Antisense oligomers for the treatment of nonsense-mediated RNA decay conditions and diseases

Abstract

Because alternative splicing events in genes can induce non-productive mRNA transcripts, which can then induce aberrant or reduced protein expression, therapeutic agents that can target alternative splicing events in genes can regulate the expression level of functional proteins and / or inhibit aberrant protein expression in patients. Such therapeutic agents can be used to treat conditions or diseases caused by protein deficiencies.

Description

[Background technology]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 381,640, filed October 31, 2022, which is incorporated herein by reference in its entirety.

[0002] Because alternative splicing events in genes can induce non-productive mRNA transcripts, which can then induce aberrant protein expression, therapeutic agents that can target alternative splicing events in genes can regulate the expression levels of functional proteins and / or inhibit aberrant protein expression in patients. Such therapeutic agents can be used to treat conditions or diseases caused by protein deficiencies. Summary of the Invention

[0003] Provided herein, in some aspects, is a method for regulating expression of a target protein in a cell, the cell comprising a pre-mRNA transcribed from a target gene and encoding the target protein, the pre-mRNA comprising an alternatively spliced ​​coding exon (ASCE), and the alternatively processed mRNA produced by splicing out the ASCE during processing of the pre-mRNA is subject to nonsense-mediated RNA decay, the method comprising contacting the cell with a therapeutic agent or a vector encoding the therapeutic agent, wherein the therapeutic agent promotes the inclusion of ASCE during processing of the pre-mRNA, thereby increasing the level of processed mRNA processed from the pre-mRNA and comprising ASCE.

[0004] Provided herein, in some aspects, are methods for treating a disease or condition or reducing the likelihood of developing a disease or condition in a subject in need thereof by modulating expression of a target protein in the subject's cells, the method comprising contacting the subject's cells with a therapeutic agent or a vector encoding the therapeutic agent, wherein the cells comprise a pre-mRNA transcribed from a target gene and encoding the target protein, the pre-mRNA comprising an alternatively spliced ​​coding exon (ASCE), wherein the alternatively processed mRNA produced by splicing out the ASCE during pre-mRNA processing is subject to nonsense-mediated RNA decay, and the therapeutic agent promotes inclusion of the ASCE during pre-mRNA processing, thereby increasing the level of processed mRNA processed from the pre-mRNA and comprising the ASCE.

[0005] In some embodiments, expression of the target protein is increased in the cell.

[0006] In some embodiments, the target gene is selected from the group consisting of: PKD1, ABCA4, FUS, CEL, and NSD1.

[0007] In some embodiments, the target protein is selected from the group consisting of polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxyl ester lipase, and nuclear receptor-associated SET domain protein 1.

[0008] In some embodiments, the therapeutic agent is: (a) binds to a targeted portion of the mRNA encoding the target protein; (b) modulates the binding of factors involved in ASCE splicing; or (c) A combination of (a) and (b) is performed.

[0009] In some embodiments, the therapeutic agent prevents binding of a factor involved in splicing of ASCE to the region of the targeted portion.

[0010] In some embodiments, the targeted moiety is proximal to the ASCE.

[0011] In some embodiments, the targeted portion is up to about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides upstream of the 5' end of ASCE.

[0012] In some embodiments, the targeted portion is at least about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, or about 1 nucleotide upstream of the 5' end of ASCE.

[0013] In some embodiments, the targeted portion is up to about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides downstream of the 3' end of ASCE.

[0014] In some embodiments, the targeted portion is at least about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, or about 1 nucleotide downstream of the 3' end of ASCE.

[0015] In some embodiments, the targeted portion is up to about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides upstream of a genomic site selected from the group consisting of GRCh38 / hg38:chr16 2092954, GRCh38 / hg38:chr1 94111438, GRCh38 / hg38:chr16 31186802, GRCh38 / hg38:chr9 133066530, and GRCh38 / hg38:chr5 177238237.

[0016] In some embodiments, the targeted portion is about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides upstream of a genomic site selected from the group consisting of: GRCh38 / hg38:chr16 2092954, GRCh38 / hg38:chr1 94111438, GRCh38 / hg38:chr16 31186802, GRCh38 / hg38:chr9 133066530, and GRCh38 / hg38:chr5 177238237.

[0017] In some embodiments, the targeted portion is up to about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides downstream of a genomic site selected from the group consisting of GRCh38 / hg38:chr16 2093093, GRCh38 / hg38:chr1 94111579, GRCh38 / hg38:chr16 31186836, GRCh38 / hg38:chr9 133066660, and GRCh38 / hg38:chr5 177238507.

[0018] In some embodiments, the targeted portion is about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides downstream of a genomic site selected from the group consisting of: GRCh38 / hg38:chr16 2093093, GRCh38 / hg38:chr1 94111579, GRCh38 / hg38:chr16 31186836, GRCh38 / hg38:chr9 133066660, and GRCh38 / hg38:chr5 177238507.

[0019] In some embodiments, the targeted moiety is located in an intronic region between the ASCE and a canonical exon region upstream of the ASCE in the mRNA encoding the target protein.

[0020] In some embodiments, the targeted moiety is located in an intronic region between the ASCE and a canonical exon region downstream of the ASCE in the mRNA encoding the target protein.

[0021] In some embodiments, the targeted portion at least partially overlaps the ASCE.

[0022] In some embodiments, the targeted portion at least partially overlaps with an intron upstream or downstream of ASCE.

[0023] In some embodiments, the targeted portion does not include the 5' exon-intron junction or the 3' exon-intron junction.

[0024] In some embodiments, the targeted moiety is within the ASCE.

[0025] In some embodiments, the targeted portion comprises about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more consecutive nucleotides of the ASCE.

[0026] In some embodiments, the mRNA encoding the target protein comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 6-10.

[0027] In some embodiments, the mRNA encoding the target protein is encoded by a gene sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1-5.

[0028] In some embodiments, the targeted portion of the mRNA comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 contiguous nucleic acids of a sequence selected from the group consisting of SEQ ID NOs: 6-10.

[0029] In some embodiments, the targeted portion of the mRNA is within an ASCE selected from the group consisting of: GRCh38 / hg38:chr16 2092954 2093093, GRCh38 / hg38:chr1 94111438 94111579, GRCh38 / hg38:chr16 31186802 31186836, GRCh38 / hg38:chr9 133066530 133066660, and GRCh38 / hg38:chr5 177238237 177238507.

[0030] In some embodiments, the targeted portion of the mRNA is upstream or downstream of an ASCE selected from the group consisting of: GRCh38 / hg38:chr16 2092954 2093093, GRCh38 / hg38:chr1 94111438 94111579, GRCh38 / hg38:chr16 31186802 31186836, GRCh38 / hg38:chr9 133066530 133066660, and GRCh38 / hg38:chr5 177238237 177238507.

[0031] In some embodiments, the targeted portion of the mRNA does not include an exon-intron junction of an ASCE selected from the group consisting of: GRCh38 / hg38:chr16 2092954 2093093, GRCh38 / hg38:chr1 94111438 94111579, GRCh38 / hg38:chr16 31186802 31186836, GRCh38 / hg38:chr9 133066530 133066660, and GRCh38 / hg38:chr5 177238237 177238507.

[0032] In some embodiments, the target protein produced is a full-length or wild-type protein.

[0033] In some embodiments, the inclusion of ASCE during processing of pre-mRNA in cells contacted with a therapeutic agent or a vector encoding a therapeutic agent is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, or about 1.1 to about 7-fold greater than the inclusion of ASCE during processing of pre-mRNA in corresponding cells not contacted with a therapeutic agent or a vector encoding a therapeutic agent. , about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times.

[0034] In some embodiments, the level of processed mRNA produced in cells contacted with a Therapeutic Agent or a vector encoding a Therapeutic Agent is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 1.1 to about 10-fold, about 1.1 to about 11-fold, about 1.1 to about 12-fold, about 1.1 to about 13-fold, about 1.1 to about 14-fold, about 1.1 to about 15-fold, about 1.1 to about 16-fold, about 1.1 to about 17-fold, about 1.1 to about 18-fold, about 1.1 to about 19-fold, about 1.1 to about 20-fold, about 1.1 to about 21-fold, about 1.1 to about 22-fold, about 1.1 to about 23-fold, about 1.1 to about 24-fold, about 1.1 to about 25-fold, about 1.1 to about 26-fold, about 1.1 to about 27-fold, about 1.1 to about 28-fold, about 1.1 to about 29-fold, about 1.1 to about 30-fold, about 1.1 to about 31-fold, about 1.1 to about 32-fold, about 1.1 to about 33-fold, about 1.1 to about 34-fold, about 1.1 to about 35-fold, about 1.1 to about 36-fold, about 1.1 to about 3 It increases by about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold.

[0035] In some embodiments, the level of target protein produced in cells contacted with a Therapeutic Agent is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold increase.

[0036] In some embodiments, the elimination of ASCE during pre-mRNA processing in cells contacted with a Therapeutic Agent is about 1.1 to about 10 times lower, about 1.5 to about 10 times lower, about 2 to about 10 times lower, about 3 to about 10 times lower, about 4 to about 10 times lower, about 1.1 to about 5 times lower, about 1.1 to about 6 times lower, about 1.1 to about 7 times lower, about 1.1 to about 8 times lower, about 1.1 to about 9 times lower, about 2 times lower, or about 3 times lower than the elimination of ASCE during pre-mRNA processing in corresponding cells not contacted with a Therapeutic Agent or a vector encoding a Therapeutic Agent. the concentration is reduced by one to about one-fifth, about one-half to about one-sixth, about one-half to about one-seventh, about one-half to about one-eighth, about one-half to about one-ninth, about one-third to about one-sixth, about one-third to about one-seventh, about one-third to about one-eighth, about one-third to about one-ninth, about one-quarter to about one-seventh, about one-quarter to about one-eighth, about one-quarter to about one-ninth, at least about 1.1 times, at least about 1.5 times, at least about half, at least about 2.5 times, at least about one-third, at least about 3.5 times, at least about one-quarter, at least about one-fifth, or at least about one-tenth.

[0037] In some embodiments, the target protein is NSD1 and the method causes modification of a histone protein in the cell.

[0038] In some embodiments, the histone protein is histone H3.

[0039] In some embodiments, the modification comprises acetylation, methylation, phosphorylation, or ubiquitination.

[0040] In some embodiments, the modification is methylation.

[0041] In some embodiments, methylation of histone proteins is increased in the cells.

[0042] In some embodiments, the methylation of histone proteins in cells contacted with a Therapeutic Agent or a vector encoding a Therapeutic Agent is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, or about 1.1 to about 8-fold higher than the methylation of histone proteins in corresponding cells not contacted with a Therapeutic Agent or a vector encoding a Therapeutic Agent. , about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times.

[0043] In some embodiments, the method further comprises assessing the mRNA level or expression level of the target protein.

[0044] In some embodiments, the disease or condition is caused by a loss-of-function mutation in the target gene.

[0045] In some embodiments, the disease or condition is associated with haploinsufficiency of a gene encoding a target protein, and the subject has a first allele that encodes a functional target protein and a second allele in which the target protein is not produced, or is produced at a reduced level, or a second allele that encodes a non-functional or partially functional target protein.

[0046] In some embodiments, the disease or condition is selected from the group consisting of: polycystic kidney disease with or without polycystic liver disease 1, autosomal dominant polycystic kidney disease, age-related macular degeneration-2, Stargardt disease 1, amyotrophic lateral sclerosis, amyotrophic lateral sclerosis with or without frontotemporal dementia 6, hereditary essential tremor 4, frontotemporal dementia, maturity-onset diabetes of the young with exocrine insufficiency type 8, maturity-onset diabetes of the young, Sotos syndrome 1, and Beckwith-Wiedemann syndrome.

[0047] In some embodiments, the disease or condition is associated with an autosomal recessive mutation in a gene encoding a target protein, and the subject has a first allele that encodes (i) the target protein is not produced or is produced at a reduced level compared to the wild-type allele, or (ii) the target protein that is produced is non-functional or is partially functional compared to the wild-type allele, and a second allele that encodes (iii) the target protein is produced at a reduced level compared to the wild-type allele and the produced target protein is at least partially functional compared to the wild-type allele, or (iv) the target protein that is produced is partially functional compared to the wild-type allele.

[0048] In some embodiments, the disease or condition is caused by a gain-of-function mutation in the target protein.

[0049] In some embodiments, the subject has an allele that causes the target protein to be produced at increased levels or encodes a mutant target protein that exhibits increased activity in the cell.

[0050] In some embodiments, the subject is a human.

[0051] In some embodiments, the subject is a non-human animal.

[0052] In some embodiments, the subject is a fetus, embryo, or child.

[0053] In some embodiments, the cells are ex vivo or are in a tissue or organ that is ex vivo.

[0054] In some embodiments, the therapeutic agent is administered to the subject by intrathecal, intracerebroventricular, intraperitoneal, intramuscular, subcutaneous, intravitreal, or intravenous injection.

[0055] In some embodiments, the method further comprises administering a second therapeutic agent to the subject.

[0056] In some embodiments, the second therapeutic agent is a small molecule.

[0057] In some embodiments, the second therapeutic agent is an antisense oligomer.

[0058] In some embodiments, the second therapeutic agent modifies intron retention.

[0059] In some embodiments, the disease or condition is a disease or condition associated with a deficiency in the amount or activity of a target protein.

[0060] In some embodiments, the disease or condition is one associated with a deficiency in the amount or activity of a protein, and the target protein functionally enhances, compensates for, replaces, or functionally interacts with the protein.

[0061] In some embodiments, the disease or condition is caused by an insufficient amount or activity of the target protein.

[0062] In some embodiments, the method further comprises evaluating the subject's genome for at least one genetic variation associated with the disease.

[0063] In some embodiments, the at least one genetic variation is within a genetic locus associated with a disease.

[0064] In some embodiments, the at least one genetic variation is in a locus associated with expression of a gene associated with the disease.

[0065] In some embodiments, the at least one genetic variation is within the locus of a gene encoding a target protein.

[0066] In some embodiments, the at least one genetic variation is in a locus associated with expression of a gene encoding a target protein.

[0067] In some embodiments, the method treats a disease or condition.

[0068] In some embodiments, the target protein is a canonical isoform of a protein.

[0069] In some embodiments, the alternatively processed mRNA produced by splicing out the ASCE comprises a premature termination codon (PTC).

[0070] In some embodiments, the agent is an antisense oligomer (ASO).

[0071] In some embodiments, the ASO is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to the targeted portion of the mRNA.

[0072] In some embodiments, the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complementary to at least eight consecutive nucleic acids of a sequence selected from the group consisting of SEQ ID NOs: 6-10.

[0073] In some embodiments, the ASO comprises backbone modifications including phosphorothioate or phosphorodiamidate linkages.

[0074] In some embodiments, the ASO comprises a phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety.

[0075] In some embodiments, the ASO comprises at least one modified sugar moiety.

[0076] In some embodiments, each sugar moiety is a modified sugar moiety.

[0077] In some embodiments, the ASO is 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, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, bases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 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, or 12 to 15 nucleobases.

[0078] In some embodiments, the target gene is NSD1 and the ASO comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 16-1748.

[0079] In some embodiments, the target gene is NSD1 and the vector encoding the agent encodes a polynucleotide comprising a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of the ASO sequences listed in Table 4, Table 5A, Table 5A-1, Table 5B, Table 5B-1, Table 5D, Table 5E, Table 5G, and Table 5G-1.

[0080] In some embodiments, the vector encoding the agent is a viral vector.

[0081] In some embodiments, the viral vector is an adenovirus-associated viral vector.

[0082] In some embodiments, the vector encoding the agent encodes a polynucleotide comprising an ASO sequence and an snRNA.

[0083] In some embodiments, the snRNA comprises a modified snRNA.

[0084] In some embodiments, the modified snRNA is a modified U1 snRNA or a modified U7 snRNA.

[0085] In some embodiments, the snRNA comprises U1 snRNA.

[0086] In some embodiments, the target gene is NSD1 and the ASO sequence comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of the ASO sequences listed in Table 4, Table 5A, Table 5B, Table 5D, Table 5E, and Table 5G.

[0087] In some embodiments, the snRNA comprises U7 snRNA.

[0088] In some embodiments, the target gene is NSD1 and the ASO comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of the ASO sequences listed in Table 4, Table 5A, Table 5A-1, Table 5B, Table 5B-1, Table 5G, and Table 5G-1.

[0089] Provided herein, in some embodiments, is a composition comprising an agent or a vector encoding the agent, wherein the agent regulates splicing of a pre-mRNA in a cell that is transcribed from a target gene and encodes a target protein, the pre-mRNA containing an alternatively spliced ​​coding exon (ASCE), and the alternatively processed mRNA produced by splicing out the ASCE during pre-mRNA processing is subject to nonsense-mediated RNA decay, and the agent promotes inclusion of the ASCE during pre-mRNA processing, thereby increasing the level of processed mRNA that is processed from the pre-mRNA and contains the ASCE.

[0090] In some embodiments, the agent increases expression of a target protein in a cell.

[0091] In some embodiments, the target gene is selected from the group consisting of: PKD1, ABCA4, FUS, CEL, and NSD1.

[0092] In some embodiments, the target protein is selected from the group consisting of polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxyl ester lipase, and nuclear receptor-associated SET domain protein 1.

[0093] In some embodiments, the agent is (a) binds to a targeted portion of the mRNA encoding the target protein; (b) modulates the binding of factors involved in ASCE splicing; or (c) A combination of (a) and (b) is performed.

[0094] In some embodiments, the agent prevents binding of a factor involved in splicing of ASCE to the region of the targeted portion.

[0095] In some embodiments, the targeted moiety is proximal to the ASCE.

[0096] In some embodiments, the targeted portion is up to about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides upstream of the 5' end of ASCE.

[0097] In some embodiments, the targeted portion is at least about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, or about 1 nucleotide upstream of the 5' end of ASCE.

[0098] In some embodiments, the targeted portion is up to about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides downstream of the 3' end of ASCE.

[0099] In some embodiments, the targeted portion is at least about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, or about 1 nucleotide downstream of the 3' end of ASCE.

[0100] In some embodiments, the targeted portion is up to about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides upstream of a genomic site selected from the group consisting of GRCh38 / hg38:chr16 2092954, GRCh38 / hg38:chr1 94111438, GRCh38 / hg38:chr16 31186802, GRCh38 / hg38:chr9 133066530, and GRCh38 / hg38:chr5 177238237.

[0101] In some embodiments, the targeted portion is about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides upstream of a genomic site selected from the group consisting of: GRCh38 / hg38:chr16 2092954, GRCh38 / hg38:chr1 94111438, GRCh38 / hg38:chr16 31186802, GRCh38 / hg38:chr9 133066530, and GRCh38 / hg38:chr5 177238237.

[0102] In some embodiments, the targeted portion is up to about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides downstream of a genomic site selected from the group consisting of GRCh38 / hg38:chr16 2093093, GRCh38 / hg38:chr1 94111579, GRCh38 / hg38:chr16 31186836, GRCh38 / hg38:chr9 133066660, and GRCh38 / hg38:chr5 177238507.

[0103] In some embodiments, the targeted portion is about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides downstream of a genomic site selected from the group consisting of: GRCh38 / hg38:chr16 2093093, GRCh38 / hg38:chr1 94111579, GRCh38 / hg38:chr16 31186836, GRCh38 / hg38:chr9 133066660, and GRCh38 / hg38:chr5 177238507.

[0104] In some embodiments, the targeted moiety is located in an intronic region between the ASCE and a canonical exon region upstream of the ASCE in the mRNA encoding the target protein.

[0105] In some embodiments, the targeted moiety is located in an intronic region between the ASCE and a canonical exon region downstream of the ASCE in the mRNA encoding the target protein.

[0106] In some embodiments, the targeted portion at least partially overlaps the ASCE.

[0107] In some embodiments, the targeted portion at least partially overlaps with an intron upstream or downstream of ASCE.

[0108] In some embodiments, the targeted portion does not include the 5' exon-intron junction or the 3' exon-intron junction.

[0109] In some embodiments, the targeted moiety is within the ASCE.

[0110] In some embodiments, the targeted portion comprises about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more consecutive nucleotides of the ASCE.

[0111] In some embodiments, the mRNA encoding the target protein comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 6-10.

[0112] In some embodiments, the mRNA encoding the target protein is encoded by a gene sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1-5.

[0113] In some embodiments, the targeted portion of the mRNA comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 contiguous nucleic acids of a sequence selected from the group consisting of SEQ ID NOs: 6-10.

[0114] In some embodiments, the targeted portion of the mRNA is within an ASCE selected from the group consisting of: GRCh38 / hg38:chr16 2092954 2093093, GRCh38 / hg38:chr1 94111438 94111579, GRCh38 / hg38:chr16 31186802 31186836, GRCh38 / hg38:chr9 133066530 133066660, and GRCh38 / hg38:chr5 177238237 177238507.

[0115] In some embodiments, the targeted portion of the mRNA is upstream or downstream of an ASCE selected from the group consisting of: GRCh38 / hg38:chr16 2092954 2093093, GRCh38 / hg38:chr1 94111438 94111579, GRCh38 / hg38:chr16 31186802 31186836, GRCh38 / hg38:chr9 133066530 133066660, and GRCh38 / hg38:chr5 177238237 177238507.

[0116] In some embodiments, the targeted portion of the mRNA does not include an exon-intron junction of an ASCE selected from the group consisting of: GRCh38 / hg38:chr16 2092954 2093093, GRCh38 / hg38:chr1 94111438 94111579, GRCh38 / hg38:chr16 31186802 31186836, GRCh38 / hg38:chr9 133066530 133066660, and GRCh38 / hg38:chr5 177238237 177238507.

[0117] In some embodiments, the target protein produced is a full-length or wild-type protein.

[0118] In some embodiments, the inclusion of ASCE during processing of pre-mRNA in cells contacted with a therapeutic agent or a vector encoding a therapeutic agent is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, or about 1.1 to about 7-fold greater than the inclusion of ASCE during processing of pre-mRNA in corresponding cells not contacted with a therapeutic agent or a vector encoding a therapeutic agent. , about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times.

[0119] In some embodiments, the level of processed mRNA produced in cells contacted with a Therapeutic Agent or a vector encoding a Therapeutic Agent is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 1.1 to about 10-fold, about 1.1 to about 11-fold, about 1.1 to about 12-fold, about 1.1 to about 13-fold, about 1.1 to about 14-fold, about 1.1 to about 15-fold, about 1.1 to about 16-fold, about 1.1 to about 17-fold, about 1.1 to about 18-fold, about 1.1 to about 19-fold, about 1.1 to about 20-fold, about 1.1 to about 21-fold, about 1.1 to about 22-fold, about 1.1 to about 23-fold, about 1.1 to about 24-fold, about 1.1 to about 25-fold, about 1.1 to about 26-fold, about 1.1 to about 27-fold, about 1.1 to about 28-fold, about 1.1 to about 29-fold, about 1.1 to about 30-fold, about 1.1 to about 31-fold, about 1.1 to about 32-fold, about 1.1 to about 33-fold, about 1.1 to about 34-fold, about 1.1 to about 35-fold, about 1.1 to about 36-fold, about 1.1 to about 3 It increases by about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold.

[0120] In some embodiments, the level of the target protein produced in cells contacted with the agent is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, or about 1.1 to about 10-fold, compared to the level of the target protein produced in corresponding cells not contacted with the therapeutic agent or a vector encoding the therapeutic agent. fold, about 2 to about 5 fold, about 2 to about 6 fold, about 2 to about 7 fold, about 2 to about 8 fold, about 2 to about 9 fold, about 3 to about 6 fold, about 3 to about 7 fold, about 3 to about 8 fold, about 3 to about 9 fold, about 4 to about 7 fold, about 4 to about 8 fold, about 4 to about 9 fold, at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 5 fold, or at least about 10 fold.

[0121] In some embodiments, the elimination of ASCE during pre-mRNA processing in cells contacted with a Therapeutic Agent is about 1.1 to about 10 times lower, about 1.5 to about 10 times lower, about 2 to about 10 times lower, about 3 to about 10 times lower, about 4 to about 10 times lower, about 1.1 to about 5 times lower, about 1.1 to about 6 times lower, about 1.1 to about 7 times lower, about 1.1 to about 8 times lower, about 1.1 to about 9 times lower, about 2 times lower, or about 3 times lower than the elimination of ASCE during pre-mRNA processing in corresponding cells not contacted with a Therapeutic Agent or a vector encoding a Therapeutic Agent. the concentration is reduced by one to about one-fifth, about one-half to about one-sixth, about one-half to about one-seventh, about one-half to about one-eighth, about one-half to about one-ninth, about one-third to about one-sixth, about one-third to about one-seventh, about one-third to about one-eighth, about one-third to about one-ninth, about one-quarter to about one-seventh, about one-quarter to about one-eighth, about one-quarter to about one-ninth, at least about 1.1 times, at least about 1.5 times, at least about half, at least about 2.5 times, at least about one-third, at least about 3.5 times, at least about one-quarter, at least about one-fifth, or at least about one-tenth.

[0122] In some embodiments, the target protein is NSD1 and the method causes modification of a histone protein in the cell.

[0123] In some embodiments, the histone protein is histone H3.

[0124] In some embodiments, the modification comprises acetylation, methylation, phosphorylation, or ubiquitination.

[0125] In some embodiments, the modification is methylation.

[0126] In some embodiments, methylation of histone proteins is increased in the cells.

[0127] In some embodiments, the methylation of histone proteins in cells contacted with a Therapeutic Agent or a vector encoding a Therapeutic Agent is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, or about 1.1 to about 8-fold higher than the methylation of histone proteins in corresponding cells not contacted with a Therapeutic Agent or a vector encoding a Therapeutic Agent. , about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times.

[0128] In some embodiments, the target protein is a canonical isoform of a protein.

[0129] In some embodiments, the alternatively processed mRNA produced by splicing out the ASCE comprises a premature termination codon (PTC).

[0130] In some embodiments, the agent is an antisense oligomer (ASO).

[0131] In some embodiments, the ASO is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to the targeted portion of the mRNA.

[0132] In some embodiments, the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complementary to at least eight consecutive nucleic acids of a sequence selected from the group consisting of SEQ ID NOs: 6-10.

[0133] In some embodiments, the ASO comprises backbone modifications including phosphorothioate or phosphorodiamidate linkages.

[0134] In some embodiments, the ASO comprises a phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety.

[0135] In some embodiments, the ASO comprises at least one modified sugar moiety.

[0136] In some embodiments, each sugar moiety is a modified sugar moiety.

[0137] In some embodiments, the ASO is 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, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, bases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 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, or 12 to 15 nucleobases.

[0138] In some embodiments, the ASO comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 16-1748.

[0139] In some embodiments, the target gene is NSD1 and the vector encoding the agent encodes a polynucleotide comprising a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of the ASO sequences listed in Table 4, Table 5A, Table 5A-1, Table 5B, Table 5B-1, Table 5D, Table 5E, Table 5G, and Table 5G-1.

[0140] In some embodiments, the vector encoding the agent is a viral vector.

[0141] In some embodiments, the viral vector is an adenovirus-associated viral vector.

[0142] In some embodiments, the vector encoding the agent encodes a polynucleotide comprising an ASO sequence and an snRNA.

[0143] In some embodiments, the snRNA comprises a modified snRNA.

[0144] In some embodiments, the modified snRNA is a modified U1 snRNA or a modified U7 snRNA.

[0145] In some embodiments, the snRNA comprises U1 snRNA.

[0146] In some embodiments, the target gene is NSD1 and the ASO sequence comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of the ASO sequences listed in Table 4, Table 5A, Table 5B, Table 5D, Table 5E, and Table 5G.

[0147] In some embodiments, the snRNA comprises U7 snRNA.

[0148] In some embodiments, the target gene is NSD1 and the ASO comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of the ASO sequences listed in Table 4, Table 5A, Table 5A-1, Table 5B, Table 5B-1, Table 5G, and Table 5G-1.

[0149] In some aspects, provided herein are compositions comprising an ASO comprising a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 16-1748.

