.

[0234] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 25. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 25.

[0235] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 26. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 26.

[0236] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 27. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 27.

[0237] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 28. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 28.

[0238] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 29. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 29.

[0239] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 30. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 30.

[0240] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 31. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 31.

[0241] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 32. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 32.

[0242] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 33. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 33.

[0243] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 34. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 34.

[0244] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 35. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 35.

[0245] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 36. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 36.

[0246] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 37. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 37.

[0247] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 38. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 38.

[0248] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 39. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 39.

[0249] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 40. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 40.

[0250] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 41. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 41.

[0251] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 42. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 42.

[0252] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 43. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 43.

[0253] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 44. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 44.

[0254] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 45. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 45.

[0255] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 46. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 46.

[0256] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 47. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 47.

[0257] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 48. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 48.

[0258] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 49. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 49.

[0259] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 50. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 50.

[0260] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 51. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 51.

[0261] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 52. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 52.

[0262] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 53. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 53.

[0263] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 54. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 54.

[0264] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 55. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 55.

[0265] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 56. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 56.

[0266] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 57. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 57.

[0267] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 58. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 58.

[0268] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 59. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 59.

[0269] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 60. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 60.

[0270] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 61. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 61.

[0271] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 62. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 62.

[0272] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 63. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 63.

[0273] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 64. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 64.

[0274] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 65. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 65.

[0275] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 66. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 66.

[0276] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 67. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 67.

[0277] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 68. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 68.

[0278] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 69. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 69.

[0279] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 70. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 70.

[0280] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 71. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 71.

[0281] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 72. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 72.

[0282] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 73. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 73.

[0283] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 74. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 74.

[0284] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 75. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 75.

[0285] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 76. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 76.

[0286] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 77. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 77.

[0287] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 78. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 78.

[0288] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 79. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 79.

[0289] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 80. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 80.

[0290] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 81. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 81.

[0291] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 82. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 82.

[0292] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 83. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 83.

[0293] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 84. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 84.

[0294] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 85. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 85.

[0295] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 86. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 86.

[0296] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 87. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 87.

[0297] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 88. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 88.

[0298] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 89. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 89.

[0299] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity with the sequence of SEQ ID NO: 90. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 90.

[0300] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity with the sequence of SEQ ID NO: 91. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 91.

[0301] In some cases, the antisense oligomers provided herein include sequences having at least 90%, 92%, 95%, 98%, or 100% identity with the sequence of SEQ ID NO: 92. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 92.

[0302] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 1 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 1 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 1 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 1 having from 0 to 1 nucleic acid substitution.

[0303] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 2 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 2 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 2 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 2 having from 0 to 1 nucleic acid substitution.

[0304] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 3 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 3 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 3 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 3 having from 0 to 1 nucleic acid substitution.

[0305] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 4 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 4 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 4 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 4 having from 0 to 1 nucleic acid substitution.

[0306] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 5 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 5 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 5 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 5 having from 0 to 1 nucleic acid substitution.

[0307] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 6 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 6 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 6 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 6 having from 0 to 1 nucleic acid substitution.

[0308] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 7 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 7 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 7 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 7 having from 0 to 1 nucleic acid substitution.

[0309] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 8 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 8 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 8 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 8 having from 0 to 1 nucleic acid substitution.

[0310] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 9 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 9 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 9 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 9 having from 0 to 1 nucleic acid substitution.

[0311] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 10 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 10 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 10 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 10 having from 0 to 1 nucleic acid substitution.

[0312] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 11 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 11 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 11 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 11 having from 0 to 1 nucleic acid substitution.

[0313] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 12 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 12 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 12 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 12 having from 0 to 1 nucleic acid substitution.

[0314] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 13 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 13 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 13 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 13 having from 0 to 1 nucleic acid substitution.

[0315] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 14 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 14 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 14 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 14 having from 0 to 1 nucleic acid substitution.

[0316] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 15 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 15 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 15 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 15 having from 0 to 1 nucleic acid substitution.

[0317] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 16 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 16 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 16 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 16 having from 0 to 1 nucleic acid substitution.

[0318] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 17 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 17 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 17 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 17 having from 0 to 1 nucleic acid substitution.

[0319] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 18 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 18 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 18 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 18 having from 0 to 1 nucleic acid substitution.

[0320] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 19 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 19 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 19 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 19 having from 0 to 1 nucleic acid substitution.

[0321] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 20 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 20 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 20 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 20 having from 0 to 1 nucleic acid substitution.

[0322] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 21 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 21 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 21 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 21 having from 0 to 1 nucleic acid substitution.

[0323] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 22 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 22 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 22 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 22 having from 0 to 1 nucleic acid substitution.

[0324] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 23 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 23 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 23 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 23 having from 0 to 1 nucleic acid substitution.

[0325] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 24 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 24 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 24 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 24 having from 0 to 1 nucleic acid substitution.

[0326] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 25 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 25 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 25 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 25 having from 0 to 1 nucleic acid substitution.

[0327] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 26 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 26 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 26 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 26 having from 0 to 1 nucleic acid substitution.

[0328] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 27 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 27 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 27 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 27 having from 0 to 1 nucleic acid substitution.

[0329] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 28 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 28 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 28 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 28 having from 0 to 1 nucleic acid substitution.

[0330] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 29 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 29 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 29 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 29 having from 0 to 1 nucleic acid substitution.

[0331] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 30 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 30 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 30 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 30 having from 0 to 1 nucleic acid substitution.

[0332] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 31 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 31 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 31 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 31 having from 0 to 1 nucleic acid substitution.

[0333] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 32 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 32 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 32 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 32 having from 0 to 1 nucleic acid substitution.

[0334] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 33 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 33 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 33 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 33 having from 0 to 1 nucleic acid substitution.

[0335] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 34 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 34 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 34 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 34 having from 0 to 1 nucleic acid substitution.

[0336] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 35 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 35 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 35 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 35 having from 0 to 1 nucleic acid substitution.

[0337] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 36 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 36 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 36 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 36 having from 0 to 1 nucleic acid substitution.

[0338] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 37 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 37 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 37 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 37 having from 0 to 1 nucleic acid substitution.

[0339] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 38 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 38 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 38 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 38 having from 0 to 1 nucleic acid substitution.

[0340] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 39 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 39 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 39 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 39 having from 0 to 1 nucleic acid substitution.

[0341] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 40 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 40 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 40 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 40 having from 0 to 1 nucleic acid substitution.

[0342] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 41 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 41 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 41 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 41 having from 0 to 1 nucleic acid substitution.

[0343] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 42 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 42 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 42 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 42 having from 0 to 1 nucleic acid substitution.

[0344] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 43 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 43 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 43 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 43 having from 0 to 1 nucleic acid substitution.

[0345] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 44 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 44 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 44 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 44 having from 0 to 1 nucleic acid substitution.

[0346] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 45 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 45 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 45 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 45 having from 0 to 1 nucleic acid substitution.

[0347] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 46 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 46 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 46 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 46 having from 0 to 1 nucleic acid substitution.

[0348] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 47 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 47 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 47 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 47 having from 0 to 1 nucleic acid substitution.

[0349] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 48 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 48 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 48 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 48 having from 0 to 1 nucleic acid substitution.

[0350] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 49 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 49 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 49 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 49 having from 0 to 1 nucleic acid substitution.

[0351] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 50 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 50 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 50 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 50 having from 0 to 1 nucleic acid substitution.

[0352] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 51 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 51 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 51 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 51 having from 0 to 1 nucleic acid substitution.

[0353] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 52 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 52 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 52 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 52 having from 0 to 1 nucleic acid substitution.

[0354] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 53 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 53 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 53 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 53 having from 0 to 1 nucleic acid substitution.

[0355] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 54 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 54 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 54 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 54 having from 0 to 1 nucleic acid substitution.

[0356] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 55 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 55 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 55 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 55 having from 0 to 1 nucleic acid substitution.

[0357] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 56 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 56 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 56 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 56 having from 0 to 1 nucleic acid substitution.

[0358] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 57 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 57 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 57 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 57 having from 0 to 1 nucleic acid substitution.

[0359] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 58 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 58 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 58 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 58 having from 0 to 1 nucleic acid substitution.

[0360] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 59 having 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 59 having 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 59 having 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 59 having 0 to 1 nucleic acid substitution.

[0361] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 60 having 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 60 having 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 60 having 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 60 having 0 to 1 nucleic acid substitution.

[0362] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 61 having 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 61 having 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 61 having 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 61 having 0 to 1 nucleic acid substitution.

[0363] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 62 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 62 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 62 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 62 having from 0 to 1 nucleic acid substitution.

[0364] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 63 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 63 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 63 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 63 having from 0 to 1 nucleic acid substitution.

[0365] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 64 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 64 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 64 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 64 having from 0 to 1 nucleic acid substitution.

[0366] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 65 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 65 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 65 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 65 having from 0 to 1 nucleic acid substitution.

[0367] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 66 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 66 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 66 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 66 having from 0 to 1 nucleic acid substitution.

[0368] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 67 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 67 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 67 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 67 having from 0 to 1 nucleic acid substitution.

[0369] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 68 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 68 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 68 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 68 having from 0 to 1 nucleic acid substitution.

[0370] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 69 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 69 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 69 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 69 having from 0 to 1 nucleic acid substitution.

[0371] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 70 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 70 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 70 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 70 having from 0 to 1 nucleic acid substitution.

[0372] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 71 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 71 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 71 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 71 having from 0 to 1 nucleic acid substitution.

[0373] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 72 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 72 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 72 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 72 having from 0 to 1 nucleic acid substitution.

[0374] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 73 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 73 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 73 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 73 having from 0 to 1 nucleic acid substitution.

[0375] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 74 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 74 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 74 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 74 having from 0 to 1 nucleic acid substitution.

[0376] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 75 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 75 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 75 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 75 having from 0 to 1 nucleic acid substitution.

[0377] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 76 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 76 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 76 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 76 having from 0 to 1 nucleic acid substitution.

[0378] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 77 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 77 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 77 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 77 having from 0 to 1 nucleic acid substitution.

[0379] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 78 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 78 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 78 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 78 having from 0 to 1 nucleic acid substitution.

[0380] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 79 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 79 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 79 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 79 having from 0 to 1 nucleic acid substitution.

[0381] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 80 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 80 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 80 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 80 having from 0 to 1 nucleic acid substitution.

[0382] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 81 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 81 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 81 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 81 having from 0 to 1 nucleic acid substitution.

[0383] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 82 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 82 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 82 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 82 having from 0 to 1 nucleic acid substitution.

[0384] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 83 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 83 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 83 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 83 having from 0 to 1 nucleic acid substitution.

[0385] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 84 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 84 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 84 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 84 having from 0 to 1 nucleic acid substitution.

[0386] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 85 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 85 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 85 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 85 having from 0 to 1 nucleic acid substitution.

[0387] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 86 having 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 86 having 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 86 having 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 86 having 0 to 1 nucleic acid substitution.

[0388] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 87 having 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 87 having 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 87 having 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 87 having 0 to 1 nucleic acid substitution.

[0389] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 88 having 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 88 having 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 88 having 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 88 having 0 to 1 nucleic acid substitution.

[0390] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 89 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 89 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 89 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 89 having from 0 to 1 nucleic acid substitution.

[0391] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 90 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 90 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 90 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 90 having from 0 to 1 nucleic acid substitution.

[0392] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 91 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 91 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 91 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 91 having from 0 to 1 nucleic acid substitution.

[0393] In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 92 having from 0 to 4 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 92 having from 0 to 3 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 92 having from 0 to 2 nucleic acid substitutions. In some cases, the antisense oligomers provided herein include the sequence of SEQ ID NO: 92 having from 0 to 1 nucleic acid substitution.

[0394] In certain embodiments, the length of the antisense oligomer targeting the target nucleic acid is from 12 to 30 subunits. In certain embodiments, the length of the antisense oligomer targeting the target nucleic acid is from 12 to 25 subunits. In certain embodiments, the length of the antisense oligomer targeting the target nucleic acid is from 12 to 22 subunits. In certain embodiments, the length of the antisense oligomer targeting the target nucleic acid is from 14 to 20 subunits. In certain embodiments, the length of the antisense oligomer targeting the target nucleic acid is from 15 to 25 subunits. In certain embodiments, the length of the antisense oligomer targeting the target nucleic acid is from 18 to 22 subunits. In certain embodiments, the length of the antisense oligomer targeting the target nucleic acid is from 19 to 21 subunits. In certain embodiments, the length of the antisense oligomer is from 8 to 80, 12 to 50, 13 to 30, 13 to 50, 14 to 30, 14 to 50, 15 to 30, 15 to 50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18 to 30, 18 to 50, 19 to 30, 19 to 50, or 20 to 30 linked subunits.

[0395] In certain embodiments, the length of the antisense oligomer targeting the target nucleic acid is 12 subunits. In certain embodiments, the length of the antisense oligomer targeting the target nucleic acid is 13 subunits. In certain embodiments, the length of the antisense oligomer targeting the target nucleic acid is 14 subunits. In certain embodiments, the length of the antisense oligomer targeting the target nucleic acid is 15 subunits. In certain embodiments, the length of the antisense oligomer targeting the target nucleic acid is 16 subunits. In certain embodiments, the length of the antisense oligomer targeting the target nucleic acid is 17 subunits. In certain embodiments, the length of the antisense oligomer targeting the target nucleic acid is 18 subunits. In certain embodiments, the length of the antisense oligomer targeting the target nucleic acid is 19 subunits. In certain embodiments, the length of the antisense oligomer targeting the target nucleic acid is 20 subunits. In certain embodiments, the length of the antisense oligomer targeting the target nucleic acid is 21 subunits. In certain embodiments, the length of the antisense oligomer targeting the target nucleic acid is 22 subunits. In certain embodiments, the length of the antisense oligomer targeting the target nucleic acid is 23 subunits. In certain embodiments, the length of the antisense oligomer targeting the target nucleic acid is 24 subunits. In certain embodiments, the length of the antisense oligomer targeting the target nucleic acid is 25 subunits. In certain embodiments, the length of the antisense oligomer targeting the target nucleic acid is 26 subunits. In certain embodiments, the length of the antisense oligomer targeting the target nucleic acid is 27 subunits. In certain embodiments, the length of the antisense oligomer targeting the target nucleic acid is 28 subunits. In certain embodiments, the length of the antisense oligomer targeting the target nucleic acid is 29 subunits. In certain embodiments, the length of the antisense oligomer targeting the target nucleic acid is 30 subunits.In certain embodiments, the length of the antisense oligomer targeting the target nucleic acid is 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked subunits, or a range defined by any two of the above values. In certain embodiments, the antisense oligomer is an antisense oligonucleotide and the linked subunits are nucleosides.

