EP3389671A1

EP3389671A1 - Antisense oligomers for treatment of alagille syndrome

Info

Publication number: EP3389671A1
Application number: EP16876499.1A
Authority: EP
Inventors: Isabel AZNAREZ; Huw M. Nash
Original assignee: Cold Spring Harbor Laboratory; Stoke Therapeutics Inc
Current assignee: Cold Spring Harbor Laboratory; Stoke Therapeutics Inc
Priority date: 2015-12-14
Filing date: 2016-12-13
Publication date: 2018-10-24
Also published as: JP2018538288A; EP3389671A4; JP2022106803A; CA3005245A1; WO2017106210A1

Abstract

Provided herein are methods and compositions for increasing the expression of JAGl, and for treating a subject in need thereof, e.g., a subject with deficient JAGl protein expression or a subject having Alagille syndrome (ALGS).

Description

ANTISENSE OLIGOMERS FOR TREATMENT OF ALAGILLE SYNDROME

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 62/267,210, filed December 14, 2015, which the application is incorporated herein by reference in its entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on December 9, 2016 is named 47991-705_601_SL.txt and is 201,192 bytes in size. The aforementioned file was created on December 9, 2016, and is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[0003] Alagille syndrome (ALGS), also known as arteriohepatic dysplasia, is a rare, debilitating, autosomal dominant, multisystem disorder (Turnpenny and Ellard, Eur. J. Hum. Gen. 2012, 20, 251-257). Patients suffer from liver damage caused by abnormalities in the bile ducts. Other effects include heart disease, vascular anomalies, skeletal anomalies, ophthalmic features, facial features, renal anomalies, growth retardation, and pancreatic insufficiency. The reported ALGS prevalence of 1 : 70,000 is thought to be an underestimate because of the variability and reduced penetrance of the condition.

[0004] Mutations of genes involved in Notch signaling have been reported to cause ALGS. Mutations in JAGl cause ALGS type 1, while mutations in NOTCH2 cause ALGS type 2, which is less prevalent than ALGS type 1. JAGl encodes JAGl protein, a cell surface ligand for the Notch transmembrane receptors. Binding of JAGl protein to the Notch receptors triggers a signaling cascade that results in transcription of genes involved in cell fate determination and differentiation.

SUMMARY OF THE INVENTION

[0005] The invention provides compositions and methods for treating Alagille syndrome, including antisense oligomers (ASOs) that promote constitutive splicing at intron splice sites of a JAGl retained-intron-containing pre-mRNA (RIC pre-mRNA). The invention further provides compositions and methods for increasing production of mature JAGl mRNA and, in turn, JAGl protein, in cells of a subject in need thereof, for example, a subject that can benefit from increased production of JAGl protein. The described methods may be used to treat subjects having Alagille Syndrome caused by a mutation(s) in the JAGl gene, including missense, splicing, frameshift and nonsense mutations, as well as whole gene deletions, that result in deficient JAGl protein production. In other embodiments, the compositions and methods of the present invention are used to treat subjects having a muscular dystrophy, who can benefit from increased production of JAGl protein.

[0006] Disclosed herein, in certain embodiments, is a method of treating Alagille syndrome in a subject in need thereof by increasing the expression of a target protein or functional RNA by cells of the subject, wherein the cells have a retained-intron-containing pre-mRNA (RIC pre- mRNA), the RIC pre-mRNA comprising a retained intron, an ex on flanking the 5' splice site, an ex on flanking the 3' splice site, and wherein the RIC pre-mRNA encodes the target protein or functional RNA, the method comprising contacting the cells of the subject with an antisense oligomer (ASO) complementary to a targeted portion of the RIC pre-mRNA encoding the target protein or functional RNA, whereby the retained intron is constitutively spliced from the RIC pre-mRNA encoding the target protein or functional RNA, thereby increasing the level of mRNA encoding the target protein or functional RNA, and increasing the expression of the target protein or functional RNA in the cells of the subject. In some embodiments, also disclosed herein is a method of increasing expression of a target protein, wherein the target protein is JAGl, by cells having a retained-intron-containing pre-mRNA (RIC pre-mRNA), the RIC pre- mRNA comprising a retained intron, an ex on flanking the 5' splice site of the retained intron, an ex on flanking the 3' splice site of the retained intron, and wherein the RIC pre-mRNA encodes JAGl protein, the method comprising contacting the cells with an antisense oligomer (ASO) complementary to a targeted portion of the RIC pre-mRNA encoding JAGl protein, whereby the retained intron is constitutively spliced from the RIC pre-mRNA encoding JAGl protein, thereby increasing the level of mRNA encoding JAGl protein, and increasing the expression of JAGl protein in the cells. In some embodiments, the target protein is JAGl . In some

embodiments, the target protein or the functional RNA is a compensating protein or a compensating functional RNA that functionally augments or replaces a target protein or functional RNA that is deficient in amount or activity in the subject. In some embodiments, the cells are in or from a subject having a condition caused by a deficient amount or activity of JAGl protein. In some embodiments, the deficient amount of the target protein is caused by haploinsufficiency of the target protein, wherein the subject has a first allele encoding a functional target protein, and a second allele from which the target protein is not produced, or a second allele encoding a nonfunctional target protein, and wherein the antisense oligomer binds to a targeted portion of a RIC pre-mRNA transcribed from the first allele. In some embodiments, the subject has a condition caused by a disorder resulting from a deficiency in the amount or function of the target protein, wherein the subject has a) a first mutant allele from which (i) the target protein is produced at a reduced level compared to production from a wild-type allele, (ii) the target protein is produced in a form having reduced function compared to an equivalent wild- type protein, or (iii) the target protein is not produced, and b) a second mutant allele from which (i) the target protein is produced at a reduced level compared to production from a wild-type allele, (ii) the target protein is produced in a form having reduced function compared to an equivalent wild-type protein, or (iii) the target protein is not produced, and wherein when the subject has a first mutant allele a.iii., the second mutant allele is b.i. or b.ii., and wherein when the subject has a second mutant allele b.iii., the first mutant allele is a.i. or a.ii., and wherein the RIC pre-mRNA is transcribed from either the first mutant allele that is a.i. or a.ii., and/or the second allele that is b.i. or b.ii. In some embodiments, the target protein is produced in a form having reduced function compared to the equivalent wild-type protein. In some embodiments, the target protein is produced in a form that is fully-functional compared to the equivalent wild- type protein. In some embodiments, the targeted portion of the RIC pre-mRNA is in the retained intron within the region +6 relative to the 5' splice site of the retained intron to -16 relative to the 3' splice site of the retained intron. In some embodiments, the targeted portion of the RIC pre-mRNA is in the retained intron within: (a) the region +6 to +500, +6 to +400, +6 to 300, +6 to 200, or +6 to +100 relative to the 5' splice site of the retained intron; or (b) the region -16 to - 500, -16 to -400, -16 to -300, -16 to -200, or -16 to -100 relative to the 3' splice site of the retained intron. In some embodiments, the targeted portion of the RIC pre-mRNA is within: (a) the region +2e to -4e in the exon flanking the 5' splice site of the retained intron; or (b) the region +2e to -4e in the exon flanking the 3' splice site of the retained intron. In some embodiments, the RIC pre-mRNA is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the RIC pre-mRNA comprises a sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 2. In some embodiments, the antisense oligomer does not increase the amount of the target protein or the functional RNA by modulating alternative splicing of pre-mRNA transcribed from a gene encoding the functional RNA or target protein. In some embodiments, the antisense oligomer does not increase the amount of the target protein or the functional RNA by modulating aberrant splicing resulting from mutation of the gene encoding the target protein or the functional RNA. In some embodiments, the RIC pre-mRNA was produced by partial splicing of a full-length pre- mRNA or partial splicing of a wild-type pre-mRNA. In some embodiments, the mRNA encoding the target protein or functional RNA is a full-length mature mRNA, or a wild-type mature mRNA. In some embodiments, the target protein produced is full-length protein, or wild- type protein. In some embodiments, the total amount of the mRNA encoding the target protein or functional RNA produced in the cell contacted with the antisense oligomer is increased 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, compared to the total amount of the mRNA encoding the target protein or functional RNA produced in a control cell. In some embodiments, the total amount of target protein produced by the cell contacted with the antisense oligomer is increased 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, compared to the total amount of target protein produced by a control cell. In some embodiments, the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage. In some embodiments, the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-0-methyl, a 2'-Fluoro, or a 2'-0-methoxyethyl moiety. In some embodiments, the antisense oligomer comprises at least one modified sugar moiety. In some embodiments, each sugar moiety is a modified sugar moiety. In some embodiments, the antisense oligomer consists of from 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 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. In some embodiments, the antisense oligomer 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 RIC pre-mRNA encoding the protein. In some embodiments, the targeted portion of the RIC pre-mRNA is within a sequence selected from SEQ ID NOs: 437-439. In some embodiments, the antisense oligomer comprises a nucleotide sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 3-436. In some embodiments, the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NOs: 3-436. In some embodiments, the cell comprises a population of RIC pre-mRNAs transcribed from the gene encoding the target protein or functional RNA, wherein the population of RIC pre-mRNAs comprises two or more retained introns, and wherein the antisense oligomer binds to the most abundant retained intron in the population of RIC pre-mRNAs. In some embodiments, the binding of the antisense oligomer to the most abundant retained intron induces splicing out of the two or more retained introns from the population of RIC pre-mRNAs to produce mRNA encoding the target protein or functional RNA. In some embodiments, the cell comprises a population of RIC pre-mRNAs transcribed from the gene encoding the target protein or functional RNA, wherein the population of RIC pre-mRNAs comprises two or more retained introns, and wherein the antisense oligomer binds to the second most abundant retained intron in the population of RIC pre-mRNAs. In some embodiments, the binding of the antisense oligomer to the second most abundant retained intron induces splicing out of the two or more retained introns from the population of RIC pre-mRNAs to produce mRNA encoding the target protein or functional RNA. In some embodiments, the condition is a disease or disorder. In some embodiments, the disease or disorder is Alagille syndrome or a muscular dystrophy. In some embodiments, the target protein and the RIC pre-mRNA are encoded by the JAG1 gene. In some embodiments, the method further comprises assessing JAG1 protein expression. In some embodiments, the antisense oligomer binds to a targeted portion of a JAG1 RIC pre-mRNA, wherein the targeted portion is within a sequence selected from SEQ ID NOS: 49, 50, 51, 52, 53, 54, 55, 56, and 57. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. In some embodiments, the subject is a fetus, an embryo, or a child. In some embodiments, the cells are ex vivo. In some embodiments, the antisense oligomer is administered by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection of the subject. In some embodiments, the 9 nucleotides at -3e to - le of the ex on flanking the 5' splice site and +1 to +6 of the retained intron, are identical to the corresponding wild-type sequence. In some embodiments, the 16 nucleotides at -15 to -1 of the retained intron and +le of the exon flanking the 3' splice site are identical to the corresponding wild-type sequence.

[0007] Disclosed herein, in certain embodiments, is an antisense oligomer as used in a method described above. [0008] Disclosed herein, in certain embodiments, is an antisense oligomer comprising a sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 3-436.

[0009] Disclosed herein, in certain embodiments, is a pharmaceutical composition comprising the antisense oligomer described above and an excipient.

[0010] Disclosed herein, in certain embodiments, is a method of treating a subject in need thereof by administering the pharmaceutical composition described above by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.

[0011] Disclosed herein, in certain embodiments, is a composition comprising an antisense oligomer for use in a method of increasing expression of a target protein or a functional RNA by cells to treat Alagille syndrome in a subject in need thereof associated with a deficient protein or deficient functional RNA, wherein the deficient protein or deficient functional RNA is deficient in amount or activity in the subject, wherein the antisense oligomer enhances constitutive splicing of a retained intron-containing pre-mRNA (RIC pre-mRNA) encoding the target protein or the functional RNA, wherein the target protein is: (a) the deficient protein; or (b) a compensating protein which functionally augments or replaces the deficient protein or in the subject; and wherein the functional RNA is: (a) the deficient RNA; or (b) a compensating functional RNA which functionally augments or replaces the deficient functional RNA in the subject; wherein the RIC pre-mRNA comprises a retained intron, an exon flanking the 5' splice site and an exon flanking the 3' splice site, and wherein the retained intron is spliced from the RIC pre-mRNA encoding the target protein or the functional RNA, thereby increasing production or activity of the target protein or the functional RNA in the subject. In some embodiments, also disclosed herein is a composition comprising an antisense oligomer for use in a method of treating a condition associated with JAGl protein in a subject in need thereof, the method comprising the step of increasing expression of JAGl protein by cells of the subject, wherein the cells have a retained-intron-containing pre-mRNA (RIC pre-mRNA) comprising a retained intron, an exon flanking the 5' splice site of the retained intron, an exon flanking the 3' splice site of the retained intron, and wherein the RIC pre-mRNA encodes the JAGl protein, the method comprising contacting the cells with the antisense oligomer, whereby the retained intron is constitutively spliced from the RIC pre-mRNA transcripts encoding JAGl protein, thereby increasing the level of mRNA encoding JAGl, and increasing the expression of JAGl protein, in the cells of the subject. In some embodiments, the condition is a disease or disorder. In some embodiments, the disease or disorder is Alagille syndrome or a muscular dystrophy. In some embodiments, the target protein and RIC pre-mRNA are encoded by the JAGl gene. In some embodiments, the antisense oligomer targets a portion of the RIC pre-mRNA that is in the retained intron within the region +6 relative to the 5' splice site of the retained intron to -16 relative to the 3' splice site of the retained intron. In some embodiments, the antisense oligomer targets a portion of the RIC pre-mRNA that is in the retained intron within: (a) the region +6 to +100 relative to the 5' splice site of the retained intron; or (b) the region -16 to -100 relative to the 3' splice site of the retained intron. In some embodiments, the antisense oligomer targets a portion of the RIC pre-mRNA that is within the region about 100 nucleotides downstream of the 5' splice site of the at least one retained intron, to about 100 nucleotides upstream of the 3' splice site of the at least one retained intron. In some embodiments, the targeted portion of the RIC pre-mRNA is within: (a) the region +2e to -4e in the ex on flanking the 5' splice site of the retained intron; or (b) the region +2e to -4e in the ex on flanking the 3' splice site of the retained intron. In some embodiments, the RIC pre-mRNA is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the RIC pre-mRNA comprises a sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 2. In some embodiments, the antisense oligomer does not increase the amount of target protein or functional RNA by modulating alternative splicing of the pre-mRNA transcribed from a gene encoding the target protein or functional RNA. In some embodiments, the antisense oligomer does not increase the amount of the functional RNA or functional protein by modulating aberrant splicing resulting from mutation of the gene encoding the target protein or functional RNA. In some embodiments, the RIC pre-mRNA was produced by partial splicing from a full- length pre-mRNA or a wild-type pre-mRNA. In some embodiments, the mRNA encoding the target protein or functional RNA is a full-length mature mRNA, or a wild-type mature mRNA. In some embodiments, the target protein produced is full-length protein, or wild-type protein. In some embodiments, the retained intron is a rate-limiting intron. In some embodiments, the retained intron is the most abundant retained intron in the RIC pre-mRNA. In some

embodiments, the retained intron is the second most abundant retained intron in the RIC pre- mRNA. In some embodiments, the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage. In some embodiments, the antisense oligomer is an antisense oligonucleotide. In some embodiments, the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-0-methyl, a 2'-Fluoro, or a 2'-0-methoxyethyl moiety. In some embodiments, the antisense oligomer comprises at least one modified sugar moiety. In some embodiments, each sugar moiety is a modified sugar moiety. In some embodiments, the antisense oligomer consists of from 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 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. In some embodiments, the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or is 100%

complementary to the targeted portion of the RIC pre-mRNA encoding the protein. In some embodiments, the antisense oligomer binds to a targeted portion of a JAG1 RIC pre-mRNA, wherein the targeted portion is in a sequence selected from SEQ ID NOs 437-439. In some embodiments, the antisense oligomer comprises a nucleotide sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 3-436. In some embodiments, the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NOs: 3-436.