[0150] In some embodiments, the ASO comprises backbone modifications including phosphorothioate or phosphorodiamidate linkages.

[0151] In some embodiments, the ASO comprises a phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety.

[0152] In some embodiments, the ASO comprises at least one modified sugar moiety.

[0153] In some embodiments, each sugar moiety is a modified sugar moiety.

[0154] In some embodiments, the ASO is 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, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, bases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 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, or 12 to 15 nucleobases.

[0155] In some aspects, provided herein are compositions comprising a vector encoding a polynucleotide comprising a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of the ASO sequences listed in Table 4, Table 5A, Table 5A-1, Table 5B, Table 5B-1, Table 5D, Table 5E, Table 5G, and Table 5G-1.

[0156] In some embodiments, the vector encoding the agent is a viral vector.

[0157] In some embodiments, the viral vector is an adenovirus-associated viral vector.

[0158] In some embodiments, the vector encoding the agent encodes a polynucleotide comprising an ASO sequence and an snRNA.

[0159] In some embodiments, the snRNA comprises a modified snRNA.

[0160] In some embodiments, the modified snRNA is a modified U1 snRNA or a modified U7 snRNA.

[0161] In some embodiments, the snRNA comprises U1 snRNA.

[0162] In some embodiments, the ASO sequence comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of the ASO sequences listed in Table 4, Table 5A, Table 5B, Table 5D, Table 5E, and Table 5G.

[0163] In some embodiments, the snRNA comprises U7 snRNA.

[0164] In some embodiments, the ASO comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of the ASO sequences listed in Table 4, Table 5A, Table 5A-1, Table 5B, Table 5B-1, Table 5G, and Table 5G-1.

[0165] In some aspects, provided herein are pharmaceutical compositions comprising a composition described herein and a pharmaceutically acceptable excipient and / or delivery vehicle.

[0166] In some aspects, provided herein are methods of treating a disease or condition or reducing the likelihood of developing a disease or condition in a subject in need thereof, the methods comprising administering to the subject a pharmaceutical composition described herein.

[0167] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein 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. [Brief explanation of the drawings]

[0168] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings.

[0169] [Figure 1A] Figure 1A shows a schematic diagram of a target pre-mRNA containing an alternatively spliced ​​coding exon (ASCE) that can be alternatively spliced ​​to produce a non-productive mRNA that undergoes nonsense-mediated RNA decay (NMD) and therapeutic drug-mediated enhancement of canonical splicing, thereby increasing the expression of a functional mRNA or full-length target mRNA for a target protein. Figure 1A shows a cell divided into nuclear and cytoplasmic compartments. In the nucleus, the pre-mRNA transcript of the target gene undergoes splicing to produce mRNA, which is transported to the cytoplasm and translated into the target protein. For this target gene, a fraction of the pre-mRNA is alternatively spliced, inducing the formation of processed mRNA (non-productive mRNA) lacking ASCE that undergoes NMD and is degraded in the cytoplasm, thus not inducing target protein production from the non-productive mRNA. [Figure 1B]Figure 1B shows a schematic diagram of a target pre-mRNA containing an alternatively spliced ​​coding exon (ASCE) that can be alternatively spliced ​​to produce a non-productive mRNA that undergoes nonsense-mediated RNA decay (NMD) and therapeutic-agent-mediated enhancement of canonical splicing, increasing expression of a functional mRNA or full-length target mRNA for a target protein. Figure 1B shows an example of the same cell separated into nuclear and cytoplasmic compartments. Treatment with a therapeutic agent, such as an antisense oligomer (ASO), promotes the inclusion of ASCE in the mRNA processed from the pre-mRNA, resulting in an increase in functional (productive) mRNA containing ASCE, which is translated into higher levels of the target protein. [Figure 1C] Figure 1C shows the difference between two alternative splicing events of a pre-mRNA transcript, where one of the alternative splicing events leads to the formation of a non-productive mRNA lacking ASCE (bottom), and the other alternative splicing event leads to the formation of a productive mRNA containing ASCE (top). [Figure 1D] Figure 1D shows the difference between two alternative splicing events of the NSD1 pre-mRNA transcript, where one of the alternative splicing events leads to the formation of a non-productive mRNA lacking ASCE (exon 8) (bottom), and the other alternative splicing event leads to the formation of a productive mRNA containing ASCE (exon 8) (top). [Figure 2A] Figure 2A shows confirmation of an exemplary alternative splicing event of ASCE in the NSD1 gene by cycloheximide treatment in astrocytes, Schwann cells, and cynomolgus monkey brain cells. Figure 2A shows a schematic diagram in which peaks corresponding to RNA sequencing reads are identified in exon 8 of NSD1 (GRCh38 / hg38:chr5 177238237:177238507). [Figure 2B]Figure 2B shows the confirmation of exemplary alternative splicing events of ASCE in the NSD1 gene by cycloheximide treatment in astrocytes, Schwann cells, and cynomolgus monkey brain cells. Figure 2B shows gel images and graphs showing that cycloheximide treatment resulted in an increase in the amount of non-productive mature NSD1 mRNA transcripts (processed NSD1 mRNAs containing premature stop codons that cause the transcripts to be targeted by NMD) in various human cells, including astrocytes, Schwann cells, HEK293 cells, SH-SY-5Y (neuroblastoma cell line) cells, and SK-N-AS (neuroblastoma cell line) cells. [Figure 2C] Figure 2C shows confirmation of exemplary alternative splicing events of ASCE in the NSD1 gene by cycloheximide treatment in astrocytes, Schwann cells, and cynomolgus monkey brain cells. Figure 2C shows gel images and graphs showing the presence of non-productive mature NSD1 mRNA transcripts in various cynomolgus monkey brain regions, including the cortex, brainstem, hippocampus, and cerebellum. [Figure 2D] FIG. 2D shows a gel image and graph demonstrating the presence of non-productive mature NSD1 mRNA transcripts in the human cortex. [Figure 3A] Figure 3A shows confirmation of the inclusion or exclusion of mouse NSD1 ASCE (mouse exon 7 corresponding to human exon 8) in NSD1 mRNA products processed from NSD1 pre-mRNA in mouse brain via in vivo or ex vivo cycloheximide treatment. Figure 3A shows a gel image showing that exclusion of mouse NSD1 ASCE (mouse exon 7 corresponding to human exon 8) in mouse brain after ex vivo cycloheximide treatment induces the formation of processed mRNA containing a premature stop codon that renders the transcript a target for NMD. [Figure 3B]Figure 3B shows confirmation of the inclusion or exclusion of mouse NSD1 ASCE (mouse exon 7 corresponding to human exon 8) in NSD1 mRNA products processed from NSD1 pre-mRNA in mouse brain via in vivo or ex vivo cycloheximide treatment. Figure 3B shows graphs of the percentage of NMD (upper panel) and fold change of NMD events (lower panel) of non-productive NSD1 mRNA products compared to productive NSD1 mRNA products from the gel image in Figure 3A. [Figure 3C] Figure 3C shows confirmation of inclusion or exclusion of ASCE of mouse NSD1 (mouse exon 7 corresponding to human exon 8) in NSD1 mRNA products processed from NSD1 pre-mRNA in mouse brain via in vivo or ex vivo cycloheximide treatment. Figure 3C shows a gel image showing that exclusion of ASCE of mouse NSD1 (mouse exon 7 corresponding to human exon 8) in mouse brain after in vivo cycloheximide treatment induces the formation of processed mRNA containing a premature stop codon that makes the transcript a target for NMD. [Figure 3D] Figure 3D shows confirmation of the inclusion or exclusion of mouse NSD1 ASCE (mouse exon 7 corresponding to human exon 8) in NSD1 mRNA products processed from NSD1 pre-mRNA in mouse brain via in vivo or ex vivo cycloheximide treatment. Figure 3D shows graphs of the percentage of NMD (left panel) and fold change of NMD events (right panel) of non-productive NSD1 mRNA products compared to productive NSD1 mRNA products from the gel image in Figure 3C. [Figure 4A]Figure 4A shows confirmation of the inclusion or exclusion of mouse NSD1 ASCE (mouse exon 7 corresponding to human exon 8) in the NSD1 mRNA product processed from the NSD1 pre-mRNA in the mouse brain via in vivo cycloheximide treatment. Figure 4A shows a gel image showing that exclusion of mouse NSD1 ASCE (mouse exon 7 corresponding to human exon 8) in the mouse brain after in vivo cycloheximide treatment induces the formation of processed mRNA containing a premature stop codon that causes the transcript to be targeted by NMD. [Figure 4B] Figure 4B shows confirmation of the inclusion or exclusion of mouse NSD1 ASCE (mouse exon 7 corresponding to human exon 8) in NSD1 mRNA products processed from NSD1 pre-mRNA in mouse brain via in vivo cycloheximide treatment. Figure 4B shows graphs of the percentage of NMD (left panel) and fold change in NMD events (right panel) of non-productive NSD1 mRNA products compared to productive NSD1 mRNA products from the gel image of Figure 4A. [Figure 5-1] Figure 5 shows an exemplary ASO walk around the human NSD1 exon 8 (GRCh38 / hg38:chr5 177238237:177238507) region. Underlined nucleotides correspond to exon skipping events, and arrows point to the 5' or 3' canonical splice site. [Figure 5-2] Figure 5 shows an exemplary ASO walk around the human NSD1 exon 8 (GRCh38 / hg38:chr5 177238237:177238507) region. Underlined nucleotides correspond to exon skipping events, and arrows point to the 5' or 3' canonical splice site. [Figure 5-3]Figure 5 shows an exemplary ASO walk around the human NSD1 exon 8 (GRCh38 / hg38:chr5 177238237:177238507) region. Underlined nucleotides correspond to exon skipping events, and arrows point to the 5' or 3' canonical splice site. [Figure 6A] Figure 6A shows a graph summarizing the changes in the levels of productive NSD1 mRNA (Figure 6A) during one ASO walk around exon 8 in HEK293 cells. [Figure 6B] Figure 6B shows a graph summarizing the changes in the levels of non-productive NSD1 mRNA (Figure 6B) during one ASO walk around exon 8 in HEK293 cells. [Figure 7A] Figure 7A shows a graph summarizing the changes in the levels of productive NSD1 mRNA (Figure 7A) in one ASO walk around exon 8. [Figure 7B] Figure 7B shows a graph summarizing the changes in the levels of non-productive NSD1 mRNA (Figure 7B) in one ASO walk around exon 8. [Figure 8] Figure 8 shows an exemplary ASO walk around the human NSD1 exon 8 (GRCh38 / hg38:chr5 177238237:177238507) region for an ASO vectorization approach using U7 snRNA. [Figure 9] Figure 9 shows an exemplary ASO walk around the human NSD1 exon 8 (GRCh38 / hg38:chr5 177238237:177238507) region for an ASO vectorization approach using U1 snRNA. [Figure 10]Figure 10 shows representative histograms of non-productive NSD1 mRNA levels in various cell lines treated with alternative NMD inhibitors. SH-SY5Y, U-87MG, HEK293, and SK-N-AS cell lines were each treated with one of three conditions: mock control (vehicle only), the NMD inhibitor cycloheximide (CHX), or the NMD inhibitor SMG1i. Treatment with SMG1i resulted in approximately 28% NSD1 non-productive mRNA levels (the percentage of non-productive NSD1 mRNA transcripts relative to the total level of all NSD1 mRNA transcripts) in U-87MG cells, approximately 19% NSD1 non-productive mRNA levels in SH-SY5Y cells, and <18% NSD1 non-productive mRNA levels in HEK293 and SK-N-AS cells. Treatment with CHX resulted in approximately 23% NSD1 non-productive mRNA in SH-SY5Y cells, approximately 15% NSD1 non-productive mRNA in U-87 MG cells, and approximately 13% NSD1 non-productive mRNA in HEK293 and SK-N-AS cells. In cells treated with vehicle (mock) alone, the percentage of non-productive RNA remained low. [Figure 11A] Figure 11A shows data demonstrating that exemplary ASOs with alternative backbone modifications have similar effects on NSD1 pre-mRNA splicing, and is a table showing the names of the ASOs, their backbone chemical structures, sequences, and lengths. [Figure 11B] Figure 11B shows data demonstrating that exemplary ASOs with alternative backbone modifications have similar effects on NSD1 pre-mRNA splicing. Figure 11B is a scatter plot showing the fold change in productive and non-productive NSD1 mRNA when various ASOs with either PMO or 2'MOE-PS backbone modifications were nucleofected into U-87 MG cells compared to cells treated with a mock control. The data in Figure 11B are normalized to the mock control. [Figure 11C]Figure 11C shows data demonstrating that exemplary ASOs with alternative backbone modifications have similar effects on NSD1 pre-mRNA splicing. Figure 11C is a histogram showing the levels of NSD1 protein present in U-87 MG cells after treatment with ASOs of various backbones (see Figure 11A) compared to cells treated with a mock control. The data in Figure 11C are normalized to the mock control. [Figure 12A] Figure 12A shows representative data demonstrating the effect of exemplary ASOs on NSD1 protein expression and H3K36me2 levels in U-87 MG cells. Figure 12A is a histogram showing the fold change in NSD1 protein in U-87 MG cells treated with various ASOs compared to cells treated with water alone. U-87 cells were nucleofected with 1 μM of each ASO, and cells were harvested 72 hours after nucleofection. NSD1 protein levels were measured by immunocapillary electrophoresis (JESS), and H3K36me2 levels were measured by AlphaLISA®. Figure 12A shows the mean ± SEM of the sum of two to three independent experiments. [Figure 12B] Figure 12B shows representative data demonstrating the effects of exemplary ASOs on NSD1 protein expression and H3K36me2 levels in U-87 MG cells. Figure 12B is a histogram showing the fold change in cellular H3K36me2 levels in U-87 MG cells treated with various ASOs compared to cells treated with water alone. U-87 cells were nucleofected with 1 μM of each ASO, and cells were harvested 72 hours after nucleofection. NSD1 protein levels were measured by immunocapillary electrophoresis (JESS), and H3K36me2 levels were measured by AlphaLISA®. Figure 12B shows the mean ± SEM of the sum of two to three independent experiments. [Figure 13A]Figure 13A shows representative data demonstrating the dose-dependent effects of exemplary ASOs on NSD1 protein expression and H3K36me2 levels in U-87 MG cells. Figure 13A is a histogram showing the fold change in NSD1 protein in U-87 MG cells treated with various dose concentrations of ASO 211 (0.25 μM, 0.5 μM, 1.0 μM, or 2.0 μM) compared to cells treated with water alone. U-87 cells were nucleofected with ASO 211 at the four tested doses (0.25 μM, 0.5 μM, 1.0 μM, or 2.0 μM), and cells were harvested 72 hours after nucleofection. NSD1 protein was measured by immunocapillary electrophoresis (JESS), and H3K36me2 levels were measured by AlphaLISA®. Total cellular histone H3 levels were also measured by AlphaLISA®. Figure 13A shows mean ± SEM from the sum of two to three independent experiments, one-way ANOVA, * p<0.05, *** p<0.01, **** p<0.001. [Figure 13B]Figure 13B shows representative data demonstrating the dose-dependent effects of exemplary ASOs on NSD1 protein expression and H3K36me2 levels in U-87 MG cells. Figure 13B is a histogram showing the fold change in cellular H3K36me2 levels in U-87 MG cells treated with various dose concentrations of ASO211 (0.25 μM, 0.5 μM, 1.0 μM, or 2.0 μM) compared to cells treated with water alone. U-87 cells were nucleofected with ASO211 at the four tested doses (0.25 μM, 0.5 μM, 1.0 μM, or 2.0 μM), and cells were harvested 72 hours after nucleofection. NSD1 protein was measured by immunocapillary electrophoresis (JESS), and H3K36me2 levels were measured by AlphaLISA®. Total cellular histone H3 levels were also measured by AlphaLISA®. Figure 13B shows mean ± SEM from the sum of two to three independent experiments, one-way ANOVA, * p<0.05, *** p<0.01, **** p<0.001. [Figure 13C] Figure 13C shows representative data demonstrating the dose-dependent effects of exemplary ASOs on NSD1 protein expression and H3K36me2 levels in U-87 MG cells. Figure 13C is a histogram showing the total histone H3 levels present in U-87 cells treated with various dose concentrations of ASO 211 compared to cells treated with water alone. U-87 cells were nucleofected with ASO 211 at four tested doses (0.25 μM, 0.5 μM, 1.0 μM, or 2.0 μM), and cells were harvested 72 hours after nucleofection. NSD1 protein was measured by immunocapillary electrophoresis (JESS), and H3K36me2 levels were measured by AlphaLISA®. Total cellular histone H3 levels were also measured by AlphaLISA®. Figure 13C shows the mean ± SEM of the sum of 2-3 independent experiments, one-way ANOVA, * p value < 0.05, *** p value < 0.01, **** p value < 0.001. DETAILED DESCRIPTION OF THE INVENTION

[0170] Certain specific details are described herein to provide a thorough understanding of various embodiments. However, those skilled in the art will understand that the present disclosure may be practiced without these details. In other instances, well-known structures have not been shown or described in detail to avoid unnecessarily obscuring the description of the embodiments. Unless the context otherwise requires, throughout the following specification and claims, the term "comprise" and its variations, such as "comprises," "comprising," etc., should be interpreted in an open and inclusive sense, i.e., "including, but not limited to." Furthermore, the headings provided herein are merely for convenience and do not describe the scope or meaning of the disclosure claimed in the claims.

[0171] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be noted that the term "or" is normally used in its sense including "and / or" unless the context clearly dictates otherwise.

[0172] The scale used herein refers to the scale of the genome reference assembly GRCh38 (Genome Research Consortium Human Assembly 38), also known as Hg38 (Human Genome Assembly 38).

[0173] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below.

[0174] Alternative splicing events in the PKD1, ABCA4, FUS, CEL, or NSD1 genes can induce non-productive mRNA transcripts, which in turn can induce reduced protein expression, and therapeutic agents that can target alternative splicing events in the PKD1, ABCA4, FUS, CEL, or NSD1 genes can regulate (e.g., increase) the expression level of functional proteins in patients. Such therapeutic agents can be used to treat conditions caused by deficiencies in the amount or activity of polycystin-1, retina-specific phospholipid transport ATPase ABCA4, RNA-binding protein FUS, bile salt-activated lipase, or H3 lysine-36-specific histone lysine N-methyltransferase.

[0175] One alternative splicing event that can lead to non-productive mRNA transcripts is the alternative splicing of coding exons (ASCE).For example, the elimination of alternative splicing of coding exons can result in processed mRNAs that are shorter than the corresponding processed mRNAs that contain ASCE (the shorter processed mRNAs are also referred to herein as "alternatively processed mRNAs").For example, the skipping of alternative splicing of coding exons can result in processed mRNAs that are shorter than the corresponding processed mRNAs that contain ASCE. For example, reducing or inhibiting splicing of the 3' splice site (e.g., canonical 3'ss) of ASCE and / or excluding alternatively spliced ​​coding exons resulting from reducing or inhibiting splicing of the 5' splice site (e.g., canonical 5'ss) of ASCE can result in a processed mRNA that is shorter than the corresponding processed mRNA that includes ASCE. The present disclosure provides compositions and methods for modulating alternative splicing of PKD1, ABCA4, FUS, CEL, or NSD1 pre-mRNA, thereby increasing the production of protein-coding mature mRNA and, thereby, the production of translated functional polycystin-1, retina-specific phospholipid transport ATPase ABCA4, RNA-binding protein FUS, bile salt-activated lipase, or H3 lysine-36-specific histone-lysine N-methyltransferase. For example, the compositions and methods provided herein can regulate the processing of PKD1, ABCA4, FUS, CEL, or NSD1 pre-mRNA by promoting or increasing splicing of the 3' splice site (e.g., canonical 3'ss) of ASCE and / or by promoting or increasing splicing of the 5' splice site (e.g., canonical 5'ss) of ASCE.For example, the compositions and methods provided herein can regulate processing of PKD1, ABCA4, FUS, CEL, or NSD1 pre-mRNA by promoting or increasing splicing of the 3' splice site of the intron upstream of ASCE and / or by promoting or increasing splicing of the 5' splice site of the intron downstream of ASCE.

[0176] These compositions and methods include antisense oligomers (ASOs) or vectors encoding ASOs that can promote constitutive splicing of PKD1, ABCA4, FUS, CEL, or NSD1 pre-mRNA. For example, these compositions and methods include ASOs or vectors encoding ASOs that can promote the inclusion of ASCE in processed mRNAs processed from PKD1, ABCA4, FUS, CEL, or NSD1 pre-mRNA. In various embodiments, functional polycystin-1, retina-specific phospholipid-transfer ATPase ABCA4, RNA-binding protein FUS, bile salt-activated lipase, or H3 lysine-36-specific histone-lysine N-methyltransferase can be increased using the methods of the present disclosure to treat conditions caused by deficiencies in the amount or activity of polycystin-1, retina-specific phospholipid-transfer ATPase ABCA4, RNA-binding protein FUS, bile salt-activated lipase, or H3 lysine-36-specific histone-lysine N-methyltransferase proteins.

[0177] As referred to herein, "polycystin-1" or "PC1," also known as autosomal dominant polycystic kidney disease 1 protein, which may be encoded by the PKD1 gene, may be a component of a heteromeric calcium-permeable ion channel formed with polycystin-2 (encoded by the PKD2 gene) that is activated by interaction with Wnt family members, e.g., WNT3A and WNT9B, may be a membrane protein involved in cell-cell or cell-matrix interactions, and regulates multiple signaling pathways to maintain normal renal tubule structure and function, and includes either recombinant or naturally occurring forms of polycystin-1 or variants or homologs thereof that have or maintain polycystin-1 activity (e.g., at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, a variant or homologue has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to native polycystin-1. In some embodiments, the polycystin-1 is substantially identical to the protein identified by UniProt reference number P98161, or a variant or homologue having substantial identity thereto.

[0178] As referred to herein, "retinal-specific phospholipid-transporting ATPase ABCA4," also known as ATP-binding cassette subfamily A member 4, RIM ABC transporter (RIM protein or RmP), retina-specific ATP-binding cassette transporter, or Stargardt disease protein, may be encoded by the ABCA4 gene (also known as ABCR), and may be a membrane-associated protein that is a member of the superfamily of ATP-binding cassette (ABC) transporters, which may be a retina-specific ABC transporter that has N-retinylidene-PE as a substrate, and which may be expressed exclusively in retinal photoreceptor cells and mediate the transport of an essential molecule, all-trans retinaldehyde (atRAL), across the photoreceptor cell membrane, and which has or maintains (e.g., at least 40%) retinal-specific phospholipid-transporting ATPase ABCA4 activity. The present invention also includes recombinant or native forms of the retina-specific phospholipid transport ATPase ABCA4, or variants or homologs thereof, having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some embodiments, the variant or homolog has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 contiguous amino acid portion) compared to the native retina-specific phospholipid transport ATPase ABCA4. In some embodiments, the retina-specific phospholipid transporting ATPase ABCA4 is substantially identical to the protein identified by UniProt reference number P78363, or a variant or homolog having substantial identity thereto.

[0179] As referred to herein, "RNA binding protein FUS," also known as FUS RNA binding protein, 75 kDa DNA-pairing protein, oncogene FUS, oncogene TLS, POMp75, or Translocated in liposarcoma protein, may be encoded by the FUS gene (also known as TLS), and may be a DNA / RNA binding protein that plays a role in various cellular processes such as transcription regulation, RNA splicing, RNA transport, DNA repair, and damage response, and includes any recombinant or naturally occurring form of the RNA binding protein FUS, or a variant or homolog thereof, that has or maintains the activity of the RNA binding protein FUS (e.g., at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variant or homolog has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to the naturally occurring RNA-binding protein FUS. In some embodiments, the RNA-binding protein FUS is substantially identical to the protein identified by UniProt reference number P35637, or a variant or homolog having substantial identity thereto.

[0180] As referred to herein, "bile salt-activated lipase," also known as carboxyl ester lipase, bile salt-stimulated lipase (BSSL), Bucelipase, cholesterol esterase, pancreatic lysophospholipase, or sterol esterase, may be encoded by the CEL gene (also known as BAL), and is capable of catalyzing the hydrolysis of a wide range of substrates, including cholesteryl esters, phospholipids, lysophospholipids, diacylglycerols and triacylglycerols, and fatty acid esters of hydroxy fatty acids (FAHFAs), and includes recombinant or naturally occurring forms of bile salt-activated lipase, or variants or homologs thereof, that have or maintain bile salt-activated lipase activity (e.g., at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, the variant or homolog has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to a naturally occurring bile salt-activated lipase. In some embodiments, the bile salt-activated lipase is substantially identical to a protein identified by UniProt reference number P19835, or a variant or homolog having substantial identity thereto.

[0181] As referred to herein, "H3 lysine-36-specific histone-lysine N-methyltransferase," also known as androgen receptor coactivator 267 kDa protein, 267 kDa androgen receptor-associated protein, H3-K36-HMTase (histone methyltransferase), lysine N-methyltransferase 3B, nuclear receptor-associated SET domain-containing protein 1 (NR-associated SET domain-containing protein), which may be encoded by the NSD1 gene (also known as ARA267 and KMT3B), may be a histone methyltransferase that dimethylates Lys-36 of histone H3 (H3K36me2), may be a transcriptional intermediary factor that can both negatively and positively affect transcription depending on cellular context, and has or maintains (e.g., at least 40%) the activity of an H3 lysine-36-specific histone-lysine N-methyltransferase. The present invention also includes recombinant or naturally occurring forms of H3 lysine-36-specific histone-lysine N-methyltransferase, or variants or homologs thereof, having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity), or variants or homologs thereof. In some embodiments, the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 contiguous amino acid portion) compared to a naturally occurring H3 lysine-36-specific histone-lysine N-methyltransferase. In some embodiments, the H3 lysine-36-specific histone-lysine N-methyltransferase is substantially identical to the protein identified by UniProt reference number Q96L73, or a variant or homolog having substantial identity thereto.

[0182] The terms "alternatively spliced ​​coding exon" or "ASCE" are used interchangeably and may refer to a coding exon (e.g., a canonical exon) that, when present in a mature RNA transcript, can prevent activation of the nonsense-mediated mRNA decay (NMD) pathway, or, when absent in a mature RNA transcript, can promote activation of the NMD pathway. In constitutive splicing events, ASCEs are not normally spliced ​​out, but ASCEs can be excluded during alternative or aberrant splicing events. Mature mRNA transcripts lacking ASCEs can be nonproductive, for example, due to frameshifts that trigger the NMD pathway. In some embodiments, ASCEs are skipped exons. In some embodiments, ASCEs are exons that induce reading frame alterations when the ASCE is not included in a mature or processed mRNA. In some embodiments, ASCEs are exons containing a number of nucleotides not evenly divisible by 3. In some embodiments, the ASCE-excluded mature or processed mRNA contains a premature stop codon (or premature termination codon (PTC)) or other sequence that promotes degradation of the ASCE-excluded mature RNA transcript. Exclusion of ASCE in the mature or processed RNA transcript can downregulate gene expression. In some embodiments, the ASCE-excluded mature or processed mRNA is generated from an alternative splicing event. For example, the ASCE-excluded mature or processed mRNA can be generated from an alternative 3' splice site event. For example, the ASCE-excluded mature or processed mRNA can be generated from an alternative 5' splice site event. For example, the ASCE-excluded mature or processed mRNA can be generated from an alternative 5' splice site event and an alternative 3' splice site event.For example, the mature mRNA or processed mRNA that excludes ASCE can be generated from exon skipping events.For example, ASCE can be a canonical exon.For example, only exons that are evenly divisible by 3 can be skipped or included in mRNA without any change in reading frame.

[0183] Alternative splicing can result in the omission of at least one ASCE in the mature mRNA transcript. The terms "mature mRNA" and "fully spliced ​​mRNA" are used interchangeably herein to describe fully processed mRNA. Mature mRNA lacking ASCE is a non-productive mRNA and can induce NMD of mature mRNA. Mature mRNA lacking ASCE may induce reduced protein expression compared to the protein expression from the corresponding mature mRNA containing ASCE.