[0396] The antisense oligomers provided herein may have nucleotides that mismatch with the target sequence. For example, an antisense oligonucleotide that is 25 nucleobases in length may have 8 or 11 mismatch bases near the ends of the antisense oligonucleotide, but can still induce specific cleavage of the target mRNA, albeit to a lesser extent than an antisense oligonucleotide that does not contain mismatches. In some cases, the antisense oligonucleotides provided herein are 12 - 30 subunits (e.g., nucleobases) in length, including those having 1 or 3 mismatches.

[0397] Chemically modified antisense oligomer In certain embodiments, the antisense oligomers provided herein have chemically modified subunits arranged in a pattern or motif to confer properties such as enhanced inhibitory activity, improved binding affinity for the target nucleic acid, or resistance to degradation by in vivo nucleases to the oligonucleotide.

[0398] In some cases, the present specification provides chimeric antisense oligomers. For example, the chimeric antisense oligomers can include at least one region modified to confer improved resistance to nuclease degradation, increased cellular uptake, improved binding affinity for a target nucleic acid, and / or improved inhibitory activity. A second region of the chimeric antisense oligomer can, optionally, function as a substrate for the cellular endonuclease RNase H that cleaves the RNA strand of an RNA:DNA duplex.

[0399] In some cases, the antisense oligomers provided herein have a gapmer motif. Antisense oligomers having a gapmer motif can be considered chimeric antisense oligomers. In a gapmer, an internal region having a plurality of nucleotides that support RNaseH cleavage can be positioned between external regions having a plurality of nucleotides that are chemically different from the nucleosides of the internal region. In the case of an antisense oligonucleotide having a gapmer motif, the gap segment can function as a substrate for endonuclease cleavage, while the wing segments contain modified nucleosides. In certain embodiments, the regions of the gapmer are distinguished by the type of sugar moiety that constitutes each different region. The types of sugar moieties used to distinguish the regions of the gapmer can include β-D-ribonucleosides, β-D-deoxyribonucleosides, 2'-modified nucleosides (such 2'-modified nucleosides can include, inter alia, 2'-MOE and 2'-O-CH3), and bicyclic sugar-modified nucleosides (such bicyclic sugar-modified nucleosides can include those having a 4'-(CH2)n-O-2' bridge (n = 1 or n = 2) and 4'-CH2-O-CH2-2'). In certain embodiments, the wings contain some modified sugar moieties, such as 2'-MOE. In certain embodiments, the wings contain some modified and unmodified sugar moieties. In certain embodiments, the wings contain various combinations of 2'-MOE nucleosides and 2'-deoxynucleosides.

[0400] Each different region can contain a uniform sugar moiety, a mutated sugar moiety, or an alternating sugar moiety. The wing-gap-wing motif is often described as "X-Y-Z", where "X" represents the length of the 5' wing, "Y" represents the length of the gap, and "Z" represents the length of the 3' wing. "X" and "Z" can contain a uniform sugar moiety, a mutated sugar moiety, or an alternating sugar moiety. In certain embodiments, "X" and "Y" contain one or more 2'-deoxynucleosides. "Y" can contain 2'-deoxynucleosides. As used herein, a gapmer described as "X-Y-Z" can have a configuration such that the gap is positioned directly adjacent to each of the 5' wing and the 3' wing. Thus, there can be no nucleotides intervening between the 5' wing and the gap, or between the gap and the 3' wing. Any of the antisense oligomers described herein can have a gapmer motif. In certain embodiments, "X" and "Z" are the same, and in other cases they are different.

[0401] In certain cases, the gapmers provided herein include, for example, 20-mers having a 5-10-5 motif in the form of "X-Y-Z" as described herein. In certain embodiments, the gapmers provided herein include, for example, 19-mers having a 5-9-5 motif in the form of "X-Y-Z" as described herein. In certain embodiments, the gapmers provided herein include, for example, 18-mers having a 5-8-5 motif in the form of "X-Y-Z" as described herein. In certain embodiments, the gapmers provided herein include, for example, 18-mers having a 4-8-6 motif in the form of "X-Y-Z" as described herein. In certain embodiments, the gapmers provided herein include, for example, 18-mers having a 6-8-4 motif in the form of "X-Y-Z" as described herein. In certain embodiments, the gapmers provided herein include, for example, 18-mers having a 5-7-6 motif in the form of "X-Y-Z" as described herein.

[0402] In some cases, the antisense oligomer comprises a 5' region (e.g., the "X" portion above) consisting of 3, 4, 5, or 6 linked nucleosides, a central region (e.g., the "Y" portion above) consisting of 8, 9, 10, 11, or 12 linked nucleosides, and a 3' region (e.g., the "Z" portion above) consisting of 3, 4, 5, or 6 linked nucleosides. In some cases, each of the 3, 4, 5, or 6 linked nucleosides in the 5' region, and each of the 3, 4, 5, or 6 linked nucleosides in the 3' region, includes a modified sugar moiety, and each of the 8, 9, 10, 11, or 12 linked nucleosides in the central region is a deoxyribonucleoside. In some cases, the modified sugar moiety includes a 2'-O-methyl moiety, a 2'-fluoro moiety, a 2'-O-methoxyethyl moiety, or a 2'-NMA moiety, or any combination thereof. In some cases, one or more nucleosides in the 5' region and the 3' region further include other modifications disclosed herein. In some cases, all nucleosides in the 5' region and the 3' region further include other modifications disclosed herein.

[0403] Complementarity The agents provided herein can have a polynucleotide sequence complementary to a target nucleic acid when a sufficient number of nucleobases in the polynucleotide sequence (e.g., an antisense oligomer) can hydrogen bond to the corresponding nucleobases of the target nucleic acid, resulting in a desired effect (e.g., antisense inhibition of a target nucleic acid such as a UBE3A nucleic acid).

[0404] Non-complementary nucleobases between a drug (e.g., an antisense oligomer) and a target nucleic acid can be tolerated as long as the drug (e.g., an antisense oligomer) can still specifically hybridize to the target nucleic acid. Further, a drug (e.g., an antisense oligomer) can hybridize to one or more segments of a target nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., loop structures, mismatches or hairpin structures).

[0405] In certain embodiments, a drug (e.g., an antisense oligomer) or a particular portion thereof provided herein is 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to a UBE3A nucleic acid, target region, target segment, or a particular portion thereof, or is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary. The percent complementarity between an antisense oligomer and a target nucleic acid can be determined using conventional methods such as the BLAST program (basic local alignment search tools) and the PowerBLAST program known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410, Zhang and Madden, Genome Res., 1997, 7, 649-656). The percent homology, sequence identity, or complementarity can be determined, for example, using the Gap program with the default settings by the algorithm of Smith and Waterman (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) (Adv. Appl. Math., 1981, 2, 482-489).

[0406] In certain embodiments, the agents (e.g., antisense oligomers) provided herein, or particular portions thereof, are completely complementary (i.e., 100% complementary) to the target nucleic acid or particular portions thereof. For example, the agents (e.g., antisense oligomers) provided herein can be completely complementary to the UBE3A nucleic acid, or target regions thereof, or target segments or target sequences thereof. As used herein, "completely complementary" can mean that each nucleobase of the antisense oligomer can base pair precisely with the corresponding nucleobase of the target nucleic acid.

[0407] The positions of non-complementary nucleobases can be at the 5' or 3' end of the antisense oligomer. Alternatively, the non-complementary nucleobase(s) can be at internal positions of the antisense oligomer. When two or more non-complementary nucleobases are present, they may or may not be contiguous (i.e., linked). In one embodiment, the non-complementary nucleobases are located in the wing segments of a gapmer antisense oligonucleotide.

[0408] In certain embodiments, antisense oligomers provided herein that are 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length, or up to 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length, contain 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer non-complementary nucleobase(s) relative to the target nucleic acid or particular portions thereof.

[0409] In certain embodiments, the antisense oligomers provided herein that are 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length, or up to 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length, comprise 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer non-complementary nucleobase(s) to the target nucleic acid or a particular portion thereof.

[0410] The agents provided herein (e.g., antisense oligomers) can also include those that are complementary to a portion of the target nucleic acid. As used herein, "portion" can refer to a defined number of consecutive (i.e., linked) nucleobases within a region or segment of the target nucleic acid. "Portion" can also refer to a defined number of consecutive nucleobases of the antisense oligomer. In certain embodiments, the agent (e.g., antisense oligomer) is complementary to at least an 8-nucleobase portion of the target segment. In certain embodiments, the agent (e.g., antisense oligomer) is complementary to at least a 9-nucleobase portion of the target segment. In certain embodiments, the agent (e.g., antisense oligomer) is complementary to at least a 10-nucleobase portion of the target segment. In certain embodiments, the agent (e.g., antisense oligomer) is complementary to at least an 11-nucleobase portion of the target segment. In certain embodiments, the agent (e.g., antisense oligomer) is complementary to at least a 12-nucleobase portion of the target segment. In certain embodiments, the agent (e.g., antisense oligomer) is complementary to at least a 13-nucleobase portion of the target segment. In certain embodiments, the agent (e.g., antisense oligomer) is complementary to at least a 14-nucleobase portion of the target segment. In certain embodiments, the agent (e.g., antisense oligomer) is complementary to at least a 15-nucleobase portion of the target segment. Also contemplated are antisense oligomers that are complementary to at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleobase portions of the target segment, or to a range defined by any two of these values.

[0411] The agents provided herein (e.g., antisense oligomers) can also have a percent identity defined with respect to a particular nucleotide sequence, SEQ ID NO, or a portion thereof. As used herein, an antisense oligomer is identical to a sequence disclosed herein if it has the same nucleic acid base pairing ability. For example, an RNA containing uracil instead of thymidine in a disclosed DNA sequence is considered identical to the DNA sequence because both uracil and thymidine pair with adenine. Shorter and longer versions of the antisense oligomers described herein, as well as oligomers having bases that are not identical to those of the antisense oligomers provided herein, are also contemplated. The non-identical bases can be adjacent to each other or dispersed throughout the antisense oligomer. The percent identity of an antisense oligomer is calculated according to the number of bases having identical base pairing to the sequence being compared.

[0412] In certain embodiments, an agent (e.g., an antisense oligomer) or a portion thereof is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to one or more agents (e.g., antisense oligomers) or SEQ ID NOs, or portions thereof, disclosed herein.

[0413] In certain embodiments, a portion of an agent (e.g., an antisense oligomer) is compared to an equal-length portion of a target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25-nucleotide portion is compared to an equal-length portion of a target nucleic acid.

[0414] In certain embodiments, a portion of an antisense oligonucleotide is compared to an equal-length portion of a target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25-nucleotide portion is compared to an equal-length portion of a target nucleic acid.

[0415] Modification In some embodiments, the antisense oligomers provided herein can have one or more chemical modifications compared to naturally occurring nucleotides (or the native form of the antisense oligomer) having the same or equivalent polynucleotide sequences. Modifications to the antisense oligomers include substitutions or changes to the internucleoside linkages, sugar moieties, or nucleobases. Modified antisense oligomers may be preferred over the native form because they have desirable properties such as improved cellular uptake, improved affinity for nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity.

[0416] Chemically modified nucleosides can be used to enhance the binding affinity of a shortened or truncated antisense oligonucleotide to its target nucleic acid. As a result, shorter antisense oligomers with such chemically modified nucleosides often give equivalent results.

[0417] A nucleoside can be a combination of a base and a sugar. The nucleobase (also called a base) portion of a nucleoside can be a native heterocyclic base portion. A nucleotide is a nucleoside that further includes a phosphate group covalently attached to the sugar portion of the nucleoside. In the case of a nucleoside containing a pentofuranosyl sugar, the phosphate group can be linked to the 2′, 3′, or 5′ hydroxyl moiety of the sugar. Oligonucleotides are formed by adjacent nucleosides covalently bonding to each other to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are generally said to form the internucleoside linkages of the oligonucleotide.

[0418] Modified internucleoside linkages The naturally occurring internucleoside linkages of RNA and DNA are 3' to 5' phosphodiester linkages. The antisense oligomers provided herein can have one or more modified, i.e., non-naturally occurring, internucleoside linkages. Antisense oligomers having one or more modified nucleotide internucleoside linkages can have desirable properties such as improved cellular uptake, improved affinity for the target nucleic acid, and increased stability in the presence of nucleases.

[0419] Oligonucleotides having modified internucleoside linkages can include internucleoside linkages that retain a phosphorus atom, as well as internucleoside linkages that do not have a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphodiester, phosphorotriester, methylphosphonate, phosphoramidate, and phosphorothioate.

[0420] In certain embodiments, antisense oligomers targeting UBE3A nucleic acids include one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside linkages are scattered throughout the antisense oligomer. In certain embodiments, the modified internucleoside linkage is a phosphorothioate linkage. In certain embodiments, each internucleoside linkage of the antisense oligomer is a phosphorothioate internucleoside linkage.

[0421] Modified sugar moiety The antisense oligomers provided herein can include one or more nucleosides having a modified sugar group. Such sugar-modified nucleosides can impart improved nuclease stability, increased binding affinity, or some other beneficial biological property to the antisense oligomer. In certain embodiments, the nucleoside includes a chemically modified ribofuranose ring moiety.

[0422] Examples of chemically modified ribofuranose rings include the addition of substituents (including 5' and 2' substituents), the formation of bicyclic nucleic acids (BNAs) by crosslinking of non-geminal ring atoms, substitution of the ribosyl ring oxygen atom with S, N(R), or C(R1)(R2) (where R, R1, and R2 are each independently H, C1-C 12 alkyl, or a protecting group), and combinations thereof, but are not limited thereto. Examples of chemically modified sugars include 2'-F-5'-methyl-substituted nucleosides (see PCT International Application WO2008 / 101157 for other disclosed 5',2'-bis-substituted nucleosides) or substitution of the ribosyl ring oxygen atom with S and further substitution at the 2' position (see U.S. Patent Application Publication US2005-0130923, published June 16, 2005) or alternatively 5'-substitution of BNA (see PCT International Application WO2007 / 134181. In this application, LNA is substituted, for example, with a 5'-methyl or 5'-vinyl group).

[0423] Examples of nucleosides having a modified sugar moiety include nucleosides containing 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH3, 2'-OCH2CH3, 2'-OCH2CH2F, 2'-NMA, and 2'-O(CH2)2OCH3 substituents, but are not limited thereto. Substituents at the 2' position can also be selected from allyl, amino, azide, thio, O-allyl, O-C1-C 10 alkyl, OCF3, OCH2F, O(CH2)2SCH3, O(CH2)2-O-N(R m )(R n ), O-CH2-C(=O)-N(R m )(R n ), and O-CH2-C(=O)-N(R l )-(CH2)2-N(R m )(R n ), and each R l , R m , and R n is independently H or substituted or unsubstituted C1-C 10 alkyl.