[0012] Disclosed herein, in certain embodients, is a pharmaceutical composition comprising the antisense oligomer of any of the compositions described above, and an excipient. In some embodiments, also described herein is a method of treating a subject in need thereof by administering the pharmaceutical composition by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.

[0013] Disclosed herein, in certain embodiments, is a pharmaceutical composition comprising: an antisense oligomer that hybridizes to a target sequence of a deficient JAG1 mRNA transcript, wherein the deficient JAG1 mRNA transcript comprises a retained intron, wherein the antisense oligomer induces splicing out of the retained intron from the deficient JAG1 mRNA transcript; and a pharmaceutical acceptable excipient. In some embodiments, the deficient JAG1 mRNA transcript is a JAG1 RIC pre-mRNA transcript. In some embodiments, the targeted portion of the JAG1 RIC pre-mRNA transcript is in the retained intron within the region +6 relative to the 5' splice site of the retained intron to -16 relative to the 3' spliced site of the retained intron. In some embodiments, the JAG1 RIC pre-mRNA transcript is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the JAG1 RIC pre-mRNA transcript comprises a sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 2. In some embodiments, the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage. In some embodiments, the antisense oligomer is an antisense oligonucleotide. In some embodiments, the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-0-methyl, a 2'-Fluoro, or a 2'-0-methoxyethyl moiety. In some embodiments, the antisense oligomer comprises at least one modified sugar moiety. In some embodiments, the antisense oligomer comprises from 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 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. In some embodiments, the antisense oligomer is at least 80%, at least 85%>, at least 90%, at least 95%, at least 98%), at least 99%, or is 100%> complementary to a targeted portion of the JAGl RIC pre- mRNA transcript. In some embodiments, the targeted portion of the JAGl RIC pre-mRNA transcript is within a sequence selected from SEQ ID NOs: 437-439. In some embodiments, the antisense oligomer comprises a nucleotide sequence that is at least about 80%>, 85%>, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 3-436. In some embodiments, the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NOs: 3-436. In some embodiments, the pharmaceutical composition is formulated for intrathecal injection, intracerebroventricular injection, intraperitoneal injection,

intramuscular injection, subcutaneous injection, or intravenous injection.

[0014] Disclosed herein, in certain embodiments, is a method of inducing processing of a deficient JAGl mRNA transcript to facilitate removal of a retained intron to produce a fully processed JAGl mRNA transcript that encodes a functional form of a tuberin protein, the method comprising: (a) contacting an antisense oligomer to a target cell of a subject; (b) hybridizing the antisense oligomer to the deficient JAGl mRNA transcript, wherein the deficient JAGl mRNA transcript is capable of encoding the functional form of tuberin protein and comprises at least one retained intron; (c) removing the at least one retained intron from the deficient JAGl mRNA transcript to produce the fully processed JAGl mRNA transcript that encodes the functional form of tuberin protein; and (d) translating the functional form of tuberin protein from the fully processed JAGl mRNA transcript. In some embodiments, the retained intron is an entire retained intron. In some embodiments, the deficient JAGl mRNA transcript is a JAGl RIC pre-mRNA transcript. [0015] Disclosed herein, in certain embodiments, is a method of treating a subject having a condition caused by a deficient amount or activity of JAG1 protein comprising: adminstering to the subject an antisense oligomer comprising a nucleotide sequence with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 3-436.

INCORPORATION BY REFERENCE

[0016] 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 DESCRIPTION OF THE DRAWINGS

[0017] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative

embodiments, in which the principles of the invention are utilized, and the accompanying drawings.

[0018] FIG. 1 illustrates a schematic representation of an exemplary retained-intron-containing (RIC) pre-mRNA transcript. The 5' splice site consensus sequence is indicated with underlined letters (letters are nucleotides; upper case: exonic portion and lower case: intronic portion) from -3e to -le and +1 to +6 (numbers labeled "e" are exonic and unlabeled numbers are intronic). The 3' splice site consensus sequence is indicated with underlined letters (letters are nucleotides; upper case: exonic portion and lower case: intronic portion) from -15 to -1 and +le (numbers labeled "e" are exonic and unlabeled numbers are intronic). Intronic target regions for ASO screening comprise nucleotides +6 relative to the 5' splice site of the retained intron (arrow at left) to -16 relative to the 3' splice site of the retained intron (arrow at right). In embodiments, intronic target regions for ASO screening comprise nucleotides +6 to +100 relative to the 5' splice site of the retained intron and -16 to -100 relative to the 3' splice site of the retained intron. Exonic target regions comprise nucleotides +2e to -4e in the ex on flanking the 5' splice site of the retained intron and +2e to -4e in the ex on flanking the 3' splice site of the retained intron. "n" or "N" denote any nucleotide, "y" denotes pyrimidine. The sequences shown represent consensus sequences for mammalian splice sites and individual introns and exons need not match the consensus sequences at every position.

[0019] FIG. 2A-FIG. 2B illustrates schematic representations of the Targeted Augmentation of Nuclear Gene Output (TANGO) approach. FIG. 2 A shows a cell divided into nuclear and cytoplasmic compartments. In the nucleus, a pre-mRNA transcript of a target gene consisting of exons (rectangles) and introns (connecting lines) undergoes splicing to generate an mRNA, and this mRNA is exported to the cytoplasm and translated into target protein. For this target gene, the splicing of intron 1 is inefficient and a retained intron-containing (RIC) pre-mRNA accumulates primarily in the nucleus, and if exported to the cytoplasm, is degraded, leading to no target protein production. FIG. 2B shows an example of the same cell divided into nuclear and cytoplasmic compartments. Treatment with an antisense oligomer (ASO) promotes the splicing of intron 1 and results in an increase in mRNA, which is in turn translated into higher levels of target protein

[0020] FIG. 3 shows intron-retention in the JAG1 gene with intron 13 detail. The

identification of intron-retention events in the JAG1 gene using RNA sequencing (RNAseq) is shown, visualized in the UCSC genome browser. The upper panel shows the read density corresponding to the JAG1 transcript expressed in THLE-3 (human liver epithelial) cells and localized in either the cytoplasmic (top) or nuclear fraction (bottom). At the bottom of this panel, a graphic representation of the JAG1 gene is shown to scale. The read density is shown as peaks. The highest read density corresponds to exons (black boxes), while no reads are observed for the majority of the introns (lines with arrow heads) in either cellular fraction. Higher read density is detected for introns 13, 18, and 23 (pointed by the arrows) in the nuclear fraction compared to the cytoplasmic fraction indicating that splicing efficiency of introns 13, 18, and 23 is low, resulting in intron retention. The retained-intron containing pre-mRNA transcripts accumulate primarily in the nucleus and are not translated into the JAG1 protein. The read density for intron 13 in THLE-3 cells is shown in detail in the lower panel indicating 12% intron retention as calculated by bioinformatic analysis.

[0021] FIG. 4 shows JAG J gene IVS 13 ASO walk. A graphic representation of the ASO walk performed for JAG1 IVS 13 targeting sequences immediately downstream of the 5' splice site or upstream of the 3' splice site using 2'-0-Me ASOs, PS backbone, is shown. ASOs were designed to cover these regions by shifting 5 nucleotides at a time. The J4G1 exon-intron structure is drawn to scale.

[0022] FIG. 5 shows JAG1 intron 13 ASO walk evaluated by radioactive RT-PCR. At the top, a schematic drawing (not to scale) of exon 13-intron 13 -exon 14 shows the primers Forward 1 (Fl) and Reverse 1 (Rl) used for the RT-PCR assay. In the middle panel, a representative gel shows radioactive RT-PCR products of JAG1 mock-treated (neg, RNAiMAX only), SMN- control ASO treated, or treated with a 2'-0-Me ASO targeting intron 13 as described in FIG. 2, at 60nM concentration in ARPE-19 cells. In the lower panel, quantification of the bands corresponding to JAG1 radioactive RT-PCR products normalized to Beta actin from two independent experiments is plotted in the bar graph as fold-change with respect to the mock- treated products. The black line labeled 1 on the Y-axis indicates a ratio of 1 (no change).

[0023] FIG. 6 shows JAG1 intron 13 ASO walk evaluated by RT-qPCR. At the top left, a schematic representation of the RT-qPCR assay shows the primer recognition sites. RT-qPCR amplification results, obtained using the same ASO transfection experiment that were evaluated by radioactive RT-PCR as shown in FIG. 3, are plotted relative to mock-treated products normalized to Beta actin (upper bar graph) or normalized to RPL32 (lower bar graph) confirming the radioactive RT-PCR results. The black line labeled 1 on the Y-axis indicates a ratio of 1 (no change).

[0024] FIG. 7 shows intron-retention in the JAG1 gene with intron 18 detail. Intron retention in the JAG1 gene was identified by RNA sequencing (RNAseq), visualized in the UCSC genome browser, as described herein in the Examples. The read density for intron 18 in TULE- 3 cells is shown in detail in the lower panel, indicating 14% intron retention as calculated by bioinformatic analysis.

[0025] FIG. 8 shows JAG1 gene IVS 18 ASO walk. A graphic representation of the ASO walk performed for JAG1 IVS 18 targeting sequences immediately downstream of the 5' splice site or upstream of the 3' splice site using 2'-0-Me ASOs, PS backbone, is shown. ASOs were designed to cover these regions by shifting 5 nucleotides at a time. The JAG1 exon-intron structure is drawn to scale.

[0026] FIG. 9 shows JAG1 intron 18 ASO walk evaluated by radioactive RT-PCR. At the top, a schematic drawing (not to scale) of exon 18 to exon 20 shows the primers Forward 1 (Fl) and Reverse 1 (Rl) used for the RT-PCR assay. In the middle panel, a representative gel shows radioactive RT-PCR products oiJAGl mock-treated (neg, RNAiMAX only), SMN-control ASO treated, or treated with a 2'-0-Me ASO targeting intron 18 as described herein in the Examples and in the description of FIG. 6, at 60nM concentration in ARPE-19 cells. Quantification of the bands corresponding to JAG1 radioactive RT-PCR products normalized to Beta actin from 2 independent experiments is plotted in the bar graph below as fold change with respect to the mock-treated products. The black line labeled 1 on the Y-axis indicates a ratio of 1 (no change).

[0027] FIG. 10 shows JAG1 intron 18 ASO walk evaluated by RT-qPCR. At the top left, a schematic representation of the RT-qPCR assay shows the primer recognition sites. RT-qPCR amplification results, obtained using the same ASO transfection experiment that were evaluated by radioactive RT-PCR as shown in FIG. 7, are plotted relative to mock-treated products normalized to Beta actin (upper bar graph) or normalized to RPL32 (lower bar graph) confirming the radioactive RT-PCR results. The black line labeled 1 on the Y-axis indicates a ratio of 1 (no change). [0028] FIG. 11 shows intron-retention in the JAGl gene with intron 23 detail. Intron retention in the JAGl gene was identified by RNA sequencing (RNAseq), visualized in the UCSC genome browser, as described herein in the Examples and in the description of FIG. 1. The read density for intron 23 in THLE-3 cells is shown in detail in the lower panel, indicating 17% intron retention as calculated by bioinformatic analysis.

[0029] FIG. 12 shows JAGl gene IVS 23 ASO walk. A graphic representation of the ASO walk performed for JAGl IVS 23 targeting sequences immediately downstream of the 5' splice site or upstream of the 3' splice site using 2'-0-Me ASOs, PS backbone, is shown. ASOs were designed to cover these regions by shifting 5 nucleotides at a time. The JAGl exon-intron structure is drawn to scale.

DETAILED DESCRIPTION OF THE INVENTION

[0030] Individual introns in primary transcripts of protein-coding genes having more than one intron are spliced from the primary transcript with different efficiencies. In most cases only the fully spliced mRNA is exported through nuclear pores for subsequent translation in the cytoplasm. Unspliced and partially spliced transcripts are detectable in the nucleus. It is generally thought that nuclear accumulation of transcripts that are not fully spliced is a mechanism to prevent the accumulation of potentially deleterious mRNAs in the cytoplasm that may be translated to protein. For some genes, splicing of the least efficient intron is a rate- limiting post-transcriptional step in gene expression, prior to translation in the cytoplasm.

[0031] Substantial levels of partially-spliced transcripts of the JAGl gene, which encodes the JAGl protein that is deficient in the debilitating genetic diseases, Alagille Syndrome, have been discovered in the nucleus of human cells. These JAGl pre-mRNA species comprise at least one retained intron. The present invention provides compositions and methods for upregulating splicing of one or more retained JAGl introns that are rate-limiting for the nuclear stages of gene expression to increase steady-state production of fully-spliced, mature mRNA, and thus, translated JAGl protein levels. These compositions and methods utilize antisense oligomers (ASOs) that promote constitutive splicing at an intron splice site of a retained-intron-containing JAGl pre-mRNA that accumulates in the nucleus. Thus, in embodiments, JAGl protein is increased using the methods of the invention to treat a condition caused by JAGl deficiency.

[0032] In other embodiments, the methods of the invention are used to increase JAGl production to treat a condition in a subject in need thereof. In embodiments, the subject has a condition in which JAGl is not necessarily deficient relative to wild-type, but where an increase in JAGl mitigates the condition nonetheless. In embodiments, the subject has a muscular dystrophy. In embodiments, the subject has a dystrophin deficiency. For example, Vieira, et al., 2015, "Jagged 1 Rescues the Duchenne Muscular Dystrophy Phenotype," Cell 163 : 1204-1213, describe findings suggesting that increasing JAGl expression in muscle can rescue dystrophin- deficient phenotypes in two different animal models for the disease.

Alagille Syndrome

[0033] Alagille syndrome (ALGS), also known as arteriohepatic dysplasia, is an autosomal dominant, multisystem disorder (Turnpenny and Ellard, 2012). The main clinical and pathological findings are liver disease due to paucity of intrahepatic bile ducts, heart disease, vascular anomalies, skeletal anomalies, ophthalmic features, facial features, renal anomalies, growth retardation, and pancreatic insufficiency. Clinical findings of ALGS are variable, with clinical diagnosis of "classic" ALGS based on the presence of 3 of 5 clinical features including anomalies of the liver, heart, vertebrae, eye, or face along with bile duct paucity (Lu et al, Am. J. Hum. Gen. 2003, 72, 1065-1070; Penton et al, Seminars Cell & Dev. Biol. 2012, 23, 450- 457). The reported ALGS prevalence of 1 :70,000 is thought to be an underestimate because of the variability and reduced penetrance of the condition.

[0034] Mutations of genes involved in Notch signaling have been reported to cause ALGS. JAGl encodes JAGl protein, a cell surface ligand for the Notch transmembrane receptors. The human genomic sequence of the JAGl gene is set forth at NCBI Gene ID 182, described by, e.g., Jurkiewicz, D., et al., 2014, "Spectrum of JAGl gene mutations in Polish patients with Alagille syndrome," J. Appl. Genetics 55:329-336, and the amino acid sequence at UniProtKB: P78504-1, Oda,T., et al., 1997, "Identification and cloning of the human homolog (JAGl) of the rat Jaggedl gene from the Alagille syndrome critical region at 20pl2," Genomics 43 (3): 376- 379, all incorporated by reference herein. The JAGl canonical mRNA sequence is set forth at NCBI Reference Sequence: NM_000214.2, described by Duryagina R, et al., 2013,

"Overexpression of Jagged-1 and its intracellular domain in human mesenchymal stromal cells differentially affect the interaction with hematopoietic stem and progenitor cells," Stem Cells Dev. 22 (20), 2736-2750, both incorporated by reference herein. Binding of JAGl ligand to Notch triggers a signaling cascade that results in transcription of genes involved in cell fate determination and differentiation. Mutations in JAGl cause ALGS type 1, the most prevalent disease type, while mutations in NOTCH2 cause ALGS type 2.