[0184] Pseudo-splice sites share the same splicing recognition sequence as true splice sites but are not used in splicing reactions. They are orders of magnitude more abundant than true splice sites in the human genome and are typically suppressed by poorly understood molecular mechanisms. Cryptic 5' splice sites have the consensus sequence NNN / GUNNNN or NNN / GCNNNN, where N is any nucleotide and / or is an exon-intron boundary. Cryptic 3' splice sites have the consensus sequence NAG / N. Their activity is positively influenced by the surrounding nucleotides, which are made more similar to the optimal consensus sequences of standard splice sites, i.e., MAG / GURAGU and YAG / G, respectively, where M is C or A, R is G or A, and Y is C or U.

[0185] Splice sites and their regulatory sequences can be readily identified by those skilled in the art using suitable publicly available algorithms, for example, those listed in Kralovicova, J. and Vorechovsky, I. (2007) Global control of aberrant splice site activation by auxiliary splicing sequences: evidence for a gradient in exon and intron definition. Nucleic Acids Res., 35, 6399-6413, (ncbi.nlm.nih.gov / pmc / articles / PMC2095810 / pdf / gkm680.pdf).

[0186] Splicing and nonsense-mediated mRNA decay The intervening sequences, or introns, are removed by a large and highly dynamic RNA-protein complex called the spliceosome, which orchestrates complex interactions between the primary transcript, small nuclear RNAs (snRNAs), and numerous proteins. The spliceosome assembles each intron in an orderly manner, starting with recognition of the 5' splice site (5'ss) by U1 snRNA or the 3' splice site (3'ss) by the U2 pathway, which involves binding of the U2 auxiliary factor (U2AF) to the 3'ss region to facilitate U2 binding to the branch point sequence (BPS). U2AF is a stable heterodimer composed of a 65-kD subunit (U2AF65) encoding U2AF-2, which binds to polypyrimidine tracts (PPTs), and a 35-kD subunit (U2AF35) encoding U2AF-1, which interacts with a highly conserved AG dinucleotide at the 3'ss to stabilize U2AF65 binding. In addition to the BPS / PPT unit and 3'ss / 5'ss, accurate splicing requires auxiliary sequences or structures that activate or repress splice site recognition, known as intronic or exonic splicing enhancers or splicing silencers. These elements enable true splice site recognition among the abundant and redundant cryptic or pseudosplice sites in the genomes of higher eukaryotes, which share sequences identical to the canonical sites but outnumber them by orders of magnitude. Although these elements often have regulatory functions, the precise mechanisms of their activation or repression are poorly understood.

[0187] The splice decision can be designed as a stochastic rather than a deterministic process, and even the most obvious splicing signals can be spliced ​​incorrectly. However, under normal conditions, pre-mRNA splicing proceeds with remarkable fidelity. This is due, in part, to the activity of adjacent cis-acting auxiliary exonic and intronic splicing regulatory elements (ESRs or ISRs). These functional elements are typically classified as either exonic or intronic splicing enhancers (ESEs or ISEs) or exonic or intronic splicing silencers (ESSs or ISSs) based on their ability to stimulate or inhibit splicing, respectively. While there is evidence that some auxiliary cis-acting elements may act by influencing the dynamics of spliceosome assembly, such as the positioning of the U1 snRNP and 5'ss complex, many elements most likely function in concert with trans-acting RNA-binding proteins (RBPs). For example, the serine-arginine-rich family of RBPs (SR proteins) is a conserved protein family that plays an important role in defining exons. SR proteins promote exon recognition by recruiting pre-spliceosomal components to adjacent splice sites or by attenuating the effects of nearby ESSs. The repressive effect of ESSs can be mediated by members of the heterologous nuclear ribonucleoprotein (hnRNP) family, which can alter the recruitment of core splicing factors to adjacent splice sites. In addition to their role in splicing regulation, silencer elements have been suggested to play a role in the suppression of pseudoexons, which are collections of decoy intron splice sites that have the typical spacing of exons but lack a functional open reading frame.ESEs and ESSs, in conjunction with their cognate trans-acting RBPs, are key components in the splicing regulatory chain that specifies how, where, and when mRNA is assembled from its precursor.

[0188] Sequences that define exon and intron boundaries are degenerate signals of varying strength that occur frequently within human genes. In multi-exon genes, multiple splice site pairs are joined in numerous different combinations, resulting in the generation of an array of diverse transcripts from a single gene. This is commonly referred to as alternative pre-mRNA splicing. While the majority of mRNA isoforms produced by alternative splicing can be exported from the nucleus and translated into functional polypeptides, the translation efficiency of various mRNA isoforms from a single gene can vary greatly. mRNA isoforms that contain premature stop codons (PTCs) or premature stop codons at least 50 bp upstream from the exon junction complex are likely to be targeted for degradation by the nonsense-mediated mRNA decay (NMD) pathway. Mutations in conventional (BPS / PPT / 3'ss / 5'ss) and auxiliary splicing motifs can lead to aberrant splicing, e.g., exon skipping, inclusion of cryptic exons (or pseudoexons), or activating splice sites, and can have a profound impact on human morbidity and mortality. Both aberrant and alternative splicing patterns can be influenced by naturally occurring DNA variants within exons and introns.

[0189] Considering that exon-intron boundaries can occur at any of three codon positions, it is clear that only a subset of alternative splicing events can maintain the canonical open reading frame. For example, only exons evenly divisible by three can be skipped or included in an mRNA without any change in the reading frame. Splicing events that do not have compatible phases will induce a frameshift. Unless reversed by downstream events, a frameshift will inevitably induce one or more PTCs, almost certainly leading to their subsequent degradation by NMD. NMD is a translation-coupled mechanism that eliminates mRNAs containing PTCs. NMD may function as a surveillance pathway present in all eukaryotes. NMD can reduce errors in gene expression by eliminating mRNA transcripts containing premature stop codons or PTCs. When these aberrant mRNAs are translated, they can potentially induce harmful gain-of-function or blockade-of-function activities in the resulting proteins. NMD targets not only transcripts with PTCs but also a wide range of mRNA isoforms expressed from many endogenous genes, suggesting that NMD is a master regulator driving both fine and coarse regulation of steady-state RNA levels in cells.

[0190] In some cases, the therapeutic agent comprises a modified snRNA, such as a modified human snRNA or mouse snRNA. In some cases, the therapeutic agent comprises a vector, such as a viral vector, encoding the modified snRNA. In some embodiments, the modified snRNA is a modified U1 snRNA (see, e.g., Alanis et al., Human Molecular Genetics, 2012, Vol. 21, No. 11 2389-2398). In some embodiments, the modified snRNA is a modified U7 snRNA (see, e.g., Gadgil et al., J Gene Med. 2021;23:e3321). Modified U7 snRNA can be generated by any method known in the art, including the method described in Meyer, K.; Schumperli, Daniel (2012), Antisense Derivatives of U7 Small Nuclear RNA as Modulators of Pre-mRNA Splicing. In: Stamm, Stefan; Smith, Christopher WJ; Luhrmann, Reinhard (eds.) Alternative pre-mRNA Splicing: Theory and Protocols (pp. 481-494), Chichester: John Wiley & Sons 10.1002 / 9783527636778.ch45, which is incorporated herein by reference in its entirety. In some embodiments, modified U7 (smOPT) does not compete with wild-type U7 (WT U7) (Stefanovic et al., 1995).

[0191] In some embodiments, the modified snRNA comprises a smOPT modification. For example, the modified snRNA can comprise the sequence AAUUUUUGGAG. For example, the sequence AAUUUUUGGAG can replace the sequence AAUUUGUCUAG in wild-type U7 snRNA to generate modified U7 snRNA (smOPT). In some embodiments, the smOPT modification of U7 snRNA renders the particle functionally inactive in histone pre-mRNA processing (Stefanovic et al., 1995). In some embodiments, modified U7 (smOPT) is expressed more stably and at higher levels in the nucleus than wild-type U7 (WT U7) (Stefanovic et al., 1995). In some embodiments, the snRNA comprises a U1 snRNP target sequence. In some embodiments, the snRNA comprises a U7 snRNP target sequence. In some embodiments, the snRNA comprises a modified U7 snRNP target sequence, and the modified U7 snRNP target sequence comprises smOPT. In some embodiments, the modified snRNA is modified to include a single-stranded nucleotide sequence that hybridizes to a pre-mRNA, such as an ASCE-containing pre-mRNA. For example, the modified snRNA can be modified to include a single-stranded nucleotide sequence that hybridizes to a PKD1, ABCA4, FUS, CEL, or NSD1 pre-mRNA. In some embodiments, the modified snRNA is designed according to the format described in Table 5C or Table 5F. In some cases, the modified snRNA containing a U7 snRNP target sequence is designed according to the format described in Table 5C. In some cases, the modified snRNA containing a U1 snRNP target sequence is designed according to the format described in Table 5F. In some embodiments, the U7 snRNP target sequence includes a single-stranded nucleotide sequence that hybridizes to a PKD1, ABCA4, FUS, CEL, or NSD1 pre-mRNA, and the single-stranded nucleotide sequence begins with the dinucleotide AA, such as the sequences in Tables 5A-1, 5B-1, and 5G-1.In some of these embodiments, when designing a single-stranded nucleotide sequence that is complementary to a target sequence in a target pre-mRNA (e.g., a PKD1, ABCA4, FUS, CEL, or NSD1 pre-mRNA), if the sequence complementary to the target sequence begins with a nucleotide other than the dinucleotide AA on the 5' end, then the dinucleotide AA will be added to its 5' end, and if the sequence complementary to the target sequence begins with a single A nucleotide on the 5' end followed by a non-A nucleotide, then a single A will be added to its 5' end. In some other cases, if the sequence complementary to the target sequence begins with the dinucleotide AA on the 5' end, then no additional A nucleotide will be added.

[0192] In some embodiments, the modified snRNA is modified to include a single-stranded nucleotide sequence that hybridizes to an ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1. In some embodiments, the modified snRNA is modified to include a single-stranded nucleotide sequence that includes one or more sequences of an ASO disclosed herein. In some embodiments, the modified snRNA is modified to include a single-stranded nucleotide sequence that hybridizes to a sequence of a mutant pre-mRNA, such as an ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1. In some embodiments, the modified snRNA is modified to include a single-stranded nucleotide sequence that includes two or more sequences that hybridize to two or more target regions of an ASCE-containing pre-mRNA, such as an ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1. For example, the modified snRNA can be modified to include a single-stranded nucleotide sequence that hybridizes to at least eight contiguous nucleic acids of an ASCE-containing pre-mRNA, such as an ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1. In some embodiments, the modified snRNA is modified to include a single-stranded nucleotide sequence that hybridizes to any of the target regions of an ASCE-containing pre-mRNA, such as an ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1 disclosed herein. In some embodiments, the modified snRNA is modified to include a single-stranded nucleotide sequence that includes two or more sequences that hybridize to two or more target regions of an ASCE-containing pre-mRNA, such as an ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1.For example, the modified snRNA can be modified to include a single-stranded nucleotide sequence that hybridizes to one or more sequences in the intron upstream of ASCE, the intron downstream of ASCE, the exon upstream of ASCE, the exon downstream of ASCE, or within ASCE of an ASCE-containing pre-mRNA, such as an ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1, or to an ASCE skipping regulatory sequence within an ASCE-containing pre-mRNA. For example, the modified snRNA can be modified to include a single-stranded nucleotide sequence that hybridizes to one or more sequences in the intron upstream of ASCE. For example, the modified snRNA can be modified to include a single-stranded nucleotide sequence that hybridizes to one or more sequences in the intron downstream of ASCE. For example, the modified snRNA can be modified to include a single-stranded nucleotide sequence that hybridizes to one or more sequences in the exon upstream of ASCE. For example, the modified snRNA can be modified to include a single-stranded nucleotide sequence that hybridizes to one or more sequences in an exon downstream of the ASCE. For example, the modified snRNA can be modified to include a single-stranded nucleotide sequence that hybridizes to one or more sequences within the ASCE.For example, the modified snRNA may be a nucleic acid sequence encoding one or more sequences within an intron upstream of the ASCE, an intron downstream of the ASCE, an exon upstream of the ASCE, an exon downstream of the ASCE, or an ASCE sequence of an ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1 (e.g., exon 38 of PKD1 (e.g., exon (GRCh38 / hg38:chr16 2092954 2093093)), exon 3 of ABCA4 (e.g., exon (GRCh38 / hg38:chr1 94111438 94111579)), exon 7 of FUS (e.g., exon (GRCh38 / hg38:chr16 31186802 31186836)), for example exon 5 of CEL (e.g., exon (GRCh38 / hg38:chr9 133066530 133066660)), for example exon 8 of NSD1 (e.g., exon (GRCh38 / hg38:chr5 177238237 177238507)). For example, the modified snRNA may be located in a region within ASCE or in a region upstream or downstream of ASCE (e.g., exon 38 of PKD1 (e.g., exon (GRCh38 / hg38:chr16 2092954 2093093)), e.g., exon 3 of ABCA4 (e.g., exon (GRCh38 / hg38:chr1 94111438 94111579)), e.g., exon 7 of FUS (e.g., exon (GRCh38 / hg38:chr16 31186802 31186836)), e.g., exon 5 of CEL (e.g., exon (GRCh38 / hg38:chr9 133066530 133066660)), for example, exon 8 of NSD1 (for example, exon (GRCh38 / hg38:chr5 177238237 177238507))).In some embodiments, the modified snRNA has a 5' region that is modified to include a single-stranded nucleotide sequence that hybridizes to an ASCE-containing pre-mRNA, such as an ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1. In some embodiments, the modified snRNA has a 3' region that is modified to include a single-stranded nucleotide sequence that hybridizes to an ASCE-containing pre-mRNA, such as an ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1.

[0193] For example, modified snRNAs may be present in regions that do not overlap with ASCE and introns upstream of ASCE (e.g., exon 38 of PKD1 (e.g., exon (GRCh38 / hg38:chr16 2092954 2093093)), exon 3 of ABCA4 (e.g., exon (GRCh38 / hg38:chr1 94111438 94111579)), exon 7 of FUS (e.g., exon (GRCh38 / hg38:chr16 31186802 31186836)), exon 5 of CEL (e.g., exon (GRCh38 / hg38:chr9 133066530)), or regions that do not overlap with ASCE (e.g., exon 38 of PKD1 (e.g., exon (GRCh38 / hg38:chr16 2092954 2093093)), exon 3 of ABCA4 (e.g., exon (GRCh38 / hg38:chr1 94111438 94111579)), exon 7 of FUS (e.g., exon (GRCh38 / hg38:chr16 31186802 31186836)), or exon 5 of CEL (e.g., exon (GRCh38 / hg38:chr9 133066530)). 133066660)), for example, exon 8 of NSD1 (for example, exon (GRCh38 / hg38:chr5 177238237 177238507))). For example, modified snRNAs may be present in regions overlapping with ASCE and introns downstream of ASCE (e.g., exon 38 of PKD1 (e.g., exon (GRCh38 / hg38:chr16 2092954 2093093)), exon 3 of ABCA4 (e.g., exon (GRCh38 / hg38:chr1 94111438 94111579)), exon 7 of FUS (e.g., exon (GRCh38 / hg38:chr16 31186802 31186836)), exon 5 of CEL (e.g., exon (GRCh38 / hg38:chr9 133066530)), and regions overlapping with ASCE downstream introns (e.g., exon 38 of PKD1 (e.g., exon (GRCh38 / hg38:chr16 2092954 2093093)), exon 4 of ABCA4 (e.g., exon (GRCh38 / hg38:chr1 94111438 94111579)), exon 5 of FUS (e.g., exon (GRCh38 / hg38:chr16 31186802 31186836)), and regions overlapping with ASCE downstream introns (e.g., exon (GRCh38 / hg38:chr9 133066530)). 133066660)), for example, exon 8 of NSD1 (for example, exon (GRCh38 / hg38:chr5 177238237 177238507))).

[0194] For example, the modified snRNA may be a target of an exon or intron sequence downstream of ASCE (e.g., exon 38 of PKD1 (e.g., exon (GRCh38 / hg38:chr16 2092954 2093093)), exon 3 of ABCA4 (e.g., exon (GRCh38 / hg38:chr1 94111438 94111579)), exon 7 of FUS (e.g., exon (GRCh38 / hg38:chr16 31186802 31186836)), or exon 5 of CEL (e.g., exon (GRCh38 / hg38:chr9 133066530)). 133066660)), for example, exon 8 of NSD1 (e.g., exon (GRCh38 / hg38:chr5 177238237 177238507))). For example, modified snRNAs may be expressed in intronic sequences downstream of ASCE (e.g., exon 38 of PKD1 (e.g., exon (GRCh38 / hg38:chr16 2092954 2093093)), exon 3 of ABCA4 (e.g., exon (GRCh38 / hg38:chr1 94111438 94111579)), exon 7 of FUS (e.g., exon (GRCh38 / hg38:chr16 31186802 31186836)), exon 5 of CEL (e.g., exon (GRCh38 / hg38:chr9 133066530)), etc.). 133066660)), for example, to contain a single-stranded nucleotide sequence that is not complementary to the 3' splice site of exon 8 of NSD1 (e.g., exon (GRCh38 / hg38:chr5 177238237 177238507)) of NSD1).For example, modified snRNAs may be expressed in intronic sequences downstream of ASCE (e.g., exon 38 of PKD1 (e.g., exon (GRCh38 / hg38:chr16 2092954 2093093)), exon 3 of ABCA4 (e.g., exon (GRCh38 / hg38:chr1 94111438 94111579)), exon 7 of FUS (e.g., exon (GRCh38 / hg38:chr16 31186802 31186836)), exon 5 of CEL (e.g., exon (GRCh38 / hg38:chr9 133066530)), etc.). 133066660)), for example, exon 8 of NSD1 (e.g., exon (GRCh38 / hg38:chr5 177238237 177238507))).

[0195] For example, modified snRNAs may be expressed in intronic sequences upstream of ASCE (e.g., exon 38 of PKD1 (e.g., exon (GRCh38 / hg38:chr16 2092954 2093093)), exon 3 of ABCA4 (e.g., exon (GRCh38 / hg38:chr1 94111438 94111579)), exon 7 of FUS (e.g., exon (GRCh38 / hg38:chr16 31186802 31186836)), exon 5 of CEL (e.g., exon (GRCh38 / hg38:chr9 133066530)), etc. 133066660)), for example, exon 8 of NSD1 (e.g., exon (GRCh38 / hg38:chr5 177238237 177238507))). For example, modified snRNAs may be expressed in intronic sequences upstream of ASCE (e.g., exon 38 of PKD1 (e.g., exon (GRCh38 / hg38:chr16 2092954 2093093)), exon 3 of ABCA4 (e.g., exon (GRCh38 / hg38:chr1 94111438 94111579)), exon 7 of FUS (e.g., exon (GRCh38 / hg38:chr16 31186802 31186836)), exon 5 of CEL (e.g., exon (GRCh38 / hg38:chr9 133066530)), etc. 133066660)), for example, to contain a single-stranded nucleotide sequence that is not complementary to a splice site of exon 8 of NSD1 (for example, exon (GRCh38 / hg38:chr5 177238237 177238507)) of NSD1).For example, modified snRNAs may be expressed in intronic sequences upstream of ASCE (e.g., exon 38 of PKD1 (e.g., exon (GRCh38 / hg38:chr16 2092954 2093093)), exon 3 of ABCA4 (e.g., exon (GRCh38 / hg38:chr1 94111438 94111579)), exon 7 of FUS (e.g., exon (GRCh38 / hg38:chr16 31186802 31186836)), exon 5 of CEL (e.g., exon (GRCh38 / hg38:chr9 133066530)), etc. 133066660)), for example, to contain a single-stranded nucleotide sequence that is not complementary to the 3' splice site of exon 8 of NSD1 (e.g., exon (GRCh38 / hg38:chr5 177238237 177238507)) of NSD1). For example, modified snRNAs may be expressed in intronic sequences upstream of ASCE (e.g., exon 38 of PKD1 (e.g., exon (GRCh38 / hg38:chr16 2092954 2093093)), exon 3 of ABCA4 (e.g., exon (GRCh38 / hg38:chr1 94111438 94111579)), exon 7 of FUS (e.g., exon (GRCh38 / hg38:chr16 31186802 31186836)), exon 5 of CEL (e.g., exon (GRCh38 / hg38:chr9 133066530)), etc. 133066660)), for example, to contain a single-stranded nucleotide sequence that is not complementary to the 5' splice site of exon 8 of NSD1 (e.g., exon (GRCh38 / hg38:chr5 177238237 177238507) of NSD1).

[0196] Methods for identifying additional ASOs that promote splicing at the canonical 3' splice site and / or that promote splicing at the canonical 5' splice site Also within the scope of the present disclosure are methods for identifying or determining therapeutic agents, such as ASOs, that promote splicing of ASCE-containing pre-mRNAs, such as ASCE-containing pre-mRNAs of PKD1, ABCA4, FUS, CEL, or NSD1, at the canonical 3' splice site of ASCE, promote splicing of the canonical 3' splice site of an intron upstream of ASCE, promote splicing of the canonical 5' splice site of ASCE, and / or promote splicing of the canonical 5' splice site of an intron downstream of ASCE. For example, the method may include identifying or determining an ASO that inhibits or reduces ASCE skipping of ASCE-containing pre-mRNAs, such as ASCE-containing pre-mRNAs of PKD1, ABCA4, FUS, CEL, or NSD1. ASOs that specifically hybridize to different nucleotides within the target region of a pre-mRNA can be screened to identify or determine ASOs that improve the rate and / or extent of splicing at the canonical 3' splice site of ASCE, the canonical 3' splice site of an intron upstream of ASCE, the canonical 5' splice site of ASCE, and / or the canonical 5' splice site of an intron downstream of ASCE, and / or that reduce the rate and / or extent of splicing at the alternative 3' splice site and / or the alternative 5' splice site of ASCE. In some embodiments, the ASO may block or interfere with the binding site of a splicing repressor / silencer. Any method known in the art can be used to identify (determine) an ASO that, when hybridized to a target region, produces a desired effect (e.g., promoting splicing at the canonical 3' splice site of ASCE, promoting splicing at the canonical 3' splice site of an intron upstream of ASCE, promoting splicing at the canonical 5' splice site of ASCE, promoting splicing at the canonical 5' splice site of an intron downstream of ASCE, protein production or production of functional RNA).These methods can also be used to identify ASOs that promote or increase inclusion of ASCE by binding to target regions adjacent to or within ASCE. Examples of methods that can be used are provided below.

[0197] A series of screenings, referred to as ASO "walks", can be carried out using ASOs designed to hybridize to target regions of pre-mRNA. For example, the ASOs used in the ASO walks can be tiled every 5 nucleotides from approximately 100 nucleotides upstream of the 3' or 5' splice site of ASCE to approximately 100 nucleotides downstream of the 3' or 5' splice site of ASCE. For example, the ASOs used in the ASO walks can be tiled every 5 nucleotides from approximately 100 nucleotides upstream of the 5' splice site of the intron following ASCE to approximately 100 nucleotides downstream of the 3' splice site of the intron following ASCE. For example, the ASOs used in the ASO walks can be tiled every 5 nucleotides from approximately 100 nucleotides upstream of the 3' splice site of the intron preceding ASCE to approximately 100 nucleotides downstream of the 5' splice site of the intron preceding ASCE. For example, the ASOs used in the ASO walk can be tiled every 5 nucleotides from approximately 100 nucleotides upstream of the 5' splice site of the intron following ASCE to approximately 100 nucleotides downstream of the 5' splice site of the intron following ASCE.For example, the ASOs used in the ASO walk can be tiled every 5 nucleotides from approximately 100 nucleotides upstream of the 3' splice site of the intron following ASCE to approximately 100 nucleotides downstream of the 3' splice site of the intron following ASCE.For example, the ASOs used in the ASO walk can be tiled every 5 nucleotides from approximately 100 nucleotides upstream of the 3' splice site of the intron preceding ASCE to approximately 100 nucleotides downstream of the 3' splice site of the intron preceding ASCE.For example, the ASOs used in the ASO walk can be tiled every 5 nucleotides from approximately 100 nucleotides upstream of the 5' splice site of the intron preceding ASCE to approximately 100 nucleotides downstream of the 5' splice site of the intron preceding ASCE.For example, ASOs used in an ASO walk can be tiled every 5 nucleotides from approximately 100 nucleotides upstream of the 3' or 5' splice site of ASCE to approximately 100 nucleotides downstream of the 3' or 5' splice site of ASCE. For example, a first ASO 15 nucleotides in length can be designed to specifically hybridize to nucleotides +6 to +20 relative to the 3' splice site of the intron preceding ASCE. A second ASO can be designed to specifically hybridize to nucleotides +11 to +25 relative to the 3' splice site of the intron preceding ASCE. ASOs are designed to span the target region of the pre-mRNA. In embodiments, ASOs can be tiled more closely, for example, every 1, 7, 8, or 9 nucleotides. Furthermore, ASOs can be tiled from 100 nucleotides downstream of the 5' splice site to 100 nucleotides upstream of the 3' splice site. In some embodiments, the ASO may be tiled from about 500 nucleotides upstream of the 3' splice site to about 500 nucleotides downstream of the 5' splice site, hi some embodiments, the ASO may be tiled from about 500 nucleotides upstream of the 3' splice site to about 500 nucleotides downstream of the 3' splice site.

[0198] One or more ASOs, or a control ASO (e.g., an ASO with a scrambled sequence, an ASO with a sequence not expected to hybridize to the target region), can be delivered, for example, by transfection, to a disease-related cell line expressing the target pre-mRNA (e.g., an ASCE-containing pre-mRNA described herein). The effect of each ASO on inhibiting exon skipping or promoting ASCE inclusion can be assessed by any method known in the art, for example, by reverse transcriptase (RT)-PCR using primers spanning the splice junction. An increase or presence of longer RT-PCR products produced using primers spanning the ASCE-containing region (e.g., including the exons flanking the ASCE) in ASO-treated cells compared to control ASO-treated cells indicates suppression of splicing out of the target ASCE. In some embodiments, the efficiency of exon skipping inhibition, the ratio of unspliced ​​to spliced ​​pre-mRNA, the reduction in splicing rate, or the reduction in the extent of splicing can be improved using the ASOs described herein. The amount of protein or functional RNA encoded by the target pre-mRNA can also be evaluated to determine whether each ASO has achieved the desired effect (e.g., improved production of functional protein). Any method known in the art for assessing and / or quantifying protein production can be used, such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA.

[0199] A second series of screens, termed ASO "microwalks," can be performed using ASOs designed to hybridize to target regions of pre-mRNA. The ASOs used in the ASO microwalks are tiled nucleotide by nucleotide to further refine the nucleotide acid sequences of pre-mRNA that, when hybridized with the ASO, promote the inclusion of ASCE in mature RNA transcripts and / or inhibit or reduce skipping of ASCE from ASCE-containing pre-mRNA transcripts.

[0200] The region defined by the ASO that promotes ASCE inclusion in the mature RNA transcript is explored in more detail by ASO “microwalks” with ASOs spaced every 1 nt, as well as longer ASOs, typically 18–25 nt.

[0201] As described above with respect to ASO walks, ASO microwalks are performed by delivering one or more ASOs, or a control ASO (e.g., an ASO with a scrambled sequence, an ASO with a sequence not expected to hybridize to the target region), for example, by transfection, into a disease-related cell line expressing the target pre-mRNA. The splicing-inducing effect of each ASO can be assessed by any method known in the art, such as by reverse transcriptase (RT)-PCR using primers spanning the ASCE described herein. An increase or presence of longer RT-PCR products produced using primers spanning the ASCE-containing region (e.g., including exons flanking the ASCE) in ASO-treated cells compared to control ASO-treated cells indicates suppression of splicing out of the target ASCE. In some embodiments, the efficiency of exon skipping inhibition, the ratio of unspliced ​​to spliced ​​pre-mRNA, the reduction in splicing rate, or the reduction in the extent of splicing can be improved using the ASOs described herein. The amount of protein or functional RNA encoded by the target pre-mRNA can also be evaluated to determine whether each ASO has achieved the desired effect (e.g., improved production of functional protein). Any method known in the art for assessing and / or quantifying protein production can be used, such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA.