[0424] As used herein, "bicyclic nucleoside" can refer to a modified nucleoside that includes a bicyclic sugar moiety. Examples of bicyclic nucleosides include, but are not limited to, nucleosides that include a bridge between the 4' ribosyl ring atom and the 2' ribosyl ring atom. In certain embodiments, the antisense oligomers provided herein include one or more bicyclic nucleosides that include a 4' to 2' bridge. Examples of such 4' to 2' bridged bicyclic nucleosides include those described in U.S. Pat. Nos. 7,399,845; 8,278,283; U.S. Patent Application Nos. 7,696,345; 7,427,672; 8,278,426; 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 7,034,133; 7,053,207; 7,399,845; 7,547,684; 7,741,457; and 7,696,345; U.S. Patent Publication No. US2008-0039618; and Chattopadhyaya et al, J. Org. Chem., 2009, 74, 118-134; Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al, Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al, J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al, J. Am. Chem. Soc, 2007, 129(26)8362-8379; Elayadi et al, Curr. Opinion Invest. Drugs, 2001, 2, 558-561; Braasch et al, Chem. Biol, 2001, 8, 1-7; and Orum et al, Curr. Opinion Mol. Ther., 2001, 3, 239-243, but are not limited thereto.Each of the aforementioned bicyclic nucleosides can be prepared to have one or more stereochemical sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose (see PCT International Application PCT / DK98 / 00393, published as WO99 / 14226 on March 25, 1999). In certain embodiments, bicyclic sugar moieties of BNA nucleosides include, but are not limited to, those described in U.S. Patent No. 11,129,844.

[0425] The synthesis and preparation of adenine, cytosine, guanine, 5-methyl-cytosine, thymine, and uracil as methyleneoxy (4’-CH2-0-2’) BNA monomers, as well as their oligomerization and nucleic acid recognition properties, are described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630).

[0426] As used herein, “4’-to-2’ bicyclic nucleoside” or “4’-to-2’ bicyclic nucleoside” can refer to a bicyclic nucleoside that includes a furanose ring, which includes a bridge connecting two carbon atoms of the furanose ring that connect the 2’ carbon atom and the 4’ carbon atom of the sugar ring.

[0427] As used herein, “monocyclic nucleoside” can refer to a nucleoside that includes a modified sugar moiety that is not a bicyclic sugar moiety. In certain embodiments, the sugar moiety or sugar moiety analog of the nucleoside is modified or substituted at any position.

[0428] As used herein, “2’-modified sugar” can mean a furanosyl sugar modified at the 2’ position. In certain embodiments, such modifications include substituents selected from halides (including, but not limited to, substituted and unsubstituted alkoxy, substituted and unsubstituted thioalkyl, substituted and unsubstituted aminoalkyl, substituted and unsubstituted alkyl, substituted and unsubstituted allyl, and substituted and unsubstituted alkynyl). In certain embodiments, the 2’ modification is O[(CH2) n O]m CH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n F, O(CH2) n ONH2, OCH2C(=O)N(H)CH3, and O(CH2) n ON[(CH2) n CH3]2 (where n and m are from 1 to about 10), and are selected from substituents including but not limited to these. Other 2'-substituents are C1-C 12 alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, CI, Br, CN, F, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving pharmacokinetic properties, or group for improving pharmacodynamic properties of antisense oligomers, and other substituents having similar properties. In certain embodiments, the modified nucleoside comprises a 2'-MOE side chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). Such 2'-MOE substitution has been described as improving binding affinity compared to unmodified nucleosides and other modified nucleosides such as 2'-O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides having a 2'-MOE substituent have also been shown to be antisense inhibitors of gene expression with promising features for in vivo use. (Martin, Helv. Chim. Acta, 1995, 78, 486-504, Altmann et al., Chimia, 1996, 50, 168-176, Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637, and Altmann et al, Nucleosides Nucleotides, 1997, 16, 917-926).

[0429] As used herein, "2'-NMA" can mean a -O-CH2-C(=O)-NH-CH3 group in place of the 2'-OH group of the ribosyl sugar moiety. A "2'-NMA sugar moiety" or "2'-NMA moiety" is a sugar moiety having a 2'-O-CH2-C(=O)-NH-CH3 group in place of the 2'-OH group of the ribosyl sugar moiety. Unless otherwise specified, the 2'-NMA sugar moiety is in the β-D configuration. "NMA" can mean O-N-methylacetamide.

[0430] As used herein, "2'-NMA nucleoside" can mean a nucleoside containing a 2'-NMA sugar moiety.

[0431] As used herein, "2'-F" can refer to a nucleoside containing a sugar having a fluoro group at the 2'-position.

[0432] As used herein, "2'-OMe" or "2'-OCH3" or "2'-O-methyl" can each refer to a nucleoside containing a sugar having an -OCH3 group at the 2'-position of the sugar ring.

[0433] As used herein, "MOE" or "2'-MOE" or "2'-OCH2CH2OCH3" or "2'-O-methoxyethyl" each refer to a nucleoside containing a sugar having an -OCH2CH2OCH3 group at the 2'-position of the sugar ring.

[0434] In certain embodiments, one or more of the plurality of nucleosides are modified. In certain embodiments, the oligonucleotide comprises one or more ribonucleosides (RNA) and / or deoxyribonucleosides (DNA). In certain embodiments, the oligonucleotide comprises a mixture of one or more ribonucleosides (RNA) and deoxyribonucleosides (DNA).

[0435] Many other bicyclic and tricyclic sugar surrogate ring systems that can be used to modify nucleosides for incorporation into antisense oligomers are also known in the art (see, for example, the review article: Leumann, Bioorg. Med. Chem., 2002, 10, 841-854). Such ring systems can undergo various additional substitutions to enhance activity.

[0436] Methods for preparing modified sugars are well known to those skilled in the art.

[0437] In nucleotides having a modified sugar moiety, the nucleobase moiety (natural, modified, or combinations thereof) is maintained for hybridization to a suitable nucleic acid target.

[0438] In certain embodiments, the antisense oligomer comprises one or more nucleosides having a modified sugar moiety. In certain embodiments, the modified sugar moiety is 2'-MOE. In certain embodiments, the 2'-MOE modified nucleosides are arranged in a gapmer motif. In certain embodiments, the modified sugar moiety is a bicyclic nucleoside having a (4'-CH(CH3)-0-2') bridging group. In certain embodiments, the (4'-CH(CH3)-0-2') modified nucleosides are arranged throughout the wings of the gapmer motif.

[0439] "5'-Methylcytosine" can mean cytosine modified with a methyl group attached to the 5'-position. 5'-Methylcytosine is a modified nucleobase.

[0440] "5'-Methyluracil" can mean uracil modified with a methyl group attached to the 5'-position. 5'-Methyluracil is a modified nucleobase.

[0441] "5'-Methylthymine" can mean thymine modified with a methyl group attached to the 5'-position. 5'-Methylthymine is a modified nucleobase.

[0442] In some cases, the antisense oligomers provided herein include 5'-methylcytosine, 5'-methyluracil, 5'-methylthymine, or combinations thereof. In some cases, each cytosine in the antisense oligomer is methylated, i.e., a methyl group is attached to the 5'-position. In some cases, each uracil in the antisense oligomer is methylated, i.e., a methyl group is attached to the 5'-position. In some cases, the antisense oligomer has 1, 2, 3, 4, 5, 6, 7, 8 or more 5'-methylcytosines. In some cases, the antisense oligomer has 1, 2, 3, 4, 5, 6, 7, 8 or more 5'-methyluracils. In some cases, the antisense oligomer has both methylcytosine and methyluracil.

[0443] Pharmaceutical Compositions and Methods of Treatment In some embodiments, provided herein are pharmaceutical compositions comprising an agent of the disclosure, such as an antisense oligomer, or a vector encoding the agent.

[0444] A pharmaceutical composition or formulation comprising an agent of the described composition, such as an antisense oligomer, or a vector encoding the agent, and used in any of the described methods, is well known in the pharmaceutical industry and can be prepared according to conventional techniques described in the published literature. In some embodiments, a pharmaceutical composition or formulation for treating a subject comprises an effective amount of any antisense oligomer described herein, or a pharmaceutically acceptable salt, solvate, hydrate, or ester thereof. A pharmaceutical formulation comprising an antisense oligomer may further comprise a pharmaceutically acceptable excipient, diluent, or carrier.

[0445] In some cases, the antisense oligomers provided herein have intermediate levels of efficiency in reducing the levels of UBE3A transcripts in cells. For example, when the antisense oligomers are provided at the maximum administration level (the maximum concentration when delivered in vitro or the maximum dose when delivered in vivo without causing significant adverse effects to the cells or the subject), the UBE3A transcript levels can be reduced by up to 75%, up to 70%, up to 65%, up to 60%, up to 55%, up to 50%, up to 45%, up to 40%, up to 35%, up to 30%, up to 20%, or from about 25% to about 75%, from about 30% to about 70%, from about 35% to about 65%, from about 40% to about 60%, or from about 45% to about 55%. Without wishing to be bound by a particular theory, the antisense oligomers provided herein having intermediate levels of UBE3A knockdown efficiency can have desirable therapeutic uses. The intermediate levels of knockdown efficiency of the antisense oligomers may indicate that the antisense oligomers target sequences that are not present in all of the UBE3A transcripts, sequences to which the antisense oligomers cannot bind at a given time point, or both. For example, the antisense oligomers provided herein that can achieve a UBE3A knockdown efficiency of up to about 50% in cells can target sequences that are present only in 50% of the UBE3A transcripts in the cells. Alternatively or additionally, it may be because the target sites of the antisense oligomers on the UBE3A transcripts are occupied by another protein complex for half of the time or are otherwise physically inaccessible to the antisense oligomers. Since abnormally reduced levels of the UBE3A protein can also lead to pathological conditions, appropriate administration of the antisense oligomers that reduce the UBE3A transcripts without causing adverse effects by overly reducing the levels of the UBE3A transcripts may be desired for therapeutic uses. In the antisense oligomers provided herein having intermediate levels of knockdown efficiency, there may be an upper limit to the amount of UBE3A knockdown by the antisense oligomers.Thus, such an upper limit of the knockdown amount of UBE3A can create a safety buffer to prevent an antisense oligomer from overly reducing UBE3A transcripts when administered to a subject in which it is necessary to reduce UBE3A levels, such as a subject with duplication, overexpression, or gain-of-function mutations of the UBE3A gene, or a subject with an increase in the activity or expression level of the UBE3A protein.

[0446] The agents (e.g., antisense oligomers) or vectors provided herein can be mixed with pharmaceutically acceptable active or inactive substances for the preparation of pharmaceutical compositions or formulations. Agents (e.g., antisense oligomers) that target UBE3A nucleic acids can be utilized in pharmaceutical compositions by combining the agent with a suitable pharmaceutically acceptable diluent or carrier. Examples of pharmaceutically acceptable diluents include phosphate buffered saline (PBS), artificial cerebrospinal fluid (aCSF), saline, or any other suitable solution.

[0447] In some cases, the compositions and methods provided herein relate to vectors encoding the agents provided herein. In some cases, the vector comprises a viral vector encoding the agent. In some cases, the viral vector comprises an adenoviral vector, an adeno-associated virus (AAV) vector, a lentiviral vector, a herpes simplex virus (HSV) viral vector, or a retroviral vector.

[0448] Pharmaceutically acceptable salts are those that, without causing excessive toxicity, irritation, allergic reactions, etc., are suitable for use in contact with the tissues of humans and lower animals and also have a reasonable benefit / risk ratio (see, e.g., S.M. Berge, et al., J. Pharmaceutical Sciences, 66:1-19 (1977), which is incorporated herein by reference for this purpose). The salts can be prepared in situ during the final isolation and purification of the compound or separately by reacting the free base form with a suitable organic acid. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed using inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or using organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or using other documented methods such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Representative alkali metal salts or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc.Additional pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0449] In some embodiments, the composition is formulated into any of a number of possible dosage forms including, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. In embodiments, the composition is formulated as a suspension in an aqueous, non-aqueous, or mixed medium. An aqueous suspension may further contain substances that increase the viscosity of the suspension, such as, for example, sodium carboxymethyl cellulose, sorbitol, and / or dextran. The suspension may also contain stabilizers. In embodiments, the pharmaceutical formulations or compositions of the present disclosure include, but are not limited to, solutions, emulsions, microemulsions, foams, or liposome-containing formulations (e.g., cationic or non-cationic liposomes).

[0450] The pharmaceutical compositions or formulations described herein can, if desired, also contain one or more penetration enhancers, carriers, excipients, or other active or inactive ingredients that are well known to those skilled in the art or described in the published literature. In embodiments, the liposomes can also include sterically stabilized liposomes, for example, liposomes that include one or more special lipids. These special lipids result in liposomes with an extended circulation lifetime. In embodiments, the sterically stabilized liposomes include one or more glycolipids or are derivatized with one or more hydrophilic polymers such as polyethylene glycol (PEG) moieties. In some embodiments, a surfactant is included in the pharmaceutical formulation or composition. The use of surfactants in pharmaceutical products, formulations, and emulsions is well known in the art. In embodiments, the present disclosure uses penetration enhancers to achieve efficient delivery of antisense oligonucleotides, for example, to facilitate diffusion across cell membranes and / or to enhance the permeability of lipophilic drugs. In some embodiments, the penetration enhancer is a surfactant, a fatty acid, a bile salt, a chelating agent, or a non-chelating non-surfactant.

[0451] In some embodiments, the pharmaceutical formulation includes multiple agents (e.g., antisense oligomers). In embodiments, the agent (e.g., antisense oligomer) or the vector encoding the agent is administered in combination with another drug or therapeutic agent.

[0452] A pharmaceutical composition containing an antisense oligomer can include any pharmaceutically acceptable salt, ester, or salt of such an ester, or any other oligonucleotide that (directly or indirectly) provides a biologically active metabolite or residue thereof when administered to an animal, including a human. Thus, for example, the present disclosure also encompasses pharmaceutically acceptable salts of antisense oligomers, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other biological equivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

[0453] The prodrug can include incorporating additional nucleosides at one or both ends of the antisense oligomer, and these additional nucleosides are cleaved by endogenous nucleases in the body to form the active antisense oligomer.