[0035] The JAGl gene consists of 26 exons and is located on chromosome 20pl l .2-20pl2. JAGl mutations in ALGS are spread across the entire protein, and there is a high de novo mutation rate of approximately 60%. 80% of mutations include frameshift, nonsense, and splice site mutations; whole gene deletions account for 7% of mutations, and missense mutations represent 12% (Lu et al, 2003). Because whole gene deletion results in a phenotype similar to that seen for truncating and missense mutations, haploinsufficiency is the likely mechanism of disease causation in the majority of cases (Lu et al, 2003; Turnpenny and Ellard, 2012). [0036] Mutations associated with the classic ALGS phenotype that includes liver disease displayed functional haploinsufficiency, resulting in cell surface concentrations of JAGl that were 50% of wild-type levels (Lu et al., 2003). By contrast, a JAGl missense mutation that results in expression of the JAG1-G274D protein has been reported to produce two protein populations, with one population exhibiting a glycosylation defect, thus preventing transport to the cell surface. The JAG1-G274D mutation was associated with a cardiac-specific phenotype in the absence of liver disease, with carriers of the mutation having more than 50% but less than 100%) of the normal concentration of JAGl on the cell surface. Thus, the developing liver may require less JAGl than the developing heart (Lu et iL, 2003).

Muscle Repair

[0037] As noted above, the potential for JAGl to rescue dystrophin-deficient phenotypes in two animal models has been described (Vieira, et al., 2015). Deficiency of muscle dystrophin in muscular dystrophies, including Duchenne Muscular Dystrophy (DMD) and Becker Muscular Dystrophy (BMD), causes progressive muscle degeneration and wasting. In DMD, death eventually results due to respiratory or cardiac failure. Although JAGl is not deficient in these conditions, increased levels of JAGl may stimulate muscle cell proliferation and repair. In embodiments, the methods and compositions of the present invention are used to increase JAGl production to stimulate muscle repair in a subject in need thereof. In embodiments, the subject has a muscular dystrophy. In embodiments, the muscular dystrophy is DMD, BMD, or limb girdle, congenital, facioscapulohumeral, myotonic, oculopharyngeal, distal, or Emery -Dreifuss muscular dystrophy. In embodiments, the subject has a dystrophin deficiency. In embodiments, the dystrophin deficiency is DMD or BMD. In embodiments, the subject has a muscular dystrophy caused by a deficiency in dysferlin (encoded by the DYSF gene), emerin (encoded by the EMD gene), DUX4, myotonin-protein kinase (MT-PK) also known as myotonic dystrophy protein kinase (MDPK) or dystrophia myotonica protein kinase (DMK) (encoded by the DMPK gene), Cellular nucleic acid-binding protein (encoded by the C BP gene), or polyadenylate- binding protein 2 (PABP-2) also known as polyadenylate-binding nuclear protein 1 (PABPNl) (encoded by the PABPNl gene). Retained Intron Containing Pre-mRNA (RIC Pre -mRNA)

[0038] In embodiments, the methods of the present invention exploit the presence of retained- intron-containing pre-mRNA (RIC pre-mRNA) transcribed from the JAGl gene and encoding JAGl protein, in the cell nucleus. Splicing of the identified JAGl RIC pre-mRNA species to produce mature, fully-spliced, JAGl mRNA, is induced using ASOs that stimulate splicing out of the retained introns. The resulting mature JAGl mRNA can be exported to the cytoplasm and translated, thereby increasing the amount of JAGl protein in the patient's cells and alleviating symptoms of Alagille syndrome. This method, described further below, is known as Targeted Augmentation of Nuclear Gene Output (TANGO).

JAGl Nuclear Transcripts

[0039] As described herein in the Examples, the JAGl gene was analyzed for intron-retention events and retention of introns 13, 18, and 23 was observed. RNA sequencing (RNAseq), visualized in the UCSC genome browser, showed JAGl transcripts expressed in THLE-3 (human liver epithelial) cells and localized in either the cytoplasmic or nuclear fraction. In both fractions, reads were not observed for the majority of the introns. However, higher read density was detected for introns 13, 18, and 23 in the nuclear fraction compared to the cytoplasmic fraction indicating that splicing efficiency of introns 13, 18, and 23 is low, resulting in intron retention. The retained-intron containing pre-mRNA transcripts accumulate primarily in the nucleus and are not translated into the JAGl protein. The read density for introns 13, 18, and 23, indicated 12%, 14%, and 17% intron retention, respectively.

[0040] Table 1 provides a non-limiting list of target sequences of a JAGl RIC pre-mRNA transcript by sequence ID, and ASOs by sequence ID, useful for increasing production of JAGl protein by targeting a region of a JAGl RIC pre-mRNA. In embodiments, other ASOs useful for this purpose are identified, using, e.g., methods described herein.

Table 1. List of targets and ASOs targeting the JAGl gene

[0041] In some embodiments, the ASOs disclosed herein target a RIC pre-mRNA transcribed from a JAGl genomic sequence. In some embodiments, the ASO targets a RIC pre-mRNA transcript from a JAGl genomic sequence comprising a retained intron. In some embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ ID NO: 1. In some embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ ID NO: 1 comprising a retained intron. In some embodiments, the ASOs disclosed herein target a JAGl RIC pre-mRNA sequence. In some embodiments, the ASO targets a JAGl RIC pre-mRNA transcript comprising a retained intron at 13, 18, 23 or a combination thereof, wherein the intron numbering correspond to the mRNA sequence at M_000214.

[0042] In some embodiments, the ASO targets a JAGl RIC pre-mRNA sequence according to SEQ ID NO: 2. In some embodiments, the ASO targets a JAG1 RIC pre-mRNA sequence according to SEQ ID NO: 2 comprising a retained intron 13, a retained intron 18, a retained intron 23, or a combination thereof. In some embodiments, the ASOs disclosed herein target SEQ ID NOs: 437-439. In some embodiments, the ASO has a sequence according to any one of SEQ ID NOs: 3-436.

[0043] In some embodiments, the ASO targets exon 18 or exon 19 of a JAGl RIC pre-mRNA comprising a retained intron 18, wherein the intron numbering correspond to the mRNA sequence at NM_000214. In some embodiments, the ASO targets an exon 18 sequence upstream (or 5') from the 5' splice site of a JAG1 RIC pre-mRNA comprising the retained intron 18. In some embodiments, the ASO targets an exon 18 sequence about 4 to about 99 nucleotides upstream (or 5') from the 5' splice site of a JAGl RIC pre-mRNA comprising the retained intron 18. In some embodiments, the ASO targets an exon 19 sequence downstream (or 3') from the 3' splice site of a JAG1 RIC pre-mRNA comprising the retained intron 18. In some embodiments, the ASO targets an exon 19 sequence about 2 to about 7 nucleotides downstream (or 3 ') from the 3' splice site of a JAGl RIC pre-mRNA comprising the retained intron 18.

[0044] In some embodiments, the ASO targets intron 18 in a JAGl RIC pre-mRNA comprising a retained intron 18, wherein the intron numbering correspond to the mRNA sequence at NM 000214. In some embodiments, the ASO targets an intron 18 sequence downstream (or 3') from the 5' splice site of a JAGl RIC pre-mRNA comprising the retained intron 18. In some embodiments, the ASO targets an intron 18 sequence about 16 to about 239 nucleotides downstream (or 3 ') from the 5' splice site of a JAGl RIC pre-mRNA comprising the retained intron 18. In some embodiments, the ASO targets an intron 18 sequence upstream (or 5') from the 3' splice site of a JAG1 RIC pre-mRNA comprising the retained intron 18. In some embodiments, the ASO targets an intron 18 sequence about 16 to about 253 nucleotides upstream (or 5') from the 3' splice site of a JAG1 RIC pre-mRNA comprising the retained intron 18.

[0045] In some embodiments, the ASO targets exon 13 or exon 14 of a JAGl RIC pre-mRNA comprising a retained intron 13, wherein the intron numbering correspond to the mRNA sequence at NM_000214. In some embodiments, the ASO targets an exon 13 sequence upstream (or 5') from the 5' splice site of a JAG1 RIC pre-mRNA comprising the retained intron 13. In some embodiments, the ASO targets an exon 13 sequence about 14 to about 131 nucleotides upstream (or 5') from the 5' splice site of a JAG1 RIC pre-mRNA comprising the retained intron 13. In some embodiments, the ASO targets an exon 14 sequence downstream (or 3') from the 3' splice site of a JAGl RIC pre-mRNA comprising the retained intron 13. In some embodiments, the ASO targets an exon 14 sequence about 2 to about 147 nucleotides downstream (or 3 ') from the 3' splice site of a JAGl RIC pre-mRNA comprising the retained intron 13.

[0046] In some embodiments, the ASO targets intron 13 in a JAGl RIC pre-mRNA comprising a retained intron 13, wherein the intron numbering correspond to the mRNA sequence at NM 000214. In some embodiments, the ASO targets an intron 13 sequence downstream (or 3') from the 5' splice site of a JAGl RIC pre-mRNA comprising the retained intron 13. In some embodiments, the ASO targets an intron 13 sequence about 16 to about 440 nucleotides downstream (or 3 ') from the 5' splice site of a JAGl RIC pre-mRNA comprising the retained intron 13. In some embodiments, the ASO targets an intron 13 sequence upstream (or 5') from the 3' splice site of a JAGl RIC pre-mRNA comprising the retained intron 13. In some embodiments, the ASO targets an intron 13 sequence about 16 to about 441 nucleotides upstream (or 5') from the 3' splice site of a JAGl RIC pre-mRNA comprising the retained intron 13.

[0047] In some embodiments, the ASO targets exon 23 or exon 24 of a JAGl RIC pre-mRNA comprising a retained intron 23, wherein the intron numbering correspond to the mRNA sequence at NM_000214. In some embodiments, the ASO targets an exon 23 sequence upstream (or 5') from the 5' splice site of a JAGl RIC pre-mRNA comprising the retained intron 23. In some embodiments, the ASO targets an exon 23 sequence about 4 to about 216 nucleotides upstream (or 5') from the 5' splice site of a JAGl RIC pre-mRNA comprising the retained intron 23. In some embodiments, the ASO targets an exon 24 sequence downstream (or 3') from the 3' splice site of a JAGl RIC pre-mRNA comprising the retained intron 23. In some embodiments, the ASO targets an exon 24 sequence about 2 to about 114 nucleotides downstream (or 3 ') from the 3' splice site of a JAGl RIC pre-mRNA comprising the retained intron 23.

[0048] In some embodiments, the ASO targets intron 23 in a JAGl RIC pre-mRNA comprising a retained intron 23, wherein the intron numbering correspond to the mRNA sequence at NM_000214. In some embodiments, the ASO targets an intron 23 sequence downstream (or 3') from the 5' splice site of a JAGl RIC pre-mRNA comprising the retained intron 23. In some embodiments, the ASO targets an intron 23 sequence about 6 to about 121 nucleotides downstream (or 3 ') from the 5' splice site of a JAGl RIC pre-mRNA comprising the retained intron 23. In some embodiments, the ASO targets an intron 23 sequence upstream (or 5') from the 3' splice site of a JAGl RIC pre-mRNA comprising the retained intron 23. In some embodiments, the ASO targets an intron 23 sequence about 16 to about 121 nucleotides upstream (or 5') from the 3' splice site of a JAGl RIC pre-mRNA comprising the retained intron 23.

[0049] It is understood that the intron numbering may change in reference to a different JAGl isoform sequence. One of skill in the art can determine the corresponding intron number in any JAGl isoform based on an intron sequence provided herein or using the intron number provided in reference to the mRNA sequence at NM_000214.2. One of skill in the art also can determine the sequences of flanking exons in any JAGl isoform for targeting using the methods of the invention, based on an intron sequence provided herein or using the intron number provided in reference to the mRNA sequence at NM_000214.2. In embodiments, the compositions and methods of the present invention are used to increase the expression of any known JAGl isoform.

JAGl Protein Expression

[0050] As described above, JAGl mutations in ALGS are spread across the entire protein, and there is a high de novo mutation rate of approximately 60%. Of the mutations characterized, 80% include frameshift, nonsense, and splice site mutations; whole gene deletions account for 7%) of mutations, and missense mutations represent 12% (Lu et al., 2003).

[0051] In embodiments, the methods described herein are used to increase the production of a functional JAGl protein. As used herein, the term "functional" refers to the amount of activity or function of a JAGl protein that is necessary to eliminate any one or more symptoms of a treated condition, e.g., Alagille syndrome or a muscular dystrophy. In embodiments, the methods are used to increase the production of a partially functional JAGl protein. As used herein, the term "partially functional" refers to any amount of activity or function of the JAGl protein that is less than the amount of activity or function that is necessary 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%, 85%, at least 90%, or at least 95% less activity relative to the fully functional protein or RNA.

[0052] In embodiments, the method is a method of increasing the expression of the JAGl protein by cells of a subject having a RIC pre-mRNA encoding the JAGl protein, wherein the subject has Alagille syndrome caused by a deficient amount of activity of JAGl protein, and wherein the deficient amount of the JAGl protein is caused by haploinsufficiency of the JAGl protein. In such an embodiment, the subject has a first allele encoding a functional JAGl protein, and a second allele from which the JAGl protein is not produced. In another such embodiment, the subject has a first allele encoding a functional JAGl protein, and a second allele encoding a nonfunctional JAGl protein. In another such embodiment, the subject has a first allele encoding a functional JAGl protein, and a second allele encoding a partially functional JAGl protein. In any of these embodiments, the antisense oligomer binds to a targeted portion of the RIC pre-mRNA transcribed from the first allele (encoding functional JAGl protein), thereby inducing constitutive splicing of the retained intron from the RIC pre- mRNA, and causing an increase in the level of mature mRNA encoding functional JAGl protein, and an increase in the expression of the JAGl protein in the cells of the subject.

[0053] In embodiments, the subject has a first allele encoding a functional JAGl protein, and a second allele encoding a partially functional JAGl protein, and the antisense oligomer binds to a targeted portion of the RIC pre-mRNA transcribed from the first allele (encoding functional JAGl protein) or a targeted portion of the RIC pre-mRNA transcribed from the second allele (encoding partially functional JAGl protein), thereby inducing constitutive splicing of the retained intron from the RIC pre-mRNA, and causing an increase in the level of mature mRNA encoding the JAGl protein, and an increase in the expression of functional or partially functional JAGl protein in the cells of the subject.

[0054] In related embodiments, the method is a method of using an ASO to increase the expression of a protein or functional RNA. In embodiments, an ASO is used to increase the expression of JAGl protein in cells of a subject having a RIC pre-mRNA encoding JAGl protein, wherein the subject has a deficiency, e.g., Alagille syndrome, in the amount or function of a JAGl protein.

[0055] In embodiments, the RIC pre-mRNA transcript that encodes the protein that is causative of the disease or condition is targeted by the ASOs described herein. In some embodiments, a RIC pre-mRNA transcript that encodes a protein that is not causative of the disease is targeted by the ASOs. For example, a disease that is the result of a mutation or deficiency of a first protein in a particular pathway may be ameliorated by targeting a RIC pre-mRNA that encodes a second protein, thereby increasing production of the second protein. In some embodiments, the function of the second protein is able to compensate for the mutation or deficiency of the first protein (which is causative of the disease or condition). In embodiments, the first protein is a protein deficient in a muscular dystrophy and the second protein is JAGl . In embodiments, the first protein is a dystrophin and the second protein is JAGl .