[0202] ASOs that hybridize to a region of a pre-mRNA and promote the inclusion of ASCE in the mature RNA transcript and / or inhibit or reduce the skipping of ASCE from ASCE-containing pre-mRNA transcripts, and increase protein production, can be tested in vivo using animal models, such as transgenic mouse models with full-length human genes knocked in, or humanized mouse models of disease. The preferred route of ASO administration can vary depending on the disease and / or the cell type to which the ASO is desired to be delivered. The ASO can be administered, for example, by intrathecal, intracerebroventricular, intraperitoneal, intramuscular, subcutaneous, or intravenous injection. After administration, the cells, tissues, and / or organs of the model animal can be evaluated to determine the effectiveness of ASO treatment, for example, by assessing splicing (e.g., efficiency, rate, extent) and protein production using methods known in the art and described herein. Animal models can also be any phenotypic or behavioral indicator of disease or disease severity.

[0203] Also within the scope of this disclosure are methods for identifying or validating ASCE in the presence of an NMD inhibitor, for example, cycloheximide. An exemplary method is provided in Example 2.

[0204] Exemplary genes encoding ASCE-containing pre-mRNAs and ASCE sequences are summarized in Tables 1 and 2 (SEQ ID NO: indicates the corresponding nucleotide sequence represented by the gene ID number (NCBI Entrez Gene Number)). The sequences of exemplary target sequences within the pre-mRNA transcripts are shown in Table 3. Exemplary ASO sequences are shown in Table 4.

[0205] [Table 1]

[0206] [Table 2]

[0207]

Table 3

[0208]

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4-5

Table 4-6

[0209]

Table 5-1

Table 5-2

Table 5-3

Table 5-4

[0210]

Table 6-1

Table 6-2

Table 6-3

[0211]

Table 7-1

Table 7-2

Table 7-3

Table 7-4

Table 7-5

Table 7-6

Table 7-7

Table 7-8

Table 7-9

Table 7-10

Table 7-11

Table 7-12

Table 7-13

Table 7-14

Table 7-15

Table 7-16

Table 7-17

Table 7-18

Table 7-19

[0212]

Table 8-1

Table 8-2

Table 8-3

Table 8-4

Table 8-5

Table 8-6

Table 8-7

Table 8-8

Table 8-9

Table 8-10

[0213]

Table 9-1

Table 9-2

[0214]

Table 10-1

Table 10-2

Table 10-3

[0215]

Table 11-1

Table 11-2

Table 11-3

Table 11-4

Table 11-5

Table 11-6

Table 11-7

Table 11-8

Table 11-9

Table 11-10

Table 11-11

Table 11-12

Table 11-13

Table 11-14

[0216] [Table 12-1] [Table 12-2]

[0217] [Table 13-1] [Table 13-2] [Table 13-3]

[0218] [Table 14-1] [Table 14-2] [Table 14-3]

[0219] Alternative splicing events in the PKD1, ABCA4, FUS, CEL, or NSD1 genes can induce nonproductive mRNA transcripts that subsequently induce abnormal protein expression, and therapeutic agents that target alternative splicing events in the PKD1, ABCA4, FUS, CEL, or NSD1 genes can regulate functional protein expression levels and / or inhibit abnormal protein expression in DS patients. Such therapeutic agents can be used to treat conditions caused by deficiencies of polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxyl ester lipase, or nuclear receptor-associated SET domain protein 1.

[0220] One alternative splicing event that can lead to nonproductive mRNA transcripts is the inclusion of extra exons in mRNA transcripts, which can trigger nonsense-mediated mRNA decay. The present disclosure provides compositions and methods that modulate alternative splicing of PKD1, ABCA4, FUS, CEL, or NSD1 to increase the production of protein-coding mature mRNA and, thereby, the production of translated functional polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxyl ester lipase, or nuclear receptor-associated SET domain protein 1. These compositions and methods include antisense oligomers (ASOs) that can cause exon skipping, e.g., pseudoexon skipping, and promote constitutive splicing of PKD1, ABCA4, FUS, CEL, or NSD1 pre-mRNA. In various embodiments, functional polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein can be increased using the methods of the present disclosure to treat a condition caused by a deficiency of polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein.

[0221] Target transcript In some embodiments, the disclosed method utilizes the presence of ASCE in pre-mRNA transcribed from the PKD1, ABCA4, FUS, CEL, or NSD1 gene. Splicing of the identified PKD1, ABCA4, FUS, CEL, or NSD1 ASCE pre-mRNA species, which produces functional mature PKD1, ABCA4, FUS, CEL, or NSD1 mRNA, can be induced using a therapeutic agent, such as an ASO, that stimulates ASCE exon skipping. Induction of exon skipping can inhibit the NMD pathway. The resulting mature PKD1, ABCA4, FUS, CEL, or NSD1 mRNA is translated normally without activating the NMD pathway, thereby increasing the amount of polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or SET domain protein 1 in patient cells, leading to a variety of diseases, including polycystic kidney disease 1 with or without polycystic liver disease, autosomal dominant polycystic kidney disease, age-related macular degeneration 2, Stargardt disease 1, amyotrophic lateral sclerosis, amyotrophic lateral sclerosis 6 with or without frontotemporal dementia, hereditary essential tremor 4, frontotemporal dementia, maturity-onset diabetes of the young with exocrine insufficiency type 8, maturity-onset diabetes of the young, Sotos syndrome 1, and Beckwith-Wiedemann syndrome. The symptoms of a condition or disease associated with a deficiency in RNA binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 may be alleviated.

[0222] In various embodiments, the present disclosure provides therapeutic agents that target PKD1, ABCA4, FUS, CEL, or NSD1 mRNA transcripts and can modulate splicing or protein expression levels. The therapeutic agents can be small molecules, polynucleotides, or polypeptides. In some embodiments, the therapeutic agents are ASOs. Various regions or sequences on PKD1, ABCA4, FUS, CEL, or NSD1 pre-mRNA can be targeted by therapeutic agents, such as ASOs. In some embodiments, the ASO targets PKD1, ABCA4, FUS, CEL, or NSD1 pre-mRNA transcripts containing ASCE. In some embodiments, the ASO targets a sequence within the ASCE of a PKD1, ABCA4, FUS, CEL, or NSD1 pre-mRNA transcript. In some embodiments, the ASO targets a sequence upstream (or 5') from the 5' end of the ASCE (3'ss) of a PKD1, ABCA4, FUS, CEL, or NSD1 pre-mRNA transcript. In some embodiments, the ASO targets a sequence downstream (or 3') from the 3' end of ASCE (5'ss) of a PKD1, ABCA4, FUS, CEL, or NSD1 pre-mRNA transcript. In some embodiments, the ASO targets a sequence within an intron adjacent to the 5' end of ASCE of a PKD1, ABCA4, FUS, CEL, or NSD1 pre-mRNA transcript. In some embodiments, the ASO targets a sequence within an intron adjacent to the 3' end of ASCE of a PKD1, ABCA4, FUS, CEL, or NSD1 pre-mRNA transcript. In some embodiments, the ASO targets a sequence comprising the ASCE-intron boundary of a PKD1, ABCA4, FUS, CEL, or NSD1 pre-mRNA transcript. The ASCE-intron boundary may refer to the junction between the intron sequence and the ASCE region. The intron sequence may be adjacent to the 5' end of ASCE or the 3' end of ASCE. In some embodiments, the ASO targets a sequence within an exon of a PKD1, ABCA4, FUS, CEL, or NSD1 pre-mRNA transcript. In some embodiments, the ASO targets a sequence within an intron of a PKD1, ABCA4, FUS, CEL, or NSD1 pre-mRNA transcript.In some embodiments, the ASO targets a sequence that includes both a portion of an intron and a portion of an exon of a PKD1, ABCA4, FUS, CEL, or NSD1 pre-mRNA transcript.

[0223] In some embodiments, the ASO targets a sequence about 4 to about 300 nucleotides upstream (or 5') from the 5' end of ASCE. In some embodiments, the ASO targets a sequence about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, or about 250 to about 300 nucleotides upstream (or 5') from the 5' end of the ASCE region. In some embodiments, the ASO may target a sequence more than 300 nucleotides upstream from the 5' end of ASCE. In some embodiments, the ASO targets a sequence about 4 to about 300 nucleotides downstream (or 3') from the 3' end of ASCE. In some embodiments, the ASO targets a sequence about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, or about 250 to about 300 nucleotides downstream from the 3' end of the ASCE. In some embodiments, the ASO targets a sequence more than 300 nucleotides downstream from the 3' end of the ASCE.

[0224] In some embodiments, the ASCE-containing pre-mRNA transcript of PKD1, ABCA4, FUS, CEL, or NSD1 is encoded by a gene sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-5. In some embodiments, the ASCE-containing pre-mRNA transcript of PKD1, ABCA4, FUS, CEL, or NSD1 comprises a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 6-10.

[0225] In some embodiments, the ASCE-containing pre-mRNA transcript of PKD1, ABCA4, FUS, CEL, or NSD1 comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 6-10. In some embodiments, the ASCE-containing pre-mRNA transcript of PKD1, ABCA4, FUS, CEL, or NSD1 is encoded by a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 1-5. In some embodiments, the targeted portion of the ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1 comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 contiguous nucleic acids of any one of SEQ ID NOs: 6-10.

[0226] In some embodiments, the ASO targets an intron upstream of ASCE, an intron downstream of ASCE, an exon upstream of ASCE, an exon downstream of ASCE, or within ASCE of an ASCE-containing pre-mRNA. In some embodiments, the ASO targets an intron upstream of ASCE, an intron downstream of ASCE, an exon upstream of ASCE, an exon downstream of ASCE, or within ASCE of an ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1.

[0227] In some embodiments, the ASO targets a sequence at least about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides upstream (or 5') from the 5' end of the ASCE. In some embodiments, the ASO targets a sequence at most about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides upstream (or 5') from the 5' end of the ASCE. In some embodiments, the ASO targets a sequence about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides upstream (or 5') of the 5' end of the ASCE.

[0228] In some embodiments, the ASO targets a sequence at least about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides downstream (or 3') from the 3' end of the ASCE. In some embodiments, the ASO targets a sequence up to about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides downstream (or 3') from the 3' end of the ASCE. In some embodiments, the ASO targets a sequence about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides downstream (or 3') from the 3' end of the ASCE.

[0229] In some embodiments, the ASO targets the ASCE-containing pre-mRNA of PKD1, where the ASCE is exon 38 of PKD1. In some embodiments, the ASO targets the ASCE-containing pre-mRNA of PKD1, where the ASCE is exon GRCh38 / hg38:chr16 2092954 2093093 of PKD1. In some embodiments, the ASO targets the ASCE-containing pre-mRNA of PKD1, where the ASCE comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 11. In some embodiments, the ASO targets an intron upstream of ASCE, an intron downstream of ASCE, an exon upstream of ASCE, an exon downstream of ASCE, or within ASCE of the ASCE-containing pre-mRNA of PKD1. In some embodiments, the ASO targets a sequence up to about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides upstream (or 5') of GRCh38 / hg38:chr16 2092954 of PKD1. In some embodiments, the ASO targets a sequence at least about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides downstream (or 3') of GRCh38 / hg38:chr16 2093093 of PKD1. In some embodiments, the ASO targets a sequence within GRCh38 / hg38:chr16 2092954 2093093 of PKD1.

[0230] In some embodiments, the ASO targets the ASCE-containing pre-mRNA of ABCA4, where ASCE is exon 3 of ABCA4. In some embodiments, the ASO targets the ASCE-containing pre-mRNA of ABCA4, where ASCE is exon GRCh38 / hg38:chr1 94111438 94111579 of ABCA4. In some embodiments, the ASO targets the ASCE-containing pre-mRNA of ABCA4, where ASCE comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 12. In some embodiments, the ASO targets an intron upstream of ASCE, an intron downstream of ASCE, an exon upstream of ASCE, an exon downstream of ASCE, or within ASCE of the ASCE-containing pre-mRNA of ABCA4. In some embodiments, the ASO targets a sequence up to about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides upstream (or 5') of GRCh38 / hg38:chr1 94111438 of ABCA4. In some embodiments, the ASO targets a sequence at least about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides downstream (or 3') of ABCA4 GRCh38 / hg38:chr1 94111579. In some embodiments, the ASO targets a sequence within ABCA4 GRCh38 / hg38:chr1 94111438 94111579.

[0231] In some embodiments, the ASO targets the ASCE-containing pre-mRNA of FUS, where ASCE is exon 7 of FUS. In some embodiments, the ASO targets the ASCE-containing pre-mRNA of FUS, where ASCE is exon GRCh38 / hg38:chr16 31186802 31186836 of FUS. In some embodiments, the ASO targets the ASCE-containing pre-mRNA of FUS, where ASCE comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 13. In some embodiments, the ASO targets an intron upstream of ASCE, an intron downstream of ASCE, an exon upstream of ASCE, an exon downstream of ASCE, or within ASCE of the ASCE-containing pre-mRNA of FUS. In some embodiments, the ASO targets a sequence up to about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides upstream (or 5') of GRCh38 / hg38:chr16 31186802 of FUS. In some embodiments, the ASO targets a sequence at least about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides downstream (or 3') of FUS GRCh38 / hg38:chr16 31186836. In some embodiments, the ASO targets a sequence within FUS GRCh38 / hg38:chr16 31186802 31186836.

[0232] In some embodiments, the ASO targets the ASCE-containing pre-mRNA of CEL, where the ASCE is exon 5 of CEL. In some embodiments, the ASO targets the ASCE-containing pre-mRNA of CEL, where the ASCE is exon GRCh38 / hg38:chr9 133066530 133066660 of CEL. In some embodiments, the ASO targets the ASCE-containing pre-mRNA of CEL, where the ASCE comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 14. In some embodiments, the ASO targets an intron upstream of ASCE, an intron downstream of ASCE, an exon upstream of ASCE, an exon downstream of ASCE, or within ASCE of the ASCE-containing pre-mRNA of CEL. In some embodiments, the ASO targets a sequence up to about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides upstream (or 5') of GRCh38 / hg38:chr9 133066530 of CEL. In some embodiments, the ASO targets a sequence at least about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides downstream (or 3') of CEL GRCh38 / hg38:chr9 133066660. In some embodiments, the ASO targets a sequence within CEL GRCh38 / hg38:chr9 133066530 133066660.

[0233] In some embodiments, the ASO targets the ASCE-containing pre-mRNA of NSD1, where the ASCE is exon 8 of NSD1. In some embodiments, the ASO targets the ASCE-containing pre-mRNA of NSD1, where the ASCE is exon GRCh38 / hg38:chr5 177238237 177238507 of NSD1. In some embodiments, the ASO targets the ASCE-containing pre-mRNA of NSD1, where the ASCE comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 15. In some embodiments, the ASO targets an intron upstream of ASCE, an intron downstream of ASCE, an exon upstream of ASCE, an exon downstream of ASCE, or within ASCE of the ASCE-containing pre-mRNA of NSD1. In some embodiments, the ASO targets a sequence up to about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides upstream (or 5') of GRCh38 / hg38:chr5 177238237 of NSD1. In some embodiments, the ASO targets a sequence at least about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides downstream (or 3') of GRCh38 / hg38:chr5 177238507 of NSD1. In some embodiments, the ASO targets a sequence within GRCh38 / hg38:chr5 177238237 177238507 of NSD1.

[0234] In some embodiments, the ASO comprises a sequence complementary to a targeted portion of an ASCE-containing pre-mRNA encoded by a gene having a sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 1-5. In some embodiments, the ASO comprises a sequence complementary to a targeted portion of an ASCE-containing pre-mRNA encoded by a gene having a sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 6-10. In some embodiments, the ASO comprises a sequence complementary to a targeted portion of an ASCE having a sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 11-15. In some embodiments, the ASO comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 16-309. In some embodiments, the ASO comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to the reverse complement of any one of SEQ ID NOs: 16-309. In some embodiments, the ASO comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to the complement of any one of SEQ ID NOs: 16-309. In some embodiments, the ASO comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 16-309 where each T is U.

[0235] In some embodiments, the ASO targets a sequence upstream from the 5' end of ASCE.

[0236] In some embodiments, the ASO targets a sequence containing an exon-intron boundary (or junction). In some embodiments, the ASO does not target a sequence containing an exon-intron boundary (or junction). In some embodiments, the ASO targets a sequence downstream from the 3' end of the ASCE. In some embodiments, the ASO targets a sequence within the ASCE.

[0237] Protein expression In some embodiments, the methods described herein are used to increase production of functional polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxyl ester lipase, or nuclear receptor-binding SET domain protein 1 protein or RNA. As used herein, the term "functional" refers to the amount of polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-associated SET domain protein 1 protein or RNA activity or function required to eliminate any one or more symptoms of the condition or disease being treated, e.g., polycystic kidney disease with or without polycystic liver disease 1, autosomal dominant polycystic kidney disease, age-related macular degeneration-2, Stargardt disease 1, amyotrophic lateral sclerosis, amyotrophic lateral sclerosis with or without frontotemporal dementia 6, hereditary essential tremor 4, frontotemporal dementia, maturity-onset diabetes of the young with exocrine insufficiency type 8, maturity-onset diabetes of the young, Sotos syndrome 1, or Beckwith-Wiedemann syndrome. In some embodiments, the methods are used to increase production of partially functional polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein or RNA. As used herein, the term "partially functional" refers to an amount of either polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein or RNA activity or function that is below the amount of activity or function required to eliminate or prevent any one or more symptoms of a disease or condition.In some embodiments, a partially functional protein or RNA will have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% less activity than a fully functional protein or RNA.

[0238] In some embodiments, a method is a method of increasing expression of polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein by cells of a subject having ASCE-containing pre-mRNA encoding the protein, wherein the subject has polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1. Patients with polycystic kidney disease 1 with or without polycystic liver disease, autosomal dominant polycystic kidney disease, age-related macular degeneration-2, Stargardt disease 1, amyotrophic lateral sclerosis, amyotrophic lateral sclerosis 6 with or without frontotemporal dementia, hereditary essential tremor 4, frontotemporal dementia, maturity-onset diabetes of the young with exocrine insufficiency type 8, maturity-onset diabetes of the young, Sotos syndrome 1, or Beckwith-Wiedemann syndrome, which are caused by deficiencies in the activity of RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1, and polycystin-1, ATP-binding cassette subfamily A member 4, FUS. The method is caused by haploinsufficiency of a protein that is an RNA binding protein, a carboxyl ester lipase, or a nuclear receptor-binding SET domain protein 1.In such embodiments, the subject has a first allele that encodes a functional polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein, and a second allele that does not produce polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein. In another such embodiment, the subject has a first allele that encodes a functional polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein, and a second allele that encodes a non-functional polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein. In another such embodiment, the subject has a first allele encoding a functional polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein, and a second allele encoding a partially functional polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein.In any of these embodiments, the antisense oligomer binds to a targeted portion of an ASCE-containing pre-mRNA transcribed from the second allele, thereby inhibiting or reducing exon skipping of ASCE from the pre-mRNA or promoting inclusion of ASCE in mature RNA processed from the ASCE-containing pre-mRNA, and causing an increase in the level of mature mRNA encoding functional polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein, and an increase in expression of polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 in the subject's cells.

[0239] In some embodiments, a method is a method of increasing expression of polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein by cells of a subject having ASCE-containing pre-mRNA encoding the protein, wherein the subject has polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1. The method includes polycystic kidney disease 1 with or without polycystic liver disease, autosomal dominant polycystic kidney disease, age-related macular degeneration-2, Stargardt disease 1, amyotrophic lateral sclerosis, amyotrophic lateral sclerosis 6 with or without frontotemporal dementia, hereditary essential tremor 4, frontotemporal dementia, maturity-onset diabetes of the young with exocrine insufficiency type 8, maturity-onset diabetes of the young, Sotos syndrome 1, or Beckwith-Wiedemann syndrome, which is caused by a deficiency in the amount of activity of RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1, and the deficiency in the amount of polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 is caused by autosomal recessive inheritance.

[0240] In some embodiments, a method is a method of increasing expression of polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein by cells of a subject having ASCE-containing pre-mRNA encoding the protein, wherein the subject has polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1. The method includes the step of: having polycystic kidney disease 1 with or without polycystic liver disease, autosomal dominant polycystic kidney disease, age-related macular degeneration-2, Stargardt disease 1, amyotrophic lateral sclerosis, amyotrophic lateral sclerosis 6 with or without frontotemporal dementia, hereditary essential tremor 4, frontotemporal dementia, maturity-onset diabetes of the young with exocrine insufficiency type 8, maturity-onset diabetes of the young, Sotos syndrome 1, or Beckwith-Wiedemann syndrome, which is caused by a deficiency in the amount of activity of RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1, and the deficiency in the amount of polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 is caused by autosomal dominant inheritance.

[0241] In some embodiments, a method is a method of increasing expression of polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein by cells of a subject having ASCE-containing pre-mRNA encoding the protein, wherein the subject has polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1. The method includes polycystic kidney disease 1 with or without polycystic liver disease, autosomal dominant polycystic kidney disease, age-related macular degeneration-2, Stargardt disease 1, amyotrophic lateral sclerosis, amyotrophic lateral sclerosis 6 with or without frontotemporal dementia, hereditary essential tremor 4, frontotemporal dementia, maturity-onset diabetes of the young with exocrine insufficiency type 8, maturity-onset diabetes of the young, Sotos syndrome 1, or Beckwith-Wiedemann syndrome, which is caused by a deficiency in the amount of activity of RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1, and the deficiency in the amount of polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 is caused by X-linked dominant inheritance.

[0242] In related embodiments, the method is a method of increasing expression of a protein or functional RNA using an ASO. In some embodiments, the ASO may be used to increase expression of polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 in cells of a subject having an ASCE-containing pre-mRNA encoding polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1, wherein the subject has an ASCE-containing pre-mRNA encoding polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1. Defects in the amount or function of RNA-binding proteins, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 include polycystic kidney disease with or without polycystic liver disease 1, autosomal dominant polycystic kidney disease, age-related macular degeneration 2, Stargardt disease 1, amyotrophic lateral sclerosis, amyotrophic lateral sclerosis with or without frontotemporal dementia 6, hereditary essential tremor 4, frontotemporal dementia, maturity-onset diabetes of the young with exocrine insufficiency type 8, maturity-onset diabetes of the young, Sotos syndrome 1, and Beckwith-Wiedemann syndrome.

[0243] In some embodiments, ASCE-containing pre-mRNA transcripts encoding proteins that cause a disease or condition are targeted by the ASOs described herein. In some embodiments, ASCE-containing pre-mRNA transcripts encoding proteins that do not cause the disease are not targeted by the ASOs. For example, a disease resulting from a mutation or deficiency of a first protein in a specific pathway can be improved by targeting an ASCE-containing pre-mRNA encoding a second protein, thereby increasing the production of the second protein. In some embodiments, the function of the second protein can compensate for the mutation or deficiency of the first protein (which is the cause of the disease or condition).

[0244] In some embodiments, the subject: (a) a first mutant allele, (i) polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxyl ester lipase, or nuclear receptor-associated SET domain protein 1 protein is produced at reduced levels compared to production from a wild-type allele; (ii) polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxyl ester lipase, or nuclear receptor-associated SET domain protein 1 protein is produced in a form that has reduced function compared to the equivalent wild-type protein; or (iii) a first mutant allele in which polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxyl ester lipase, or nuclear receptor-associated SET domain protein 1 protein or functional RNA is not produced; and (b) a second mutant allele, (i) polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxyl ester lipase, or nuclear receptor-associated SET domain protein 1 protein is produced at reduced levels compared to production from a wild-type allele; (ii) polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxyl ester lipase, or nuclear receptor-associated SET domain protein 1 protein is produced in a form that has reduced function compared to the equivalent wild-type protein; or (iii) a second mutant allele that results in the absence of polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxyl ester lipase, or nuclear receptor-associated SET domain protein 1 protein; and The ASCE-containing pre-mRNA is transcribed from a first allele and / or a second allele. In these embodiments, the ASO binds to a targeted portion of the ASCE-containing pre-mRNA transcribed from the first allele or the second allele, thereby promoting the inclusion of an ASCE exon in the processed mRNA processed from the ASCE-containing pre-mRNA, resulting in increased levels of mRNA encoding polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1, and increased expression of the target protein or functional RNA in the subject's cells. In these embodiments, the target protein or functional RNA whose expression level is increased as a result of reducing or inhibiting ASCE exon skipping from the ASCE-containing pre-mRNA can be in a form that has reduced function compared to the equivalent wild-type protein (partially functional) or has complete function compared to the equivalent wild-type protein (fully functional).

[0245] In some embodiments, the level of mRNA encoding polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 is increased by 1.1 to 10 fold when compared to the amount of mRNA encoding polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 produced in control cells, e.g., cells that are not treated with an antisense oligomer or that are treated with an antisense oligomer that does not bind to a targeted portion of the ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1.

[0246] In some embodiments, a subject treated using the methods of the present disclosure expresses a partially functional polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein from one allele, where the partially functional polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein may be caused by a frameshift mutation, a nonsense mutation, a missense mutation, or a partial gene deletion. In some embodiments, subjects treated using the methods of the present disclosure express non-functional polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein from one allele, which may be caused by a frameshift mutation, a nonsense mutation, a missense mutation, or a partial gene deletion in one allele. In some embodiments, subjects treated using the methods of the present disclosure have a deletion of the entire PKD1, ABCA4, FUS, CEL, or NSD1 gene in one allele.

[0247] Exon inclusion As used herein, an "ASCE-containing pre-mRNA" is a pre-mRNA transcript that contains at least one alternatively spliced ​​coding exon. Alternative or aberrant splicing can result in the omission of at least one ASC in the mature mRNA transcript. The terms "mature mRNA" and "fully spliced ​​mRNA" are used interchangeably herein to describe fully processed mRNA. The inclusion of at least one pseudoexon can result in a non-productive mRNA and can induce NMD of the mature mRNA. ASCE-containing mature mRNAs can induce aberrant protein expression.

[0248] In some embodiments, the included pseudoexon is the most abundant pseudoexon in a population of ASCE-containing pre-mRNAs transcribed from a gene encoding a target protein in a cell. In some embodiments, the included pseudoexon is the most abundant pseudoexon in a population of ASCE-containing pre-mRNAs transcribed from a gene encoding a target protein in a cell, where the population of ASCE-containing pre-mRNAs contains two or more included pseudoexons. In some embodiments, an antisense oligomer targeted to the most abundant pseudoexon in a population of ASCE-containing pre-mRNAs encoding a target protein induces exon skipping of one or more pseudoexons in the population, including the pseudoexon targeted or bound by the antisense oligomer. In some embodiments, the targeted region is within a pseudoexon that is the most abundant pseudoexon in ASCE-containing pre-mRNAs encoding polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxyl ester lipase, or nuclear receptor-binding SET domain protein 1.

[0249] The degree of exon inclusion can be expressed as a percentage of exon inclusion, for example, as the percentage of transcripts that contain a given pseudoexon. Briefly, the percentage of exon inclusion can be calculated as the percentage of the amount of RNA transcripts with exon inclusion relative to the sum of the average amount of RNA transcripts with exon inclusion and the average amount of RNA transcripts with exon exclusion.