[0454] The antisense oligomers disclosed herein can be covalently attached to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the resulting antisense oligomer. Conjugate groups can include cholesterol moieties and lipid moieties. Further conjugate groups can include carbohydrates, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes.

[0455] The antisense oligomers of the present disclosure can be modified to have one or more stabilizing groups generally attached to one or both ends of the antisense oligomer to enhance properties such as nuclease stability. Stabilizing groups include cap structures. These terminal modifications can protect antisense oligomers with terminal nucleic acids from exonuclease degradation and can assist in delivery and / or localization within cells. The cap can be present at the 5' end (5' cap) or the 3' end (3' cap), or at both ends. Cap structures can include, for example, inverted deoxyabasic caps. Further 3' and 5' stabilizing groups that can be used to cap one or both ends of the antisense oligomer to confer nuclease stability can include those disclosed in International Publication No. WO 03 / 004602 published on January 16, 2003.

[0456] Any of the compositions provided herein can be administered to an individual. "Individual" can be used interchangeably with "subject" or "patient". An individual can be a mammal, such as a human, or a non-human primate, rodent, rabbit, rat, mouse, horse, donkey, goat, cat, dog, cow, pig, or sheep. In embodiments, the individual is a human. In embodiments, the individual is a fetus, embryo, or pediatric patient. In some embodiments, the compositions provided herein are administered to cells ex vivo.

[0457] In some embodiments, the compositions provided herein are administered to an individual as a method of treating a disease or disorder. In certain embodiments, the individual has a neurological disorder. In some cases, the individual has a disease or disorder associated with an excessive expression level or activity level of the UBE3A protein. In some cases, the diseases or disorders to which the compositions provided herein are applicable are associated with duplication, overexpression, or gain-of-function mutations of the UBE3A gene, such as duplication of chromosome 15q11.2-q13.1, Dup15q syndrome.

[0458] Dup15q syndrome is one of the most common genetic mutations associated with autism spectrum disorder (ASD) related to duplication of chromosome 15q11.2-q13.1. This chromosomal region contains the imprinted Prader-Willi / Angelman syndrome critical region (PWACR), ubiquitin protein ligase E3A (UBE3A), small nuclear ribonucleoprotein polypeptide N (SNRPN), and three GABAA receptor genes (GABRB3, GABRA5, and GABRG3). Dup15q syndrome can include two major types of duplications of 15q11.2 - 13.1, namely, (1) isodicentric chromosome 15 (idic(15)) that results in two additional maternally-derived copies on an extra chromosome containing the proximal regions of 15p and 15q11, most commonly resulting in four copies of this region, or (2) interstitial 15q duplication where one extra copy of the 15q11.2 - q13.1 region occurs on the same chromosome arm (usually resulting in three copies of that region and having an overall milder phenotype). Duplications of 15q11.2 - q13.1 pose a strong risk of autism spectrum disorder, epilepsy, and intellectual disability to patients. Studies have shown that patients with Dup15q syndrome may exhibit many symptoms including a specific behavioral profile such as relative weaknesses in the areas of motor skills, facial expressions, social smiling, and reciprocal social interactions. Patients with Dup15q syndrome may also exhibit a characteristic electroencephalogram (EEG) signature in the form of high-amplitude spontaneous beta frequency (12 - 30 Hz) oscillations.

[0459] In certain embodiments, provided herein is a method for prophylactically reducing UBE3A expression in an individual. Certain embodiments include treating an individual in need of treatment by administering to the individual a therapeutically effective amount of an agent that targets UBE3A nucleic acid (e.g., an antisense oligomer) or a vector encoding the agent.

[0460] In some embodiments, the individual has a genetic disease, such as any of the diseases described herein. In some embodiments, the individual is at risk of having a disease, such as any of the diseases described herein. In some embodiments, the individual has an increased risk of having a disease or disorder caused by insufficient amount of a protein or insufficient activity of a protein. If the individual has an increased risk of having a disease or disorder caused by insufficient amount of a protein or insufficient activity of a protein, the method includes preventative or prophylactic treatment. For example, due to a family history of a certain disease, an individual may have an increased risk of having such a disease or disorder. Typically, an individual at increased risk of having such a disease or disorder benefits from preventative treatment (e.g., by preventing or delaying the onset or progression of the disease or disorder). In an embodiment, the fetus is treated in utero by administering to the fetus directly or indirectly (e.g., via the mother) a therapeutic effective amount of an antisense oligomer, such as a drug, or a vector encoding the drug.

[0461] In one embodiment, administration of a therapeutically effective amount of an antisense oligomer targeting UBE3A nucleic acid is performed concomitantly with monitoring of UBE3A levels in the individual, in order to determine the individual's response to the administration of the antisense oligomer. The individual's response to the administration of the antisense oligomer can be used by a physician to determine the amount and duration of therapeutic intervention.

[0462] In certain embodiments, administration of an antisense oligomer targeting UBE3A nucleic acid reduces processed mRNA encoding UBE3A protein (e.g., UBE3A mRNA) and / or protein expression by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or by a range defined by any two of these values.

[0463] In some cases, the level of processed mRNA encoding UBE3A protein (e.g., UBE3A mRNA) in cells contacted with an agent or vector is about 10% to about 99%, about 10% to about 95%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, about 50% to about 99%, about 50% to about 95%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 99%, about 60% to about 95%, about 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 70% to about 99%, about 70% to about 95%, about 70% to about 90%, about 70% to about 80%, about 80% to about 99%, about 80% to about 95%, about 80% to about 90%, about 90% to about 99%, about 90% to about 95%, or about 95% to about 99% lower compared to cells that are not contacted with the agent or vector but are otherwise the same.

[0464] In some embodiments, the level of processed mRNA encoding the UBE3A protein (e.g., UBE3A mRNA) in cells contacted with the agent or vector is at least about 10%, 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%, or at least about 99% lower compared to cells that are not contacted with the agent or vector but are otherwise the same.

[0465] In some cases, the methods and compositions provided herein reduce the level of UBE3A protein in cells. In some embodiments, the level of UBE3A protein in cells contacted with an agent or vector is about 10% to about 99%, about 10% to about 95%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, about 50% to about 99%, about 50% to about 95%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 99%, about 60% to about 95%, about 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 70% to about 99%, about 70% to about 95%, about 70% to about 90%, about 70% to about 80%, about 80% to about 99%, about 80% to about 95%, about 80% to about 90%, about 90% to about 99%, about 90% to about 95%, or about 95% to about 99% lower compared to cells that are not contacted with the agent or vector but are otherwise the same. In some cases, the level of UBE3A protein in cells contacted with an agent or vector is at least about 10%, 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%, or at least about 99% lower compared to cells that are not contacted with the agent or vector but are otherwise the same.

[0466] In certain embodiments, administration of an antisense oligomer targeting UBE3A nucleic acid improves the cognitive function of an animal. In certain embodiments, administration of the UBE3A antisense oligomer improves the cognitive function by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or by a range defined by any two of these values.

[0467] In certain embodiments, administration of an antisense oligomer targeting UBE3A nucleic acid improves the motor function of an animal. In certain embodiments, administration of the UBE3A antisense oligomer improves the cognitive function by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or by a range defined by any two of these values.

[0468] In certain embodiments, administration of an antisense oligomer targeting UBE3A nucleic acid improves the anxiety of an animal. In certain embodiments, administration of the UBE3A antisense oligomer improves anxiety by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or a range defined by any two of these values. In certain embodiments, administration of an antisense oligomer targeting UBE3A nucleic acid improves the social interaction of an animal. In certain embodiments, administration of the UBE3A antisense oligomer improves social interaction by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or a range defined by any two of these values. In certain embodiments, administration of an antisense oligomer targeting UBE3A nucleic acid reduces seizures. In certain embodiments, administration of the UBE3A antisense oligomer reduces seizures by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or a range defined by any two of these values. In certain embodiments, administration of an antisense oligomer targeting UBE3A nucleic acid normalizes EEG discharges.

[0469] In certain embodiments, a pharmaceutical composition comprising an antisense oligomer targeting UBE3A is used in the preparation of a medicament for treating a patient having or at risk of having a disease including Dup15q syndrome.

[0470] The route suitable for administration of the agent (e.g., antisense oligomer) or vector encoding the agent of the present invention can vary depending on the cell type to which delivery of the agent or vector is desired. The agent or vector encoding the agent of the present disclosure can be administered parenterally to a patient, for example, by intrathecal injection, intraventricular injection, intracisternal injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.

[0471] In embodiments, the antisense oligomer is administered with one or more agents that can facilitate penetration of the subject antisense oligomer across the blood-brain barrier by any method known in the art. For example, delivery of an agent by administration of an adenoviral vector to motor neurons in muscle tissue is described in U.S. Patent No. 6,632,427, which is incorporated herein by reference. Direct delivery of a vector to the brain, such as the striatum, thalamus, hippocampus, or substantia nigra, is described, for example, in U.S. Patent No. 6,756,523, which is incorporated herein by reference.

[0472] In some embodiments, the antisense oligomer is linked or conjugated to an agent that provides desirable pharmaceutical or pharmacodynamic properties. In embodiments, the antisense oligomer is coupled to a substance known in the art to facilitate penetration or transport across the blood-brain barrier, such as an antibody to the transferrin receptor. In embodiments, for example, the antisense oligonucleotide is linked to a viral vector to make the antisense oligomer more effective or to increase transport across the blood-brain barrier. In embodiments, osmotic blood-brain barrier disruption is facilitated by injection of saccharides (such as meso-erythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose, dulcitol, myo-inositol, L(-) fructose, D(-) mannitol, D(+) glucose, D(+) arabinose, D(-) arabinose, cellobiose, D(+) maltose, D(+) raffinose, L(+) rhamnose, D(+) melibiose, D(-) ribose, adonitol, D(+) arabitol, L(-) arabitol, D(+) fucose, L(-) fucose, D(-) lyxose, L(+) lyxose, and L(-) lyxose), amino acids (such as glutamine, lysine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, leucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine, and taurine). Methods and substances for enhancing blood-brain barrier penetration are described, for example, in U.S. Patent Nos. 9,193,969, 4,866,042, 6,294,520, and 6,936,589, which are each incorporated herein by reference.

[0473] In some embodiments, the subject being treated using the methods and compositions is known in the art and is evaluated for improvement of the condition using any method known and described in the art.

[0474] The effects of the agents (e.g., antisense oligomers) provided herein on the level, activity or expression of UBE3A nucleic acid or UBE3A protein can be tested in vitro in various cell types. Exemplary cell types include, but are not limited to, Hela cells, HS02 cells, 293T cells, HepG2 cells, Hep3B cells, and primary hepatocytes.

[0475] In some embodiments, provided herein is a method of treating cells with an agent (e.g., an antisense oligomer). When the cells reach a confluence density of about 60-80% during culture, the cells can be treated with an agent (e.g., an antisense oligomer) or a vector encoding the agent.

[0476] For example, the antisense oligomer can be introduced into cultured cells with the aid of the cationic lipid transfection reagent LIPOFECTIN (Invitrogen, Carlsbad, CA). The antisense oligomer is mixed with LIPOFECTIN in OPTI-MEM 1 (Invitrogen, Carlsbad, CA) to achieve the desired final concentration of the antisense oligomer and a LIPOFECTIN concentration that can range from 2-12 μg / mL per 100 nM antisense oligonucleotide. The antisense oligomer can also be introduced into cultured cells with the aid of LIPOFECTAMINE (Invitrogen, Carlsbad, CA). The antisense oligomer is mixed with LIPOFECTAMINE in OPTI-MEM 1 reduced serum medium (Invitrogen, Carlsbad, CA) to achieve the desired final concentration of the antisense oligomer and a LIPOFECTAMINE concentration that can range from 2-12 μg / mL per 100 nM antisense oligonucleotide. Another reagent that can be used to introduce the antisense oligomer into cultured cells is TURBOFECT (Thermo Scientific, Carlsbad, CA).

[0477] In some cases, antisense oligomers are introduced into cultured cells by electroporation.

[0478] Antisense inhibition of UBE3A nucleic acid can be evaluated by measuring UBE3A protein level or UBE3A mRNA transcript level. The UBE3A mRNA transcript level can be measured by conventional techniques in the art such as real-time PCR. The protein level of UBE3A can be evaluated or quantified by various methods well-known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assay, protein activity assay (e.g., caspase activity assay), immunohistochemistry, immunocytochemistry, or fluorescence-activated cell sorting (FACS). Target-specific antibodies can be identified and obtained from various sources such as the MSRS antibody catalog (Aerie Corporation, Birmingham, MI), or can be prepared by conventional monoclonal or polyclonal antibody production methods well-known in the art.

[0479] In vivo testing of antisense oligomers Antisense oligomers, such as antisense oligonucleotides, are tested in animals to evaluate their ability to inhibit the expression of UBE3A and to bring about phenotypic changes such as improvement in behavior, motor function, and cognition. In certain embodiments, motor function is measured by analysis of gait initiation, rotarod, grip strength, pole climbing, open field performance, beam walk, and hindlimb footprint tests in animals. In certain embodiments, behavior is measured by elevated plus maze and three-chamber social interaction. The tests can be performed in normal animals or experimental models. After a period of treatment with antisense oligonucleotides, RNA can be isolated from CNS tissue or CSF, and changes in UBE3A nucleic acid expression can be measured.

[0480] Exemplary embodiments [1] A method for reducing the expression of UBE3A protein in mammalian cells having duplication, overexpression, or gain-of-function mutations of the UBE3A gene encoding the UBE3A protein, comprising contacting the mammalian cells with a drug or a vector encoding the drug, wherein the drug reduces the level of processed mRNA encoding the UBE3A protein in the mammalian cells.

[0481] [2] The method according to paragraph [1], wherein the drug comprises a polynucleotide sequence that is at least 80% complementary to at least 8 consecutive nucleic acids of the sequence set forth in any one of SEQ ID NOs: 93 to 120.

[0482] [3] The method according to paragraph [1], wherein the drug comprises a polynucleotide sequence that is at least 80% complementary to at least 8 consecutive nucleic acids of each mRNA transcript listed in Table 2.

[0483] [4] The method according to any one of paragraphs [1] to [3], wherein the drug comprises an antisense oligomer.

[0484] [5] The method according to paragraph [4], wherein the drug comprises an antisense oligomer having at least 80% identity with the sequence set forth in any one of SEQ ID NOs: 1 to 92.

[0485] [6] A method for regulating the expression of the UBE3A gene encoding the UBE3A protein in mammalian cells, comprising contacting the mammalian cells with a drug or a vector encoding the drug, wherein the drug comprises a polynucleotide sequence comprising an antisense oligomer having at least 80% identity with the sequence set forth in any one of SEQ ID NOs: 1 to 92.