[0056] In embodiments, the subject has:

[0057] a. a first mutant allele from which

i) the JAGl protein is produced at a reduced level compared to production from a wild-type allele,

ii) the JAGl protein is produced in a form having reduced function

compared to an equivalent wild-type protein, or iii) the JAGl protein or functional RNA is not produced; and b.a second mutant allele from which

ii) the JAGl protein is produced in a form having reduced function

compared to an equivalent wild-type protein, or

iii) the JAGl protein is not produced, and

[0058] wherein the RIC pre-mRNA is transcribed from the first allele and/or the second allele. In these embodiments, the ASO binds to a targeted portion of the RIC pre-mRNA transcribed from the first allele or the second allele, thereby inducing constitutive splicing of the retained intron from the RIC pre-mRNA, and causing an increase in the level of mRNA encoding JAGl protein and an increase in the expression of the target protein or functional RNA in the cells of the subject. In these embodiments, the target protein or functional RNA having an increase in expression level resulting from the constitutive splicing of the retained intron from the RIC pre- mRNA is either in a form having reduced function compared to the equivalent wild-type protein (partially-functional), or having full function compared to the equivalent wild-type protein (fully-functional).

[0059] In embodiments, the level of mRNA encoding JAGl protein is increased 1.1 to 10-fold, when compared to the amount of mRNA encoding JAGl that is produced in a control cell, e.g., one that is not treated with the anti sense oligomer or one that is treated with an anti sense oligomer that does not bind to the targeted portion of the JAGl RIC pre-mRNA.

[0060] In embodiments, the condition caused by a deficient amount or activity of JAGl protein is not a condition caused by alternative or aberrant splicing of the retained intron to which the ASO is targeted. In embodiments, the condition caused by a deficient amount or activity of the JAGl protein is not a condition caused by alternative or aberrant splicing of any retained intron in a RIC pre-mRNA encoding the JAGl protein. In embodiments, alternative or aberrant splicing may occur in a pre-mRNA transcribed from the gene, however the compositions and methods of the invention do not prevent or correct this alternative or aberrant splicing.

[0061] In embodiments, a subject treated using the methods of the invention expresses a partially functional JAGl protein from one allele, wherein the partially functional JAGl protein is caused by a frameshift mutation, a nonsense mutation, a missense mutation, or a partial gene deletion. In embodiments, a subject treated using the methods of the invention expresses a nonfunctional JAGl protein from one allele, wherein the nonfunctional JAGl protein is caused by a frameshift mutation, a nonsense mutation, a missense mutation, a partial gene deletion, in one allele. In embodiments, a subject treated using the methods of the invention has a JAGl whole gene deletion, in one allele.

[0062] In embodiments, the subject has a JAGl missense mutation selected from G274D, L37S, R184H, P163L, P871R, C234Y, C664S, P810L, or R937Q. In embodiments, the subject has JAGl deletion mutation 1485-1486delCT. In embodiments, the subject has JAGl duplication mutation 414-415dupGT. In embodiments, a subject having any JAGl mutation known in the art and described in the literature, e.g., by Lu, et al., 2003, Penton, et al., 2012, referenced above, is treated using the methods and compositions of the present invention.

Use of TANGO for Increasing JAGl Protein Expression

[0063] As described above, in embodiments, Targeted Augmentation of Nuclear Gene Output (TANGO) is used in the methods of the invention to increase expression of a JAGl protein. In these embodiments, a retained-intron-containing pre-mRNA (RIC pre-mRNA) encoding JAG 1 protein is present in the nucleus of a cell. Cells having a JAGl RIC pre-mRNA that comprises a retained intron, an ex on flanking the 5' splice site, and an exon flanking the 3' splice site, encoding the JAGl protein, are contacted with antisense oligomers (ASOs) that are

complementary to a targeted portion of the RIC pre-mRNA. Hybridization of the ASOs to the targeted portion of the RIC pre-mRNA results in enhanced splicing at the splice site (5' splice site or 3' splice site) of the retained intron and subsequently increases target protein production.

[0064] The terms "pre-mRNA," and "pre-mRNA transcript" may be used interchangeably and refer to any pre-mRNA species that contains at least one intron. In embodiments, pre-mRNA or pre-mRNA transcripts comprise a 5'-7-methylguanosine cap and/or a poly-A tail. In

embodiments, pre-mRNA or pre-mRNA transcripts comprise both a 5'-7-methylguanosine cap and a poly-A tail. In some embodiments, the pre-mRNA transcript does not comprise a 5 '-7- methylguanosine cap and/or a poly-A tail. A pre-mRNA transcript is a non-productive messenger RNA (mRNA) molecule if it is not translated into a protein (or transported into the cytoplasm from the nucleus).

[0065] As used herein, a "retained-intron-containing pre-mRNA" ("RIC pre-mRNA") is a pre- mRNA transcript that contains at least one retained intron. The RIC pre-mRNA contains a retained intron, an exon flanking the 5' splice site of the retained intron, an exon flanking the 3' splice site of the retained intron, and encodes the target protein. An "RIC pre-mRNA encoding a target protein" is understood to encode the target protein when fully spliced. A "retained intron" is any intron that is present in a pre-mRNA transcript when one or more other introns, such as an adjacent intron, encoded by the same gene have been spliced out of the same pre- mRNA transcript. In some embodiments, the retained intron is the most abundant intron in RIC pre-mRNA encoding the target protein. In embodiments, the retained intron is the most abundant intron in a population of RIC pre-mRNAs transcribed from the gene encoding the target protein in a cell, wherein the population of RIC pre-mRNAs comprises two or more retained introns. In embodiments, an antisense oligomer targeted to the most abundant intron in the population of RIC pre-mRNAs encoding the target protein induces splicing out of two or more retained introns in the population, including the retained intron to which the antisense oligomer is targeted or binds. In embodiments, a mature mRNA encoding the target protein is thereby produced. The terms "mature mRNA," and "fully-spliced mRNA," are used

interchangeably herein to describe a fully processed mRNA encoding a target protein (e.g., mRNA that is exported from the nucleus into the cytoplasm and translated into target protein) or a fully processed functional RNA. The term "productive mRNA," also can be used to describe a fully processed mRNA encoding a target protein. In embodiments, the targeted region is in a retained intron that is the most abundant intron in a population of RIC pre-mRNA encoding the JAG1 protein. In embodiments, the most retained intron in a population of RIC pre-mRNA encoding the JAG1 protein is intron 23.

[0066] In embodiments, a retained intron is an intron that is identified as a retained intron based on a determination of 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%, retention. In embodiments, a retained intron is an intron that is identified as a retained intron based on a determination 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 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%, 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 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 70%), about 15% to about 65%, about 15% to about 60%, about 15% to about 65%, about 15%) to about 60%), about 15% to about 55%, about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, about 20% to about 100%, about 20% to about 95%, about 20% to about 90%, about 20% to about 85%, about 20% to about 80%, about 20% to about 75%, about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 65%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to 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 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%, retention. ENCODE data (described by, e.g., Tilgner, et al., 2012, "Deep sequencing of subcellular RNA fractions shows splicing to be predominantly co- transcriptional in the human genome but inefficient for IncRNAs," Genome Research

22(9): 1616-25) can be used to aid in identifying retained introns.

[0067] As used herein, the term "comprise" or variations thereof such as "comprises" or "comprising" are to be read to indicate the inclusion of any recited feature (e.g. in the case of an antisense oligomer, a defined nucleobase sequence) but not the exclusion of any other features. Thus, as used herein, the term "comprising" is inclusive and does not exclude additional, unrecited features (e.g. in the case of an antisense oligomer, the presence of additional, unrecited nucleobases).

[0068] In embodiments of any of the compositions and methods provided herein, "comprising" may be replaced with "consisting essentially of " or "consisting of." The phrase "consisting essentially of is used herein to require the specified feature(s) (e.g. nucleobase sequence) as well as those which do not materially affect the character or function of the claimed invention. As used herein, the term "consisting" is used to indicate the presence of the recited feature (e.g. nucleobase sequence) alone (so that in the case of an antisense oligomer consisting of a specified nucleobase sequence, the presence of additional, unrecited nucleobases is excluded).

[0069] In embodiments, the targeted region is in a retained intron that is the second most abundant intron in a population of RIC pre-mRNA encoding the JAG1 protein. For example, the second most abundant retained intron may be targeted rather than the most abundant retained intron due to the uniqueness of the nucleotide sequence of the second most abundant retained intron, ease of ASO design to target a particular nucleotide sequence, and/or amount of increase in protein production resulting from targeting the intron with an ASO. In embodiments, the retained intron is the second most abundant intron in a population of RIC pre-mRNAs transcribed from the gene encoding the target protein in a cell, wherein the population of RIC pre-mRNAs comprises two or more retained introns. In embodiments, an antisense oligomer targeted to the second most abundant intron in the population of RIC pre-mRNAs encoding the target protein induces splicing out of two or more retained introns in the population, including the retained intron to which the antisense oligomer is targeted or binds. In embodiments, fully- spliced (mature) RNA encoding the target protein is thereby produced. In embodiments, the second-most retained intron in a population of RIC pre-mRNA encoding the JAG1 protein is intron 18. In embodiments, the second-most retained intron in a population of RIC pre-mRNA encoding the JAG1 protein is intron 13.

[0070] In embodiments, an ASO is complementary to a targeted region that is within a non- retained intron in a RIC pre-mRNA. In embodiments, the targeted portion of the RIC pre- mRNA is within: the region +6 to +100 relative to the 5' splice site of the non-retained intron; or the region -16 to -100 relative to the 3' splice site of the non-retained intron. In embodiments, the targeted portion of the RIC pre-mRNA is within the region +100 relative to the 5' splice site of the non-retained intron to -100 relative to the 3' splice site of the non-retained intron. As used to identify the location of a region or sequence, "within" is understood to include the residues at the positions recited. For example, a region +6 to +100 includes the residues at positions +6 and +100. In embodiments, fully-spliced (mature) RNA encoding the target protein is thereby produced.

[0071] In embodiments, the retained intron of the RIC pre-mRNA is an inefficiently spliced intron. As used herein, "inefficiently spliced" may refer to a relatively low frequency of splicing at a splice site adjacent to the retained intron (5' splice site or 3' splice site) as compared to the frequency of splicing at another splice site in the RIC pre-mRNA. The term "inefficiently spliced" may also refer to the relative rate or kinetics of splicing at a splice site, in which an "inefficiently spliced" intron may be spliced or removed at a slower rate as compared to another intron in a RIC pre-mRNA.

[0072] In embodiments, the 9-nucleotide sequence at -3e to - le of the exon flanking the 5' splice site and +1 to +6 of the retained intron is identical to the corresponding wild-type sequence. In embodiments, the 16 nucleotide sequence at -15 to -1 of the retained intron and +le of the exon flanking the 3' splice site is identical to the corresponding wild-type sequence. As used herein, the "wild-type sequence" refers to the nucleotide sequence for the JAG1 gene in the published reference genome deposited in the NCBI repository of biological and scientific information (operated by National Center for Biotechnology Information, National Library of Medicine, 8600 Rockville Pike, Bethesda, MD USA 20894). As used herein, the "wild-type sequence" refers to the canonical sequence available at NCBI Gene ID 182. Also used herein, a nucleotide position denoted with an "e" indicates the nucleotide is present in the sequence of an exon {e.g., the exon flanking the 5' splice site or the exon flanking the 3 ' splice site). [0073] The methods involve contacting cells with an ASO that is complementary to a portion of a pre-mRNA encoding JAG1 protein, resulting in increased expression of JAG1. As used herein, "contacting" or administering to cells refers to any method of providing an ASO in immediate proximity with the cells such that the ASO and the cells interact. A cell that is contacted with an ASO will take up or transport the ASO into the cell. The method involves contacting a condition or disease-associated or condition or disease-relevant cell with any of the ASOs described herein. In some embodiments, the ASO may be further modified or attached (e.g., covalently attached) to another molecule to target the ASO to a cell type, enhance contact between the ASO and the condition or disease-associated or condition or disease-relevant cell, or enhance uptake of the ASO.

[0074] As used herein, the term "increasing protein production" or "increasing expression of a target protein" means enhancing the amount of protein that is translated from an mRNA in a cell. A "target protein" may be any protein for which increased expression/production is desired.

[0075] In embodiments, contacting a cell that expresses a JAGl RIC pre-mRNA with an ASO that is complementary to a targeted portion of the JAG1 RIC pre-mRNA transcript results in a measurable increase in the amount of the JAG1 protein {e.g., a target protein) encoded by the pre-mRNA. Methods of measuring or detecting production of a protein will be evident to one of skill in the art and include any known method, for example, Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA.

[0076] In embodiments, contacting cells with an ASO that is complementary to a targeted portion of a JAG1 RIC pre-mRNA transcript results in an increase in the amount of JAG1 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 the protein produced by a cell in the absence of the ASO/absence of treatment. In embodiments, the total amount of JAG1 protein produced by the cell to which the antisense oligomer was contacted is increased 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, compared to the amount of target protein produced by an control compound. A control compound can be, for example, an oligonucleotide that is not complementary to the targeted portion of the RIC pre-mRNA.

[0077] In some embodiments, contacting cells with an ASO that is complementary to a targeted portion of a JAGl RIC pre-mRNA transcript results in an increase in the amount of mRNA encoding JAGl, including the mature mRNA encoding the target protein. In some

embodiments, the amount of mRNA encoding JAGl protein, or the mature mRNA encoding the JAGl protein, 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 the protein produced by a cell in the absence of the ASO/absence of treatment. In embodiments, the total amount of the mRNA encoding JAGl protein, or the mature mRNA encoding JAGl protein produced in the cell to which the antisense oligomer was contacted is increased 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 compared to the amount of mature RNA produced in an untreated cell, e.g., an untreated cell or a cell treated with a control compound. A control compound can be, for example, an oligonucleotide that is not complementary to the targeted portion of the JAGl RIC pre-mRNA.

Constitutive Splicing of a Retained Intron from a RIC pre-mRNA

[0078] The methods and antisense oligonucleotide compositions provided herein are useful for increasing the expression of JAGl protein in cells, for example, in a subject having Alagille syndrome caused by a deficiency in the amount or activity of JAGl protein, by increasing the level of mRNA encoding JAGl protein, or the mature mRNA encoding JAGl protein. In particular, the methods and compositions as described herein induce the constitutive splicing of a retained intron from a JAGl RIC pre-mRNA transcript encoding JAGl protein, thereby increasing the level of mRNA encoding JAGl protein, or the mature mRNA encoding JAGl protein and increasing the expression of JAGl protein.

[0079] Constitutive splicing of a retained intron from a RIC pre-mRNA correctly removes the retained intron from the RIC pre-mRNA, wherein the retained intron has wild-type splice sequences. Constitutive splicing, as used herein, does not encompass splicing of a retained intron from a RIC pre-mRNA transcribed from a gene or allele having a mutation that causes alternative splicing or aberrant splicing of a pre-mRNA transcribed from the gene or allele. For example, constitutive splicing of a retained intron, as induced using the methods and antisense oligonucleotides provided herein, does not correct aberrant splicing in or influence alternative splicing of a pre-mRNA to result in an increased expression of a target protein or functional RNA.