[0250] In some embodiments, the ASCE is an exon identified as an ASCE based on a determination that the exclusion is at least about 5%, 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%, or at least about 50%. In embodiments, the ASCE is selected from the group consisting of: about 5% to about 100%, about 5% to about 95%, about 5% to about 90%, about 5% to about 85%, about 5% to about 80%, about 5% to about 75%, about 5% to about 70%, about 5% to about 65%, about 5% to about 60%, about 5% to about 55%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 10% to about 100%, about 10% to about 95%, and about 10% to about 90% , about 10% to about 85%, about 10% to about 80%, about 10% to about 75%, about 10% to about 70%, about 10% to about 65%, about 10% to about 60%, about 10% to about 55%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 15% to about 100%, about 15% to about 95%, about 15% to about 90%, about 15% to about 85%, about 15% to about 80%, about 15% to about 75%, about 15% to about 7 0%, approximately 15% to approximately 65%, approximately 15% to approximately 60%, approximately 15% to approximately 55%, approximately 15% to approximately 50%, approximately 15% to approximately 45%, approximately 15% to approximately 40%, approximately 15% to approximately 35%, approximately 15% to approximately 30%, approximately 15% to approximately 25%, approximately 20% to approximately 100%, approximately 20% to approximately 95%, approximately 20% to approximately 90%, approximately 20% to approximately 85%, approximately 20% to approximately 80%, approximately 20% to approximately 75%, approximately 20% to approximately 70%, approximately 20% to approximately 65%, approximately 20% to approximately 60%, approximately 20% to approximately 55%, approximately 20% to approximately 50%, approximately 20% to The exons identified as ASCE are based on a determination that the exons are about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 25% to about 100%, about 25% to about 95%, about 25% to about 90%, about 25% to about 85%, about 25% to about 80%, about 25% to about 75%, about 25% to about 70%, about 25% to about 65%, about 25% to about 60%, about 25% to about 55%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, or about 25% to about 35%.ENCODE data (described in Tilgner, et al., 2012, "Deep sequencing of subcellular RNA fractions shows splicing to be predominantly co-transcriptional in the human genome but inefficient for lncRNAs," Genome Research 22(9):1616-25) can be used to help identify exon inclusion or exclusion.

[0251] In some embodiments, contacting cells with an ASO complementary to a targeted portion of a PKD1, ABCA4, FUS, CEL, or NSD1 pre-mRNA transcript results in an increase in the amount of polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxyl ester lipase, or nuclear receptor-associated SET domain protein 1 protein produced by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000% compared to the amount of protein produced by cells in the absence / untreated of the ASO. In some embodiments, the amount of polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein produced by cells contacted with the antisense oligomer is increased by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000% compared to the amount of protein produced by cells in the absence / untreated of the ASO. The total protein amount of RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 is about 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, or about 50% to about 100% compared to the amount of target protein produced by the control compound. , about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, by at least about 10%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%.In some embodiments, the total amount of polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 produced by a cell contacted with an antisense oligomer is increased by about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about The increase may be 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold. The control compound may be, for example, an oligonucleotide that is not complementary to the targeted portion of the pre-mRNA.

[0252] In some embodiments, contacting a cell with an ASO that is complementary to a targeted portion of a PKD1, ABCA4, FUS, CEL, or NSD1 pre-mRNA transcript results in an increase in the amount of PKD1, ABCA4, FUS, CEL, or NSD1 mRNA, including the mature mRNA encoding the target protein. In some embodiments, the amount of mRNA encoding polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1, or the amount of mature mRNA encoding polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1, is increased by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000% compared to the amount of protein produced by cells in the absence / untreated cells of the ASO.In some embodiments, the total amount of mRNA encoding polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1, or the total amount of mature mRNA encoding polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1, produced in a cell contacted with an antisense oligomer is reduced by about 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about 150%, about 20% to about 200%, about 20% to about 3 ... An increase of 0% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 10%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%.In some embodiments, the total amount of mRNA encoding polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1, or the total amount of mature mRNA encoding polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1, produced in a cell contacted with an antisense oligomer is increased by about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 1.1 to about 10-fold, about 1.1 to about 11-fold, about 1.1 to about 12-fold, about 1.1 to about 13-fold, about 1.1 to about 14-fold, about 1.1 to about 15-fold, about 1.1 to about 16-fold, about 1.1 to about 17-fold, about 1.1 to about 18-fold, about 1.1 to about 20-fold, about 1.1 to about 21-fold, about 1.1 to about 22-fold, about 1.1 to about 23-fold, about 1.1 to about 24-fold, about 1.1 to about 25-fold, about 1.1 to about 26-fold, about 1.1 to about 27-fold, about 1.1 to about 28-fold, about 1.1 to about 29-fold, about 1.1 to about 30-fold, about 1.1 to about 3 The control compound can increase by 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold. The control compound can be, for example, an oligonucleotide that is not complementary to the targeted portion of the ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1.

[0253] An ASCE can be any length. An ASCE can include a canonical exon. An ASCE can include the entire sequence of a canonical exon. In some embodiments, an ASCE can be 5 to 10 nucleotides in length, 10 to 15 nucleotides in length, 15 to 20 nucleotides in length, 20 to 25 nucleotides in length, 25 to 30 nucleotides in length, 30 to 35 nucleotides in length, 35 to 40 nucleotides in length, 40 to 45 nucleotides in length, 45 to 50 nucleotides in length, 50 to 55 nucleotides in length, 55 to 60 nucleotides in length, 60 to 65 nucleotides in length, 65 to 70 nucleotides in length, 70 to 75 nucleotides in length, 75 to 80 nucleotides in length, 80 to 85 nucleotides in length, 85 to 90 nucleotides in length, 90 to 95 nucleotides in length, or 95 to 100 nucleotides in length. In some embodiments, an ASCE can be at least 10 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, or at least 100 nucleotides in length. In some embodiments, an ASCE can be 100-200 nucleotides, 200-300 nucleotides, 300-400 nucleotides, 400-500 nucleotides, 500-600 nucleotides, 600-700 nucleotides, 700-800 nucleotides, 800-900 nucleotides, or 900-1,000 nucleotides in length. In some embodiments, an ASCE can be greater than 1,000 nucleotides in length.

[0254] The elimination of ASCE can induce frameshifting and the introduction of a premature termination codon (PIC) in the mature mRNA transcript, which makes the transcript a target for NMD. A mature mRNA transcript lacking ASCE can be a non-productive mRNA transcript that does not induce protein expression. The PIC can be located at any position downstream of the exon upstream of ASCE in the pre-mRNA. In some embodiments, the PIC can be located at any exon downstream of the exon upstream of ASCE in the pre-mRNA.

[0255] Treatment drugs In various embodiments of the present disclosure, compositions and methods, including therapeutic agents, are provided for regulating the expression level of ABCA4, FUS, CEL, or NSD1 proteins. In some embodiments, provided herein are compositions and methods for regulating alternative splicing of PKD1, ABCA4, FUS, CEL, or NSD1 pre-mRNA. In some embodiments, provided herein are compositions and methods for promoting the inclusion of ASCE during splicing of PKD1, ABCA4, FUS, CEL, or NSD1 pre-mRNA, for example, inhibiting ASCE skipping of ASCE during splicing of PKD1, ABCA4, FUS, CEL, or NSD1 pre-mRNA.

[0256] The therapeutic agent disclosed herein can be an NMD inhibitor. The therapeutic agent can include a polynucleic acid polymer.

[0257] According to one aspect of the present disclosure, provided herein is a method for treating or preventing a condition or disease associated with a deficiency of functional polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein, comprising administering an ASCE inhibitor to a subject to increase the level of functional polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein, wherein the agent binds to a region of a pre-mRNA transcript and reduces the inclusion of ASCE in the mature transcript. For example, provided herein are methods for treating or preventing a condition associated with a deficiency of functional polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein, comprising administering an ASCE inhibitor to a subject to increase the level of functional polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein, wherein the agent binds to a region of pre-mRNA containing ASCE.For example, provided herein are methods for treating or preventing a condition associated with a deficiency of functional polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-associated SET domain protein 1, comprising administering an ASCE inhibitor to a subject to increase the level of functional polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-associated SET domain protein 1, wherein the agent inhibits an ASCE-containing region of pre-mRNA (e.g., ASCE of PKD1 (GRCh38 / hg38:chr16 2092954 2093093), ASCE of ABC4 (GRCh38 / hg38:chr1 94111438 94111579), ASCE of FUS (GRCh38 / hg38:chr16 31186802), 31186836), ASCE of CEL (GRCh38 / hg38:chr9 133066530 133066660), ASCE of NSD1 (GRCh38 / hg38:chr5 177238237 177238507)).

[0258] When referring to promoting the inclusion of ASCE in mature mRNA, this promotion can be complete, for example, 100%, or partial. The promotion can be clinically significant. The promotion / correction can be compared to the level of ASCE inclusion in untreated subjects, or compared to the amount of ASCE inclusion in a similar subject population. The promotion / correction can be at least 10% more ASCE inclusion compared to the average subject, or subject before treatment. The promotion can be at least 20% more ASCE inclusion compared to the average subject, or subject before treatment. The promotion can be at least 40% more ASCE inclusion compared to the average subject, or subject before treatment. The promotion can be at least 50% more ASCE inclusion compared to the average subject, or subject before treatment. The promotion can be at least 60% more ASCE inclusion compared to the average subject, or subject before treatment. The promotion can be at least 80% more ASCE inclusion compared to the average subject, or subject before treatment. The promotion can be the inclusion of at least 90% more ASCEs compared to the average subject, or compared to the subject before receiving treatment.

[0259] When referring to increasing the level of active polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein, the increase can be clinically significant. The increase can be compared to the level of active polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein in a subject not receiving treatment, or compared to the amount of active polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein in a similar subject population. The increase can be at least 10% higher active polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein compared to the average subject or subjects before treatment. The increase can be at least 20% more active polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein compared to the average subject or pre-treatment subject. The increase can be at least 40% more active polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein compared to the average subject or pre-treatment subject. The increase can be at least 50% more active polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein compared to the average subject or pre-treatment subject.The increase can be at least 80% more active polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein compared to the average subject or the subject before treatment. The increase can be at least 100% more active polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein compared to the average subject or the subject before treatment. The increase can be at least 200% more active polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxylester lipase, or nuclear receptor-binding SET domain protein 1 protein compared to the average subject or the subject before treatment. The increase can be at least 500% higher activity of polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxyl ester lipase, or nuclear receptor-binding SET domain protein 1 protein compared to the average subject or the subject before treatment.

[0260] In embodiments where the ASCE inhibitor comprises a polynucleic acid polymer, the polynucleic acid polymer can be about 50 nucleotides in length. The polynucleic acid polymer can be about 45 nucleotides in length. The polynucleic acid polymer can be about 40 nucleotides in length. The polynucleic acid polymer can be about 35 nucleotides in length. The polynucleic acid polymer can be about 30 nucleotides in length. The polynucleic acid polymer can be about 24 nucleotides in length. The polynucleic acid polymer can be about 25 nucleotides in length. The polynucleic acid polymer can be about 20 nucleotides in length. The polynucleic acid polymer can be about 19 nucleotides in length. The polynucleic acid polymer can be about 18 nucleotides in length. The polynucleic acid polymer can be about 17 nucleotides in length. The polynucleic acid polymer can be about 16 nucleotides in length. The polynucleic acid polymer can be about 15 nucleotides in length. The polynucleic acid polymer can be about 14 nucleotides in length. The polynucleic acid polymer can be about 13 nucleotides in length. The polynucleic acid polymer can be about 12 nucleotides in length. The polynucleic acid polymer can be about 11 nucleotides in length. The polynucleic acid polymer can be about 10 nucleotides in length. A polynucleic acid polymer can be about 10 to about 50 nucleotides in length. A polynucleic acid polymer can be about 10 to about 45 nucleotides in length. A polynucleic acid polymer can be about 10 to about 40 nucleotides in length. A polynucleic acid polymer can be about 10 to about 35 nucleotides in length. A polynucleic acid polymer can be about 10 to about 30 nucleotides in length. A polynucleic acid polymer can be about 10 to about 25 nucleotides in length. A polynucleic acid polymer can be about 10 to about 20 nucleotides in length. A polynucleic acid polymer can be about 15 to about 25 nucleotides in length. A polynucleic acid polymer can be about 15 to about 30 nucleotides in length. A polynucleic acid polymer can be about 12 to about 30 nucleotides in length.

[0261] The sequence of the polynucleic acid polymer can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% complementary to the target sequence of an mRNA transcript, e.g., a partially processed mRNA transcript. The sequence of the polynucleic acid polymer can be 100% complementary to the target sequence of a pre-mRNA transcript.

[0262] The sequence of the polynucleic acid polymer may have no more than four mismatches with respect to the target sequence of the pre-mRNA transcript. The sequence of the polynucleic acid polymer may have no more than three mismatches with respect to the target sequence of the pre-mRNA transcript. The sequence of the polynucleic acid polymer may have no more than two mismatches with respect to the target sequence of the pre-mRNA transcript. The sequence of the polynucleic acid polymer may have no more than one mismatch with respect to the target sequence of the pre-mRNA transcript. The sequence of the polynucleic acid polymer may have no mismatches with respect to the target sequence of the pre-mRNA transcript.

[0263] Polynucleic acid polymer can specifically hybridize with the target sequence of mRNA precursor transcript.For example, polynucleic acid polymer can have 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence complementarity with the target sequence of mRNA precursor transcript.Hybridization can be carried out under highly stringent hybridization conditions.

[0264] The polynucleic acid polymer comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 16-309. The polynucleic acid polymer may comprise a sequence having 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 16-309.

[0265] When referring to a polynucleic acid polymer sequence, one of skill in the art will understand that one or more substitutions, optionally two substitutions, may be tolerated within the sequence to maintain the ability to hybridize to a target sequence or, if the substitution is within the target sequence, to maintain the ability to be recognized as the target sequence. References to sequence identity may be determined by BLAST sequence alignment using standard / default parameters. For example, a sequence may have 99% identity and still function in accordance with the present disclosure. In other embodiments, a sequence may have 98% identity and still function in accordance with the present disclosure. In another embodiment, a sequence may have 95% identity and still function in accordance with the present disclosure. In another embodiment, a sequence may have 90% identity and still function in accordance with the present disclosure.

[0266] Antisense oligomers Provided herein are compositions containing antisense oligomers that induce exon skipping by binding to a targeted portion of an ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1. As used herein, the terms "ASO" and "antisense oligomer" are used interchangeably and refer to an oligomer, such as a polynucleotide, that contains nucleobases that hybridize to a target nucleic acid sequence (e.g., an ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1) via Watson-Crick base pairing or wobble base pairing (GU). ASOs can be strictly complementary to the target sequence or nearly complementary (e.g., sufficiently complementary to bind to the target sequence and enhance splicing at the splice site). ASOs are designed to bind (hybridize) and maintain a hybridized state to a target nucleic acid (e.g., a targeted portion of a pre-mRNA transcript) under physiological conditions. Typically, when an ASO hybridizes to a site other than the intended (target) nucleic acid sequence, it hybridizes to a limited number of sequences that are not the target nucleic acid (it hybridizes to a small number of sites other than the target nucleic acid). The design of the ASO can take into account the occurrence of the nucleic acid sequence of the target portion of the mRNA precursor transcript, or the occurrence of sufficiently similar nucleic acid sequences in other locations in the genome or cell's mRNA precursor or transcriptome, so that the possibility of the ASO binding to other sites and causing "off-target" effects is limited. Any antisense oligomer known in the art can be used to carry out the methods described herein, for example, any antisense oligomer described in PCT Application No. PCT / US2014 / 054151, published as International Publication No. WO 2015 / 035091, entitled "Reducing Nonsense-Mediated mRNA Decay," which is incorporated herein by reference.

[0267] In some embodiments, the ASO "specifically hybridizes" or is "specific" for a target nucleic acid or targeted portion of an ASCE-containing pre-mRNA. Typically, such hybridization occurs at temperatures substantially above 37°C, preferably at least 50°C, and typically between 60°C and approximately 90°C. m Preferably, such hybridization corresponds to stringent hybridization conditions. At a given ionic strength and pH, T m is the temperature at which 50% of the target sequence hybridizes to a complementary oligonucleotide.

[0268] Oligomers, such as oligonucleotides, are "complementary" to each other when hybridization occurs between two single-stranded polynucleotides in an antiparallel configuration. A double-stranded polynucleotide can be "complementary" to another polynucleotide when hybridization can occur between one of the strands of a first polynucleotide and one of the strands of a second polynucleotide. Complementarity (the degree to which one polynucleotide is complementary to another) can be quantified in terms of the proportion (e.g., percentage) of bases in opposing strands that are expected to form hydrogen bonds with each other according to generally accepted base-pairing rules. The sequence of an antisense oligomer (ASO) does not need to be 100% complementary to the sequence of its target nucleic acid in order to hybridize. In certain embodiments, ASOs may contain at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence complementarity to a target region within a targeted nucleic acid sequence. For example, an ASO in which 18 of 20 nucleobases of an oligomeric compound are complementary to a target region and thereby specifically hybridize would exhibit 90% complementarity. In this example, the remaining non-complementary nucleobases may be clustered with complementary nucleobases or may be interspersed with complementary nucleobases, and need not be contiguous with each other or with complementary nucleobases. The percent complementarity of an ASO with a target nucleic acid region can be routinely determined using the BLAST (basic local alignment search tool) and PowerBLAST programs known in the art (Altschul, et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

[0269] An ASO does not need to hybridize to all nucleobases in a target sequence; the hybridizing nucleobases may be contiguous or non-contiguous. An ASO may hybridize across one or more segments of a pre-mRNA transcript such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop or hairpin structure may be formed). In certain embodiments, an ASO hybridizes to non-contiguous nucleobases in a target pre-mRNA transcript. For example, an ASO may hybridize to nucleobases in a pre-mRNA transcript that are separated by one or more nucleobases to which the ASO does not hybridize.

[0270] The ASOs described herein contain nucleobases complementary to those present in the target portion of an ASCE-containing pre-mRNA. The term ASO embodies oligonucleotides and any other oligomeric molecules that contain nucleobases capable of hybridizing to complementary nucleobases on a target mRNA but do not contain sugar moieties, such as peptide nucleic acids (PNAs). ASOs can contain natural nucleotides, nucleotide analogs, modified nucleotides, or any combination of two or three of the foregoing. The term "natural nucleotides" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" includes nucleotides with modified or substituted sugar groups and / or modified backbones. In some embodiments, all of the nucleotides in an ASO are modified nucleotides. Chemical modifications of ASOs, or components of ASOs, that are compatible with the methods and compositions described herein will be apparent to those skilled in the art and can be found, for example, in U.S. Pat. No. 8,258,109 B2, U.S. Pat. No. 5,656,612, U.S. Patent Application Publication No. 2012 / 0190728, and Dias and Stein, Mol. Cancer Ther. 2002, 347-355, which are incorporated herein by reference in their entireties.

[0271] The one or more nucleobases of the ASO can be any naturally occurring unmodified nucleobase, such as adenine, guanine, cytosine, thymine, and uracil, or any synthetic or modified nucleobase sufficiently similar to an unmodified nucleobase so as to be capable of hydrogen bonding with a nucleobase present on the target pre-mRNA. Examples of modified nucleobases include, but are not limited to, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine.

[0272] The ASOs described herein also include a backbone structure that connects the components of the oligomer. The terms "backbone structure" and "oligomeric linkage" can be used interchangeably and can refer to the linkage between the monomers of the ASO. In natural oligonucleotides, the backbone includes a 3'-5' phosphodiester bond that connects the sugar moieties of the oligomer. The backbone structure or oligomeric linkage of the ASOs described herein can include (but is not limited to) phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoramidate, etc. For example, LaPlanche, et al., Nucleic Acids Res.14:9081(1986); Stec, et al., J.Am.Chem.Soc.106:6077(1984), Stein, et al., Nucleic Acids Res.16:3209(1988), Zon, et al., Anti-Cancer Drug Design 6:539 (1991), Zon, et al., Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)), Stec, et al., U.S. Patent No. 5,151,510, Uhlmann and Peyman, Chemical Reviews 90:543 (1990). In some embodiments, the backbone structure of the ASO does not contain phosphorus, but contains peptide bonds, such as in peptide nucleic acids (PNAs), or linking groups including carbamate groups, amide groups, and linear and cyclic hydrocarbon groups. In some embodiments, the backbone modification is a phosphorothioate bond. In some embodiments, the backbone modification is a phosphoramidate bond.

[0273] In some embodiments, the stereochemistry at each phosphorus internucleotide linkage in the ASO backbone is random. In some embodiments, the stereochemistry at each phosphorus internucleotide linkage in the ASO backbone is controlled and not random. For example, U.S. Patent Application Publication No. 2014 / 0194610, "Methods for the Synthesis of Functionalized Nucleic Acids," describes methods for independently selecting the handedness of chirality at each phosphorus atom in a nucleic acid oligomer. In some embodiments, ASOs used in the methods of the present disclosure, including but not limited to any of the ASOs described herein in Tables 4, 5A, 5A-1, 5B, 5B-1, 5D, 5E, 5G, and 5G-1, include ASOs with non-random phosphorus internucleotide linkages. In some embodiments, compositions used in the methods of the present disclosure include pure diastereomeric ASOs. In some embodiments, the compositions used in the methods of the present disclosure comprise an ASO having a diastereomeric purity of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100%.

[0274] In some embodiments, the ASO has a non-random mixture of Rp and Sp configurations at its phosphorus internucleotide linkages. For example, it has been suggested that a mixture of Rp and Sp configurations is required in antisense oligonucleotides to achieve a balance between good activity and nuclease stability (Wan, et al., 2014, "Synthesis, biophysical properties and biological activity of second-generation antisense oligonucleotides containing chiral phosphorothioate linkages," Nucleic Acids Research 42(22):13456-13468, incorporated herein by reference). In some embodiments, the ASOs used in the methods of the disclosure, including but not limited to any of the ASOs described herein in SEQ ID NOs: 16-309, comprise about 5-100% Rp, at least about 5% Rp, at least about 10% Rp, at least about 15% Rp, at least about 20% Rp, at least about 25% Rp, at least about 30% Rp, at least about 35% Rp, at least about 40% Rp, at least about 45% Rp, at least about 50% Rp, at least about 55% Rp, at least about 60% Rp, at least about 65% Rp, at least about 70% Rp, at least about 75% Rp, at least about 80% Rp, at least about 85% Rp, at least about 90% Rp, or at least about 95% Rp, with the remainder as Sp, or comprise about 100% Rp.In some embodiments, the ASOs used in the methods of the present disclosure, including, but not limited to, any of the ASOs described herein, comprising a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 consecutive nucleic acids of any one of SEQ ID NOS: 16-309, can have an Rp of about 10% to about 100%, about 15% to about 100%, about 20% to about 100%, about 25% to about 100%, about 30% to about 100%, about 35% to about 100%, about 40% to about 100%, about 45% to about 100%, about 50% to about 100%, about 55% to about 100%, about 60% to about 100%, about 65% to about 100%, about 70% to about 100%, about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, about 95% to about 100%, about 97% to about 100%, about 10 ... Rp, about 45% to about 100% Rp, about 50% to about 100% Rp, about 55% to about 100% Rp, about 60% to about 100% Rp, about 65% to about 100% Rp, about 70% to about 100% Rp, about 75% to about 100% Rp, about 80% to about 100% Rp, about 85% to about 100% Rp, about 90% to about 100% Rp, or about 95% to about 100% Rp, about 20% to about 80% Rp, about 25% to about 75% Rp, about 30% to about 70% Rp, about 40% to about 60% Rp, or about 45% to about 55% Rp, with the remainder being Sp.

[0275] In some embodiments, the ASOs used in the methods of the disclosure, including but not limited to any of the ASOs described herein, comprising a sequence complementary to a sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 consecutive nucleic acids of any one of SEQ ID NOs: 6-10, can be about 5-100% Sp, at least about 5% Sp, at least about 10% Sp, at least about 15% Sp, at least about 20% Sp, at least about 30% Sp, at least about 40% Sp, at least about 50% Sp, at least about 60% Sp, at least about 70% Sp, at least about 80% Sp, at least about 90% Sp, at least about 10 ...100% Sp, at least about 100% Sp, at least about 100% Sp, at least about 100% Sp, at least about 100% Sp, at least about 100% Sp, at least about 100% Sp, at least about 100% Sp, at least about 100% Sp, at least about 100% Sp, at least about 100% Sp, at least about 100% Sp, at least about 100% Sp, at least about 100% Sp, at least about 100% Sp, at least about 100% Sp The composition may contain about 25% Sp, at least about 30% Sp, at least about 35% Sp, at least about 40% Sp, at least about 45% Sp, at least about 50% Sp, at least about 55% Sp, at least about 60% Sp, at least about 65% Sp, at least about 70% Sp, at least about 75% Sp, at least about 80% Sp, at least about 85% Sp, at least about 90% Sp, or at least about 95% Sp, with the remainder as Rp, or about 100% Sp. In embodiments, the ASOs used in the methods of the present disclosure, including, but not limited to, any of the ASOs described herein, comprising a sequence complementary to a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 consecutive nucleic acids of any one of SEQ ID NOS: 6-10, can be selected from the group consisting of about 10% to about 100% Sp, about 15% to about 100% Sp, about 20% to about 100% Sp, about 25% to about 100% Sp, about 30% to about 100% Sp, about 35% to about 100% Sp, about 40% to about 100% Sp, about 50% to about 100% Sp, about 60% to about 100% Sp, about 70% to about 100% Sp, about 80% to about 100% Sp, about 90% to about 100% Sp, about 110% to about 110% Sp, about 120% to about 120% Sp, about 130% to about 130% Sp, about 140% to about 140% Sp, about 150% to about 150% Sp, about 20% to about 100% Sp, about 25% to about 100% Sp, about 30% to about 100% Sp, about 35% to about 100% Sp, about 40% to about 100% Sp, about 16% to about 160% Sp, about 18% to about 180% Sp, about 19% to about 190% Sp, about 21% to about 210% Sp, about 22% to about 220% Sp, about 23% to about 230% Sp, about 24% to about 00% Sp, about 45% to about 100% Sp, about 50% to about 100% Sp, about 55% to about 100% Sp, about 60% to about 100% Sp, about 65% to about 100% Sp, about 70% to about 100% Sp, about 75% to about 100% Sp, about 80% to about 100% Sp, about 85% to about 100% Sp, about 90% to about 100% Sp, or about 95% to about 100% Sp, about 20% to about 80% Sp, about 25% to about 75% Sp, about 30% to about 70% Sp, about 40% to about 60% Sp, or about 45% to about 55% Sp, with the remainder being Rp.

[0276] Any of the ASOs described herein may contain a sugar moiety containing a ribose or deoxyribose found in natural nucleotides, or a modified sugar moiety or sugar analog containing a morpholine ring. Non-limiting examples of modified sugar moieties include 2'-substituted sugars such as 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'F, N3'->P5' phosphoramidate, 2'dimethylaminooxyethoxy, 2'dimethylaminoethoxyethoxy, 2'-guanidinium, 2'-O-guanidinium ethyl, carbamate-modified sugars, and bicyclic modified sugars. In some embodiments, the sugar moiety modification is selected from 2'-O-Me, 2'F, and 2'MOE. In some embodiments, the sugar moiety modification is an extra crosslink, such as, for example, a locked nucleic acid (LNA). In some embodiments, the sugar analog contains a morpholine ring, such as, for example, phosphorodiamidate morpholino (PMO). In some embodiments, the sugar moiety comprises a ribofuransyl or 2'deoxyribofuransyl modification. In some embodiments, the sugar moiety comprises a 2'4'-constrained 2'O-methyloxyethyl (cMOE) modification. In some embodiments, the sugar moiety comprises a cEt 2',4'-constrained 2'-O-ethyl BNA modification. In some embodiments, the sugar moiety comprises a tricycloDNA (tcDNA) modification. In some embodiments, the sugar moiety comprises an ethylene nucleic acid (ENA) modification. In some embodiments, the sugar moiety comprises an MCE modification. Modifications are known in the art and are described in the literature, for example, by Jarver, et al., 2014, "A Chemical View of Oligonucleotides for Exon Skipping and Related Drug Applications," Nucleic Acid Therapeutics 24(1):37-47, which is incorporated by reference for the present purposes herein.