[0486] [7] The method according to any one of paragraphs [4] to [6], wherein the antisense oligomer comprises a backbone modification, a modified sugar moiety, or a combination thereof.

[0487] [8] The method according to any one of paragraphs [4] to [7], wherein the antisense oligomer contains phosphorothioate bonds or phosphorodiamidate bonds.

[0488] [9] The method according to any one of paragraphs [4] to [8], wherein the antisense oligomer contains phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl moiety, 2'-fluoro moiety, 2'-O-methoxyethyl moiety, or 2'-NMA moiety.

[0489]

[10] The method according to any one of paragraphs [4] to [9], wherein the antisense oligomer contains at least one modified sugar moiety.

[0490]

[11] The method according to any one of paragraphs [4] to

[10] , wherein the antisense oligomer contains at least 1, 2, 3, 4, 5, or 6 modified nucleosides at the 5'-end of the antisense oligomer.

[0491]

[12] The method according to any one of paragraphs [4] to

[10] , wherein the antisense oligomer contains 1, 2, 3, 4, 5, or 6 modified nucleosides at the 5'-end of the antisense oligomer.

[0492]

[13] The method according to any one of paragraphs [4] to

[10] , wherein the antisense oligomer contains 1, 2, 3, 4, 5, or 6 2'-O-methoxyethyl modified nucleosides at the 5'-end of the antisense oligomer.

[0493]

[14] The method according to any one of paragraphs [4] to

[13] , wherein the antisense oligomer contains at least 1, 2, 3, 4, 5, or 6 modified nucleosides at the 3'-end of the antisense oligomer.

[0494]

[15] The method according to any one of paragraphs [4] to

[13] , wherein the antisense oligomer contains 1, 2, 3, 4, 5, or 6 modified nucleosides at the 3'-end of the antisense oligomer.

[0495]

[16] The method according to any one of paragraphs [4] to

[13] , wherein the antisense oligomer contains 1, 2, 3, 4, 5, or 6 2'-O-methoxyethyl-modified nucleosides at the 3'-end of the antisense oligomer.

[0496]

[17] The method according to any one of paragraphs [4] to

[16] , wherein the antisense oligomer contains 3, 4, 5, or 6 2'-O-methoxyethyl-modified nucleosides at the 5'-end of the antisense oligomer, contains 3, 4, 5, or 6 2'-O-methoxyethyl-modified nucleosides at the 3'-end of the antisense oligomer, and contains phosphorothioate bonds between any two adjacent nucleosides of the antisense oligomer.

[0497]

[18] The antisense oligomer is a 5'-region consisting of 3, 4, 5, or 6 linked nucleosides, a central region consisting of 8, 9, 10, 11, or 12 linked nucleosides, and a 3'-region consisting of 3, 4, 5, or 6 linked nucleosides, and each of the 3, 4, 5, or 6 linked nucleosides in the 5'-region and each of the 3, 4, 5, or 6 linked nucleosides in the 3'-region contain a modified sugar moiety, and each of the 8, 9, 10, 11, or 12 linked nucleosides in the central region is a deoxyribonucleoside, the method according to any one of paragraphs [4] to

[16] .

[0498]

[19] The method according to any one of paragraphs [4] to

[18] , wherein the antisense oligomer consists of 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, 12 to 15 nucleobases, 15 to 50 nucleobases, 15 to 40 nucleobases, 15 to 35 nucleobases, 15 to 30 nucleobases, 15 to 25 nucleobases, 15 to 20 nucleobases, 15 to 19 nucleobases, 15 to 18 nucleobases, 15 to 16 nucleobases, 16 to 20 nucleobases, 16 to 19 nucleobases, 16 to 18 nucleobases, 17 to 20 nucleobases, 17 to 19 nucleobases, or 18 to 20 nucleobases.

[0499]

[20] The method according to any one of paragraphs [4] to

[16] , wherein the antisense oligomer is a modified oligonucleotide comprising the sequence set forth in any one of SEQ ID NOs: 154 to 189 or 192 to 247.

[0500]

[21] The method according to any one of paragraphs [4] to

[16] , wherein the antisense oligomer is a modified oligonucleotide consisting of the sequence set forth in any one of SEQ ID NOs: 154 to 189 or 192 to 247.

[0501]

[22] The method according to any one of paragraphs [1] to [6], wherein the vector comprises a viral vector encoding the agent.

[0502]

[23] The method according to paragraph

[22] , wherein the viral vector comprises an adenovirus vector, an adeno-associated virus (AAV) vector, a lentivirus vector, a herpes simplex virus (HSV) vector, or a retrovirus vector.

[0503]

[24] The method according to any one of paragraphs [4] to

[23] , wherein the antisense oligomer comprises a sequence having at least 90% identity with the sequence set forth in any one of SEQ ID NOs: 1 to 92.

[0504]

[25] The method according to any one of paragraphs [4] to

[23] , wherein the antisense oligomer comprises a sequence having 100% identity with the sequence set forth in any one of SEQ ID NOs: 1 to 92.

[0505]

[26] The method according to any one of paragraphs [4] to

[23] , wherein the antisense oligomer consists of a sequence having at least 80% identity with the sequence set forth in any one of SEQ ID NOs: 1 to 92.

[0506]

[27] The method according to any one of paragraphs [4] to

[23] , wherein the antisense oligomer consists of a sequence having at least 90% identity with the sequence set forth in any one of SEQ ID NOs: 1 to 92.

[0507]

[28] The method according to any one of paragraphs [6] to

[28] , wherein the antisense oligomer consists of a sequence having 100% identity with the sequence set forth in any one of SEQ ID NOs: 1 to 92.

[0508]

[29] The method according to any one of paragraphs [6] to

[28] , wherein the method reduces the level of processed mRNA encoding the UBE3A protein in the mammalian cell.

[0509]

[30] The level of the processed mRNA encoding the UBE3A protein in the mammalian cells contacted with the agent or the vector is about 10% to about 99%, about 10% to about 95%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, about 50% to about 99%, about 50% to about 95%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 99%, about 60% to about 95%, about 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 70% to about 99%, about 70% to about 95%, about 70% to about 90%, about 70% to about 80%, about 80% to about 99%, about 80% to about 95%, about 80% to about 90%, about 90% to about 99%, about 90% to about 95%, or about 95% to about 99% lower as compared to cells that are not contacted with the agent or the vector but are otherwise the same. The method according to any one of paragraphs [1] to [5] or paragraphs [7] to

[29] .

[0510]

[31] The level of the processed mRNA encoding the UBE3A protein in the mammalian cells contacted with the agent or the vector is at least about 10%, 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%, or at least about 99% lower as compared to mammalian cells that are not contacted with the agent or the vector but are otherwise the same. The method according to any one of paragraphs [1] to [5] or paragraphs [7] to

[29] .

[0511]

[32] The method according to any one of paragraphs [1] to

[31] , wherein the method reduces the level of the UBE3A protein in the mammalian cell.

[0512]

[33] The level of the UBE3A protein in the mammalian cell contacted with the agent or the vector is about 10% to about 99%, about 10% to about 95%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, about 50% to about 99%, about 50% to about 95%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 99%, about 60% to about 95%, about 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 70% to about 99%, about 70% to about 95%, about 70% to about 90%, about 70% to about 80%, about 80% to about 99%, about 80% to about 95%, about 80% to about 90%, about 90% to about 99%, about 90% to about 95%, or about 95% to about 99% lower as compared to a cell that is not contacted with the agent or the vector but is otherwise the same. The method according to paragraph

[32] .

[0513]

[34] The method according to paragraph

[32] , wherein the level of the UBE3A protein in the mammalian cell contacted with the agent or the vector is at least about 10%, 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%, or at least about 99% lower compared to cells that are not contacted with the agent or the vector but are otherwise the same.

[0514]

[35] The method according to any one of paragraphs [1] to

[34] , wherein the method comprises contacting the agent or the vector with a population of mammalian cells.

[0515]

[36] The method according to paragraph

[35] , wherein the agent reduces the level of the processed mRNA encoding the UBE3A protein in the population of mammalian cells by up to about 75%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, or up to about 20% compared to a population of mammalian cells that are not contacted with the agent or the vector but are otherwise the same.

[0516]

[37] The method according to paragraph

[35] or

[36] , wherein the agent reduces the level of the UBE3A protein in the population of mammalian cells by up to about 75%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, or up to about 20% compared to a population of cells that are not contacted with the agent or the vector but are otherwise the same.

[0517]

[38] The method according to any one of paragraphs [1] to

[37] , wherein the mammalian cells are ex vivo.

[0518]

[39] The method according to any one of paragraphs [1] to

[37] , wherein the mammalian cells are in vivo.

[0519]

[40] The method according to any one of paragraphs [1] to

[39] , wherein the genome of the mammalian cell has a duplication of a genomic region containing the UBE3A gene encoding the UBE3A protein.

[0520]

[41] The method according to any one of paragraphs [1] to

[39] , wherein the mammalian cell is a human cell and the genome of the mammalian cell has a duplication of chromosome 15q11.2 - q13.1.

[0521]

[42] The method according to any one of paragraphs [1] to

[38] , wherein the mammalian cell is obtained from a human subject suffering from Dup15q syndrome or is a descendant of sample cells obtained from the human subject.

[0522]

[43] An antisense oligomer comprising a sequence having at least 80% identity with the sequence set forth in any one of SEQ ID NOs: 1 - 92.

[0523]

[44] The antisense oligomer according to paragraph

[43] , wherein the antisense oligomer comprises a backbone modification, a modified sugar moiety, or a combination thereof.

[0524]

[45] The antisense oligomer according to paragraph

[43] or

[44] , wherein the antisense oligomer comprises a phosphorothioate bond or a phosphorodiamidate bond.

[0525]

[46] The antisense oligomer according to any one of paragraphs

[43] to

[45] , wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl moiety, a 2'-fluoro moiety, a 2'-O-methoxyethyl moiety, or a 2'-NMA moiety.

[0526]

[47] The antisense oligomer according to any one of paragraphs

[43] to

[46] , wherein the antisense oligomer comprises at least one modified sugar moiety.

[0527]

[48] The antisense oligomer according to any one of paragraphs

[43] to

[47] , wherein the antisense oligomer contains at least 1, 2, 3, 4, 5, or 6 modified nucleosides at the 5'-end of the antisense oligomer.

[0528]

[49] The antisense oligomer according to any one of paragraphs

[43] to

[47] , wherein the antisense oligomer contains 1, 2, 3, 4, 5, or 6 modified nucleosides at the 5'-end of the antisense oligomer.

[0529]

[50] The antisense oligomer according to any one of paragraphs

[43] to

[47] , wherein the antisense oligomer contains 1, 2, 3, 4, 5, or 6 2'-O-methoxyethyl modified nucleosides at the 5'-end of the antisense oligomer.

[0530]

[51] The antisense oligomer according to any one of paragraphs

[43] to

[50] , wherein the antisense oligomer contains at least 1, 2, 3, 4, 5, or 6 modified nucleosides at the 3'-end of the antisense oligomer.

[0531]

[52] The antisense oligomer according to any one of paragraphs

[43] to

[50] , wherein the antisense oligomer contains 1, 2, 3, 4, 5, or 6 modified nucleosides at the 3'-end of the antisense oligomer.

[0532]

[53] The antisense oligomer according to any one of paragraphs

[43] to

[50] , wherein the antisense oligomer contains 1, 2, 3, 4, 5, or 6 2'-O-methoxyethyl modified nucleosides at the 3'-end of the antisense oligomer.

[0533]

[54] The antisense oligomer according to any one of paragraphs

[43] to

[53] , wherein the antisense oligomer contains 3, 4, 5, or 6 2'-O-methoxyethyl-modified nucleosides at the 5'-end of the antisense oligomer, contains 3, 4, 5, or 6 2'-O-methoxyethyl-modified nucleosides at the 3'-end of the antisense oligomer, and contains phosphorothioate bonds between any two adjacent nucleosides of the antisense oligomer.

[0534]

[55] The antisense oligomer comprises a 5'-region consisting of 3, 4, 5, or 6 linked nucleosides, a central region consisting of 8, 9, 10, 11, or 12 linked nucleosides, and a 3'-region consisting of 3, 4, 5, or 6 linked nucleosides, and each of the 3, 4, 5, or 6 linked nucleosides in the 5'-region and each of the 3, 4, 5, or 6 linked nucleosides in the 3'-region contains a modified sugar moiety, and each of the 8, 9, 10, 11, or 12 linked nucleosides in the central region is a deoxyribonucleoside, the antisense oligomer according to any one of paragraphs

[43] to

[53] .

[0535]

[56] The antisense oligomer consists of 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, 12 to 15 nucleobases, 15 to 50 nucleobases, 15 to 40 nucleobases, 15 to 35 nucleobases, 15 to 30 nucleobases, 15 to 25 nucleobases, 15 to 20 nucleobases, 15 to 19 nucleobases, 15 to 18 nucleobases, 15 to 16 nucleobases, 16 to 20 nucleobases, 16 to 19 nucleobases, 16 to 18 nucleobases, 17 to 20 nucleobases, 17 to 19 nucleobases, or 18 to 20 nucleobases, and is the antisense oligomer according to any one of paragraphs

[43] to

[55] .

[0536]

[57] The antisense oligomer contains a sequence having at least 90% identity with the sequence described in any one of SEQ ID NOs: 1 to 92, and is the antisense oligomer according to any one of paragraphs

[43] to

[56] .

[0537]

[58] The antisense oligomer contains a sequence having 100% identity with the sequence described in any one of SEQ ID NOs: 1 to 92, and is the antisense oligomer according to any one of paragraphs

[43] to

[56] .

[0538]

[59] The antisense oligomer consists of a sequence having at least 80% identity with the sequence described in any one of SEQ ID NOs: 1 to 92, and is the antisense oligomer according to any one of paragraphs

[43] to

[56] .

[0539]

[60] The antisense oligomer according to any one of paragraphs

[43] to

[56] , comprising a sequence having at least 90% identity with the sequence set forth in any one of SEQ ID NOs: 1 to 92.

[0540]

[61] The antisense oligomer according to any one of paragraphs

[43] to

[56] , comprising a sequence having 100% identity with the sequence set forth in any one of SEQ ID NOs: 1 to 92.

[0541]

[62] The antisense oligomer according to any one of paragraphs

[43] to

[56] , which is a modified oligonucleotide containing the sequence set forth in any one of SEQ ID NOs: 154 to 189 or 192 to 247.