[0080] In embodiments, constitutive splicing correctly removes a retained intron from a JAGl RIC pre-mRNA, wherein the JAGl RIC pre-mRNA is transcribed from a wild-type gene or allele, or a polymorphic gene or allele, that encodes a fully-functional target protein or functional RNA, and wherein the gene or allele does not have a mutation that causes alternative splicing or aberrant splicing of the retained intron.

[0081] In some embodiments, constitutive splicing of a retained intron from a, JAGl RIC pre- mRNA encoding JAGl protein correctly removes a retained intron from a JAGl RIC pre-mRNA encoding JAGl protein, wherein the JAGl RIC pre-mRNA is transcribed from a gene or allele from which the target gene or functional RNA is produced at a reduced level compared to production from a wild-type allele, and wherein the gene or allele does not have a mutation that causes alternative splicing or aberrant splicing of the retained intron. In these embodiments, the correct removal of the constitutively spliced retained intron results in production of target protein or functional RNA that is functional when compared to an equivalent wild-type protein or functional RNA.

[0082] In other embodiments, constitutive splicing correctly removes a retained intron from a JAGl RIC pre-mRNA, wherein the JAGl RIC pre-mRNA is transcribed from a gene or allele that encodes a target protein or functional RNA produced in a form having reduced function compared to an equivalent wild-type protein or functional RNA, and wherein the gene or allele does not have a mutation that causes alternative splicing or aberrant splicing of the retained intron. In these embodiments, the correct removal of the constitutively spliced retained intron results in production of partially functional target protein, or functional RNA that is partially functional when compared to an equivalent wild-type protein or functional RNA.

[0083] "Correct removal" of the retained intron by constitutive splicing refers to removal of the entire intron, without removal of any part of an exon.

[0084] In embodiments, an antisense oligomer as described herein or used in any method described herein does not increase the amount of mRNA encoding JAGl protein or the amount of JAGl protein by modulating alternative splicing or aberrant splicing of a pre-mRNA transcribed from the JAGl gene. Modulation of alternative splicing or aberrant splicing can be measured using any known method for analyzing the sequence and length of RNA species, e.g., by RT-PCR and using methods described elsewhere herein and in the literature. In embodiments, modulation of alternative or aberrant splicing is determined based on an increase or decrease in the amount of the spliced species of interest of at least 10% or 1.1 -fold. In embodiments, modulation is determined based on an increase or decrease at a level that is at least 10% to 100% or 1.1 to 10-fold, as described herein regarding determining an increase in mRNA encoding JAGl protein in the methods of the invention.

[0085] In embodiments, the method is a method wherein the JAGl RIC pre-mRNA was produced by partial splicing of a wild-type JAGl pre-mRNA. In embodiments, the method is a method wherein the JAGl RIC pre-mRNA was produced by partial splicing of a full-length wild-type JAGl pre-mRNA. In embodiments, the JAGl RIC pre-mRNA was produced by partial splicing of a full-length JAGl pre-mRNA. In these embodiments, a full-length JAGl pre-mRNA may have a polymorphism in a splice site of the retained intron that does not impair correct splicing of the retained intron as compared to splicing of the retained intron having the wild-type splice site sequence.

[0086] In embodiments, the mRNA encoding JAGl protein is a full-length mature mRNA, or a wild-type mature mRNA. In these embodiments, a full-length mature mRNA may have a polymorphism that does not affect the activity of the target protein or the functional RNA encoded by the mature mRNA, as compared to the activity of JAGl protein encoded by the wild-type mature mRNA.

Antisense Oligomers

[0087] One aspect of the present disclosure is a composition comprising antisense oligomers that enhances splicing by binding to a targeted portion of a JAGl RIC pre-mRNA. As used herein, the terms "ASO" and "antisense oligomer" are used interchangeably and refer to an oligomer such as a polynucleotide, comprising nucleobases, that hybridizes to a target nucleic acid (e.g., a JAGl RIC pre-mRNA) sequence by Watson-Crick base pairing or wobble base pairing (G-U). The ASO may have exact sequence complementary to the target sequence or near complementarity (e.g., sufficient complementarity to bind the target sequence and enhancing splicing at a splice site). ASOs are designed so that they bind (hybridize) to a target nucleic acid (e.g., a targeted portion of a pre-mRNA transcript) and remain hybridized under physiological conditions. Typically, if they hybridize to a site other than the intended (targeted) nucleic acid sequence, they hybridize to a limited number of sequences that are not a target nucleic acid (to a few sites other than a target nucleic acid). Design of an ASO can take into consideration the occurrence of the nucleic acid sequence of the targeted portion of the pre- mRNA transcript or a sufficiently similar nucleic acid sequence in other locations in the genome or cellular pre-mRNA or transcriptome, such that the likelihood the ASO will bind other sites and cause "off-target" effects is limited. Any antisense oligomers known in the art, for example in PCT Application No. PCT/US2014/054151, published as WO 2015/035091, titled "Reducing Nonsense-Mediated mRNA Decay," can be used to practice the methods described herein.

[0088] In some embodiments, ASOs "specifically hybridize" to or are "specific" to a target nucleic acid or a targeted portion of a RIC pre-mRNA. Typically such hybridization occurs with a Tm substantially greater than 37°C, preferably at least 50°C, and typically between 60°C to approximately 90°C. Such hybridization preferably corresponds to stringent hybridization conditions. At a given ionic strength and pH, the Tm is the temperature at which 50% of a target sequence hybridizes to a complementary oligonucleotide.

[0089] Oligomers, such as oligonucleotides, are "complementary" to one another when hybridization occurs in an antiparallel configuration between two single-stranded

polynucleotides. A double-stranded polynucleotide can be "complementary" to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second. Complementarity (the degree to which one polynucleotide is complementary with another) is quantifiable in terms of the proportion (e.g., the 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) need not be 100% complementary to that of its target nucleic acid to hybridize. In certain embodiments, ASOs can comprise 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 the target nucleic acid sequence to which they are targeted. For example, an ASO in which 18 of 20 nucleobases of the oligomeric compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent

complementarity. In this example, the remaining noncomplementary nucleobases may be clustered together or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. Percent complementarity of an ASO with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) 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).

[0090] An ASO need not hybridize to all nucleobases in a target sequence and the nucleobases to which it does hybridize may be contiguous or noncontiguous. ASOs may hybridize over 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 structure or hairpin structure may be formed). In certain embodiments, an ASO hybridizes to noncontiguous nucleobases in a target pre-mRNA transcript. For example, an ASO can hybridize to nucleobases in a pre-mRNA transcript that are separated by one or more nucleobase(s) to which the ASO does not hybridize. [0091] The ASOs described herein comprise nucleobases that are complementary to nucleobases present in a target portion of a RIC pre-mRNA. The term ASO embodies oligonucleotides and any other oligomeric molecule that comprises nucleobases capable of hybridizing to a complementary nucleobase on a target mRNA but does not comprise a sugar moiety, such as a peptide nucleic acid (PNA). The ASOs may comprise naturally-occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination of two or three of the preceding. The term "naturally occurring nucleotides" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" includes nucleotides with modified or substituted sugar groups and/or having a modified backbone. In some embodiments, all of the nucleotides of the ASO are modied nucleotides. Chemical modifications of ASOs or components of ASOs that are compatible with the methods and compositions described herein will be evident to one of skill in the art and can be found, for example, in U.S. Patent No.

8,258,109 B2, U.S. Patent No. 5,656,612, U.S. Patent Publication No. 2012/0190728, and Dias and Stein, Mol. Cancer Ther. 2002, 1 , 347-355, herein incorporated by reference in their entirety.

[0092] The nucleobase of an ASO may be any naturally occurring, unmodified nucleobase such as adenine, guanine, cytosine, thymine and uracil, or any synthetic or modified nucleobase that is sufficiently similar to an unmodified nucleobase such that it is capable of hydrogen bonding with a nucleobase present on a target pre-mRNA. Examples of modified nucleobases include, without limitation, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5- m ethyl cytosine, and 5 -hydroxymethoyl cytosine.

[0093] The ASOs described herein also comprise a backbone structure that connects the components of an oligomer. The term "backbone structure" and "oligomer linkages" may be used interchangeably and refer to the connection between monomers of the ASO. In naturally occurring oligonucleotides, the backbone comprises a 3 '-5' phosphodiester linkage connecting sugar moieties of the oligomer. The backbone structure or oligomer linkages of the ASOs described herein may include (but are not limited to) phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoramidate, and the like. See e.g., 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. Pat. No. 5, 151,510; Uhlmann and Peyman, Chemical Reviews 90:543 (1990). In some embodiments, the backbone structure of the ASO does not contain phosphorous but rather contains peptide bonds, for example in a peptide nucleic acid (PNA), or linking groups including carbamate, amides, and linear and cyclic hydrocarbon groups. In some embodiments, the backbone modification is a phosphorothioate linkage. In some embodiments, the backbone modification is a phosphoramidate linkage.

[0094] In embodiments, the stereochemistry at each of the phosphorus internucleotide linkages of the ASO backbone is random. In embodiments, the stereochemistry at each of the

phosphorus internucleotide linkages of the ASO backbone is controlled and is not random. For example, U.S. Pat. App. Pub. No. 2014/0194610, "Methods for the Synthesis of Functionalized Nucleic Acids," incorporated herein by reference, describes methods for independently selecting the handedness of chirality at each phosphorous atom in a nucleic acid oligomer. In

embodiments, an ASO used in the methods of the invention, including, but not limited to, any of the ASOs set forth herein in Table 1, comprises an ASO having phosphorus internucleotide linkages that are not random. In embodiments, a composition used in the methods of the invention comprises a pure diastereomeric ASO. In embodiments, a composition used in the methods of the invention comprises an ASO that has 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%.

[0095] In embodiments, the ASO has a nonrandom mixture of Rp and Sp configurations at its phosphorus internucleotide linkages. For example, it has been suggested that a mix of Rp and Sp 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

embodiments, an ASO used in the methods of the invention, including, but not limited to, any of the ASOs set forth herein in Table 1, comprises 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 Sp, or about 100% Rp. In embodiments, an ASO used in the methods of the invention, including, but not limited to, any of the ASOs set forth herein in Table 1, comprises about 10% to about 100% Rp, about 15% to about 100% Rp, about 20% to about 100% Rp, about 25% to about 100% Rp, about 30% to about 100% Rp, about 35% to about 100% Rp, about 40% to about 100% 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 Sp.

[0096] In embodiments, an ASO used in the methods of the invention, including, but not limited to, any of the ASOs set forth herein in Table 1, comprises 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 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 Rp, or about 100% Sp. In embodiments, an ASO used in the methods of the invention, including, but not limited to, any of the ASOs set forth herein in Table 1, comprises 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 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 Rp.

[0097] Any of the ASOs described herein may contain a sugar moiety that comprises ribose or deoxyribose, as present in naturally occurring nucleotides, or a modified sugar moiety or sugar analog, including a morpholine ring. Non-limiting examples of modified sugar moieties include 2' substitutions such as 2'-0-methyl (2'-0-Me), 2'-0-methoxyethyl (2'MOE), 2'-0-aminoethyl, 2'F; N3'->P5' phosphoramidate, 2'dimethylaminooxyethoxy, 2'dimethylaminoethoxyethoxy, 2'-guanidinidium, 2'-0-guanidinium ethyl, carbamate modified sugars, and bicyclic modified sugars. In some embodiments, the sugar moiety modification is selected from 2'-0-Me, 2'F, and 2'MOE. In some embodiments, the sugar moiety modification is an extra bridge bond, such as in a locked nucleic acid (LNA). In some embodiments the sugar analog contains a morpholine ring, such as phosphorodiamidate morpholino (PMO). In some embodiments, the sugar moiety comprises a ribofuransyl or 2'deoxyribofuransyl modification. In some embodiments, the sugar moiety comprises 2' 4' -constrained 2'0-methyloxyethyl (cMOE) modifications. In some embodiments, the sugar moiety comprises cEt 2', 4' constrained 2'-0 ethyl BNA modifications. In some embodiments, the sugar moiety comprises tricycloDNA (tcDNA) modifications. In some embodiments, the sugar moiety comprises ethylene nucleic acid (ENA) modifications. In some embodiments, the sugar moiety comprises MCE modifications. Modifications are known in the art and described in the literature, e.g., by Jarver, et al., 2014, "A Chemical View of Oligonucleotides for Exon Skipping and Related Drug Applications," Nucleic Acid Therapeutics 24(1): 37-47, incorporated by reference for this purpose herein.

[0098] In some examples, each monomer of the ASO is modified in the same way, for example each linkage of the backbone of the ASO comprises a phosphorothioate linkage or each ribose sugar moiety comprises a 2'O-methyl modification. Such modifications that are 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 desired, for example, an ASO may comprise a combination of phosphorodiamidate linkages and sugar moieties comprising morpholine rings (morpholinos). Combinations of different modifications to an ASO are referred to as "mixed modifications" or "mixed chemistries."

[0099] In some embodiments, the ASO comprises one or more backbone modification. In some embodiments, the ASO comprises one or more sugar moiety modification. In some

embodiments, the ASO comprises one or more backbone modification and one or more sugar moiety modification. In some embodiments, the ASO comprises 2'MOE modifications 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 or any component of an ASO (e.g., a nucleobase, sugar moiety, backbone) described herein may be modified in order to achieve desired properties or activities of the ASO or reduce undesired properties or activities of the ASO. For example, an ASO or one or more component of any ASO may be modified to enhance binding affinity to a target sequence on a pre-mRNA transcript; reduce binding to any non-target sequence; reduce degradation by cellular nucleases (i.e., RNase H); improve uptake of the ASO into a cell and/or into the nucleus of a cell; alter the pharmacokinetics or pharmacodynamics of the ASO; and modulate the half-life of the ASO.

[0100] In some embodiments, the ASOs are comprised of 2'-0-(2-methoxyethyl) (MOE) phosphorothioate-modified nucleotides. ASOs comprised of such nucleotides are especially well-suited to the methods disclosed herein; oligomers having such modifications have been shown to have significantly enhanced resistance to nuclease degradation and increased bioavailability, making them suitable, for example, for oral delivery in some embodiments described herein. See e.g., Geary et al., J Pharmacol Exp Ther. 2001; 296(3):890-7; Geary et al., J Pharmacol Exp Ther. 2001; 296(3):898-904.

[0101] Methods of synthesizing ASOs will be known to one of skill in the art. Alternatively or in addition, ASOs may be obtained from a commercial source.

[0102] Unless specified otherwise, the left-hand end of single-stranded nucleic acid (e.g., pre- mRNA transcript, oligonucleotide, ASO, etc.) sequences is the 5' end and the left-hand direction of single or double-stranded nucleic acid sequences is referred to as the 5' direction. Similarly, the right-hand end or direction of a nucleic acid sequence (single or double stranded) is the 3' end or 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 end of an mRNA is where the initiation or start codon is located, while the 3 ' end or direction is where the termination codon is located. In some aspects, nucleotides that are upstream of a reference point in a nucleic acid may be designated by a negative number, while nucleotides that are downstream of a reference point may be designated by a positive number. For example, a reference point (e.g., an exon- ex on junction in mRNA) may be designated as the "zero" site, and a nucleotide that is directly adjacent and upstream of the reference point is designated "minus one," e.g., while a nucleotide that is directly adjacent and downstream of the reference point is designated "plus one," e.g., "+1."