[0277] In some embodiments, each monomer of an ASO is modified in the same manner, e.g., each linkage in the backbone of the ASO contains a phosphorothioate linkage, or each ribose sugar moiety contains a 2'O-methyl modification. Such modifications present on each of the monomer components of an ASO are referred to as "uniform modifications." In some examples, a combination of different modifications may be desirable; for example, an ASO may contain a combination of phosphorodiamidate linkages and sugar moieties containing morpholine rings (morpholinos). A combination of different modifications to an ASO is referred to as a "mixed modification" or "mixed chemistry."

[0278] In some embodiments, the ASO comprises one or more backbone modifications. In some embodiments, the ASO comprises one or more sugar moiety modifications. In some embodiments, the ASO comprises one or more backbone modifications and one or more sugar moiety modifications. In some embodiments, the ASO comprises a 2'MOE modification and a phosphorothioate backbone. In some embodiments, the ASO comprises a phosphorodiamidate morpholino (PMO). In some embodiments, the ASO comprises a peptide nucleic acid (PNA). Any of the ASOs described herein, or any component of the ASO (e.g., nucleobase, sugar moiety, backbone), can be modified to achieve a desirable characteristic or activity of the ASO or to reduce an undesirable characteristic or activity of the ASO. For example, an ASO, or one or more components of any ASO, can be modified to improve binding affinity for a target sequence on a pre-mRNA transcript, reduce binding to any non-target sequences, reduce degradation by cellular nucleases (i.e., RNase H), improve uptake of the ASO into cells and / or the cell nucleus, alter the pharmacokinetics or pharmacology of the ASO, and / or modulate the half-life of the ASO.

[0279] In some embodiments, ASOs are composed of 2'-O-(2-methoxyethyl) (MOE) phosphorothioate modified nucleotides. ASOs composed of such nucleotides are particularly suitable for the methods disclosed herein, as oligomers with such modifications have been shown to significantly improve resistance to nuclease degradation and increase bioavailability, making ASOs suitable for oral delivery in some embodiments of the present invention. See, for example, Geary, et al., J Pharmacol Exp Ther. 2001; 296(3):890-7; Geary, et al., J Pharmacol Exp Ther. 2001; 296(3):898-904.

[0280] Methods for synthesizing ASOs will be known to those of skill in the art. Alternatively, or in addition, ASOs can be obtained from commercial sources.

[0281] Unless otherwise specified, the left-hand end of a single-stranded nucleic acid sequence (e.g., a pre-mRNA transcript, an oligonucleotide, an ASO, etc.) is the 5'-end, and the left-hand direction of a single-stranded or double-stranded nucleic acid sequence is referred to as the 5'-direction. Similarly, the right-hand end or direction of a nucleic acid sequence (single-stranded or double-stranded) is the 3'-end or 3'-direction. Generally, a region or sequence that is 5' to a reference point in a nucleic acid is referred to as "upstream," and a region or sequence that is 3' to a reference point in a nucleic acid is referred to as "downstream." Generally, the 5'-direction or 5'-end of an mRNA is where the start codon is located, while the 3'-end or 3'-direction is where the stop codon is located. In some embodiments, nucleotides upstream of the reference point in a nucleic acid may be referred to as negative numbers, while nucleotides downstream of the reference point may be referred to as positive numbers. For example, a reference point (e.g., an exon-exon junction in an mRNA) may be referred to as the "zero" site, and the nucleotide immediately adjacent to and upstream of the reference point is referred to as "minus one," e.g., "-1," while the nucleotide immediately adjacent to and downstream of the reference point is referred to as "plus one," e.g., "+1."

[0282] In some embodiments, the ASO is complementary to (and binds to) a targeted portion of an ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1 that is downstream (3' direction) of the ASCE 5' splice site (or the 3' end of ASCE) in the ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1 (e.g., in the direction designated by positive numbers relative to the 5' splice site). In some embodiments, the ASO is complementary to a targeted portion of an ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1 that is within a region of about +1 to about +500 relative to the ASCE 5' splice site (or the 3' end). In some embodiments, the ASO may be complementary to a targeted portion of the ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1 within the region of nucleotides +6 to approximately +40,000 relative to the 5' splice site (or 3' end) of the ASCE.In some embodiments, the ASO has a splice site (or 3' end) of ASCE of about +1 to about +40,000, about +1 to about +30,000, about +1 to about +20,000, about +1 to about +15,000, about +1 to about +10,000, about +1 to about +5,000, about +1 to about +4,000, about +1 to about +3,000, about +1 to about +2,000, about +1 to about +1,000, about +1 to about +2,000, about +1 to about +1,000, or about +1 +1 to about +500, about +1 to about +490, about +1 to about +480, about +1 to about +470, about +1 to about +460, about +1 to about +450, about +1 to about +440, about +1 to about +430, about +1 to about +420, about +1 to about +410, about +1 to about +400, about +1 to about +390, about +1 to about +380, about +1 to about +370, about +1 to about +360, about +1 to about +350, about +1 to about +340, about +1 to about +330, about +1 to about +320, about +1 to about +310, about +1 to about +300, about +1 to about +290, about +1 to about +280, about +1 to about +270, about +1 to about +260, about +1 to about +250, about +1 to about +240, about +1 to about +230, about +1 to about +220, about +1 to about +210, about +1 to about +200, about +1 to about +190, about +1 to about +180, about +1 to about +170, It is complementary to a target portion within a region of about +1 to about +160, about +1 to about +150, about +1 to about +140, about +1 to about +130, about +1 to about +120, about +1 to about +110, about +1 to about +100, about +1 to about +90, about +1 to about +80, about +1 to about +70, about +1 to about +60, about +1 to about +50, about +1 to about +40, about +1 to about +30, or about +1 to about +20. In some embodiments, the ASO is complementary to a target portion within the region of about +1 to about +100, about +100 to about +200, about +200 to about +300, about +300 to about +400, or about +400 to about +500 relative to the 5' splice site (or 3' end) of the ASCE.

[0283] In some embodiments, the ASO is complementary to (and binds to) a targeted portion of the ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1 that is upstream (5' direction) of the 5' splice site (or 3' end) of the ASCE (e.g., in the direction designated by negative numbers relative to the 5' splice site). In some embodiments, the ASO is complementary to a targeted portion of the ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1 that is within the region of about -4 to about -270 relative to the 5' splice site (or 3' end) of the ASCE. In some embodiments, the ASO may be complementary to a targeted portion of the ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1 within the region of nucleotides -1 to -40,000 relative to the 5' splice site (or 3' end) of the ASCE.In some embodiments, the ASO has a nucleotide sequence of about -1 to about -40,000, about -1 to about -30,000, about -1 to about -20,000, about -1 to about -15,000, about -1 to about -10,000, about -1 to about -5,000, about -1 to about -4,000, about -1 to about -3,000, about -1 to about -2,000, about -1 to about -1,000, or about - 1 to about -500, about -1 to about -490, about -1 to about -480, about -1 to about -470, about -1 to about -460, about -1 to about -450, about -1 to about -440, about -1 to about -430, about -1 to about -420, about -1 to about -410, about -1 to about -400, about -1 to about -390, about -1 to about -380, about -1 to about -370, about -1 to about -360, about -1 to about -350, about -1 to about -340, about -1 to about -330, about -1 to about -320, about -1 to about -310, about -1 to about -300, about -1 to about -290, about -1 to about -280, about -1 to about -270, about -1 to about -260, about -1 to about -250, about -1 to about -240, about -1 to about -230, about -1 to about -220, about -1 to about -210, about -1 to about -200, about -1 to about -190, about -1 to about -180, about -1 to about -170, It is complementary to a target portion within a region of about -1 to about -160, about -1 to about -150, about -1 to about -140, about -1 to about -130, about -1 to about -120, about -1 to about -110, about -1 to about -100, about -1 to about -90, about -1 to about -80, about -1 to about -70, about -1 to about -60, about -1 to about -50, about -1 to about -40, about -1 to about -30, or about -1 to about -20.

[0284] In some embodiments, the ASO is complementary to a region targeted by the ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1 that is upstream (5' direction) of the 3' splice site (or 5' end) of ASCE (e.g., in the direction designated by negative numbers). In some embodiments, the ASO is complementary to a portion targeted by the ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1 within a region of about -1 to about -500 relative to the 3' splice site (or 5' end) of ASCE. In some embodiments, the ASO is complementary to a portion targeted by the ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1 within a region of about -1 to about -40,000 relative to the 3' splice site of ASCE.In some embodiments, the ASO has a nucleotide sequence of about -1 to about -40,000, about -1 to about -30,000, about -1 to about -20,000, about -1 to about -15,000, about -1 to about -10,000, about -1 to about -5,000, about -1 to about -4,000, about -1 to about -3,000, about -1 to about -2,000, about -1 to about -1,000, or about -1 to about -500 relative to the 3' splice site of ASCE. , about -1 to about -490, about -1 to about -480, about -1 to about -470, about -1 to about -460, about -1 to about -450, about -1 to about -440, about -1 to about -430, about -1 to about -420, about -1 to about -410, about -1 to about -400, about -1 to about -390, about -1 to about -380, about -1 to about -370, about -1 to about -360, about -1 to about -350, about -1 to about -340, about -1 to about - 330, about -1 to about -320, about -1 to about -310, about -1 to about -300, about -1 to about -290, about -1 to about -280, about -1 to about -270, about -1 to about -260, about -1 to about -250, about -1 to about -240, about -1 to about -230, about -1 to about -220, about -1 to about -210, about -1 to about -200, about -1 to about -190, about -1 to about -180, about -1 to about -170, about - The ASO is complementary to a target portion within the range of about -1 to about -160, about -1 to about -150, about -1 to about -140, about -1 to about -130, about -1 to about -120, about -1 to about -110, about -1 to about -100, about -1 to about -90, about -1 to about -80, about -1 to about -70, about -1 to about -60, about -1 to about -50, about -1 to about -40, about -1 to about -30, or about -1 to about -20. In some embodiments, the ASO is complementary to a target portion within the range of about -1 to about -100, about -100 to about -200, about -200 to about -300, about -300 to about -400, or about -400 to about -500 relative to the 3' splice site of ASCE.

[0285] In some embodiments, the ASO is complementary to a region targeted by the ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1 that is downstream (3' direction) of the 3' splice site (5' end) of ASCE (e.g., in the direction designated by positive numbers) in the ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1. In some embodiments, the ASO is complementary to a portion targeted by the ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1 within the region of about +1 to about +40,000 relative to the ASCE 3' splice site. In some embodiments, the ASO has a splice site of ASCE of about +1 to about +40,000, about +1 to about +30,000, about +1 to about +20,000, about +1 to about +15,000, about +1 to about +10,000, about +1 to about +5,000, about +1 to about +4,000, about +1 to about +3,000, about +1 to about +2,000, about +1 to about +1,000, about +1 to about +500, about +1 +1 to about +490, about +1 to about +480, about +1 to about +470, about +1 to about +460, about +1 to about +450, about +1 to about +440, about +1 to about +430, about +1 to about +420, about +1 to about +410, about +1 to about +400, about +1 to about +390, about +1 to about +380, about +1 to about +370, about +1 to about +360, about +1 to about +350, about +1 to about +340, about +1 to about +330, about + +1 to about +320, about +1 to about +310, about +1 to about +300, about +1 to about +290, about +1 to about +280, about +1 to about +270, about +1 to about +260, about +1 to about +250, about +1 to about +240, about +1 to about +230, about +1 to about +220, about +1 to about +210, about +1 to about +200, about +1 to about +190, about +1 to about +180, about +1 to about +170, about +1 to about +160, about It is complementary to a target portion within the region of +1 to about +150, about +1 to about +140, about +1 to about +130, about +1 to about +120, about +1 to about +110, about +1 to about +100, about +1 to about +90, about +1 to about +80, about +1 to about +70, about +1 to about +60, about +1 to about +50, about +1 to about +40, about +1 to about +30, or about +1 to about +20, or about +1 to about +10.

[0286] In some embodiments, the targeted portion of the ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1 is within the region from +100 relative to the 5' splice site (3' end) of ASCE to -100 relative to the 3' splice site (5' end) of ASCE. In some embodiments, the targeted portion of the ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1 is within ASCE. In some embodiments, the targeted portion of the ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1 includes the boundary between ASCE and an intron. In some embodiments, the targeted portion of the ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1 does not include the boundary between ASCE and an intron.

[0287] ASOs can be of any length suitable for specific binding and effective splicing reduction. In some embodiments, the ASO consists of 8 to 50 nucleobases. For example, the ASO can be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, or 50 nucleobases in length. In some embodiments, the ASO consists of more than 50 nucleobases. In some embodiments, the ASO is 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, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, Nucleic acid bases, 10-35 nucleic acid bases, 10-30 nucleic acid bases, 10-25 nucleic acid bases, 10-20 nucleic acid bases, 10-15 nucleic acid bases, 11-50 nucleic acid bases, 11-40 nucleic acid bases, 11-35 nucleic acid bases, 11-30 nucleic acid bases, 11-25 nucleic acid bases, 11-20 nucleic acid bases, 11-15 nucleic acid bases, 12-50 nucleic acid bases, 12-40 nucleic acid bases, 12-35 nucleic acid bases, 12-3 0 nucleobases, 12-25 nucleobases, 12-20 nucleobases, 12-15 nucleobases, 13-50 nucleobases, 13-40 nucleobases, 13-35 nucleobases, 13-30 nucleobases, 13-25 nucleobases, 13-20 nucleobases, 14-50 nucleobases, 14-40 nucleobases, 14-35 nucleobases, 14-30 nucleobases, 14-25 nucleobases, 14-20 nucleobases The length may be 15 to 50 nucleobases, 15 to 40 nucleobases, 15 to 35 nucleobases, 15 to 30 nucleobases, 15 to 25 nucleobases, 15 to 20 nucleobases, 20 to 50 nucleobases, 20 to 40 nucleobases, 20 to 35 nucleobases, 20 to 30 nucleobases, 20 to 25 nucleobases, 25 to 50 nucleobases, 25 to 40 nucleobases, 25 to 35 nucleobases, or 25 to 30 nucleobases.In some embodiments, the ASO is 18 nucleotides in length, In some embodiments, the ASO is 15 nucleotides in length, In some embodiments, the ASO is 25 nucleotides in length.

[0288] In some embodiments, two or more ASOs are used that have different chemical structures but are complementary to the same target portion of the ASCE-containing pre-mRNA, hi some embodiments, two or more ASOs are used that are complementary to different target portions of the ASCE-containing pre-mRNA.

[0289] In some embodiments, the antisense oligonucleotides of the present disclosure are chemically linked to one or more moieties or conjugates, such as targeting moieties or other conjugates that improve the activity or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties, cholesteryl moieties, aliphatic chains, such as dodecanediol or undecyl residues, polyamines or polyethylene glycol chains, or adamantane acetic acid. Oligonucleotides containing lipophilic moieties and preparation methods are described in the published literature. In embodiments, the antisense oligomer is conjugated to a moiety, including, but not limited to, abasic nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates, such as N-acetylgalactosamine (GalNAc), N-Ac-glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate), lipids, or polyhydrocarbon compounds. Conjugates can be attached to one or more of any nucleotides comprising an antisense oligomer at any of several positions on the sugar, base, or phosphate group, using, for example, a linker, as understood in the art and described in the literature. Linkers can include bivalent or trivalent branched linkers. In embodiments, the conjugate is attached to the 3' end of the antisense oligomer. For methods of preparing oligonucleotide conjugates, see, e.g., U.S. Pat. No. 8,450,467, "Carbohydrate conjugates as delivery agents for oligonucleotides," which is incorporated herein by reference.

[0290] In some embodiments, the nucleic acid targeted by the ASO is an ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1 expressed in a cell, such as a eukaryotic cell. In some embodiments, the term "cell" may refer to a population of cells. In some embodiments, the cell is in a subject. In some embodiments, the cell is isolated from a subject. In some embodiments, the cell is ex vivo. In some embodiments, the cell is a cell or cell line associated with a condition or disease. In some embodiments, the cell is in vitro (e.g., in cell culture).

[0291] Pharmaceutical Composition Pharmaceutical preparations or pharmaceutical formulations for use in any of the described methods, including the described compositions, such as antisense oligonucleotides, can be prepared according to conventional techniques well known in the pharmaceutical industry and described in published literature.In embodiments, the pharmaceutical composition or formulation for treating a subject comprises an effective amount of any antisense oligomer described herein, or its pharmaceutically acceptable salt, solvate, hydrate or ester.The pharmaceutical preparations comprising antisense oligomers can further comprise pharmaceutically acceptable excipients, diluents or carriers.

[0292] Pharmaceutically acceptable salts are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., and are commensurate with 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. Salts may be used in conjunction with other salts during the final isolation and purification of the compounds. It can be prepared in situ, or can be prepared separately by reacting its free base form with a suitable organic acid. Examples of pharmaceutically acceptable non-toxic acid addition salts include salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or salts of amino groups formed with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or salts of amino groups formed by other established 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, etc. phosphate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, 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 or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc.Further pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as, for example, halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkylsulfonates, and arylsulfonates.

[0293] In some embodiments, the compositions are formulated into any of many possible dosage forms, including, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. In some embodiments, the compositions are formulated as suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran. Suspensions may contain stabilizers. In embodiments, 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).

[0294] The pharmaceutical compositions or formulations described herein may optionally include one or more penetration enhancers, carriers, excipients, or other active or inactive ingredients known to those of skill in the art or described in the published literature. In embodiments, liposomes also include sterically stabilized liposomes, such as liposomes containing one or more specialized lipids. These specialized lipids result in liposomes with extended circulation lifetimes. In embodiments, sterically stabilized liposomes contain 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 pharmaceuticals, formulations, and emulsions is well known in the art. In embodiments, the present disclosure employs a penetration enhancer to effect efficient delivery of antisense oligonucleotides, e.g., to aid diffusion across cell membranes and / or increase the permeability of lipophilic drugs. In some embodiments, the penetration enhancer is a surfactant, fatty acid, bile salt, chelating agent, or non-chelating non-surfactant.

[0295] In some embodiments, the pharmaceutical formulation comprises more than one antisense oligonucleotide. In embodiments, the antisense oligonucleotide is administered in combination with another drug or therapeutic agent.

[0296] Combination therapy In some embodiments, the ASOs disclosed herein can be used in combination with one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents can include small molecules. For example, the one or more additional therapeutic agents can include small molecules described in International Publication Nos. 2016128343A1, 2017053982A1, 2016196386A1, 201428459A1, 201524876A2, 2013119916A2, and 2014209841A2, each of which is incorporated by reference in its entirety.

[0297] Targeted treatment 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, e.g., a human, or an animal such as 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 child. In other embodiments, the individual can be another eukaryotic organism, such as a plant. In some embodiments, the compositions provided herein are administered to cells ex vivo.

[0298] In some embodiments, the compositions provided herein are administered to an individual as a method of treating a disease or disorder. 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 is at high risk of having a disease or disorder caused by insufficient protein amount or protein activity. When an individual is at "high risk" of having a disease or disorder caused by insufficient protein amount or protein activity, the method involves prevention or prophylactic treatment. For example, an individual may be at high risk of having such a disease or disorder because of a family history of the disease. Typically, individuals at high risk of having such a disease or disorder benefit from prophylactic treatment (e.g., by preventing or delaying the onset or progression of the disease or disorder). In embodiments, a fetus is treated in utero, for example, by administering an ASO composition to the fetus directly or indirectly (e.g., via the mother).

[0299] The suitable route for administering ASO of the present disclosure can vary depending on the cell type that ASO is desired to be delivered to.Multiple tissues and organs are affected by Dravet syndrome, and the most significantly affected tissue is the brain.ASO of the present disclosure can be administered parenterally to patients, for example, by intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection or intravenous injection.

[0300] In embodiments, antisense oligonucleotides are administered together with one or more agents capable of promoting penetration of the subject antisense oligonucleotides across the blood-brain barrier by any method known in the art. For example, delivery of agents by administration of adenoviral vectors to motor neurons in muscle tissue is described in U.S. Patent No. 6,632,427, "Adenoviral-vector-mediated gene transfer into medullary motor neurons," which is incorporated herein by reference. Delivery of vectors directly to the brain, for example, the striatum, thalamus, hippocampus, or substantia nigra, is described in, for example, U.S. Patent No. 6,756,523, "Adenovirus vectors for the transfer of foreign genes into the cells of the central nervous system, particularly in the brain," which is incorporated herein by reference.

[0301] In some embodiments, the antisense oligonucleotide is coupled or conjugated to an agent that provides desirable pharmaceutical or pharmacological properties. In embodiments, the antisense oligonucleotide is coupled to an agent known in the art, such as an antibody against the transferrin receptor, that enhances penetration or transport across the blood-brain barrier. In embodiments, the antisense oligonucleotide is coupled to a viral vector, for example, to enhance the effectiveness of the antisense compound or increase transport across the blood-brain barrier. In embodiments, disruption of the permeable blood-brain barrier is achieved by the addition of sugars, such as mesoerythritol, 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, adonisol, or the like. This is supported by infusion of thorbitol, D(+)arabitol, L(-)arabitol, D(+)fucose, L(-)fucose, D(-)lyxose, L(+)lyxose, and L(-)lyxose, or 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 materials for enhancing penetration of the blood-brain barrier are described, for example, in U.S. Pat. No. 9,193,969, entitled "Compositions and methods for selective delivery of oligonucleotide molecules to specific neuron types," U.S. Pat. No. 4,866,042, entitled "Method for the delivery of genetic material across the blood-brain barrier," U.S. Pat. No. 6,294,520, entitled "Material for passage through the blood-brain barrier," and U.S. Pat. No. 6,936,589, entitled "Parenteral delivery systems," each of which is incorporated herein by reference.

[0302] In some embodiments, the ASOs of the present disclosure are conjugated to dopamine reuptake inhibitors (DRIs), selective serotonin reuptake inhibitors (SSRIs), noradrenaline reuptake inhibitors (NRIs), norepinephrine-dopamine reuptake inhibitors (NDRIs), and serotonin-norepinephrine-dopamine reuptake inhibitors (SNDRIs), for example, using the methods described in U.S. Pat. No. 9,193,969, which is incorporated herein by reference.

[0303] In some embodiments, subjects treated using the methods and compositions are assessed for improvement in their condition using any method known and described in the art.

[0304] How to identify additional ASOs to facilitate ASCE inclusion Also within the scope of the present disclosure are methods for identifying or determining ASOs that promote the inclusion of ASCE in processed mRNA that is processed from a pre-mRNA that contains ASCE. Also within the scope of the present disclosure are methods for identifying or determining ASOs that promote the inclusion of ASCE in ASCE-containing pre-mRNAs of PKD1, ABCA4, FUS, CEL, or NSD1. Also within the scope of the present disclosure are methods for identifying or determining ASOs that promote the inclusion of ASCE in processed mRNA that is processed from a pre-mRNA that contains ASCE.

[0305] For example, the method may involve identifying or determining an ASO that promotes the inclusion of ASCE in an ASCE-containing pre-mRNA of PKD1, ABCA4, FUS, CEL, or NSD1. ASOs that specifically hybridize to various nucleotides within the target region of the pre-mRNA may be screened to identify or determine an ASO that improves the rate and / or extent of splicing of the target intron. In some embodiments, the ASO may block or interfere with the binding site of a splicing repressor / silencer. Any method known in the art may be used to identify (determine) an ASO that produces a desired effect (e.g., exon inclusion, protein or functional RNA production) when hybridized to the target region of an exon. These methods may also be used to identify ASOs that promote exon inclusion of an excluded exon by binding to a targeted region in an intron adjacent to the excluded exon or to a targeted region in a non-excluded exon. Examples of methods that may be used are provided below.

[0306] A series of screens, referred to as an ASO "walk," can be performed using ASOs designed to hybridize to target regions of pre-mRNA. For example, the ASOs used in the ASO walk can be tiled every 5 nucleotides from approximately 100 nucleotides upstream of the 3' splice site adjacent to the ASCE (e.g., a portion of the intron sequence located upstream of the target / ASCE) to approximately 100 nucleotides downstream of the 3' splice site adjacent to the target / ASCE, and / or from approximately 100 nucleotides upstream of the 5' splice site adjacent to the ASCE to approximately 100 nucleotides downstream of the 5' splice site adjacent to the target / ASCE (e.g., a portion of the intron sequence located downstream of the target / ASCE). For example, a first ASO 15 nucleotides in length can be designed to specifically hybridize to nucleotides -6 to -20 relative to the 3' splice site adjacent to the target / ASCE. A second ASO can be designed to specifically hybridize to nucleotides -1 to -15 relative to the 3' splice site adjacent to the target / ASCE. ASO is designed to span the target region of pre-mRNA.In some embodiments, ASO can be more carefully tiled, for example, every 1, 2, 3, or 4 nucleotides.Furthermore, ASO can be tiled from 100 nucleotides downstream of the 5' splice site to 100 nucleotides upstream of the 3' splice site.In some embodiments, ASO can be tiled from about 1000 or 500 nucleotides upstream of the 3' splice site to about 1000 or 500 nucleotides downstream of the 5' splice site.In some embodiments, ASO can be tiled from about 1000 or 500 nucleotides upstream of the 3' splice site to about 1000 or 500 nucleotides downstream of the 3' splice site.

[0307] One or more ASOs, or a control ASO (e.g., an ASO with a scrambled sequence, an ASO with a sequence not expected to hybridize to the target region), are delivered, for example, by transfection, to a disease-related cell line expressing a target pre-mRNA (e.g., an ASCE-containing pre-mRNA described herein). The effect of exon inclusion of each ASO can be assessed by any method known in the art, for example, by reverse transcriptase (RT)-PCR using primers spanning the splice junction, as described in Example 3. An increase or presence of longer RT-PCR products produced using primers spanning a region containing the ASCE (e.g., including the flanking introns of the ASCE) in ASO-treated cells compared to control ASO-treated cells indicates reduced splicing out of the target ASCE. In some embodiments, the exon inclusion efficiency, the ratio of unspliced ​​to spliced ​​pre-mRNA, the splicing rate, or the extent of splicing can be modulated using the ASOs described herein. The amount of protein or functional RNA encoded by the target mRNA precursor can also be evaluated to determine whether each ASO has achieved the desired effect (e.g., improved production of functional protein). For example, any method known in the art for assessing and / or quantifying protein production, such as Western blotting, Jess blotting, flow cytometry, immunofluorescence microscopy, and ELISA, can be used.

[0308] A second series of screens, termed ASO "microwalks," can be performed using ASOs designed to hybridize to target regions of pre-mRNA. The ASOs used in the ASO microwalk are tiled nucleotide by nucleotide to further refine the nucleotide sequence of the pre-mRNA that results in exon inclusion (or reduced splicing of the ASO) when hybridized with the ASO.

[0309] The region defined by the ASO that reduces splicing of the target exon is explored in more detail by ASO "microwalks," involving ASOs spaced every 1 nt, as well as longer ASOs, typically 18-25 nt.

[0310] As described above with respect to ASO walks, ASO microwalks are performed by delivering one or more ASOs, or a control ASO (e.g., an ASO with a scrambled sequence, an ASO with a sequence not expected to hybridize to the target region), for example, by transfection, into a disease-related cell line expressing the target pre-mRNA. The splicing-inducing effect of each ASO can be assessed by any method known in the art, for example, by reverse transcriptase (RT)-PCR using primers spanning the ASCE as described herein (see, e.g., Example 5). An increase or presence of longer RT-PCR products produced using primers spanning the ASCE in ASO-treated cells compared to control ASO-treated cells indicates enhanced exon inclusion. In some embodiments, the exon inclusion efficiency, the ratio of unspliced ​​to spliced ​​pre-mRNA, the splicing rate, or the extent of splicing can be modulated using the ASOs described herein. The amount of protein or functional RNA encoded by the target mRNA precursor can also be evaluated to determine whether each ASO has achieved the desired effect (e.g., improved production of functional protein). For example, any method known in the art for assessing and / or quantifying protein production, such as Western blotting, Jess blotting, flow cytometry, immunofluorescence microscopy, and ELISA, can be used.