[0542]

[63] The antisense oligomer according to any one of paragraphs

[43] to

[56] , which is a modified oligonucleotide consisting of the sequence set forth in any one of SEQ ID NOs: 154 to 189 or 192 to 247.

[0543]

[64] The antisense oligomer according to any one of paragraphs

[43] to

[63] , which is configured to reduce the level of processed mRNA transcripts encoding the UBE3A protein in a population of mammalian cells when contacted with the population.

[0544]

[65] The antisense oligomer is configured to reduce the level of the processed mRNA encoding the UBE3A protein in the population by about 10% to about 99%, about 10% to about 95%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, about 50% to about 99%, about 50% to about 95%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 99%, about 60% to about 95%, about 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 70% to about 99%, about 70% to about 95%, about 70% to about 90%, about 70% to about 80%, about 80% to about 99%, about 80% to about 95%, about 80% to about 90%, about 90% to about 99%, about 90% to about 95%, or about 95% to about 99% compared to a population of mammalian cells that is otherwise the same but not in contact with the antisense oligomer. The antisense oligomer described in paragraph

[64] .

[0545]

[66] The antisense oligomer is configured to reduce the level of the processed mRNA encoding the UBE3A protein in the population by at least about 10%, 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%, or at least about 99% compared to a population of mammalian cells that is otherwise the same but not in contact with the antisense oligomer. The antisense oligomer described in paragraph

[64] .

[0546]

[67] The antisense oligomer is configured to reduce the level of the processed mRNA encoding the UBE3A protein in the population by up to about 75%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, or up to about 20% compared to a population of mammalian cells that is otherwise the same but has not been contacted with the antisense oligomer. The antisense oligomer according to any one of paragraphs

[64] to

[66] .

[0547]

[68] The antisense oligomer is configured to reduce the level of the UBE3A protein in the population of mammalian cells. The antisense oligomer according to any one of paragraphs

[64] to

[67] .

[0548]

[69] The antisense oligomer is configured to reduce the level of the UBE3A protein in the population by about 10% to about 99%, about 10% to about 95%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, about 50% to about 99%, about 50% to about 95%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 99%, about 60% to about 95%, about 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 70% to about 99%, about 70% to about 95%, about 70% to about 90%, about 70% to about 80%, about 80% to about 99%, about 80% to about 95%, about 80% to about 90%, about 90% to about 99%, about 90% to about 95%, or about 95% to about 99% compared to a population of mammalian cells that is otherwise the same but has not been contacted with the antisense oligomer. The antisense oligomer according to paragraph

[68] .

[0549]

[70] The antisense oligomer is configured to reduce the level of the UBE3A protein in the population by at least about 10%, 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%, or at least about 99% as compared to a population of mammalian cells that is otherwise the same but has not been contacted with the antisense oligomer. The antisense oligomer according to paragraph

[68] .

[0550]

[71] The antisense oligomer is configured to reduce the level of the UBE3A protein in the population by up to about 75%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, or up to about 20% as compared to a population of mammalian cells that is otherwise the same but has not been contacted with the antisense oligomer. The antisense oligomer according to any one of paragraphs

[64] to

[70] .

[0551]

[72] The mammalian cells are ex vivo. The antisense oligomer according to any one of paragraphs

[64] to

[71] .

[0552]

[73] The mammalian cells are in vivo. The antisense oligomer according to any one of paragraphs

[64] to

[71] .

[0553]

[74] The genome of the mammalian cells has a duplication of a genomic region containing the UBE3A gene that encodes the UBE3A protein. The antisense oligomer according to any one of paragraphs

[64] to

[73] .

[0554]

[75] The antisense oligomer according to any one of paragraphs

[64] to

[74] , wherein the mammalian cell is a human cell.

[0555]

[76] The antisense oligomer according to paragraph

[75] , wherein the genome of the mammalian cell has a duplication in chromosome 15q11.2 to q13.1.

[0556]

[77] The antisense oligomer according to any one of paragraphs

[64] to

[76] , wherein the mammalian cell is obtained from a human subject suffering from Dup15q syndrome or is a descendant of sample cells obtained from the human subject.

[0557]

[78] (a) A pharmaceutically acceptable excipient or carrier, and (b) The antisense oligomer according to any one of paragraphs

[43] to

[77] , a pharmaceutical composition comprising.

[0558]

[79] (a) A pharmaceutically acceptable excipient or carrier, and (b) A pharmaceutical composition comprising an agent configured to reduce the level of processed mRNA transcript encoding the UBE3A protein in the mammalian cell when contacted with the mammalian cell or a vector encoding the agent.

[0559]

[80] The pharmaceutical composition according to paragraph

[79] , wherein the agent comprises a polynucleotide sequence that is at least 80% complementary to at least 8 consecutive nucleic acids of the sequence set forth in any one of SEQ ID NOs: 93 to 120.

[0560]

[81] The pharmaceutical composition according to paragraph

[79] , wherein the agent comprises a polynucleotide sequence that is at least 80% complementary to at least 8 consecutive nucleic acids of each mRNA transcript listed in Table 2.

[0561]

[82] The method according to any one of paragraphs

[79] to

[81] , wherein the agent comprises an antisense oligomer.

[0562]

[83] The pharmaceutical composition according to paragraph

[82] , wherein the antisense oligomer has at least 80% identity with the sequence described in any one of SEQ ID NOs: 1 to 92.

[0563]

[84] The pharmaceutical composition according to any one of paragraphs

[79] to

[83] , comprising the vector, wherein the vector comprises a viral vector encoding the agent.

[0564]

[85] The pharmaceutical composition according to paragraph

[84] , wherein the viral vector comprises an adenovirus vector, an adeno-associated virus (AAV) vector, a lentivirus vector, a herpes simplex virus (HSV) vector, or a retrovirus vector.

[0565]

[86] The pharmaceutical composition according to any one of paragraphs

[82] to

[85] , wherein the antisense oligomer comprises a sequence having at least 90% identity with the sequence described in any one of SEQ ID NOs: 1 to 92.

[0566]

[87] The pharmaceutical composition according to any one of paragraphs

[82] to

[85] , wherein the antisense oligomer comprises a sequence having 100% identity with the sequence described in any one of SEQ ID NOs: 1 to 92.

[0567]

[88] The pharmaceutical composition according to any one of paragraphs

[82] to

[85] , wherein the antisense oligomer consists of a sequence having at least 80% identity with the sequence described in any one of SEQ ID NOs: 1 to 92.

[0568]

[89] The pharmaceutical composition according to any one of paragraphs

[82] to

[85] , wherein the antisense oligomer consists of a sequence having at least 90% identity with the sequence described in any one of SEQ ID NOs: 1 to 92.

[0569]

[90] The pharmaceutical composition according to any one of paragraphs

[82] to

[85] , wherein the antisense oligomer consists of a sequence having 100% identity with the sequence set forth in any one of SEQ ID NOs: 1 to 92.

[0570]

[91] The pharmaceutical composition according to paragraph

[82] , wherein the antisense oligomer is a modified oligonucleotide comprising the sequence set forth in any one of SEQ ID NOs: 154 to 189 or 192 to 247.

[0571]

[92] The pharmaceutical composition according to paragraph

[82] , wherein the antisense oligomer is a modified oligonucleotide consisting of the sequence set forth in any one of SEQ ID NOs: 154 to 189 or 192 to 247.

[0572]

[93] The pharmaceutical composition according to any one of paragraphs

[79] to

[92] , wherein the agent is configured to reduce the level of the processed mRNA encoding the UBE3A protein in the population when contacted with the population of mammalian cells.

[0573]

[94] The pharmaceutical composition according to paragraph

[93] , wherein the antisense oligomer is configured to reduce the level of the processed mRNA encoding the UBE3A protein in the population by about 10% to about 99%, about 10% to about 95%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, about 50% to about 99%, about 50% to about 95%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 99%, about 60% to about 95%, about 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 70% to about 99%, about 70% to about 95%, about 70% to about 90%, about 70% to about 80%, about 80% to about 99%, about 80% to about 95%, about 80% to about 90%, about 90% to about 99%, about 90% to about 95%, or about 95% to about 99% compared to a population of mammalian cells that are otherwise the same but not in contact with the agent or the vector.

[0574]

[95] The pharmaceutical composition according to paragraph

[93] , wherein the agent is configured to reduce the level of the processed mRNA encoding the UBE3A protein in the population by at least about 10%, 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%, or at least about 99% compared to a population of mammalian cells that are otherwise the same but not in contact with the agent or the vector.

[0575]

[96] The pharmaceutical composition according to any one of paragraphs

[93] to

[95] , wherein the agent is configured to reduce the level of the processed mRNA encoding the UBE3A protein in the population by up to about 75%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, or up to about 20% compared to a population of mammalian cells that is otherwise the same but has not been contacted with the antisense oligomer.

[0576]

[97] The pharmaceutical composition according to any one of paragraphs

[93] to

[96] , wherein the agent is configured to reduce the level of the UBE3A protein in the population.

[0577]

[98] The pharmaceutical composition according to paragraph

[97] , wherein the agent is configured to reduce the level of the UBE3A protein in the population by about 10% to about 99%, about 10% to about 95%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, about 50% to about 99%, about 50% to about 95%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 99%, about 60% to about 95%, about 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 70% to about 99%, about 70% to about 95%, about 70% to about 90%, about 70% to about 80%, about 80% to about 99%, about 80% to about 95%, about 80% to about 90%, about 90% to about 99%, about 90% to about 95%, or about 95% to about 99% compared to a population of mammalian cells that is otherwise the same but has not been contacted with the agent or the vector.

[0578]

[99] The pharmaceutical composition according to paragraph

[97] , wherein the agent is configured to reduce the level of the UBE3A protein in the population by at least about 10%, 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%, or at least about 99% as compared to a population of mammalian cells that is otherwise the same but has not been contacted with the agent or the vector.

[0579]

[0100] The pharmaceutical composition according to any one of paragraphs

[97] to

[99] , wherein the agent is configured to reduce the level of the UBE3A protein in the population by up to about 75%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, or up to about 20% as compared to a population of mammalian cells that is otherwise the same but has not been contacted with the antisense oligomer.

[0580]

[0101] The pharmaceutical composition according to any one of paragraphs

[79] to

[0100] , wherein the mammalian cells are ex vivo.

[0581]

[0102] The pharmaceutical composition according to any one of paragraphs

[79] to

[0100] , wherein the mammalian cells are in vivo.

[0582]

[0103] The pharmaceutical composition according to any one of paragraphs

[79] to

[0102] , wherein the genome of the mammalian cells has a duplication of a genomic region containing the UBE3A gene encoding the UBE3A protein.

[0583]

[0104] The pharmaceutical composition according to any one of paragraphs

[79] to

[0103] , wherein the mammalian cells are human cells.

[0584] The pharmaceutical composition according to paragraph

[0104] , wherein the genome of the mammalian cell has a duplication of chromosome 15q11.2-q13.1.

[0585] The pharmaceutical composition according to any one of paragraphs

[79] to

[99] , wherein the mammalian cell is obtained from a human subject suffering from Dup15q syndrome or is a descendant of sample cells obtained from the human subject.

[0586] The pharmaceutical composition according to any one of paragraphs

[78] to

[0106] , wherein the pharmaceutical composition is formulated for intracerebroventricular injection, intraperitoneal injection, intramuscular injection, intrathecal injection, intracisternal injection, subcutaneous injection, oral administration, synovial injection, intravitreal administration, subretinal injection, topical application, transplantation, or intravenous injection.

[0587] The pharmaceutical composition according to any one of paragraphs

[78] to

[0106] , wherein the pharmaceutical composition is formulated for intrathecal injection.

[0588] The pharmaceutical composition according to any one of paragraphs

[78] to

[0108] , wherein the pharmaceutically acceptable excipient or carrier contains artificial cerebrospinal fluid.

[0589] The pharmaceutical composition according to any one of paragraphs

[78] to

[0109] , wherein the pharmaceutical composition further comprises a second therapeutic agent.

[0590] The pharmaceutical composition according to paragraph

[0110] , wherein the second therapeutic agent comprises a small molecule, an antisense oligomer, or a gene editing molecule.

[0591] A method for treating a disease or condition of a subject or reducing the likelihood of onset thereof by reducing the expression of UBE3A protein in cells of the subject in need thereof, the method comprising contacting the cells of the subject with the pharmaceutical composition according to any one of paragraphs 78 to 111.

[0592]

[0113] The method according to paragraph

[0112] , wherein the disease or condition is related to overexpression of the UBE3A gene encoding the UBE3A protein or a gain-of-function mutation.

[0593]

[0114] The method according to paragraph

[0112] or

[0113] , wherein the genome of the cells of the subject has at least one extra copy of the UBE3A gene encoding the UBE3A protein.

[0594]

[0115] The method according to paragraph

[0112] or

[0114] , wherein the genome of the cells of the subject has a duplication of a genomic region containing the UBE3A gene encoding the UBE3A protein.

[0595]

[0116] The method according to paragraph

[0112] or

[0115] , wherein the genome of the cells of the subject has a duplication of chromosome 15q11.2 - q13.1.

[0596]

[0117] The method according to any one of paragraphs

[0112] to

[0116] , wherein the disease or condition includes Dup15q syndrome, autism spectrum disorder, epilepsy, or intellectual disability.

[0597]

[0118] The method according to any one of paragraphs

[0112] to

[0117] , wherein the subject is a human.

[0598]

[0119] The method according to any one of paragraphs

[0112] to

[0118] , wherein the subject is a fetus, embryo, or pediatric patient.

[0599]

[0120] The method according to any one of paragraphs

[0112] to

[0119] , wherein the cells are ex vivo.

[0600] The method according to any one of paragraphs

[0112] to

[0119] , comprising administering the pharmaceutical composition to the subject by intracerebroventricular injection, intraperitoneal injection, intramuscular injection, intrathecal injection, intracisternal injection, subcutaneous injection, oral administration, synovial injection, intravitreal administration, subretinal injection, topical application, transplantation, or intravenous injection.

[0601] The method according to any one of paragraphs

[0112] to

[0119] , comprising administering the pharmaceutical composition to the subject by intrathecal injection.

[0602] The method according to any one of paragraphs

[0112] to

[0122] , wherein the method treats the disease or condition.

Examples

[0603] The present disclosure is more specifically described by the following examples. However, it should be understood that the present disclosure is not limited in any way by these examples.