[0103] In other embodiments, the ASOs are complementary to (and bind to) a targeted portion of a JAGl RIC pre-mRNA that is downstream (in the 3' direction) of the 5' splice site of the retained intron in a JAGl RIC pre-mRNA (e.g., the direction designated by positive numbers relative to the 5' splice site) (FIG. 1). In some embodiments, the ASOs are complementary to a targeted portion of the JAGl RIC pre-mRNA that is within the region +6 to +500, +6 to +400, +6 to +300, +6 to +200, or +6 to +100 relative to the 5' splice site of the retained intron. In some embodiments, the ASO is not complementary to nucleotides +1 to +5 relative to the 5' splice site (the first five nucleotides located downstream of the 5' splice site). In some embodiments, the ASOs may be complementary to a targeted portion of a JAGl RIC pre-mRNA that is within the region between nucleotides +6 and +50 relative to the 5' splice site of the retained intron. In some aspects, the ASOs are complementary to a targeted portion that is within the region +6 to +90, +6 to +80, +6 to +70, +6 to +60, +6 to +50, +6 to +40, +6 to +30, or +6 to +20 relative to 5' splice site of the retained intron.

[0104] In some embodiments, the ASOs are complementary to a targeted region of a JAGl RIC pre-mRNA that is upstream (5' relative) of the 3 ' splice site of the retained intron in a JAGl RIC pre-mRNA (e.g., in the direction designated by negative numbers) (FIG. 1). In some embodiments, the ASOs are complementary to a targeted portion of the JAGl RIC pre-mRNA that is within the region -16 to -500, -16 to -400, -16 to -300, -6 to -200, or -16 to -100 relative to the 3' splice site of the retained intron. In some embodiments, the ASO is not complementary to nucleotides -1 to -15 relative to the 3' splice site (the first 15 nucleotides located upstream of the 3' splice site). In some embodiments, the ASOs are complementary to a targeted portion of the JAGl RIC pre-mRNA that is within the region -16 to -50 relative to the 3' splice site of the retained intron. In some aspects, the ASOs are complementary to a targeted portion that is within the region -16 to -90, -16 to -80, -16 to -70, -16 to -60, -16 to -50, -16 to -40, or -16 to -30 relative to 3' splice site of the retained intron.

[0105] In embodiments, the targeted portion of the JAGl RIC pre-mRNA is within the region +6 relative to the 5' splice site of the retained intron to -16 relative to the 3 ' splice site of the retained intron.

[0106] In some embodiments, the ASOs are complementary to a targeted portion of a JAGl RIC pre-mRNA that is within the ex on flanking the 5' splice site (upstream) of the retained intron (FIG. 1). In some embodiments, the ASOs are complementary to a targeted portion of the JAGl RIC pre-mRNA that is within the region +2e to -4e in the exon flanking the 5' splice site of the retained intron. In some embodiments, the ASOs are not complementary to nucleotides - le to -3e relative to the 5' splice site of the retained intron. In some embodiments, the ASOs are complementary to a targeted portion of the JAGl RIC pre-mRNA that is within the region -4e to-lOOe, -4e to -90e, -4e to -80e, -4e to -70e, -4e to -60e, -4e to -50e, -4 to -40e, -4e to -30e, or - 4e to -20e relative to the 5' splice site of the retained intron.

[0107] In some embodiments, the ASOs are complementary to a targeted portion of a JAGl RIC pre-mRNA that is within the exon flanking the 3' splice site (downstream) of the retained intron (FIG. 1). In some embodiments, the ASOs are complementary to a targeted portion to the JAGl RIC pre-mRNA that is within the region +2e to -4e in the exon flanking the 3' splice site of the retained intron. In some embodiments, the ASOs are not complementary to nucleotide +le relative to the 3' splice site of the retained intron. In some embodiments, the ASOs are complementary to a targeted portion of the JAGl RIC pre-mRNA that is within the region+2e to +100e, +2e to +90e, +2e to +80e, +2e to +70e, +2e to +60e, +2e to +50e, +2e to +40e, +2e to +30e, or +2 to +20e relative to the 3' splice site of the retained intron. The ASOs may be of any length suitable for specific binding and effective enhancement of splicing. In some

embodiments, the ASOs consist of 8 to 50 nucleobases. For example, the ASO may 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 ASOs consist of more than 50 nucleobases. In some embodiments, the ASO is from 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 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, 12 to 15 nucleobases, 13 to 50 nucleobases, 13 to 40 nucleobases, 13 to 35 nucleobases, 13 to 30 nucleobases, 13 to 25 nucleobases, 13 to 20 nucleobases, 14 to 50 nucleobases, 14 to 40 nucleobases, 14 to 35 nucleobases, 14 to 30 nucleobases, 14 to 25 nucleobases, 14 to 20 nucleobases, 15 to 50 nucleobases, 15 to 40 nucleobases, 15 to 35 nucleobases, 15 to 30 nucleobases, 15 to 25 nucleobases, 15 to 20 nucleobases, 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 length. In some embodiments, the ASOs are 30 nucleotides in length. In some embodiments, the ASOs are 29 nucleotides in length. In some embodiments, the ASOs are 28 nucleotides in length. In some embodiments, the ASOs are 27 nucleotides in length. In some embodiments, the ASOs are 26 nucleotides in length. In some embodiments, the ASOs are 25 nucleotides in length. In some embodiments, the ASOs are 24 nucleotides in length. In some embodiments, the ASOs are 23 nucleotides in length. In some embodiments, the ASOs are 22 nucleotides in length. In some embodiments, the ASOs are 21 nucleotides in length. In some embodiments, the ASOs are 20 nucleotides in length. In some embodiments, the ASOs are 19 nucleotides in length. In some embodiments, the ASOs are 18 nucleotides in length. In some embodiments, the ASOs are 17 nucleotides in length. In some embodiments, the ASOs are 16 nucleotides in length. In some embodiments, the ASOs are 15 nucleotides in length. In some embodiments, the ASOs are 14 nucleotides in length. =In some embodiments, the ASOs are 13 nucleotides in length. In some embodiments, the ASOs are 12 nucleotides in length. In some embodiments, the ASOs are 1 1 nucleotides in length. In some embodiments, the ASOs are 10 nucleotides in length.

[0108] In some embodiments, two or more ASOs with different chemistries but complementary to the same targeted portion of the RIC pre-mRNA are used. In some embodiments, two or more ASOs that are complementary to different targeted portions of the RIC pre-mRNA are used.

[0109] In embodiments, the antisense oligonucleotides of the invention are chemically linked to one or more moieties or conjugates, e.g., a targeting moiety or other conjugate that enhances the activity or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, a lipid moiety, e.g., as a cholesterol moiety, a cholesteryl moiety, an aliphatic chain, e.g., dodecandiol or undecyl residues, a polyamine or a polyethylene glycol chain, or adamantane acetic acid. Oligonucleotides comprising lipophilic moieties, and preparation methods have been described in the published literature. In embodiments, the antisense oligonucleotide is conjugated with a moiety including, but not limited to, an abasic nucleotide, a polyether, a polyamine, a polyamide, a peptides, a carbohydrate, e.g., N-acetylgalactosamine (GalNAc), N- Ac-Glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate), a lipid, or a

polyhydrocarbon compound. Conjugates can be linked to one or more of any nucleotides comprising the antisense oligonucleotide at any of several positions on the sugar, base or phosphate group, as understood in the art and described in the literature, e.g., using a linker. Linkers can include a bivalent or trivalent branched linker. In embodiments, the conjugate is attached to the 3' end of the antisense oligonucleotide. Methods of preparing oligonucleotide conjugates are described, e.g., in U.S. Pat. No. 8,450,467, "Carbohydrate conjugates as delivery agents for oligonucleotides," incorporated by reference herein.

[0110] In some embodiments, the nucleic acid to be targeted by an ASO is a JAG1 RIC pre- mRNA 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 condition or disease-relevant cell or a cell line. In some embodiments, the cell is in vitro {e.g., in cell culture).

Pharmaceutical Compositions

[0111] Pharmaceutical compositions or formulations comprising the antisense oligonucleotide of the described compositions and for use in any of the described methods can be prepared according to conventional techniques well known in the pharmaceutical industry and described in the published literature. In embodiments, a pharmaceutical composition or formulation for treating a subject comprises an effective amount of any antisense oligomer as described above, or a pharmaceutically acceptable salt, solvate, hydrate or ester thereof, and a pharmaceutically acceptable diluent. The antisense oligomer of a pharmaceutical formulation may further comprise a pharmaceutically acceptable excipient, diluent or carrier.

[0112] 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), incorporated herein by reference for this purpose. The salts can be prepared in situ during the final isolation and purification of the compounds, or separately by reacting the free base function with a suitable organic acid. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other documented methodologies such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate,

camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemi sulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, 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 salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further

pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.

[0113] In embodiments, the compositions are formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. In 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. The suspension may also contain stabilizers. In embodiments, a pharmaceutical formulation or composition of the present invention includes, but is not limited to, a solution, emulsion, microemulsion, foam or liposome-containing formulation (e.g., cationic or noncationic liposomes).

[0114] The pharmaceutical composition or formulation of the present invention may comprise one or more penetration enhancer, carrier, excipients or other active or inactive ingredients as appropriate and well known to those of skill in the art or described in the published literature. In embodiments, liposomes also include sterically stabilized liposomes, e.g., liposomes comprising one or more specialized lipids. These specialized lipids result in liposomes with enhanced circulation lifetimes. In embodiments, a sterically stabilized liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. In embodiments, a surfactant is included in the pharmaceutical formulation or compositions. The use of surfactants in drug products, formulations and emulsions is well known in the art. In embodiments, the present invention employs a penetration enhancer to effect the efficient delivery of the antisense oligonucleotide, e.g., to aid diffusion across cell membranes and /or enhance the permeability of a lipophilic drug. In embodiments, the penetration enhancers is a surfactant, fatty acid, bile salt, chelating agent, or non-chelating nonsurfactant.

[0115] In embodiments, the pharmaceutical formulation comprises multiple antisense oligonucleotides. In embodiments, the antisense oligonucleotide is administered in combination with another drug or therapeutic agent. In embodiments, the antisense oligonucleotide is administered with one or more agents capable of promoting penetration of the subject antisense oligonucleotide across the blood-brain barrier by any method known in the art. For example, delivery of agents by administration of an adenovirus vector to motor neurons in muscle tissue is described in U.S. Pat. No. 6,632,427, "Adenoviral -vector-mediated gene transfer into medullary motor neurons," incorporated herein by reference. Delivery of vectors directly to the brain, e.g., the striatum, the thalamus, the hippocampus, or the substantia nigra, is described, e.g., in U.S. Pat. No. 6,756,523, "Adenovirus vectors for the transfer of foreign genes into cells of the central nervous system particularly in brain," incorporated herein by reference.

[0116] In embodiments, the antisense oligonucleotides are linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties. In embodiments, the antisense oligonucleotide is coupled to a substance, known in the art to promote penetration or transport across the blood-brain barrier, e.g., an antibody to the transferrin receptor. In embodiments, the antisense oligonucleotide is linked with a viral vector, e.g., to render the antisense compound more effective or increase transport across the blood-brain barrier. In embodiments, osmotic blood brain barrier disruption is assisted by infusion of sugars, e.g., meso erythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose, dulcitol, myo-inositol, L(-) fructose, D(-) mannitol, D(+) glucose, D(+) arabinose, D(-) arabinose, cellobiose, D(+) maltose, D(+) raffinose, L(+) rhamnose, D(+) melibiose, D(-) ribose, adonitol, D(+) arabitol, L(-) arabitol, D(+) fucose, L(-) fucose, D(-) lyxose, L(+) lyxose, and L(-) lyxose, or amino acids, e.g., 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 blood brain barrier penetration are described, e.g., in U.S. Pat. No. 4,866,042, "Method for the delivery of genetic material across the blood brain barrier," U.S. Pat. No. 6,294,520, "Material for passage through the blood-brain barrier," and U.S. Pat. No. 6,936,589, "Parenteral delivery systems," each incorporated herein by reference. [0117] In embodiments, the antisense oligonucleotides of the invention are chemically linked to one or more moieties or conjugates, e.g., a targeting moiety or other conjugate that enhances the activity or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, a lipid moiety, e.g., as a cholesterol moiety, a cholesteryl moiety, an aliphatic chain, e.g., dodecandiol or undecyl residues, a polyamine or a polyethylene glycol chain, or adamantane acetic acid. Oligonucleotides comprising lipophilic moieties, and preparation methods have been described in the published literature. In embodiments, the antisense oligonucleotide is conjugated with a moiety including, but not limited to, an abasic nucleotide, a polyether, a polyamine, a polyamide, a peptides, a carbohydrate, e.g., N-acetylgalactosamine (GalNAc), N- Ac-Glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate), a lipid, or a

Treatment of Subjects

[0118] Any of the compositions provided herein may be administered to an individual.

"Individual" may be used interchangeably with "subject" or "patient." An individual may be a mammal, for example a human or animal such as a non-human primate, a rodent, a rabbit, a rat, a mouse, a horse, a donkey, a goat, a cat, a dog, a cow, a pig, or a sheep. In embodiments, the individual is a human. In embodiments, the individual is a fetus, an embryo, or a child. In other embodiments, the individual may be another eukaryotic organism, such as a plant. In some embodiments, the compositions provided herein are administered to a cell ex vivo.

[0119] 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 the disease, such as any of the diseases described herein. In some embodiments, the individual is at increased risk of having a disease or disorder caused by insufficient amount of a protein or insufficient activity of a protein. If an individual is "at an increased risk" of having a disease or disorder caused insufficient amount of a protein or insufficient activity of a protein, the method involves preventative or prophylactic treatment. For example, an individual may be at an increased risk of having such a disease or disorder because of family history of the disease. Typically, individuals at an increased 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).

[0120] Suitable routes for administration of ASOs of the present invention may vary depending on cell type to which delivery of the ASOs is desired. Multiple tissues and organs are affected by Alagille syndrome, with the liver being the most significantly affected tissue. The ASOs of the present invention may be administered to patients parenterally. In embodiments, the ASOs of the present invention are administered to patients by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. In embodiments, delivery is to the heart or liver. In embodiments, a fetus is treated in utero, e.g., by administering the ASO composition to the fetus directly or indirectly (e.g., via the mother).

[0121] In the treatment of subjects having muscular dystrophy, the compositions of the present invention may be provided to muscle cells by any suitable means, including direct

administration (e.g., locally by injection or topical administration at a treatment site) or systemically (e.g., parenterally or orally), intranasally, orally, or by inhalational, enteral, topical, intrauterine, vaginal, sublingual, rectal, intramuscular, intrapleural, intraventricular,

intraperitoneal, ophthalmic, intravenous, or subcutaneous means.

Methods of identifying additional ASOs that enhance splicing

[0122] Also within the scope of the present invention are methods for identifying (determining) additional ASOs that enhance splicing of a JAG1 RIC pre-mRNA, specifically at the target intron. ASOs that specifically hybridize to different nucleotides within the target region of the pre-mRNA may be screened to identify (determine) ASOs that improve the rate and/or extent of splicing of the target intron. In some embodiments, the ASO may block or interfere with the binding site(s) of a splicing repressor(s)/silencer. Any method known in the art may be used to identify (determine) an ASO that when hybridized to the target region of the intron results in the desired effect {e.g., enhanced splicing, protein or functional RNA production). These methods also can be used for identifying ASOs that enhance splicing of the retained intron by binding to a targeted region in an exon flanking the retained intron, or in a non-retained intron. An example of a method that may be used is provided below.