[0311] ASOs that hybridize to regions of pre-mRNA and result in increased exon inclusion and protein production can be tested in vivo using animal models, such as transgenic mouse models with full-length human genes knocked in or humanized mouse models of disease. The preferred route for ASO administration can vary depending on the disease and / or the cell type to which the ASO is desired to be delivered. ASOs can be administered, for example, by intrathecal, intracerebroventricular, intraperitoneal, intramuscular, subcutaneous, or intravenous injection. After administration, the cells, tissues, and / or organs of the model animal can be evaluated to determine the effectiveness of ASO treatment by assessing splicing (e.g., efficiency, speed, extent) and protein production, for example, by methods known in the art and described herein. Animal models can also be any phenotypic or behavioral indicator of disease or disease severity.

[0312] Also within the scope of this disclosure are methods for identifying or validating ASCE in the presence of an NMD inhibitor, for example, cycloheximide. An exemplary method is provided in Example 2. [Example]

[0313] The present disclosure will be further illustrated by the following examples, but it should be understood that the present disclosure is not limited to these examples in any way.

[0314] Example 1. Identifying NMD-induced exon inclusion events in transcripts by RNAseq using next-generation sequencing Whole-transcriptome shotgun sequencing was performed using next-generation sequencing to reveal a snapshot of the transcripts produced by the genes described herein and identify ASCE inclusion events. To this end, polyA+ RNA was isolated from nuclear and cytoplasmic fractions of human cells, and cDNA libraries were constructed using Illumina's TruSeq Stranded mRNA Library Prep Kit. Libraries were sequenced using a paired-end method, yielding 100-nucleotide reads that mapped to the human genome (GRCh38 / hg38 assembly).

[0315] Example 2. Confirmation of ASCE via cycloheximide treatment RT-PCR analysis using RNA extracts from human and mouse cells after DMSO or cycloheximide treatment and primers in the exons (e.g., a forward primer complementary to exon 7 and a reverse primer complementary to exon 9) confirmed the presence of bands corresponding to NMD-induced exon exclusion events. Treatment of cells with cycloheximide to inhibit NMD can induce an increase in products corresponding to NMD-induced exon exclusion events in the cytoplasmic fraction. RT-PCR and quantification of the cassette exon (exon 8: GRCh38 / hg38:chr5 177238237:177238507) in NSD1 RNA was performed. Densitometric analysis of the bands on the RT-PCR product images was performed to calculate the percentage of ASCE inclusion of the total transcript. Figures 2A-D show confirmation of an exemplary alternative splicing event in the NSD1 gene via cycloheximide treatment in various human cells, as well as the presence of nonproductive NSD1 mRNA transcripts in cynomolgus monkey brain regions and human cortex. Figure 2A shows a schematic diagram in which peaks corresponding to RNA sequencing reads are identified in exon 8 of NSD1 (GRCh38 / hg38:chr5 177238237:177238507). Figure 2B shows gel images and graphs demonstrating that cycloheximide treatment resulted in an increase in the amount of nonproductive mature NSD1 mRNA transcripts (processed NSD1 mRNA containing a premature stop codon that renders the transcript targetable for NMD) in various human cells, including astrocytes, Schwann cells, HEK293 cells, SH-SY-5Y (neuroblastoma cell line), and SK-N-AS (neuroblastoma cell line) cells. Figure 2C shows a gel image and a graph showing the presence of non-productive mature NSD1 mRNA transcripts in various cynomolgus monkey brain regions, including the cortex, brainstem, hippocampus, and cerebellum. Figure 2D shows a gel image and a graph showing the presence of non-productive mature NSD1 mRNA transcripts in the human cortex.

[0316] Example 3. Confirmation of ASCE via cycloheximide treatment in mice RT-PCR analysis using total RNA from mouse brain regions (cortex, deep structures, cerebellum, and brainstem) after in vivo or ex vivo DMSO or cycloheximide treatment and primers in the exons (e.g., a forward primer complementary to mouse exon 6 and a reverse primer complementary to mouse exon 8) confirmed the presence of a band corresponding to the ASCE exclusion event (Figures 3A-3D). Figure 3A shows gel images demonstrating that exclusion of ASCE of mouse NSD1 (mouse exon 7 corresponding to human exon 8) in mouse brain after ex vivo cycloheximide or DMSO treatment induces the formation of a processed mRNA containing a premature stop codon that renders the transcript targetable for NMD. Figure 3B shows graphs of the percentage of NMD (top panel) and fold change in NMD events (bottom panel) of non-productive NSD1 mRNA products compared to productive NSD1 mRNA, according to densitometric analysis of bands from the gel image in Figure 3A to calculate the percentage of ASCE. The fold change in the bottom panel of Figure 3B was calculated as the fold change in the percentage of NMD between DMSO-treated and cycloheximide-treated samples, i.e., the percentage of NMD in the cycloheximide-treated samples divided by the percentage of NMD in the corresponding DMSO-treated samples, for each indicated brain region. Figure 3C shows gel images demonstrating that exclusion of ASCE (mouse exon 7 corresponding to human exon 8) of mouse NSD1 induces the formation of processed mRNA containing a premature stop codon that renders the transcript eligible for NMD in mouse brains treated with cycloheximide in vivo for 3, 6, or 12 hours. Figure 3D shows graphs of the percentage of NMD (left panel) and fold change of NMD events (right panel) of non-productive NSD1 mRNA products compared to productive NSD1 mRNA according to densitometric analysis of bands from the gel image in Figure 3C to calculate the percentage of ASCE.The fold changes in the right panel of Figure 3D were calculated as the fold change in percentage of NMD between saline- and cycloheximide-treated samples, i.e., the percentage of NMD in the cycloheximide-treated sample divided by the percentage of NMD in the corresponding saline-treated sample, for each indicated brain region.

[0317] Figures 4A-4B show confirmation of the inclusion or exclusion of mouse NSD1 ASCE (mouse exon 7 corresponding to human exon 8) in NSD1 mRNA products processed from NSD1 precursor mRNA in mouse brain via in vivo cycloheximide treatment. Figure 4A shows a gel image demonstrating that exclusion of mouse NSD1 ASCE (mouse exon 7 corresponding to human exon 8) in mouse brain after in vivo cycloheximide treatment induces the formation of processed mRNA containing a premature stop codon, rendering the transcript a target for NMD. Figure 4B shows graphs of the percentage of NMD (left panel) and fold change in NMD events (right panel) of non-productive NSD1 mRNA products compared to productive NSD1 mRNA, according to densitometric analysis of bands from the gel image in Figure 4A to calculate the percentage of ASCE. The fold changes in the right panel of Figure 4B were calculated as the fold change in the percentage of NMD between saline- and cycloheximide-treated samples, i.e., the percentage of NMD in the 60 mg / kg or 120 mg / kg cycloheximide-treated samples divided by the percentage of NMD in the corresponding saline-treated sample.

[0318] Example 4. ASO walk of the ASCE region Using 2'-MOE ASOs in a PS backbone, ASO walks were performed for ASCE regions targeting sequences upstream of the canonical 3' splice site beyond the 3' splice site, skipped exons (e.g., exon 8) beyond the 5' splice site, and downstream of the 5' splice site. ASOs can be designed to cover these regions by shifting five nucleotides at a time, or by shifting any predetermined number of nucleotides at a time. In some embodiments, ASO walks can be performed for ASCE regions targeting sequences that do not extend beyond the 3' splice site and / or do not extend beyond the 5' splice site. Figure 5 shows an exemplary ASO walk for an exemplary ASCE region. Shaded nucleotides in Figure 5 correspond to exon skipping events, and arrows indicate canonical splice sites.

[0319] Example 5. ASO walk of the ASCE region assessed by RT-PCR The ASO walk sequence can be evaluated, for example, by RT-PCR. PAGE can be used to show the SYBR-safe stained RT-PCR products of mock-treated cells or ASO-treated cells targeting the ASCE region described herein at a concentration of 20 μM, due to gymnotic uptake in human / mouse cells. The products corresponding to exon exclusion and full-length can be quantified, and the percentage of NMD can be plotted. The full-length product can be normalized to an internal standard.

[0320] In one experiment, HEK293 cells were transfected with an exemplary ASO according to some embodiments of the present disclosure at a concentration of 80 nM for 24 hours. Figure 6A shows a graph summarizing the changes in productive NSD1 mRNA levels during one ASO walk around the cassette exon (exon 8). Figure 6B shows a graph summarizing the changes in non-productive NSD1 mRNA levels during one ASO walk around the cassette exon (exon 8).

[0321] In another experiment, an exemplary ASO according to some embodiments of the present disclosure was used at a concentration of 5 mM to perform an ASO microwalk by nucleofecting SH-SY-5Y cells for 24 hours. Figure 7A shows a graph summarizing the changes in productive NSD1 mRNA levels during one ASO walk around the cassette exon (exon 8). Figure 7B shows a graph summarizing the changes in non-productive NSD1 mRNA levels during one ASO walk around the cassette exon (exon 8).

[0322] Example 6. Walk of vectored ASO in the ASCE region of NSD1 ASO walks are performed on ASCE regions targeting sequences upstream of the canonical 3' splice site beyond the 3' splice site, skipped exons (e.g., exon 8) beyond the 5' splice site, and downstream of the 5' splice site to identify vectored ASOs that can prevent non-productive AS events (e.g., promote ASCE inclusion in processed mRNA). Systematic vectored ASO walks can be performed every 5 nt or 2 nt along the AS event of interest. These vectored ASOs can be expressed as modified U1 snRNA or U7 snRNA from vectors containing the ASO sequence as their targeting sequence. Figure 8 shows a systematic vectored ASO walk along the AS event of NSD1 pre-mRNA for vectored ASOs expressed as modified U7 snRNA. Figure 9 shows a systematic vectored ASO walk along the AS event of NSD1 pre-mRNA for vectored ASOs expressed as U1 snRNA. RT-PCR analysis from transfected cell lines can identify several vectored ASOs that induce a reduction in AS in NSD1 mRNA (e.g., promoting the inclusion of ASCE in processed mRNA) and an increase in productive mRNA. The observed increase in NSD1 productive mRNA can be confirmed by TaqMan qPCR. The fold change in AS can be plotted against the increase in productive mRNA (measured by qPCR) to demonstrate that the vectored ASO is mechanistically functioning.

[0323] Example 7. Non-productive mRNA levels of NSD1 in various cell lines Alternative NMD inhibitors (i.e., non-ASOs) were tested in various cell lines to assess baseline differences in levels of non-productive RNA across different cell types.

[0324] The cell lines used were SH-SY5Y, U-87MG, HEK293, and SK-N-AS. SH-SY5Y is a subcloned cell line derived from a neuroblastoma cell line derived from a metastatic bone tumor. U-87MG is a cell line isolated from a malignant glioma exhibiting epithelial morphology. HEK (Human Embryonic Kidney) 293 is a cell line routinely used in basic biotechnology research. SK-N-AS cells are human neuroblastoma cells derived from neuroblastoma cells.

[0325] The NMD inhibitors tested included SMG1 Nonsense-Mediated MRNA Decay Associated PI3K Related Kinase inhibitor (SMG1i) and cycloheximide (CHX). SMG1i is an inhibitor of the nonsense-mediated mRNA decay (NMD) regulator SMG1 and was originally designed to target multiple myeloma. SMG1i was used as the NMD inhibitor and tested alongside CHX, a standard mRNA translation inhibitor also known to inhibit NMD. The effects of the NMD inhibitors were measured in various cell lines to determine whether the cell lines had different baseline levels of nonproductive RNA, which is interpreted as equivalent to NMD events. Each of the four cell lines (SH-SY5Y, U-87 MG, HEK293, and SK-N-AS) was incubated with a negative control (mock) and NMD inhibitors (CHX at a final concentration of 50 μg / ml and SMG1i at a final concentration of 1 μM) for three hours to assess baseline levels of non-productive NSD1 mRNA (Figure 10). After treatment with either the water-only mock control, CHX, or SMG1i, cells from each cell line were harvested, and RNA was extracted and quantified. Treatment with SMG1i resulted in approximately 28% NSD1 non-productive mRNA (the percentage of non-productive NSD1 mRNA transcripts relative to the total level of all NSD1 mRNA transcripts) in U-87 MG cells, approximately 19% in SH-SY5Y cells, and <18% in HEK293 and SK-N-AS cells. Treatment with CHX resulted in approximately 23% NSD1 non-productive mRNA in SH-SY5Y cells, approximately 15% in U-87 MG cells, and approximately 13% in HEK293 and SK-N-AS cells. In cells treated with water (mock) alone, the percentage of non-productive RNA remained low.

[0326] Example 8. Effect of exemplary chemically modified ASOs The effects of ASOs with modified backbone chemistries on U-87 MG cells were determined. U-87 MG cells were treated with either (1) ASOs with phosphorodiamidate morpholino (PMO) modifications or (2) ASOs with 2'-O-methoxyethyl modifications and a phosphorothioate backbone (2'MOE-PS) (Figure 11A, Table 6).

[0327] NSD1 mRNA levels were assessed 24 hours after nucleofection into U-87 MG cells with either 2 μM PMO-modified ASO or 1 μM 2'MOE-PS-modified ASO, and the fold change was quantified for productive and non-productive mRNA transcripts compared to mock controls (Figure 11B). All results were normalized to the mock control. PMO-containing ASO 1749 corresponds in sequence to 2'MOE-PS-containing ASO 1752. Both chemically modified ASO 1749 and ASO 1752 resulted in at least a 1.1-fold increase in productive NSD1 mRNA compared to mock controls. ASO 1749 resulted in an approximately 0.4-fold reduction in non-productive NSD1 mRNA compared to a water-only mock control, and ASO 1752 resulted in an approximately 0.3-fold reduction in non-productive NSD1 mRNA. PMO-containing ASO 1750 corresponds in sequence to 2'MOE-PS-containing ASO 1754. PMO-containing ASO 1750 resulted in at least an approximately 1.2-fold increase in productive NSD1 mRNA compared to a mock control and an approximately 0.3-fold reduction in non-productive NSD1 mRNA. 2'MOE-PS-containing ASO 1754 resulted in at least an approximately 1.1-fold increase in productive NSD1 mRNA compared to a mock control and an approximately 0.2-fold reduction in non-productive NSD1 mRNA. PMO-containing ASO 1751 corresponds in sequence to 2'MOE-PS-containing ASO 1755. PMO-containing ASO 1751 resulted in at least a 1.2-fold increase in productive NSD1 mRNA and an approximately 0.3-fold decrease in non-productive NSD1 mRNA compared to the mock control. 2'MOE-PS-containing ASO 1755 resulted in no change in productive NSD1 mRNA but an approximately 0.3-fold decrease in non-productive NSD1 mRNA compared to the mock control. 2'MOE-PS-containing ASO 1753 resulted in at least a 1.2-fold increase in productive NSD1 mRNA and an approximately 0.3-fold decrease in non-productive NSD1 mRNA compared to the mock control.Overall, when cells were treated with either 2 μM of ASO with PMO modification or 1 μM of ASO with 2'MOE-PS modification, the levels of productive NSD1 mRNA increased and non-productive NSD1 mRNA decreased compared to mock controls.

[0328] NSD1 protein levels were assessed 72 hours after nucleofection into U-87 MG cells with either 2 μM ASO with PMO modification or 1 μM ASO with 2′MOE-PS modification and compared with mock controls ( FIG. 11C ). All results were normalized to mock controls. 2′MOE-PS-containing ASO 1752 resulted in at least a 1.3-fold increase in NSD1 protein compared with mock controls. PMO-containing ASO 1750 resulted in at least a 1.1-fold increase in NSD1 protein compared with water-only mock controls. 2′MOE-PS-containing ASO 1754 resulted in at least a 1.2-fold increase in NSD1 protein compared with mock controls. PMO-containing ASO 1751 resulted in at least a 1.2-fold increase in NSD1 protein compared with mock controls. The 2'MOE-PS-containing ASO 1755 resulted in at least a 1.1-fold increase in NSD1 protein compared to the mock control. The 2'MOE-PS-containing ASO 1753 resulted in at least a 1.2-fold increase in NSD1 protein compared to the mock control. In general, when cells were treated with either 2 μM of ASO with PMO modification or 1 μM of ASO with 2'MOE-PS modification, NSD1 protein levels increased compared to the mock control. Thus, the effects of the MOE-PS ASO hits are directly transferable (i.e., behave similarly to ASOs) to ASOs with alternative backbones, such as those modified with PMO. [Table 15]

[0329] Example 9. Upregulation of NSD1 protein by exemplary ASOs resulted in a general increase in H3K36me2 levels H3K36me2 is an epigenetic modification on histone H3, and NSD1 is a histone methyltransferase that can mediate the dimethylation of histone H3 at residue K36 (H3K36me2). NSD1-mediated H3K36me2 may contribute to the recruitment of DNA methyltransferases and the maintenance of DNA methylation in intergenic regions. Therefore, we investigated the levels of H3K36me2 to determine whether upregulation of NSD1 protein by ASO promotes elevated H3K36me2 levels in U-87 MG cells. All results in this example include data derived from two to three independent experiments. All results for each assay are normalized to the water-only control and are the mean ± SEM.

[0330] ASOs were evaluated in U-87 MG cells to assess their efficacy in increasing NSD1 protein expression and H3K36me2 levels. U-87 MG cells were nucleofected with 1 μM of one of four exemplary ASOs (ASO 214, ASO 210, ASO 211, or ASO 215) and harvested 72 hours after nucleofection. NSD1 protein levels for each of the four exemplary ASOs were measured by immunocapillary electrophoresis (JESS) and compared with the water-only control ( FIG. 12A ). When U-87 MG cells were treated with ASO 210, ASO 211, or ASO 215, NSD1 protein levels were higher than those of the water-only control, whereas when U-87 MG cells were treated with ASO 214, NSD1 protein levels were slightly lower than those of the water-only control. Treatment with ASO 210 resulted in an approximately 1.25-fold increase in NSD1 protein levels. Treatment with ASO 211 resulted in an approximately 1.3-fold increase in NSD1 protein levels. Treatment with ASO 215 resulted in an approximately 1.15-fold increase in NSD1 protein levels. Treatment with ASO 214 reduced NSD1 protein levels to approximately 0.96-fold compared to water-only controls.

[0331] Cellular H3K36me2 levels, as measured by AlphaLISA® assay, increased after treatment with all four ASOs (Figure 12B). H3K36me2 levels were approximately 1.41-fold higher in cells treated with ASO214. Compared to the H3K36me2 levels in cells treated with water alone, H3K36me2 levels were approximately 1.39-fold higher in cells treated with ASO 210, approximately 1.38-fold higher in cells treated with ASO 211, and approximately 1.35-fold higher in cells treated with ASO 215. In summary, the four tested ASOs induced both increased NSD1 protein levels and elevated cellular H3K36me2 levels in U-87 MG cells.

[0332] Example 10. ASO 211 resulted in a dose-dependent increase in global H3K36me2 ASO 211 was further evaluated to determine whether varying dosages affected NSD1 protein expression and levels of H3K36me2 in U-87 MG cells.

[0333] U-87 MG cells were nucleofected with one of four doses (0.25 μM, 0.5 μM, 1.0 μM, or 2.0 μM) of the hit ASO (ASO 211) or a water-only control. Cells were harvested 72 hours after nucleofection, and cellular levels of NSD1 protein, H3K36me2, and total histone 3 (H3) were quantified. All results in this example include data from two to three independent experiments. All results for each assay were normalized to the water-only control. Results are shown as mean ± SEM by one-way ANOVA; * indicates p<0.05, *** indicates p<0.01, and **** indicates p<0.001.

[0334] NSD1 protein levels were measured by immuno-capillary electrophoresis (JESS). NSD1 protein levels were higher at all tested concentrations of ASO 211 than in the water-only control (Figure 13A). Specifically, ASO 211 at 0.25 μM resulted in an approximately 1.1-fold increase in NSD1 protein levels compared to the water-only control, 0.5 μM resulted in an approximately 1.2-fold increase, 1.0 μM resulted in an approximately 1.4-fold increase, and 2.0 μM resulted in an approximately 1.1-fold increase. Treatment of U-87 cells with 1.0 μM ASO 211 resulted in the highest fold increase (approximately 1.4-fold) in NSD1 protein compared to the remaining three ASO concentrations. Treatment of U-87 cells with 0.5 μM ASO 211 resulted in the second highest fold increase (approximately 1.2-fold) in NSD1 protein compared to the three other ASO concentrations. Treatment of U-87 cells with either the highest or lowest concentrations of ASO 211 (0.25 μM and 2.0 μM) resulted in the smallest fold increase in NSD1 protein (approximately 1.1-fold) compared to concentrations in between of the other ASOs tested.

[0335] As measured by AlphaLISA® assay, cellular H3K36me2 levels increased after treatment with ASO 211 at all tested concentrations ( FIG. 13B ). H3K36me2 levels increased approximately 1.3-fold in cells treated with 1.0 μM ASO 211 compared to cells treated with water alone. H3K36me2 levels were approximately 1.1-fold higher when cells were treated with either 0.25 μM or 0.5 μM ASO 211 than in cells treated with water alone. H3K36me2 levels were approximately 1.2-fold higher when cells were treated with 2.0 μM ASO 211 than in cells treated with water alone. Lower dose concentrations of ASO generally resulted in smaller fold changes in cellular H3K36me2 levels.

[0336] To rule out the possibility that the observed effects on NSD1 protein expression and H3K36me2 levels were due to changes in cellular levels of histone H3, we also measured total H3 levels using the AlphaLISA® assay (Figure 13C). Histone H3 levels were found to be similar across all experimental conditions, regardless of whether water or ASO 211 at any concentration was used. Therefore, it is likely that the modulation of NSD1 protein expression and H3K36me2 levels by ASO211 was not caused by changes in total histone H3 levels, but rather by the presence of the ASO itself and the experimental concentrations tested. In summary, ASO211 was found to increase global H3K36me2 levels in a dose-dependent manner.

[0337] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present disclosure. It is understood that various alternatives to the embodiments of the present disclosure may be employed in practicing the disclosure. The following claims define the scope of the disclosure, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. A method for regulating expression of a target protein in a cell, the cell comprising a pre-mRNA transcribed from a target gene and encoding the target protein, the pre-mRNA comprising an alternatively spliced ​​coding exon (ASCE), and wherein alternatively processed mRNA produced by splicing out ASCE during processing of the pre-mRNA is subject to nonsense-mediated RNA decay, the method comprising contacting the cell with a therapeutic agent or a vector encoding the therapeutic agent, wherein the therapeutic agent promotes inclusion of ASCE during processing of the pre-mRNA, thereby increasing the level of processed mRNA processed from the pre-mRNA and comprising ASCE.

2. 1. A method of treating a disease or condition or reducing the likelihood of developing a disease or condition in a subject in need thereof by modulating expression of a target protein in the cells of the subject, the method comprising contacting the cells of the subject with a therapeutic agent or a vector encoding the therapeutic agent, wherein the cells comprise a pre-mRNA transcribed from a target gene and encoding the target protein, the pre-mRNA comprising an alternatively spliced ​​coding exon (ASCE), wherein alternatively processed mRNA produced by splicing out the ASCE during processing of the pre-mRNA undergoes nonsense-mediated RNA decay, and the therapeutic agent promotes inclusion of the ASCE during processing of the pre-mRNA, thereby increasing the level of processed mRNA that is processed from the pre-mRNA and comprises the ASCE.

3. The method of claim 1 or 2, wherein expression of the target protein is increased in the cell.

4. 4. The method of any one of claims 1 to 3, wherein the target gene is selected from the group consisting of: PKD1, ABCA4, FUS, CEL, and NSD1.

5. 5. The method of any one of claims 1 to 4, wherein the target protein is selected from the group consisting of polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxyl ester lipase, and nuclear receptor-binding SET domain protein 1.

6. The therapeutic agent is (a) binding to a targeted portion of the mRNA encoding the target protein; (b) modulating the binding of a factor involved in the splicing of the ASCE; or 6. The method of claim 1, wherein (c) a combination of (a) and (b) is performed.

7. The method of claim 6, wherein the therapeutic agent prevents binding of the factor involved in splicing to the region of the targeted portion of the ASCE.

8. 7. The method of claim 6, wherein the targeting moiety is proximal to the ASCE.

9. The method of claim 6, wherein the targeted portion is up to about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides upstream of the 5' end of the ASCE.

10. The method of claim 6, wherein the targeted portion is at least about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, or about 1 nucleotide upstream of the 5' end of the ASCE.

11. The method of claim 6, wherein the targeted portion is up to about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides downstream of the 3' end of the ASCE.

12. The method of claim 6, wherein the targeted portion is at least about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, or about 1 nucleotide downstream of the 3' end of the ASCE.

13. The method of claim 6, wherein the targeted portion is up to about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides upstream of a genomic site selected from the group consisting of: GRCh38 / hg38:chr16 2092954, GRCh38 / hg38:chr1 94111438, GRCh38 / hg38:chr16 31186802, GRCh38 / hg38:chr9 133066530, and GRCh38 / hg38:chr5 177238237.

14. The method of claim 6, wherein the targeted portion is about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides upstream of a genomic site selected from the group consisting of: GRCh38 / hg38:chr16 2092954, GRCh38 / hg38:chr1 94111438, GRCh38 / hg38:chr16 31186802, GRCh38 / hg38:chr9 133066530, and GRCh38 / hg38:chr5 177238237.

15. The method of claim 6, wherein the targeted portion is up to about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides downstream of a genomic site selected from the group consisting of: GRCh38 / hg38:chr16 2093093, GRCh38 / hg38:chr1 94111579, GRCh38 / hg38:chr16 31186836, GRCh38 / hg38:chr9 133066660, and GRCh38 / hg38:chr5 177238507.

16. The method of claim 6, wherein the targeted portion is about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides downstream of a genomic site selected from the group consisting of: GRCh38 / hg38:chr16 2093093, GRCh38 / hg38:chr1 94111579, GRCh38 / hg38:chr16 31186836, GRCh38 / hg38:chr9 133066660, and GRCh38 / hg38:chr5 177238507.

17. The method of claim 6, wherein the targeted portion is located in an intron region between the ASCE and a canonical exon region upstream of the ASCE of the mRNA encoding the target protein.

18. The method of claim 6, wherein the targeted portion is located in an intron region between the ASCE and a canonical exon region downstream of the ASCE of the mRNA encoding the target protein.

19. 7. The method of claim 6, wherein the targeting portion at least partially overlaps with the ASCE.

20. The method of claim 6, wherein the targeted portion at least partially overlaps with an intron upstream or downstream of the ASCE.

21. The method of claim 6, wherein the targeted portion does not include a 5' exon-intron junction or a 3' exon-intron junction.

22. The method of claim 6 , wherein the target moiety is within the ASCE.

23. 7. The method of claim 6, wherein the targeted portion comprises about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more consecutive nucleotides of the ASCE.

24. 24. The method of any one of claims 1 to 23, wherein the mRNA encoding the target protein comprises a sequence having at least about 80%, 85%, 90%, 95%, 97% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 6-10.

25. 24. The method of any one of claims 1 to 23, wherein the mRNA encoding the target protein is encoded by a gene sequence having at least about 80%, 85%, 90%, 95%, 97% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1-5.

26. 24. The method of any one of claims 1 to 23, wherein the targeted portion of the mRNA comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 consecutive nucleic acids of a sequence selected from the group consisting of SEQ ID NOs: 6-10.

27. 27. The method of any one of claims 1-26, wherein the targeted portion of mRNA is within an ASCE selected from the group consisting of: GRCh38 / hg38:chr16 2092954 2093093, GRCh38 / hg38:chr1 94111438 94111579, GRCh38 / hg38:chr16 31186802 31186836, GRCh38 / hg38:chr9 133066530 133066660, and GRCh38 / hg38:chr5 177238237 177238507.

28. 27. The method of any one of claims 1 to 26, wherein the targeted portion of an mRNA is upstream or downstream of an ASCE selected from the group consisting of: GRCh38 / hg38:chr16 2092954 2093093, GRCh38 / hg38:chr1 94111438 94111579, GRCh38 / hg38:chr16 31186802 31186836, GRCh38 / hg38:chr9 133066530 133066660, and GRCh38 / hg38:chr5 177238237 177238507.