[0604] Example 1: Knockdown of the UBE3A gene in human cells. In one experiment, the effect of the compound molecules according to some embodiments of the present disclosure on human UBE3A mRNA transcripts was examined. The compound molecules are the modified oligonucleotides listed in Table 3. Among the compound molecules examined, there were 22 test oligonucleotides according to some embodiments of the present disclosure, one template-free control (NTC) that functions as a negative control ("Ncontrol"), and one positive control oligonucleotide ("Pcontrol") whose knockdown effect on the UBE3A gene is known. As shown in the table, " / 52MOErX / " represents a 2-O-methoxyethyl-modified ribonucleoside X at the 5'-end of the oligonucleotide, where X is A, T, G, or methylated cytosine (MeC), " / i2MOErX / " represents an internal 2-O-methoxyethyl-modified ribonucleoside X, where X is A, T, G, or MeC, " / 32MOErX / " represents a 2-O-methoxyethyl-modified ribonucleoside X at the 3'-end of the oligonucleotide, where X is A, T, G, or MeC, and the other nucleosides A, T, G, and C are deoxyribonucleosides, and "*" represents a phosphorothioate bond between two adjacent nucleosides.

[0605]

Table 3-1

[0606]

Table 3-2

[0607]

Table 3-3

[0608]

Table 3-4

[0609]

Table 3-5

[0610]

Table 4-1

[0611]

Table 4-2

[0612]

Table 4-3

[0613]

Table 4-4

[0614]

Table 4-5

[0615]

Table 4-6

[0616]

Table 4-7

[0617] Experiments were conducted using HS02 (primary human fibroblasts) in a 96-well plate. 8000 cells / well were seeded in the 96-well plate. Twenty-four ASOs targeting UBE3A (22 exemplary ASOs + 2 controls) were tested in an 8-point concentration-response curve (CRC) format, for example, eight different concentrations of each ASO were tested, and the response to ASO treatment was measured and plotted against the different concentrations. The two control ASOs included a 100 nM positive control ("Pcontrol") to evaluate the success of ASO delivery and a 100 nM negative control ("Ncontrol") which is an ASO targeting a different gene, TUG1. For each cell plate, a well for receiving only the transfection reagent ("TF only") was also secured as another negative control for quality control (QC). All ASOs were delivered by Lipofectamine 2000 transfection (0.2 μl / well). The 22 exemplary ASOs were added to the cell medium at concentrations of 100 nM, 31.62 nM, 10 nM, 3.16 nM, 1.0 nM, 0.32 nM, 0.10 nM, or 0.032 nM.

[0618] Each ASO source plate was tested on duplicate cell plates in the same experiment. Lysates from each cell well tested in duplicate were measured on the same qPCR plate.

[0619] After treating each cell well with the respective ASO for 24 hours, nuclear counting and potency measurement were performed. To count the number of nuclei of ASO-treated cells, 20 μl / well of Hoechst 33342 (50 μg / mL, nuclear stain) was added for 30 minutes. After staining, the cells were imaged by live imaging using a 10x objective lens on an IN Cell Analyzer 2200 (GE Healthcare), 4 fields of view were captured per well, and automated analysis of the captured images was performed using Columbus software (PerkinElmer).

[0620] After imaging, cells were lysed using a lysis solution containing DNAseI (25 μl / well) and stop solution (2.5 μl / well) from the TaqMan one-step Cells-to-Ct kit. Next, qPCR assays were performed on the cell lysates. 15% of the cell lysate from each well was added to a qPCR reaction using TaqMan Master Mix from the TaqMan one-step Cells-to-Ct kit.

[0621] In the TaqMan assay, Hs UBE3A assay 3 (FAM) was multiplexed with Hs HPRT1 (VIC). When the TaqMan qPCT assay was completed, -ΔΔCt the 2−ΔΔCt method was used to calculate the amount of UBE3A transcript, normalized to the measurement of the internal reference HPRT1, and then the change in gene expression was measured compared to the control condition (100% expression).

[0622] Cell loss under the lipofectamine-only condition (TF only, orange) was less than 0%, indicating some toxicity under the condition transfected with ASO. To account for these differences, the data were normalized to the TUG1 ASO negative CTRL for each plate. Cell loss was less than 20% for all test ASOs except for four wells, indicating low overall toxicity.

[0623] Figures 1A - 1D show the CRC of each tested ASO, including 22 exemplary ASOs, and the potency (percent knockdown of UBE3A mRNA transcript) of Pcontrol and Ncontrol.

[0624] Table 5 summarizes the average UBE3A knockdown rate and the respective EC50 of each of the tested ASOs, including Pcontrol and Ncontrol. The EC50 (half maximal effective concentration) was calculated using non-linear regression (4-parameter) analysis. There were several (8 cases) ASOs for which the EC50 value could not be calculated because the CRC did not result in an S-curve. They are indicated as n.d. (not determined) in the table.

[0625]

Table 5

[0626] Example 2: In vivo ASO tolerance of antisense oligonucleotides (ASO) by intracerebroventricular (ICV) administration. This example shows experiments conducted to test the in vivo tolerance of specific exemplary ASOs of the present disclosure in mice.

[0627] The effects of the exemplary ASO were examined by an in vivo experiment with intracerebroventricular (ICV) administration to mice. Adult C57BL / 6 mice were surgically implanted with ICV cannulas. After recovery from the surgery, on day 0, the mice were given an ICV injection of ASO or Dulbecco's phosphate buffered saline (DPBS). Observations were made before administration, and at 1 hour, 2 hours, 4 hours, 24 hours, and 7 days after administration. The animals were sacrificed 2 weeks after administration. Terminal plasma, brain, liver, heart, and kidney tissues were collected and stored.

[0628] In this study, adult male C57Bl / 6 mice were used. Upon arrival, the animals were placed individually in polycarbonate cages and acclimated for at least 4 days before the start of the study. The animals were housed on a 12 / 12 hour light / dark cycle in conditions maintaining room temperature (22 ± 2 °C) and humidity (about 50%). The animals had free access to food and water. The experiments were conducted according to a protocol approved by the Institutional Animal Care and Use Committee of Charles River Laboratories South San Francisco.

[0629] The dosing formulations were prepared at appropriate concentrations to meet the target dose levels (e.g., 400 μg / mouse, 500 μg / mouse) for each mouse. All ASOs were formulated in DPBS and stored at -80 °C until the day of administration. Each animal test condition included 6 animals, and except for groups 4, 14, and 25 that were excluded from the study due to solubility issues, a single-dose intracerebroventricular injection of 5 μl of the test sample was administered to each animal according to the grouping shown in Table 6. Group 1 was a control condition receiving only DPBS, and group 2 was a positive control receiving tominersen, a known antisense oligonucleotide therapeutic agent for reducing the expression levels of huntingtin protein (HTT) and mutant huntingtin protein (mHTT). Groups 3 - 26 were experimental conditions using the designated ASOs.

[0630]

Table 6-1

[0631]

Table 6-2

[0632] Surgical treatment ICV cannula insertion surgery The mice were anesthetized using isoflurane (2%, 800 ml / min O2). Bupivacaine was used for local analgesia and carprofen was used for perioperative / postoperative analgesia. The animals were placed in a stereotaxic frame (KOPF® Laboratory Instruments, USA). The surgery was performed using aseptic techniques. The anteroposterior axis (AP), mediolateral axis (ML), and dorsoventral axis (DV) were set to zero at bregma. The following coordinates were used for the ICV injection cannula (PLASTICS ONE®, Roanoke, Virginia): AP -0.3 mm from the dura, lateral -1.0, DV -2.2 mm. The position of the drill hole was specified and the hole was drilled. The drill was only used to penetrate the bone and the injection cannula was lowered into the lateral ventricle. Two screws and dental acrylic were used to fix the ICV cannula. After surgery, the animals were given food and water ad libitum. Any animals showing abnormal or adverse signs were not used in the study.

[0633] Preoperative and postoperative care Carprofen (5 mg / kg SC) was administered before surgery and carprofen (0.067 mg / ml) was added to the drinking water and given to the animals for 3 days after surgery. The animals were carefully monitored once a day on the day of surgery and for an additional 3 days after surgery.

[0634] ICV injection (day 0): The animals were administered according to the conditions shown in Table 7 for at least 7 days after surgery. The animals were administered in a conscious state. The injection cannula was inserted into the ICV cannula and the test substance was injected at a rate of 0.08 μl / sec. The injection cannula was left in the guide cannula for an additional 30 seconds before removing and replacing the dummy stylet.

[0635]

Table 7

[0636] Behavior Observation On the administration day, the animals were observed and scored according to Table 8 before administration, 1 hour, 2 hours, 4 hours, 24 hours, and 1 week after administration. Detailed clinical observations including walking and other hind limb defects were recorded. Adverse events included ataxia, seizures, changes in walking or coordinated movement, and weight loss (more than 15%).

[0637]

Table 8

[0638] Post - administration Humane Endpoint After administration, if the animals met the criteria shown in Table 9, they were euthanized before the scheduled euthanasia time point.

[0639]

Table 9

[0640] Endpoint Sampling Scheduled Euthanasia and Sample Collection Two weeks after administration, the animals were deeply anesthetized with isoflurane. Next, blood was collected, and after the animals were perfused with PBS transcardially, tissues were collected.

[0641] Early Euthanasia and Sample Collection If it was necessary to euthanize the animals before the scheduled euthanasia time point, tissues were collected. Tissues were not collected from animals found dead (FD).

[0642] Statistical Analysis and Graphical Representation of Data Data was plotted using Prism 9 for Windows (GraphPad Software, Inc.). Mixed-effect analysis was used to evaluate body weight over time between groups. Tukey's post hoc test was applied for comparison of individual groups. Statistical significance was set at p < 0.05.

[0643] In vivo results Three ASOs (Compound_22, Compound_32, Compound_21) did not dissolve during formulation. Therefore, these three treatment groups were not dosed as planned. The other six ASOs (Tomisen, Compound_23, Compound_25, Compound_26, Compound_12, and Compound_89) were soluble only at 80 mg / ml. Therefore, animals in these six treatment groups were dosed with 400 μg of ASO per mouse instead of 500 μg of ASO per mouse as planned. All other ASO administrations were as planned.

[0644] Table 10 shows the number of animals that reached the criteria for early euthanasia (early takedown, or eTD) after dosing, or animals found dead (FD) in each treatment group.

[0645] Table 11 shows behavioral observations at baseline 1 hour, 2 hours, 4 hours, 24 hours, and 1 week after dosing. Abbreviations for the behavioral scoring criteria can be found in Table 8. Items marked with an asterisk indicate that the animal was found dead 1 week later.

[0646] [Table 10]

[0647] [Table 11-1]

[0648] [Table 11-2]

[0649]

Table 11-3

[0650]

Table 11-4

[0651]

Table 11-5

[0652]

Table 11-6

[0653]

Table 11-7

[0654]

Table 11-8

[0655]

Table 11-9

[0656]

Table 11-10

[0657]

Table 11-11

[0658]

Table 11-12

[0659]

Table 11-13

[0660] Example 3: UBE3A knockdown by ASO at the mRNA and protein levels in F-Dup neurons This example shows an experiment conducted to test the effect of certain exemplary ASOs of the present disclosure on UBE3A mRNA levels and protein expression levels in neuronal cells derived from human induced pluripotent stem cells (iPSCs) obtained by dedifferentiating cells from Dup15q patients (F-Dup neurons).

[0661] After treating neuronal cells with 10 exemplary ASOs of the present invention, UBE3A knockdown was evaluated at the mRNA and protein levels. Human induced pluripotent stem cell-derived (hiPSC-derived) F-Dup neurons and corrected neurons (neuronal cells derived from human iPSCs that are from Dup15q patients but genetically “corrected” to remove the extra chromosome) were seeded in 96-well plates at 150,000 cells / well. POS1 and POS2 targeting UBE3A were used as positive controls for qPCR. Scr GFP_ASO, scr P1, and NEAT1 ASO were used as negative controls in F-Dup and corrected untreated cells. scr Pq was used as a negative control for qPCR only.

[0662] The ASOs were delivered by gymnosis. Control ASOs were used at a concentration of 5 μM for qPCR. Test ASOs and control ASOs were delivered at concentrations of approximately 6.33 μM and 20 μM. Each ASO plate source was tested in duplicate on the same cell plate, and lysates from each cell well were tested in technical duplicates on the same qPCR plate and further tested in technical singlicates in separate runs.

[0663] hiPSC-derived F-Dup neurons and corrected neurons were cultured for a total of approximately 21 days, and the culture medium was refreshed every 2 or 3 days. From day 7 of culture, the cells were treated with ASO for 7 days, and then the cells were cultured in a culture medium without ASO supplementation for an additional 7 days, including the day of transition from ASO supplementation to no ASO supplementation. These ASO-treated cells were then harvested on day 14 of culture, or 7 days after the first ASO treatment, and then harvested every 2 or 3 days for a total of 3 lysate harvests. Specifically, cell lysates were harvested for qPCR and protein analysis on days 7, 10, and 14 from the first day of ASO treatment, or on days corresponding to 14, 17, and 21 days of the total culture time. The washout stage of the culture began after the end of ASO treatment and the transition to the use of non-ASO culture medium and continued for 7 days.

[0664] Preparation of cell lysates Lysis was performed with 30 μl of buffer per well according to the conditions using RIPA Lysis and Extraction Buffer (Thermo Fisher catalog number 89900) supplemented with Halt Protease Inhibitor Cocktail (Thermo Fisher catalog number 1861278). Preparation of lysates for qPCR analysis included using lysates supplemented with DNaseI (25 μl / well) and stop solution (2.5 μl / well) from the Invitrogen Cells-to-CT 1-Step TaqMan kit (Thermo Fisher catalog number A25602).

[0665] Protein quantification The total protein levels of cell lysates were quantified by assessing protein yields with the bicinchoninic acid (BCA) assay, which provides colorimetric detection and quantification of total protein. Two biological replicates were evaluated for each test ASO, positive and negative control ASOs, and untreated cells. UBE3A protein levels were quantified by Jess Western blot. The primary antibody used was the Proteintech UBE3A antibody (Proteintech catalog number 10344-1-AP) from rabbit host, diluted 1:10 with 10% goat serum. The secondary antibody used was an anti-rabbit secondary HRP antibody and was at 1×. Each sample was normalized against the final concentration of total protein.

[0666] Sample preparation for qPCR analysis Fifteen percent of the amount of lysate prepared from each sample was used in qPCR reactions using TaqMan Master Mix from the Invitrogen Cells-to-CT 1-Step TaqMan kit. A FAM-labeled human UBE3A probe was used simultaneously with an Applied Biosystems™ human RPLP0 (large ribosomal protein) endogenous control (VIC™ / TAMRA probe, Primer Limited).