[0123] A round of screening, referred to as an ASO "walk" may be performed using ASOs that have been designed to hybridize to a target region of a pre-mRNA. 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 retained intron {e.g., a portion of sequence of the exon located upstream of the target/retained intron) to approximately 100 nucleotides downstream of the 5' splice site of the target/retained intron and/or from approximately 100 nucleotides upstream of the 3' splice site of the retained intron to approximately 100 nucleotides downstream of the 3 ' splice site of the target/retained intron (e.g., a portion of sequence of the ex on located

downstream of the target/retained intron). For example, a first ASO of 15 nucleotides in length may be designed to specifically hybridize to nucleotides +6 to +20 relative to the 5' splice site of the target/retained intron. A second ASO is designed to specifically hybridize to nucleotides +1 1 to +25 relative to the 5' splice site of the target/retained intron. ASOs are designed as such spanning the target region of the pre-mRNA. In embodiments, the ASOs can be tiled more closely, e.g., every 1, 2, 3, or 4 nucleotides. Further, the ASOs can be tiled from 100 nucleotides downstream of the 5 ' splice site, to 100 nucleotides upstream of the 3 ' splice site.

[0124] One or more ASOs, or a control ASO (an ASO with a scrambled sequence, sequence that is not expected to hybridize to the target region) are delivered, for example by transfection, into a disease-relevant cell line that expresses the target pre-mRNA (e.g., the RIC pre-mRNA described elsewhere herein). The splicing-inducing effects of each of the ASOs may be assessed by any method known in the art, for example by reverse transcriptase (RT)-PCR using primers that span the splice junction, as described herein (see "Identification of intron-retention events"). A reduction or absence of the RT-PCR product produced using the primers spanning the splice junction in ASO-treated cells as compared to in control ASO-treated cells indicates that splicing of the target intron has been enhanced. In some embodiments, the splicing efficiency, the ratio of spliced to unspliced pre-mRNA, the rate of splicing, or the extent of splicing may be improved using the ASOs described herein. The amount of protein or functional RNA that is encoded by the target pre-mRNA can also be assessed to determine whether each ASO achieved the desired effect (e.g., enhanced protein production). Any method known in the art for assessing and/or quantifying protein production, such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA, can be used.

[0125] A second round of screening, referred to as an ASO "micro-walk" may be performed using ASOs that have been designed to hybridize to a target region of a pre-mRNA. The ASOs used in the ASO micro-walk are tiled every 1 nucleotide to further refine the nucleotide acid sequence of the pre-mRNA that when hybridized with an ASO results in enhanced splicing.

[0126] Regions defined by ASOs that promote splicing of the target intron are explored in greater detail by means of an ASO "micro-walk", involving ASOs spaced in 1-nt steps, as well as longer ASOs, typically 18-25 nt.

[0127] As described for the ASO walk above, the ASO micro-walk is performed by delivering one or more ASOs, or a control ASO (an ASO with a scrambled sequence, sequence that is not expected to hybridize to the target region), for example by transfection, into a disease-relevant cell line that expresses the target pre-mRNA. The splicing-inducing effects of each of the ASOs may be assessed by any method known in the art, for example by reverse transcriptase (RT)- PCR using primers that span the splice junction, as described herein (see "Identification of intron-retention events"). A reduction or absence of the RT-PCR product produced using the primers spanning the splice junction in ASO-treated cells as compared to in control ASO-treated cells indicates that splicing of the target intron has been enhanced. In some embodiments, the splicing efficiency, the ratio of spliced to unspliced pre-mRNA, the rate of splicing, or the extent of splicing may be improved using the ASOs described herein. The amount of protein or functional RNA that is encoded by the target pre-mRNA can also be assessed to determine whether each ASO achieved the desired effect (e.g., enhanced protein production). Any method known in the art for assessing and/or quantifying protein production, such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA, can be used.

[0128] ASOs that when hybridized to a region of a pre-mRNA result in enhanced splicing and increased protein production may be tested in vivo using animal models, for example transgenic mouse models in which the full-length human gene has been knocked-in or in humanized mouse models of disease. Suitable routes for administration of ASOs may vary depending on the disease and/or the cell types to which delivery of the ASOs is desired. ASOs may be

administered, for example, by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. Following administration, the cells, tissues, and/or organs of the model animals may be assessed to determine the effect of the ASO treatment by for example evaluating splicing (efficiency, rate, extent) and protein production by methods known in the art and described herein. The animal models may also be any phenotypic or behavioral indication of the disease or disease severity.

[0129] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

EXAMPLES

[0130] The present invention will be more specifically illustrated by the following Examples. However, it should be understood that the present invention is not limited by these examples in any manner. Example 1: Identification of intron retention events in JAG1 transcripts by RNAseq using next generation sequencing

[0131] Whole transcriptome shotgun sequencing was carried out using next generation sequencing to reveal a snapshot of transcripts produced by the JAG1 gene to identify intron- retention events. For this purpose, polyA+ RNA from nuclear and cytoplasmic fractions of THLE-3 (human liver epithelial) cells was isolated cDNA libraries constructed using Dlumina's TruSeq Stranded mRNA library Prep Kit. The libraries were pair-end sequenced resulting in 100-nucleotide reads that were mapped to the human genome (Feb. 2009, GRCh37/hgl9 assembly). The sequencing results for JAG1 are shown in FIG. 3. Briefly, FIG. 3 shows the mapped reads visualized using the UCSC genome browser (operated by the UCSC Genome Informatics Group (Center for Biomolecular Science & Engineering, University of California, Santa Cruz, 1156 High Street, Santa Cruz, CA 95064) and described by, e.g., Rosenbloom, et al., 2015, "The UCSC Genome Browser database: 2015 update," Nucleic Acids Research 43, Database Issue, doi: 10.1093/nar/gkul 177) and the coverage and number of reads can be inferred by the peak signals. The height of the peaks indicates the level of expression given by the density of the reads in a particular region. A schematic representation of JAG1 (drawn to scale) is provided by the UCSC genome browser (below the read signals) so that peaks can be matched to JAG1 exonic and intronic regions. Based on this display, we identified three introns (13, 18, and 23, indicated by arrows) that have high read density in the nuclear fraction of THLE-3 cells, but have very low to no reads in the cytoplasmic fraction of these cells (as shown for intron 13 in the bottom diagram of FIG. 3, for intron 18 in the bottom diagram of FIG. 7, and for intron 23 in the bottom diagram of FIG. 11). This indicates that these introns are retained and that the intron- 13, intron- 18, and intron-23 containing transcripts remain in the nucleus, and suggests that these retained JAG1 RIC pre-mRNAs are non-productive, as they are not exported out to the cytoplasm.

Example 2: Design of ASO-walk targeting intron 13 of JAG1

[0132] An ASO walk was designed to target intron 13 using the method described herein (FIG. 4; Table 2). A region immediately downstream of the intron 13 5' splice site spanning nucleotides +6 to +69 and a region immediately upstream of intron 13 3' splice site spanning nucleotides -16 to -68 of the intron were targeted with 2'-0-Me RNA, PS backbone, 18-mer ASOs shifted by 5-nucleotide intervals (with the exception of 1 ASO, JAGl-IVS 13+52) (FIG. 4; Table 2). Example 3: Improved splicing efficiency via ASO-targeting of JAGl intron 13 increases transcript levels

[0133] To determine whether we can achieve an increase in JAGl expression by improving splicing efficiency of JAGl intron 13 using ASOs we used the method described herein (FIG. 5). To this end, ARPE-19 cells were mock-transfected, or transfected with each of the targeting ASOs described in FIG. 4 and Table 2, or a non-targeting SMN-ASO control, independently, using RNAiMAX (RiM) (Invitrogen) delivery reagents. Experiments were performed using 60 nM ASOs (as indicated in FIG. 5) for 48 hrs. Radioactive RT-PCR results show that several targeting ASOs (+6, SEQ ID NO: 135; +11, SEQ ID NO: 136; +21, SEQ ID NO: 138; +26, SEQ ID NO: 139; +31, SEQ ID NO: 140; +36, SEQ ID NO: 141; +52; -16, SEQ ID NO: 301; -36, SEQ ID NO: 297; -41, SEQ ID NO: 296; -46, SEQ ID NO: 295; and -51 SEQ ID NO: 294) increase JAGl transcript level compared to the mock-transfected or non-targeting ASO (FIG. 5). Intensities of the bands corresponding to the JAGl PCR products from targeting-ASO- transfected cells were normalized to Beta actin and plotted relative to the normalized JAGl PCR product from mock-treated cells. Results of this analysis indicate that several targeting ASOs increase JAGl transcript level 2 - 6 fold (FIG. 5). These results were confirmed by RT-qPCR using primers elsewhere in the JAGl transcript, showing the same trend of JAGl upregulation evidenced by the fold-change plots in FIG. 6 (top, normalized to Beta actin, bottom normalized to RPL32). Altogether, these results confirm that improving the splicing efficiency of a rate limiting intron in the JAGl gene using ASOs leads to an increase in gene expression.

Example 4: Design of an ASO-walk targeting intron 18 of JAGl

[0134] An ASO walk was designed to target intron 18 using the method described herein (FIG. 8, Table 2). A region immediately downstream of intron 18 5' splice site spanning nucleotides +15 to +67 and a region immediately upstream of intron 18 3' splice site spanning nucleotides - 16 to -68 of the intron were targeted with 2'-0-Me RNA, PS backbone, 18-mer ASOs shifted by 5-nucleotide intervals (FIG. 8, Table 2).

Example 5: Improved splicing efficiency via ASO-targeting of JAGl intron 18 increases transcript levels

[0135] To determine whether an increase in JAGl expression could be achieved by improving splicing efficiency of JAGl intron 18 using ASOs, the method described herein (FIG. 9) was used. To this end, ARPE-19 cells were mock-transfected, transfected with each of the targeting ASOs described in FIG. 8 and Table 2, or transfected with a non-targeting SMN-ASO control, independently, using RNAiMAX (RiM) (Invitrogen) delivery reagents. Experiments were performed using 60 nM ASOs (as indicated in FIG. 5) for 48 hrs. Radioactive RT-PCR results show that several targeting ASOs (+15; +20; and -26, SEQ ID NO: 107) increase JAGl transcript level compared to the mock-transfected or non-targeting ASO (FIG. 9). Intensities of the bands corresponding to the JAGl PCR products from targeting-ASO-transfected cells were normalized to Beta actin and plotted relative to the normalized JAGl PCR product from mock- treated cells. Results of this analysis indicate that three targeting ASOs increase JAGl transcript level at least 1.5 fold (FIG. 9). These results were confirmed by RT-qPCR using primers elsewhere in the JAGl transcript, showing the same trend of JAGl upregulation evidenced by the fold-change plots in FIG. 10 (top, normalized to Beta actin, bottom normalized to RPL32). Altogether, these results confirm that improving the splicing efficiency of a rate limiting intron in the JAGl gene using ASOs leads to an increase in gene expression.

[0136] A second method can be used to determine whether an increase in JAGl target gene intron splicing efficiency can be achieved with ASOs. In brief, ARPE-19 cells, a human retinal epithelium cell line (American Type Culture Collection (ATCC), USA), or Huh-7, a human hepatoma cell line (NIBIOHN, Japan), or SK-N-AS, a human neuroblastoma cell line (ATCC) are mock-transfected, or transfected with the targeting ASOs described in Tables 1 and 2. Cells are transfected using Lipofectamine RNAiMax transfection reagent (Thermo Fisher) according to vendor's specifications. ASOs are plated in 96-well tissue culture plates and combined with RNAiMax diluted in Opti-MEM. Cells are detached using trypsin and resuspended in full medium, and approximately 25,000 cells are added the ASO-transfection mixture. Transfection experiments are carried out in triplicate plate replicates. Final ASO concentration is 80 nM. Media is changed 6h post-transfection, and cells harvested at 24h, using the Cells-to-Ct lysis reagent, supplemented with DNAse (Thermo Fisher), according to vendor's

specifications. cDNA is generated with Cells-to-Ct RT reagents (Thermo Fisher) according to vendor's specifications. To quantify the amount of splicing at the intron of interest, quantitative PCR is carried out using Taqman assays with probes spanning the corresponding exon-exon junction (Thermo Fisher). Taqman assays are carried out according to vendor's specifications, on a QuantStudio 7 Flex Real-Time PCR system (Thermo Fisher). Target gene assay values are normalized to RPL32 (deltaCt) and plate-matched mock transfected samples (delta-delta Ct), generating fold-change over mock quantitation (2^A-(delta-deltaCt).

Example 6: Design of ASO-walk targeting intron 23 of JAGl

[0137] An ASO walk was designed to target intron 23 using the method described herein (FIG. 12, Table 2). A region immediately downstream of intron 23 5' splice site spanning nucleotides +6 to +58 and a region immediately upstream of intron 23 3' splice site spanning nucleotides -16 to -68 of the intron were targeted with 2'-0-Me RNA, PS backbone, 18-mer ASOs shifted by 5- nucleotide intervals (FIG. 12; Table 2). Table 2: ASOs targeting the JAGl Gene

Claims

CLAIMS What is claimed is:

1. A method of treating Alagille syndrome in a subject in need thereof by increasing the expression of a target protein or functional RNA by cells of the subject, wherein the cells have a retained-intron-containing pre-mRNA (RIC pre-mRNA), the RIC pre-mRNA comprising a retained intron, an ex on flanking the 5' splice site, an ex on flanking the 3' splice site, and wherein the RIC pre-mRNA encodes the target protein or functional RNA, the method comprising contacting the cells of the subject with an antisense oligomer (ASO) complementary to a targeted portion of the RIC pre-mRNA encoding the target protein or functional RNA, whereby the retained intron is constitutively spliced from the RIC pre-mRNA encoding the target protein or functional RNA, thereby increasing the level of mRNA encoding the target protein or functional RNA, and increasing the expression of the target protein or functional RNA in the cells of the subject.

2. A method of increasing expression of a target protein, wherein the target protein is JAGl, by cells having a retained-intron-containing pre-mRNA (RIC pre-mRNA), the RIC pre- mRNA comprising a retained intron, an ex on flanking the 5' splice site of the retained intron, an ex on flanking the 3' splice site of the retained intron, and wherein the RIC pre-mRNA encodes JAGl protein, the method comprising contacting the cells with an antisense oligomer (ASO) complementary to a targeted portion of the RIC pre-mRNA encoding JAGl protein, whereby the retained intron is constitutively spliced from the RIC pre-mRNA encoding JAGl protein, thereby increasing the level of mRNA encoding JAGl protein, and increasing the expression of JAGl protein in the cells.

3. The method of claim 1, wherein the target protein is JAGl .

4. The method of claim 1, wherein the target protein or the functional RNA is a compensating protein or a compensating functional RNA that functionally augments or replaces a target protein or functional RNA that is deficient in amount or activity in the subject.

5. The method of claim 2, wherein the cells are in or from a subject having a condition caused by a deficient amount or activity of JAGl protein.

6. The method of any one of claims 1 to 5, wherein the deficient amount of the target protein is caused by haploinsufficiency of the target protein, wherein the subject has a first allele encoding a functional target protein, and a second allele from which the target protein is not produced, or a second allele encoding a nonfunctional target protein, and wherein the antisense oligomer binds to a targeted portion of a RIC pre-mRNA transcribed from the first allele.

7. The method of any one of claims 1 to 5, wherein the subject has a condition caused by a disorder resulting from a deficiency in the amount or function of the target protein, wherein the subject has

a. a first mutant allele from which

i. the target protein is produced at a reduced level compared to production from a wild-type allele,

ii. the target protein is produced in a form having reduced function compared to an equivalent wild-type protein, or

iii. the target protein is not produced, and

b. a second mutant allele from which

iii. the target protein is not produced, and

wherein when the subject has a first mutant allele a.iii., the second mutant allele is b.i. or b.ii., and wherein when the subject has a second mutant allele b.iii., the first mutant allele is a.i. or a.ii., and wherein the RIC pre-mRNA is transcribed from either the first mutant allele that is a.i. or a.ii., and/or the second allele that is b.i. or b.ii.