29. 27. The method of any one of claims 1 to 26, wherein the targeted portion of the mRNA does not include an exon-intron junction of ASCE selected from the group consisting of: GRCh38 / hg38:chr16 2092954 2093093, GRCh38 / hg38:chr1 94111438 94111579, GRCh38 / hg38:chr16 31186802 31186836, GRCh38 / hg38:chr9 133066530 133066660, and GRCh38 / hg38:chr5 177238237 177238507.

30. 30. The method of any one of claims 1 to 29, wherein the target protein produced is a full-length or wild-type protein.

31. Inclusion of the ASCE during processing of the pre-mRNA in a cell contacted with the therapeutic agent or the vector encoding the therapeutic agent is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 10 ...

31. The method of any one of claims 1 to 30, wherein the IL-16 expression level is increased by about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold.

32. The level of the processed mRNA produced in a cell contacted with the therapeutic agent or the vector encoding the therapeutic agent is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 1.1 to about 10-fold, about 1.1 to about 11-fold, about 1.1 to about 12-fold, about 1.1 to about 13-fold, about 1.1 to about 14-fold, about 1.1 to about 15-fold, about 1.1 to about 16-fold, about 1.1 to about 17-fold, about 1.1 to about 18-fold, about 1.1 to about 19-fold, about 1.1 to about 20-fold, about 1.1 to about 21-fold, about 1.1 to about 22-fold, about 1.1 to about 23-fold, about 1.1 to about 24-fold, about 1.1 to about 25-fold, about 1.1 to about 26-fold, about 1.1 to about 27-fold, about 1.1 to about 28-fold, about 1.1 to about 29-fold, about 1.1 to about 30-fold, about 1.1 to about 31-fold, about 1.1 to about 32-fold, about 1.1 to about 33-fold, about 1.1 to about 34-fold, about 1.1 to about 35-fold, about 1.1 to about 36-fold, about 1.1 to about 37-fold, about 1.

32. The method of any one of claims 1 to 31, wherein the IL-16 expression level is increased by up to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold.

33. the level of the target protein produced in cells contacted with the therapeutic agent is about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times higher than the level of the target protein produced in corresponding cells not contacted with the therapeutic agent or the vector encoding the therapeutic agent; 33. The method of any one of claims 1 to 32, wherein the IL-16 expression level is increased by about 2 to about 6 fold, about 2 to about 7 fold, about 2 to about 8 fold, about 2 to about 9 fold, about 3 to about 6 fold, about 3 to about 7 fold, about 3 to about 8 fold, about 3 to about 9 fold, about 4 to about 7 fold, about 4 to about 8 fold, about 4 to about 9 fold, at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 5 fold, or at least about 10 fold.

34. The elimination of ASCE during processing of the pre-mRNA in cells contacted with the therapeutic agent is about 1.1-fold to about 10-fold, about 1.5-fold to about 10-fold, about 2-fold to about 10-fold, about 3-fold to about 10-fold, about 4-fold to about 10-fold, about 1.1-fold to about 5-fold, about 1.1-fold to about 6-fold, about 1.1-fold to about 7-fold, about 1.1-fold to about 8-fold, about 1.1-fold to about 9-fold, or about 2-fold to about 5-fold lower than the elimination of ASCE during processing of the pre-mRNA in corresponding cells not contacted with the therapeutic agent or the vector encoding the therapeutic agent.

34. The method of any one of claims 1 to 33, wherein the ionic strength is reduced by a factor of 1 / 2 to about 6, 1 / 2 to about 7, 1 / 2 to about 8, 1 / 2 to about 9, 1 / 3 to about 6, 1 / 3 to about 7, 1 / 3 to about 8, 1 / 3 to about 9, 1 / 4 to about 7, 1 / 4 to about 8, 1 / 4 to about 9, at least about 1.1, at least about 1.5, at least about 2, at least about 2.5, at least about 3, at least about 3.5, at least about 4, at least about 5, or at least about 10.

35. 35. The method of any one of claims 1 to 34, wherein the target protein is NSD1 and the method causes modification of a histone protein in the cell.

36. 36. The method of claim 35, wherein the histone protein is histone H3.

37. 37. The method of claim 35 or 36, wherein the modification comprises acetylation, methylation, phosphorylation, or ubiquitination.

38. 37. The method of claim 35 or 36, wherein the modification is methylation.

39. 39. The method of claim 38, wherein the methylation of the histone protein is increased in the cell.

40. the methylation of the histone protein in cells contacted with the therapeutic agent or the vector encoding the therapeutic agent is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, compared to the methylation of the histone protein in corresponding cells not contacted with the therapeutic agent or the vector encoding the therapeutic agent; 39. The method of claim 38, wherein the increase is by about 1.1 to about 9 fold, about 2 to about 5 fold, about 2 to about 6 fold, about 2 to about 7 fold, about 2 to about 8 fold, about 2 to about 9 fold, about 3 to about 6 fold, about 3 to about 7 fold, about 3 to about 8 fold, about 3 to about 9 fold, about 4 to about 7 fold, about 4 to about 8 fold, about 4 to about 9 fold, at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 5 fold, or at least about 10 fold.

41. 41. The method of any one of claims 1 to 40, wherein the method further comprises assessing the mRNA level or expression level of the target protein.

42. 42. The method of any one of claims 2 to 41, wherein the disease or condition is caused by a loss-of-function mutation in the target gene.

43. 42. The method of any one of claims 2 to 41, wherein the disease or condition is associated with haploinsufficiency of the gene encoding the target protein, and the subject has a first allele that encodes a functional target protein and a second allele in which the target protein is not produced or is produced at a reduced level, or a second allele that encodes a non-functional or partially functional target protein.

44. 44. The method of any one of claims 2 to 43, wherein the disease or condition is selected from the group consisting of polycystic kidney disease with or without polycystic liver disease 1, autosomal dominant polycystic kidney disease, age-related macular degeneration-2, Stargardt disease 1, amyotrophic lateral sclerosis, amyotrophic lateral sclerosis with or without frontotemporal dementia 6, hereditary essential tremor 4, frontotemporal dementia, maturity-onset diabetes of the young with exocrine insufficiency type 8, maturity-onset diabetes of the young, Sotos syndrome 1, and Beckwith-Wiedemann syndrome.

45. 41. The method of any one of claims 2 to 40, wherein the disease or condition is associated with an autosomal recessive mutation in a gene encoding the target protein, and the subject has a first allele that encodes (i) the target protein is not produced or is produced at a reduced level compared to the wild-type allele, or (ii) the produced target protein is non-functional or partially functional compared to the wild-type allele, and a second allele that (iii) the target protein is produced at a reduced level compared to the wild-type allele and the produced target protein is at least partially functional compared to the wild-type allele, or (iv) the produced target protein is partially functional compared to the wild-type allele.

46. 41. The method of any one of claims 2 to 40, wherein the disease or condition is caused by a gain-of-function mutation in the target protein.

47. 47. The method of claim 46, wherein the subject has an allele in which the target protein is produced at increased levels or an allele that encodes a mutant target protein that exhibits increased activity in the cell.

48. 48. The method of any one of claims 2 to 47, wherein the subject is a human.

49. 48. The method of any one of claims 2 to 47, wherein the subject is a non-human animal.

50. 48. The method of any one of claims 2 to 47, wherein the subject is a fetus, embryo, or child.

51. 48. The method of any one of claims 2 to 47, wherein the cells are ex vivo or in a tissue or organ that is ex vivo.

52. 48. The method of any one of claims 2 to 47, wherein the therapeutic agent is administered to the subject by intrathecal, intracerebroventricular, intraperitoneal, intramuscular, subcutaneous, intravitreal, or intravenous injection.

53. 48. The method of any one of claims 2 to 47, wherein the method further comprises administering a second therapeutic agent to the subject.

54. 54. The method of claim 53, wherein the second therapeutic agent is a small molecule.

55. 54. The method of claim 53, wherein the second therapeutic agent is an antisense oligomer.

56. 54. The method of claim 53, wherein the second therapeutic agent modifies intron retention.

57. 57. The method of any one of claims 2 to 56, wherein the disease or condition is a disease or condition associated with a deficiency in the amount or activity of the target protein.

58. 58. The method of any one of claims 2 to 57, wherein the disease or condition is a disease or condition associated with a deficiency in the amount or activity of a protein, and the target protein functionally enhances, compensates for, replaces, or functionally interacts with the protein.

59. 58. The method of any one of claims 2 to 57, wherein the disease or condition is caused by an insufficient amount or activity of the target protein.

60. 60. The method of any one of claims 2 to 59, wherein the method further comprises evaluating the subject's genome for at least one genetic variation associated with the disease.

61. 61. The method of claim 60, wherein at least one genetic mutation is within the locus of a gene associated with the disease.

62. 61. The method of claim 60, wherein at least one genetic variation is within a locus associated with expression of a gene associated with the disease.

63. 61. The method of claim 60, wherein at least one genetic mutation is within the locus of a gene encoding the target protein.

64. 61. The method of claim 60, wherein at least one genetic variation is within a locus associated with expression of a gene encoding the target protein.

65. 65. The method of any one of claims 2 to 64, wherein the method treats the disease or condition.

66. 66. The method of any one of claims 1 to 65, wherein the target protein is a canonical isoform of the protein.

67. 67. The method of any one of claims 1 to 66, wherein the alternatively processed mRNA produced by splicing out the ASCE comprises a premature termination codon (PTC).

68. 68. The method of any one of claims 1 to 67, wherein the agent is an antisense oligomer (ASO).

69. 69. The method of claim 68, wherein the ASO is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to the targeted portion of the mRNA.

70. 70. The method of claim 68 or 69, wherein the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complementary to at least eight contiguous nucleic acids of a sequence selected from the group consisting of SEQ ID NOs: 6-10.

71. 71. The method of any one of claims 68 to 70, wherein the ASO comprises backbone modifications including phosphorothioate or phosphorodiamidate linkages.

72. 71. The method of any one of claims 68 to 70, wherein the ASO comprises a phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety.

73. 71. The method of any one of claims 68 to 70, wherein the ASO comprises at least one modified sugar moiety.

74. 74. The method of claim 73, wherein each sugar moiety is a modified sugar moiety.

75. The ASO may be 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, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 75. The method of any one of claims 68 to 74, consisting of 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 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, or 12 to 15 nucleobases.

76. 75. The method of any one of claims 68 to 74, wherein the target gene is NSD1 and the ASO comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 16-1748.

77. 68. The method of any one of claims 1 to 67, wherein the target gene is NSD1 and the vector encoding the agent encodes a polynucleotide comprising a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of the ASO sequences listed in Table 4, Table 5A, Table 5A-1, Table 5B, Table 5B-1, Table 5D, Table 5E, Table 5G, and Table 5G-1.

78. 78. The method of claim 77, wherein the vector encoding the agent is a viral vector.

79. 79. The method of claim 78, wherein the viral vector is an adenovirus-associated viral vector.

80. 68. The method of any one of claims 1 to 67, wherein the vector encoding the agent encodes a polynucleotide comprising an ASO sequence and snRNA.

81. 81. The method of claim 80, wherein the snRNA comprises a modified snRNA.

82. 82. The method of claim 81, wherein the modified snRNA is a modified U1 snRNA or a modified U7 snRNA.

83. 83. The method of any one of claims 80 to 82, wherein the snRNA comprises U1 snRNA.

84. 84. The method of claim 83, wherein the target gene is NSD1 and the ASO sequence comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of the ASO sequences listed in Table 4, Table 5A, Table 5B, Table 5D, Table 5E, and Table 5G.

85. 83. The method of any one of claims 80 to 82, wherein the snRNA comprises U7 snRNA.

86. 86. The method of claim 85, wherein the target gene is NSD1 and the ASO comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of the ASO sequences listed in Table 4, Table 5A, Table 5A-1, Table 5B, Table 5B-1, Table 5G, Table 5G-1.

87. A composition comprising a drug or a vector encoding a drug, wherein the drug regulates the splicing of a pre-mRNA in a cell that is transcribed from a target gene and encodes a target protein, the pre-mRNA containing an alternatively spliced ​​coding exon (ASCE), and the alternatively processed mRNA produced by splicing out the ASCE during processing of the pre-mRNA is subject to nonsense-mediated RNA decay, and the drug promotes the inclusion of the ASCE during processing of the pre-mRNA, thereby increasing the level of processed mRNA that is processed from the pre-mRNA and contains the ASCE.

88. 88. The composition of claim 87, wherein the agent increases expression of the target protein in the cell.

89. The composition of claim 87 or 88, wherein the target gene is selected from the group consisting of: PKD1, ABCA4, FUS, CEL, and NSD1.

90. 90. The composition of any one of claims 87 to 89, wherein the target protein is selected from the group consisting of polycystin-1, ATP-binding cassette subfamily A member 4, FUS RNA-binding protein, carboxyl ester lipase, and nuclear receptor-binding SET domain protein 1.

91. The drug is (a) binding to a targeted portion of the mRNA encoding the target protein; (b) modulating the binding of a factor involved in the splicing of the ASCE; or (c) combining (a) and (b),

92. The composition of claim 91, wherein the agent prevents binding of a factor involved in splicing of the ASCE to the region of the targeted portion.

93. 92. The composition of claim 91, wherein the targeting moiety is proximal to the ASCE.

94. The composition of claim 91, wherein the targeted portion is up to about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides upstream of the 5' end of the ASCE.

95. The composition of claim 91, wherein the targeted portion is at least about 1500, about 1000, about 800, about 700, about 600, about 500, about 400, about 300, about 200, about 100, about 80, about 70, about 60, about 50, about 40, about 30, about 20, about 10, about 5, about 4, about 2, or about 1 nucleotide upstream of the 5' end of the ASCE.

96. The composition of claim 91, wherein the targeted portion is up to about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides downstream of the 3' end of the ASCE.

97. The composition of claim 91, wherein the targeted portion is at least about 1500, about 1000, about 800, about 700, about 600, about 500, about 400, about 300, about 200, about 100, about 80, about 70, about 60, about 50, about 40, about 30, about 20, about 10, about 5, about 4, about 2, or about 1 nucleotide downstream of the 3' end of the ASCE.

98. The composition of claim 91, wherein the targeted portion is up to about 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides upstream of a genomic site selected from the group consisting of: GRCh38 / hg38:chr16 2092954, GRCh38 / hg38:chr1 94111438, GRCh38 / hg38:chr16 31186802, GRCh38 / hg38:chr9 133066530, and GRCh38 / hg38:chr5 177238237.

99. The composition of claim 91, wherein the targeted portion is about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides upstream of a genomic site selected from the group consisting of: GRCh38 / hg38:chr16 2092954, GRCh38 / hg38:chr1 94111438, GRCh38 / hg38:chr16 31186802, GRCh38 / hg38:chr9 133066530, and GRCh38 / hg38:chr5 177238237.

100. The composition of claim 91, wherein the targeted portion is up to about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides downstream of a genomic site selected from the group consisting of: GRCh38 / hg38:chr16 2093093, GRCh38 / hg38:chr1 94111579, GRCh38 / hg38:chr16 31186836, GRCh38 / hg38:chr9 133066660, and GRCh38 / hg38:chr5 177238507.

101. The composition of claim 91, wherein the targeted portion is about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides downstream of a genomic site selected from the group consisting of: GRCh38 / hg38:chr16 2093093, GRCh38 / hg38:chr1 94111579, GRCh38 / hg38:chr16 31186836, GRCh38 / hg38:chr9 133066660, and GRCh38 / hg38:chr5 177238507.

102. The composition of claim 91, wherein the targeting portion is located in an intron region between the ASCE and a canonical exon region upstream of the ASCE of the mRNA encoding the target protein.

103. The composition of claim 91, wherein the targeting portion is located in an intron region between the ASCE and a canonical exon region downstream of the ASCE of the mRNA encoding the target protein.

104. 92. The composition of claim 91, wherein the targeting portion at least partially overlaps with the ASCE.

105. 92. The composition of claim 91, wherein the targeted portion at least partially overlaps with an intron upstream or downstream of the ASCE.

106. 92. The composition of claim 91, wherein the targeted portion does not include a 5' exon-intron junction or a 3' exon-intron junction.

107. 92. The composition of claim 91, wherein the targeting moiety is within the ASCE.

108. 92. The composition of claim 91, wherein the targeting portion comprises about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more consecutive nucleotides of the ASCE.

109. 109. The composition of any one of claims 87-108, wherein the mRNA encoding the target protein comprises a sequence having at least about 80%, 85%, 90%, 95%, 97% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 6-10.

110. 109. The composition of any one of claims 87 to 108, wherein the mRNA encoding the target protein is encoded by a gene sequence having at least about 80%, 85%, 90%, 95%, 97% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1-5.

111. 111. The composition of any one of claims 87 to 110, wherein the targeted portion of an mRNA comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 consecutive nucleic acids of a sequence selected from the group consisting of SEQ ID NOs: 6-10.

112. The composition of any one of claims 87-111, wherein the targeted portion of the mRNA is within an ASCE selected from the group consisting of: GRCh38 / hg38:chr16 2092954 2093093, GRCh38 / hg38:chr1 94111438 94111579, GRCh38 / hg38:chr16 31186802 31186836, GRCh38 / hg38:chr9 133066530 133066660, and GRCh38 / hg38:chr5 177238237 177238507.

113. The composition of any one of claims 87-111, wherein the targeted portion of the mRNA is upstream or downstream of an ASCE selected from the group consisting of: GRCh38 / hg38:chr16 2092954 2093093, GRCh38 / hg38:chr1 94111438 94111579, GRCh38 / hg38:chr16 31186802 31186836, GRCh38 / hg38:chr9 133066530 133066660, and GRCh38 / hg38:chr5 177238237 177238507.

114. 112. The composition of any one of claims 87-111, wherein the targeted portion of the mRNA does not include an exon-intron junction of ASCE selected from the group consisting of: GRCh38 / hg38:chr16 2092954 2093093, GRCh38 / hg38:chr1 94111438 94111579, GRCh38 / hg38:chr16 31186802 31186836, GRCh38 / hg38:chr9 133066530 133066660, and GRCh38 / hg38:chr5 177238237 177238507.

115. 115. The composition of any one of claims 87 to 114, wherein the target protein produced is a full-length or wild-type protein.

116. Inclusion of the ASCE during processing of the pre-mRNA in a cell contacted with the therapeutic agent or the vector encoding the therapeutic agent is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, or ... or about 2 to about 10-fold, or about 2 to about 10-fold, or about 3 to about 10-fold, or about 4 to about 10-fold, or about 1.1 to about 5-fold, about 1.1 to about 6-fold, about fold, about 1.1 to about 9 fold, about 2 to about 5 fold, about 2 to about 6 fold, about 2 to about 7 fold, about 2 to about 8 fold, about 2 to about 9 fold, about 3 to about 6 fold, about 3 to about 7 fold, about 3 to about 8 fold, about 3 to about 9 fold, about 4 to about 7 fold, about 4 to about 8 fold, about 4 to about 9 fold, at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 5 fold, or at least about 10 fold.

117. The level of processed mRNA produced in the cells contacted with the therapeutic agent or the vector encoding the therapeutic agent is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 1.1 to about 10-fold, about 1.1 to about 11-fold, about 1.1 to about 12-fold, about 1.1 to about 13-fold, about 1.1 to about 14-fold, about 1.1 to about 15-fold, about 1.1 to about 16-fold, about 1.1 to about 17-fold, about 1.1 to about 18-fold, about 1.1 to about 19-fold, about 1.1 to about 20-fold, about 1.1 to about 21-fold, about 1.1 to about 22-fold, about 1.1 to about 23-fold, about 1.1 to about 24-fold, about 1.1 to about 25-fold, about 1.1 to about 26-fold, about 1.1 to about 27-fold, about 1.1 to about 28-fold, about 1.1 to about 29-fold, about 1.1 to about 30-fold, about 1.1 to about 31-fold, about 1.1 to about 32-fold, about 1.1 to about 33-fold, about 1.1 to about 34-fold, about 1.1 to about 35-fold, about 1.1 to about 36-fold, about 1.1 to about 37-fold, about 1.1 117. The composition of any one of claims 87 to 116, wherein the ELISA assay is increased by about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold.

118. The level of the target protein produced in cells contacted with the therapeutic agent is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 118. The composition of any one of claims 87 to 117, wherein the IL-16 concentration is increased by up to about 6 fold, about 2 to about 7 fold, about 2 to about 8 fold, about 2 to about 9 fold, about 3 to about 6 fold, about 3 to about 7 fold, about 3 to about 8 fold, about 3 to about 9 fold, about 4 to about 7 fold, about 4 to about 8 fold, about 4 to about 9 fold, at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 5 fold, or at least about 10 fold.

119. The elimination of ASCE during processing of the pre-mRNA in cells contacted with the therapeutic agent is about 1.1 fold to about 10 fold, about 1.5 fold to about 10 fold, about 2 fold to about 10 fold, about 3 fold to about 10 fold, about 4 fold to about 10 fold, about 1.1 fold to about 5 fold, about 1.1 fold to about 6 fold, about 1.1 fold to about 7 fold, about 1.1 fold to about 8 fold, about 1.1 fold to about 9 fold, about 2 fold to about 5 fold, about 119. The composition of any one of claims 87 to 118, wherein the composition is reduced by a factor of from 2 to about 6, from about 2 to about 7, from about 2 to about 8, from about 2 to about 9, from about 3 to about 6, from about 3 to about 7, from about 3 to about 8, from about 3 to about 9, from about 4 to about 7, from about 4 to about 8, from about 4 to about 9, at least about 1.1, at least about 1.5, at least about 2, at least about 2.5, at least about 3, at least about 3.5, at least about 4, at least about 5, or at least about 10.

120. 120. The composition of any one of claims 87 to 119, wherein the target protein is NSD1 and the method causes modification of a histone protein in the cell.

121. 121. The composition of claim 120, wherein the histone protein is histone H3.

122. 122. The composition of claim 120 or 121, wherein the modification comprises acetylation, methylation, phosphorylation, or ubiquitination.

123. 122. The composition of claim 120 or 121, wherein the modification is methylation.

124. The composition of claim 123, wherein the methylation of the histone protein is increased in the cell.

125. The methylation of histone proteins in the cells contacted with the therapeutic agent or the vector encoding the therapeutic agent is about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 1.1 to about 10-fold, about 1.1 to about 11-fold, about 1.1 to about 12-fold, about 1.1 to about 13-fold, about 1.1 to about 14-fold, about 1.1 to about 15-fold, about 1.1 to about 16-fold, about 1.1 to about 17-fold, about 1.1 to about 18-fold, about 1.1 to about 19-fold, about 1.1 to about 20-fold, about 1.1 to about 21-fold, about 1.1 to about 22-fold, about 1.1 to about 23-fold, about 1.1 to about 24-fold, about 1.1 to about 25-fold, about 1.1 to about 26-fold, about 1.1 to about 27-fold, about 1.1 to about 28-fold, about 1.1 to about 29-fold, about 1.1 to about 30-fold, about 1.1 to about 31-fold, about 1.1 to about 32-fold, about 1.1 to about 33-fold, about 1.1 to about 34-fold, about 1.1 to about 35-fold, about 1.1 to about 36-fold, about 1.1 to about 37-fold, about 1 124. The composition of claim 123, wherein the IL-16 concentration is increased by up to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold.

126. 126. The composition of any one of claims 87 to 125, wherein the target protein is the canonical isoform of the protein.

127. 127. The composition of any one of claims 87 to 126, wherein the alternatively processed mRNA produced by splicing out the ASCE comprises a premature termination codon (PTC).

128. 128. The composition of any one of claims 87 to 127, wherein the agent is an antisense oligomer (ASO).

129. The composition of claim 128, wherein the ASO is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to the targeted portion of the mRNA.

130. 130. The composition of claim 128 or 129, wherein the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complementary to at least eight contiguous nucleic acids of a sequence selected from the group consisting of SEQ ID NOs: 6-10.

131. 131. The composition of any one of claims 128 to 130, wherein the ASO comprises backbone modifications including phosphorothioate or phosphorodiamidate linkages.

132. 131. The composition of any one of claims 128 to 130, wherein the ASO comprises a phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety.

133. 131. The composition of any one of claims 128 to 130, wherein the ASO comprises at least one modified sugar moiety.

134. 134. The composition of claim 133, wherein each sugar moiety is a modified sugar moiety.

135. The ASO may be 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, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 135. The composition of any one of claims 128 to 134, consisting of up to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 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, or 12 to 15 nucleobases.

136. The composition of any one of claims 128 to 135, wherein the ASO comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 16-1748.

137. 120. The composition of any one of claims 87 to 119, wherein the target gene is NSD1 and the agent vector encoding the agent encodes a polynucleotide comprising a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of the ASO sequences listed in Table 4, Table 5A, Table 5A-1, Table 5B, Table 5B-1, Table 5D, Table 5E, Table 5G, and Table 5G-1.

138. The composition of claim 137, wherein the vector encoding the agent is a viral vector.

139. 139. The composition of claim 138, wherein the viral vector is an adenovirus-associated viral vector.

140. 120. The composition of any one of claims 87 to 119, wherein the vector encoding the agent encodes a polynucleotide comprising an ASO sequence and snRNA.

141. 141. The composition of claim 140, wherein the snRNA comprises a modified snRNA.

142. 142. The composition of claim 141, wherein the modified snRNA is a modified U1 snRNA or a modified U7 snRNA.

143. 143. The composition of any one of claims 140 to 142, wherein the snRNA comprises U1 snRNA.

144. The composition of claim 143, wherein the target gene is NSD1 and the ASO sequence comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of the ASO sequences listed in Table 4, Table 5A, Table 5B, Table 5D, Table 5E, and Table 5G.

145. 143. The composition of any one of claims 140 to 142, wherein the snRNA comprises U7 snRNA.

146. The composition of claim 145, wherein the target gene is NSD1 and the ASO comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of the ASO sequences listed in Table 4, Table 5A, Table 5A-1, Table 5B, Table 5B-1, Table 5G, and Table 5G-1.

147. A composition comprising an ASO comprising a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 16-1748.

148. The composition of claim 147, wherein the ASO comprises backbone modifications including phosphorothioate or phosphorodiamidate linkages.

149. 148. The composition of claim 147, wherein the ASO comprises a phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety.

150. The composition of claim 147, wherein the ASO comprises at least one modified sugar moiety.

151. 151. The composition of claim 150, wherein each sugar moiety is a modified sugar moiety.

152. The ASO may be 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, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 152. The composition of any one of claims 147 to 151, consisting of up to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 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, or 12 to 15 nucleobases.

153. A composition comprising a vector encoding a polynucleotide comprising a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of the ASO sequences listed in Table 4, Table 5A, Table 5A-1, Table 5B, Table 5B-1, Table 5D, Table 5E, Table 5G, and Table 5G-1.

154. The composition of claim 153, wherein the vector encoding the agent is a viral vector.

155. 155. The composition of claim 154, wherein the viral vector is an adenovirus-associated viral vector.

156. 156. The composition of any one of claims 153 to 155, wherein the vector encoding the agent encodes a polynucleotide comprising an ASO sequence and snRNA.

157. 157. The composition of claim 156, wherein the snRNA comprises a modified snRNA.

158. 158. The composition of claim 157, wherein the modified snRNA is a modified U1 snRNA or a modified U7 snRNA.

159. 159. The composition of any one of claims 156 to 158, wherein the snRNA comprises U1 snRNA.

160. The composition of claim 159, wherein the ASO sequence comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of the ASO sequences listed in Table 4, Table 5A, Table 5B, Table 5D, Table 5E, and Table 5G.

161. 159. The composition of any one of claims 156 to 158, wherein the snRNA comprises U7 snRNA.

162. The composition of claim 161, wherein the ASO comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of the ASO sequences listed in Table 4, Table 5A, Table 5A-1, Table 5B, Table 5B-1, Table 5G, and Table 5G-1.

163. 163. A pharmaceutical composition comprising the composition of any one of claims 87 to 162 and a pharmaceutically acceptable excipient and / or delivery vehicle.

164. 164. A method of treating a disease or condition or reducing the likelihood of developing a disease or condition in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 163.

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