[0667] Experimental setup Ten ASOs in each cell plate were tested in duplicate at two concentrations (6.3 μM and 20 μM). Seven cell plates were used. To normalize the qPCR data for each plate during data analysis, an ASO control was included in all plates at one concentration (5 μM). NEAT1 ASO, F-Dup untreated, and scrambled untreated were used as reference points for evaluating ASO-induced knockdown. All test ASOs at both concentrations and one set of controls (POS1, POS2, NEAT1 ASO, scr GFP_ASO, scr P1, F-Dup untreated, and scrambled untreated) were processed on the ProteinSimple Jess automated Western blot device from Bio-Techne for protein quantification by protein separation and immunodetection.

[0668] Assay performance of control and test ASOs across the qPCR plate on the 7th and 10th days of treatment (mRNA) The UBE3A assay consistently showed high UBE3A knockdown levels and limited variability in the control ASO population. The success of ASO delivery was confirmed by UBE2A knockdown of over 65% and over 40% by POS1 and POS2, respectively, but no UBE3A mRNA knockdown was obtained with the NEAT1 ASO. The positive control ASO showed similar levels of UBE3A mRNA knockdown on both the 7th and 10th days of treatment, thus indicating a clear and stable effect.

[0669] UBE3A knockdown at the mRNA and protein levels normalized to NEAT1 ASO on the 7th, 10th, and 14th days of ASO treatment Treatment of the gymnosis with ASO targeting UBE3A resulted in concentration-dependent UBE3A protein knockdown for the test ASO normalized to NEAT1 ASO on the 7th, 10th, and 14th days of ASO treatment (corresponding to washout days 0, 3, and 7, or culture days 14, 17, and 21). The UBE3A mRNA knockdown effect only increased slightly on the 10th day, suggesting that intracellular ASO may have been abundant on the 7th day at these concentrations (6.3 μM and 20 μM) (Figures 3A - 3B). Compound_25 resulted in the highest UBE3A mRNA knockdown, while Compound_11 and Compound_20 resulted in the lowest UBE3A mRNA knockdown.

[0670] All test ASOs induced concentration - and time - dependent UBE3A protein knockdown, corroborating the effects observed at the mRNA level. The data shown in the figures of this example represent the mean of technical singlicates per condition + / − SD (biological replicates n = 2) corresponding to each treatment day.

[0671] The UBE3A protein knockdown gradually increased as the ASO treatment duration increased. By the 10th day of treatment, all 10 exemplary ASOs were able to knockdown 50 - 80% of the UBE3A protein at 20 μM. By the 14th day of treatment, all exemplary test ASOs induced more than 60% UBE3A protein knockdown at 20 μM.

[0672] No data points were obtained for compound_34 (mRNA and protein) and compound_17 (protein) at 6.3 μM on the 10th day of treatment. No data points were collected for compound_31 and compound_34 (protein) at 20 μM on the 14th day of treatment. No data points were collected for NT modification (mRNA) at 6.3 μM on the 7th day of treatment.

[0673] Comparison of UBE3A mRNA and protein expression levels between F-Dup and corrected neurons The relative UBE3A mRNA expression levels between F-Dup 1-8 cells and F-Dup 1-8 corrected cells on the 11th day of differentiation (corresponding to the day before the start of ASO treatment) showed a difference of approximately 2.5-fold (Figure 4A). The relative UBE3A protein expression levels between F-Dup 1-8 cells and F-Dup 1-8 corrected cells on the 11th day of differentiation (corresponding to the day before the start of ASO treatment) showed a difference of approximately 1.25-fold (Figure 4B).

[0674] On the 19th, 22nd, and 26th days of differentiation (corresponding to the 14th, 17th, and 21st days of culture), or on the 0th, 3rd, and 7th days after the ASO treatment was completed and non-ASO culture medium was used, the expression of UBE3A mRNA and UBE3A protein was evaluated. UBE3A mRNA was overall increased in F-Dup 1-8 cells. More specifically, in F-Dup 1-8 cells, the expression of UBE3A mRNA was increased by about 2-fold compared to F-Dup 1-8 corrected cells (Figure 4C), while for the UBE3A protein expression level, there was a smaller fold change between the two cell lines (about 1.24-fold to about 1.6-fold) (Figure 4D). This difference in relative mRNA-to-protein expression can be explained by different regulatory pathways of the additional copies of UBE3A, or a short protein half-life that prevents excessive accumulation in the cell.

[0675] The data shown in Figures 4A - 4D represent the average of technical duplicates + / - SD (biological replicates n = 2 - 4) for each condition corresponding to each differentiation day. Data for mRNA from corrected neurons were not collected on the 19th day of differentiation.

[0676] UBE3A protein expression levels on the 7th and 10th days of ASO treatment ASO treatment by gymnosis using an ASO targeting UBE3A resulted in concentration - responsive and time - dependent UBE3A protein knockdown for an exemplary test ASO normalized against NEAT1 ASO (Figures 5A - 5B). Protein expression levels were normalized against total protein output. Data represent the mean of technical singlicates per condition+ / -SD (biological replicates n = 2) corresponding to each treatment day (NT = untreated). Data points were not collected for 6.3 μM of Compound_34 on day 10 of treatment, or for Compound_17 on days 10 and 14 of treatment. Data points were not collected for 20 μM of Compound_31 or Compound_34 on day 14 of treatment. D7, D10, and D14 in the figure represent day 7, day 10, and day 14 (treatment days 7, 10, 14) after ASO treatment, respectively.

[0677]

Table 12

[0678]

Table 13

[0679]

Table 14

[0680]

Table 15

[0681] Preferred embodiments of the present disclosure are shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Those skilled in the art will envision numerous variations, modifications, and substitutions without departing from the present disclosure. It should be understood that various alternative forms of the embodiments of the present disclosure may be used in practicing the present disclosure. The following claims define the scope of the present disclosure, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. A pharmaceutical composition for use in a method for reducing the expression of the UBE3A protein in mammalian cells having duplication, overexpression, or gain-of-function mutations of the UBE3A gene encoding the UBE3A protein, wherein the pharmaceutical composition is formulated to come into contact with the mammalian cells, and the pharmaceutical composition comprises an antisense oligomer or a vector encoding the antisense oligomer, wherein the antisense oligomer reduces the level of mRNA encoding the UBE3A protein in the mammalian cells.

2. The pharmaceutical composition according to claim 1, wherein the antisense oligomer comprises a polynucleotide sequence that is at least 80% complementary to at least eight consecutive nucleotides in the sequence described in any one of SEQ ID NOs: 93 to 153.

3. The pharmaceutical composition according to claim 1, wherein the antisense oligomer includes a sequence having at least 80%, at least 90%, or 100% identity with the sequence described in any one of SEQ ID NOs: 17, 36, 5, 9, 12, 28, 29, 32, 1-4, 6-8, 10, 11, 13-16, 18-27, 30, 31, 35, and 37-92.

4. The pharmaceutical composition comprises the antisense oligomer, and the antisense oligomer is Includes skeletal modifications, modified sugar moieties, or combinations thereof, The aforementioned skeletal modification includes a phosphorothioate bond or a phosphorodiamidate bond. The pharmaceutical composition according to claim 1, wherein the modified sugar portion comprises a phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, a 2'-O-methyl portion, a 2'-fluoro portion, a 2'-O-methoxyethyl portion, or a 2'-NMA portion.

5. The pharmaceutical composition according to claim 4, wherein the antisense oligomer comprises 3, 4, 5, or 6 2'-O-methoxyethyl modified nucleosides at the 5' end of the antisense oligomer, 3, 4, 5, or 6 2'-O-methoxyethyl modified nucleosides at the 3' end of the antisense oligomer, and a phosphorothioate bond between any two adjacent nucleosides of the antisense oligomer.

6. The antisense oligomer is (a) A 5' region consisting of 3, 4, 5, or 6 linked nucleosides, (b) A central region consisting of 8, 9, 10, 11, or 12 linked nucleosides, (c) A 3' region consisting of 3, 4, 5, or 6 linked nucleosides, The pharmaceutical composition according to claim 4, wherein each of the 3, 4, 5, or 6 linked nucleosides in the 5' region and each of the 3, 4, 5, or 6 linked nucleosides in the 3' region contains a modified sugar moiety, and each of the 8, 9, 10, 11, or 12 linked nucleosides in the central region is a deoxyribonucleoside.

7. The antisense oligomer is composed of 8 to 50 nucleic acid bases, 8 to 40 nucleic acid bases, 8 to 35 nucleic acid bases, 8 to 30 nucleic acid bases, 8 to 25 nucleic acid bases, 8 to 20 nucleic acid bases, 8 to 15 nucleic acid bases, 10 to 50 nucleic acid bases, 10 to 40 nucleic acid bases, 10 to 35 nucleic acid bases, 10 to 30 nucleic acid bases, 10 to 25 nucleic acid bases, 10 to 20 nucleic acid bases, 10 to 15 nucleic acid bases, 12 to 50 nucleic acid bases, 12 to 40 nucleic acid bases, 12 to 35 nucleic acid bases, 12 to 30 nucleic acid bases, and 12 to 25 nucleic acid bases. The pharmaceutical composition according to claim 4, comprising: 12 to 20 nucleic acid bases, 12 to 15 nucleic acid bases, 15 to 50 nucleic acid bases, 15 to 40 nucleic acid bases, 15 to 35 nucleic acid bases, 15 to 30 nucleic acid bases, 15 to 25 nucleic acid bases, 15 to 20 nucleic acid bases, 15 to 19 nucleic acid bases, 15 to 18 nucleic acid bases, 15 to 16 nucleic acid bases, 16 to 20 nucleic acid bases, 16 to 19 nucleic acid bases, 16 to 18 nucleic acid bases, 17 to 20 nucleic acid bases, 17 to 19 nucleic acid bases, or 18 to 20 nucleic acid bases.

8. The antisense oligomer is (a) A 5' region consisting of five linked nucleosides, (b) A central region consisting of 10 linked nucleosides, (c) A 3' region consisting of five linked nucleosides, Each nucleoside bond of the aforementioned antisense oligomer contains a phosphorothioate bond. Each of the five linked nucleosides in the 5' region and each of the five linked nucleosides in the 3' region contains a modified sugar moiety. The modified sugar portion is the 2'-O-methoxyethyl portion, Each cytosine of the five linked nucleosides in the 5' region and each cytosine of the five linked nucleosides in the 3' region are 5-methylcytosine. Each uracil of the five linked nucleosides in the 5' region and each uracil of the five linked nucleosides in the 3' region are 5-methyluracil. The pharmaceutical composition according to claim 3, wherein each of the 10 linked nucleosides in the central region is a deoxyribonucleoside.

9. The pharmaceutical composition according to claim 8, wherein the antisense oligomer is a modified oligonucleotide containing the sequence described in any one of SEQ ID NOs: 170, 189, 158, 165, 181, 182, 185, 154-157, 159-164, 166-169, 171-180, 183, 184, 186-188, and 192-247, or the sequence described in any one of Tables 3 and 4.

10. The pharmaceutical composition according to claim 9, wherein the antisense oligomer is a modified oligonucleotide containing the sequence described in SEQ ID NO: 170 or SEQ ID NO:

189.

11. The pharmaceutical composition according to claim 1, wherein the genome of the mammalian cell has overlapping genomic regions including the UBE3A gene encoding the UBE3A protein.

12. The mammalian cell is a human cell, and the genome of the mammalian cell has duplication of chromosomes 15q11.2 to q13.1, and / or The pharmaceutical composition according to claim 1, wherein the mammalian cells are obtained from a human subject suffering from Dup15q syndrome, or are descendants of sample cells obtained from the human subject.

13. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is formulated for intracranial administration.

14. The level of the mRNA encoding the UBE3A protein in the mammalian cells that have come into contact with the antisense oligomer or the vector encoding the antisense oligomer is approximately 10% to 99%, approximately 10% to 95%, approximately 10% to 90%, approximately 10% to 80%, approximately 10% to 70%, approximately 10% to 60%, approximately 10% to 50%, approximately 10% to 40%, approximately 10% to 30%, approximately 10% to 20%, approximately 20% to 90%, approximately 20% to 80%, approximately 20% to 70%, approximately 20% to 60%, approximately 20% to 50%, approximately 20% to 40%, approximately 20% % to approximately 30%, approximately 30% to approximately 80%, approximately 30% to approximately 70%, approximately 30% to approximately 60%, approximately 30% to approximately 50%, approximately 40% to approximately 70%, approximately 40% to approximately 60%, approximately 40% to approximately 50%, approximately 50% to approximately 99%, approximately 50% to approximately 95%, approximately 50% to approximately 90%, approximately 50% to approximately 80%, approximately 50% to approximately 70%, approximately 50% to approximately 60%, approximately 60% to approximately 99%, approximately 60% to approximately 95%, approximately 6 A pharmaceutical composition according to any one of claims 1 to 13, which decreases by 0% to approximately 90%, approximately 60% to approximately 80%, approximately 60% to approximately 70%, approximately 70% to approximately 99%, approximately 70% to approximately 95%, approximately 70% to approximately 90%, approximately 70% to approximately 80%, approximately 80% to approximately 99%, approximately 80% to approximately 95%, approximately 80% to approximately 90%, approximately 90% to approximately 99%, approximately 90% to approximately 95%, or approximately 95% to approximately 99%.

15. The pharmaceutical composition reduces the level of the UBE3A protein in the mammalian cells, The levels of the UBE3A protein in mammalian cells that have come into contact with the antisense oligomer or the vector encoding the antisense oligomer are approximately 10% to 99%, 10% to 95%, 10% to 90%, 10% to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 90%, 20% to 80%, 20% to 70%, 20% to 60%, 20% to 50%, 20% to 40%, and 20% to 30% compared to cells that are otherwise identical but not in contact with the antisense oligomer or the vector encoding the antisense oligomer. Approximately 30% to 80%, approximately 30% to 70%, approximately 30% to 60%, approximately 30% to 50%, approximately 40% to 70%, approximately 40% to 60%, approximately 40% to 50%, approximately 50% to 99%, approximately 50% to 95%, approximately 50% to 90%, approximately 50% to 80%, approximately 50% to 70%, approximately 50% to 60%, approximately 60% to 99%, approximately 60% to 95%, approximately 60% to A pharmaceutical composition according to any one of claims 1 to 13, which reduces by approximately 90%, approximately 60% to approximately 80%, approximately 60% to approximately 70%, approximately 70% to approximately 99%, approximately 70% to approximately 95%, approximately 70% to approximately 90%, approximately 70% to approximately 80%, approximately 80% to approximately 99%, approximately 80% to approximately 95%, approximately 80% to approximately 90%, approximately 90% to approximately 99%, approximately 90% to approximately 95%, or approximately 95% to approximately 99%.

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