8. The method of claim 7, wherein the target protein is produced in a form having reduced function compared to the equivalent wild-type protein.

9. The method of claim 7, wherein the target protein is produced in a form that is fully- functional compared to the equivalent wild-type protein.

10. The method of any one of claims 1 to 9, wherein the targeted portion of the RIC pre- mRNA is in the retained intron within the region +6 relative to the 5' splice site of the retained intron to -16 relative to the 3' splice site of the retained intron.

11. The method of any one of claims 1 to 9, wherein the targeted portion of the RIC pre- mRNA is in the retained intron within: (a) the region +6 to +500, +6 to +400, +6 to 300, +6 to 200, or +6 to +100 relative to the 5' splice site of the retained intron; or

(b) the region -16 to -500, -16 to -400, -16 to -300, -16 to -200, or -16 to -100 relative to the 3' splice site of the retained intron.

12. The method of any one of claims 1 to 9, wherein the targeted portion of the RIC pre- mRNA is within:

(a) the region +2e to -4e in the exon flanking the 5' splice site of the retained intron; or

(b) the region +2e to -4e in the exon flanking the 3' splice site of the retained intron.

13. The method of any one of claims 1 to 12, wherein the RIC pre-mRNA is encoded by genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1.

14. The method of any one of claims 1 to 13, wherein the RIC pre-mRNA comprises a sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 2.

15. The method of any one of claims 1 to 14, wherein the antisense oligomer does not increase the amount of the target protein or the functional RNA by modulating alternative splicing of pre-mRNA transcribed from a gene encoding the functional RNA or target protein.

16. The method of any one of claims 1 to 15, wherein the antisense oligomer does not increase the amount of the target protein or the functional RNA by modulating aberrant splicin resulting from mutation of the gene encoding the target protein or the functional RNA.

17. The method of any one of claims 1 to 16, wherein the RIC pre-mRNA was produced by partial splicing of a full-length pre-mRNA or partial splicing of a wild-type pre-mRNA.

18. The method of any one of claims 1 to 17, wherein the mRNA encoding the target protein or functional RNA is a full-length mature mRNA, or a wild-type mature mRNA.

19. The method of any one of claims 1 to 18, wherein the target protein produced is full- length protein, or wild-type protein.

20. The method of any one of claims 1 to 19, wherein the total amount of the mRNA encoding the target protein or functional RNA produced in the cell contacted with the antisense oligomer is increased 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, compared to the total amount of the mRNA encoding the target protein or functional RNA produced in a control cell.

21. The method of any one of claims 1 to 20, wherein the total amount of target protein produced by the cell contacted with the antisense oligomer is increased 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, compared to the total amount of target protein produced by a control cell.

22. The method of any one of claims 1 to 21, wherein the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.

23. The method of any one of claims 1 to 22, wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-0-methyl, a 2'-Fluoro, or a 2'-0-methoxyethyl moiety.

24. The method of any one of claims 1 to 23, wherein the antisense oligomer comprises at least one modified sugar moiety.

25. The method of claim 24, wherein each sugar moiety is a modified sugar moiety.

26. The method of any one of claims 1 to 25, wherein the antisense oligomer consists of from 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 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.

27. The method of any one of claims 1 to 26, wherein the antisense oligomer 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 RIC pre-mRNA encoding the protein.

28. The method of any one of claims 1 to 27, wherein the targeted portion of the RIC pre- mRNA is within a sequence selected from SEQ ID NOs: 437-439.

29. The method of any one of claims 1 to 28, wherein the antisense oligomer comprises a nucleotide sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 3-436.

30. The method of any one of claims 1 to 28, wherein the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NOs: 3-436.

31. The method of any one of claims 1 to 30, wherein the cell comprises a population of RIC pre-mRNAs transcribed from the gene encoding the target protein or functional RNA, wherein the population of RIC pre-mRNAs comprises two or more retained introns, and wherein the antisense oligomer binds to the most abundant retained intron in the population of RIC pre- mRNAs.

32. The method of claim 31, whereby the binding of the antisense oligomer to the most abundant retained intron induces splicing out of the two or more retained introns from the population of RIC pre-mRNAs to produce mRNA encoding the target protein or functional RNA.

33. The method of any one of claims 1 to 30, wherein the cell comprises a population of RIC pre-mRNAs transcribed from the gene encoding the target protein or functional RNA, wherein the population of RIC pre-mRNAs comprises two or more retained introns, and wherein the anti sense oligomer binds to the second most abundant retained intron in the population of RIC pre-mRNAs.

34. The method of claim 33, whereby the binding of the antisense oligomer to the second most abundant retained intron induces splicing out of the two or more retained introns from the population of RIC pre-mRNAs to produce mRNA encoding the target protein or functional RNA.

35. The method of any one of claims 5 to 34, wherein the condition is a disease or disorder.

36. The method of claim 35, wherein the disease or disorder is Alagille syndrome or a muscular dystrophy.

37. The method of claim 36, wherein the target protein and the RIC pre-mRNA are encoded by the JAGl gene.

38. The method of any one of claims 1 to 37, wherein the method further comprises assessing JAGl protein expression.

39. The method of any one of claims 1 to 38, wherein the antisense oligomer binds to a targeted portion of a JAGl RIC pre-mRNA, wherein the targeted portion is within a sequence selected from SEQ ID NOS: 49, 50, 51, 52, 53, 54, 55, 56, and 57.

40. The method of any one of claims 1 to 39, wherein the subject is a human.

41. The method of any one of claims 1 to 39, wherein the subject is a non-human animal.

42. The method of any one of claims 1 to 40, wherein the subject is a fetus, an embryo, or a child.

43. The method of any one of claims 1 to 41, wherein the cells are ex vivo.

44. The method of any one of claims 1 to 41, wherein the antisense oligomer is administered by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection of the subject.

45. The method of any one of claims 1 to 44, wherein the 9 nucleotides at -3e to -le of the ex on flanking the 5' splice site and +1 to +6 of the retained intron, are identical to the corresponding wild-type sequence.

46. The method of any one of claims 1 to 45, wherein the 16 nucleotides at -15 to -1 of the retained intron and +le of the exon flanking the 3 ' splice site are identical to the corresponding wild-type sequence.

47. An antisense oligomer as used in a method of any one of claims 1 to 39.

48. An antisense oligomer comprising a sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 3-436.

49. A pharmaceutical composition comprising the antisense oligomer of claim 47 or 48 and an excipient.

50. A method of treating a subject in need thereof by administering the pharmaceutical composition of claim 49 by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.

51. A composition comprising an antisense oligomer for use in a method of increasing expression of a target protein or a functional RNA by cells to treat Alagille syndrome in a subject in need thereof associated with a deficient protein or deficient functional RNA, wherein the deficient protein or deficient functional RNA is deficient in amount or activity in the subject, wherein the antisense oligomer enhances constitutive splicing of a retained intron-containing pre-mRNA (RIC pre-mRNA) encoding the target protein or the functional RNA, wherein the target protein is:

(a) the deficient protein; or

(b) a compensating protein which functionally augments or replaces the deficient protein or in the subject; and wherein the functional RNA is:

(a) the deficient RNA; or

(b) a compensating functional RNA which functionally augments or replaces the deficient functional RNA in the subject; wherein the RIC pre-mRNA comprises a retained intron, an exon flanking the 5' splice site and an exon flanking the 3' splice site, and wherein the retained intron is spliced from the RIC pre- mRNA encoding the target protein or the functional RNA, thereby increasing production or activity of the target protein or the functional RNA in the subject.

52. A composition comprising an antisense oligomer for use in a method of treating a condition associated with JAG1 protein in a subject in need thereof, the method comprising the step of increasing expression of JAG1 protein by cells of the subject, wherein the cells have a retained-intron-containing pre-mRNA (RIC pre-mRNA) comprising a retained intron, an exon flanking the 5' splice site of the retained intron, an exon flanking the 3' splice site of the retained intron, and wherein the RIC pre-mRNA encodes the JAG1 protein, the method comprising contacting the cells with the antisense oligomer, whereby the retained intron is constitutively spliced from the RIC pre-mRNA transcripts encoding JAG1 protein, thereby increasing the level of mRNA encoding JAG1, and increasing the expression of JAG1 protein, in the cells of the subject.

53. The composition of claim 52, wherein the condition is a disease or disorder.

54. The composition of claim 53, wherein the disease or disorder is Alagille syndrome or a muscular dystrophy.

55. The composition of claim 54, wherein the target protein and RIC pre-mRNA are encoded by the JAG1 gene.

56. The composition of any one of claims 51 to 55, wherein the antisense oligomer targets a portion of the RIC pre-mRNA that is in the retained intron within the region +6 relative to the 5' splice site of the retained intron to -16 relative to the 3' splice site of the retained intron.

57. The composition of any one of claims 51 to 56, wherein the antisense oligomer targets a portion of the RIC pre-mRNA that is in the retained intron within:

(a) the region +6 to +100 relative to the 5' splice site of the retained intron; or

(b) the region -16 to -100 relative to the 3' splice site of the retained intron.

58. The composition of any one of claims 51 to 57, wherein the antisense oligomer targets a portion of the RIC pre-mRNA that is within the region about 100 nucleotides downstream of the 5' splice site of the at least one retained intron, to about 100 nucleotides upstream of the 3' splice site of the at least one retained intron.

59. The composition of any one of claims 51 to 57, wherein the targeted portion of the RIC pre-mRNA is within:

(a) the region +2e to -4e in the exon flanking the 5' splice site of the retained intron; or (b) the region +2e to -4e in the exon flanking the 3' splice site of the retained intron.

60. The composition of any one of claims 51 to 59, wherein the RIC pre-mRNA is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1.

61. The composition of any one of claims 51 to 60, wherein the RIC pre-mRNA comprises a sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 2.

62. The composition of any one of claims 51 to 61, wherein the antisense oligomer does not increase the amount of target protein or functional RNA by modulating alternative splicing of the pre-mRNA transcribed from a gene encoding the target protein or functional RNA.

63. The composition of any one of claims 51 to 62, wherein the antisense oligomer does not increase the amount of the functional RNA or functional protein by modulating aberrant splicing resulting from mutation of the gene encoding the target protein or functional RNA.

64. The composition of any one of claims 51 to 63, wherein the RIC pre-mRNA was produced by partial splicing from a full-length pre-mRNA or a wild-type pre-mRNA.

65. The composition of any one of claims 51 to 64, wherein the mRNA encoding the target protein or functional RNA is a full-length mature mRNA, or a wild-type mature mRNA.

66. The composition of any one of claims 51 to 65, wherein the target protein produced is full-length protein, or wild-type protein.

67. The composition of any one of claims 51 to 66, wherein the retained intron is a rate- limiting intron.

68. The composition of any one of claims 51 to 67 wherein the retained intron is the most abundant retained intron in the RIC pre-mRNA.

69. The composition of any one of claims 51 to 67, wherein the retained intron is the second most abundant retained intron in the RIC pre-mRNA.

70. The composition of any one of claims 51 to 69, wherein the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.

71. The composition of any one of claims 51 to 70 wherein the antisense oligomer is an antisense oligonucleotide.

72. The composition of any one of claims 51 to 71, wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'- O-methyl, a 2'-Fluoro, or a 2'-0-methoxyethyl moiety.

73. The composition of any one of claims 51 to 72, wherein the antisense oligomer comprises at least one modified sugar moiety.

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

75. The composition of any one of claims 51 to 74, wherein the antisense oligomer consists of from 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 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. The composition of any one of claims 51 to 75, wherein the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or is 100% complementary to the targeted portion of the RIC pre-mRNA encoding the protein.

77. The composition of any one of claims 51 to 76, wherein the antisense oligomer binds to a targeted portion of a JAG1 RIC pre-mRNA, wherein the targeted portion is in a sequence selected from SEQ ID NOs 437-439.

78. The composition of any one of claims 51 to 77, wherein the antisense oligomer comprises a nucleotide sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 3-436.

79. The composition of any one of claims 51 to 77, wherein the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NOs: 3-436.

80. A pharmaceutical composition comprising the antisense oligomer of any of the compositions of claims 51 to 79, and an excipient.

81. A method of treating a subj ect in need thereof by administering the pharmaceutical composition of claim 80 by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.

82. A pharmaceutical composition comprising:

an antisense oligomer that hybridizes to a target sequence of a deficient JAGl mRNA transcript, wherein the deficient JAGl mRNA transcript comprises a retained intron, wherein the antisense oligomer induces splicing out of the retained intron from the deficient JAGl mRNA transcript; and a pharmaceutical acceptable excipient.

83. The pharmaceutical composition of claim 82, wherein the deficient JAGl mRNA transcript is a JAGl RIC pre-mRNA transcript.

84. The pharmaceutical composition of claim 82 or 83, wherein the targeted portion of the JAGl RIC pre-mRNA transcript is in the retained intron within the region +6 relative to the 5' splice site of the retained intron to -16 relative to the 3' spliced site of the retained intron.

85. The pharmaceutical composition of any one of claims 82-84, wherein the JAGl RIC pre-mRNA transcript is encoded by a genetic sequence with at least about 80%, 85%>, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1.

86. The pharmaceutical composition of any one of claims 82-85, wherein the JAGl RIC pre-mRNA transcript comprises a sequence with at least about 80%>, 85%>, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 2.

87. The pharmaceutical composition of any one of claims 82-86, wherein the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.

88. The pharmaceutical composition of any one of claims 82-87, wherein the antisense oligomer is an antisense oligonucleotide.

89. The pharmaceutical composition of any one of claims 82-88, wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-0-methyl, a 2'-Fluoro, or a 2'-0-methoxyethyl moiety.

90. The pharmaceutical composition of any one of claims 82-89, wherein the antisense oligomer comprises at least one modified sugar moiety.

91. The pharmaceutical composition of any one of claims 82-90, wherein the antisense oligomer comprises from 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 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.

92. The pharmaceutical composition of any one of claims 82-91, wherein the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or is 100%) complementary to a targeted portion of the JAGl RIC pre-mRNA transcript.

93. The pharmaceutical composition of any one of claims 82-92, wherein the targeted portion of the JAGl RIC pre-mRNA transcript is within a sequence selected from SEQ ID NOs: 437-439.

94. The pharmaceutical composition of any one of claims 82-93, wherein the antisense oligomer comprises a nucleotide sequence that is at least about 80%>, 85%>, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 3-436.

95. The pharmaceutical composition of any one of claims 82-93, wherein the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NOs: 3-436.

96. The pharmaceutical composition of any one of the claims 82-95, wherein the pharmaceutical composition is formulated for intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.

97. A method of inducing processing of a deficient JAGl mRNA transcript to facilitate removal of a retained intron to produce a fully processed JAGl mRNA transcript that encodes a functional form of a tuberin protein, the method comprising:

a) contacting an antisense oligomer to a target cell of a subject; b) hybridizing the antisense oligomer to the deficient JAGl mRNA transcript, wherein the deficient JAGl mRNA transcript is capable of encoding the functional form of tuberin protein and comprises at least one retained intron;

c) removing the at least one retained intron from the deficient JAGl mRNA transcript to produce the fully processed JAGl mRNA transcript that encodes the functional form of tuberin protein; and

d) translating the functional form of tuberin protein from the fully processed JAGl mRNA transcript.

98. The method of claim 97, wherein the retained intron is an entire retained intron.

99. The method of claim 97 or 98, wherein the deficient JAGl mRNA transcript is a JAGl RIC pre-mRNA transcript.

100. A method of treating a subject having a condition caused by a deficient amount or activity of JAGl protein comprising:

adminstering to the subject an antisense oligomer comprising a nucleotide sequence with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 3-436.