JP2019500350A - Antisense oligomers for the treatment of tuberous sclerosis - Google Patents

Abstract

Described herein are methods for increasing TSC2 expression and for treating a subject in need, eg, a subject having deficient tuberin protein expression or a subject having tuberous sclerosis, and A composition is provided. [Selection] Figure 1

Description

  This application claims the benefit of US Provisional Patent Application No. 62 / 267,212, filed December 14, 2015, which is hereby incorporated by reference in its entirety.

<Sequence Listing>
This application includes a sequence listing, which is submitted in ASCII format via EFS-Web, which is incorporated herein by reference in its entirety. A copy of ASCII created on December 12, 2016 is 47991-706_601_SL. The file name is txt and has a size of 1,663,708 bytes. The aforementioned file was created on December 12, 2016, and is incorporated herein by reference in its entirety.

  Tuberous sclerosis (TSC) is a disorder characterized by the growth of benign tumors in multiple organ systems (Au, K., et al., J. Child Neurol., 2004, 19: 699-709). Central nervous system (CNS) tumors are the leading cause of morbidity and mortality, followed by kidney disease. Patients may suffer from brain abnormalities that can include stroke, intellectual disability, and developmental delays, as well as skin, lung, kidney, and heart abnormalities. Disorders occur in as many as 25,000 to 40,000 individuals in the United States and approximately 1 to 2 million individuals worldwide, with an estimated morbidity rate of 1 in 6,000 newborns Presumed.

  TSC is a genetic disease with an autosomal dominant inheritance pattern caused by inherited defects or de novo mutations that occur on the two genes TSC1 and TSC2. TSC is present if only one of the genes is affected. The TSC1 gene on chromosome 9 produces a protein called hamartin. The TSC2 gene, discovered in 1993, is on chromosome 16 and produces the protein tuberin. Scientists believe that these proteins act in the complex as growth inhibitors by inhibiting the activation of a dominant evolutionary conserved kinase called mTOR. Loss of regulation of mTOR occurs in cells lacking either hamartin or tuberin, which causes abnormal differentiation and development, as seen in TSC brain lesions, and the generation of expanded cells.

  The present invention provides compositions and methods for treating tuberous sclerosis, the composition comprising constitutive splicing at the intron splice site of the retained intron-containing mRNA precursor (RIC mRNA precursor) of TSC2. An antisense oligomer (ASO) that promotes The present invention further provides compositions and methods for increasing production of mature TSC2 mRNA and, in turn, TSC2 protein in a subject in need, eg, cells of a subject that can benefit from increased production of TSC2 protein. I will provide a. In addition to mutations in the TSC2 gene, including missense, splicing, frameshift and nonsense mutations, the above method results in tuberous sclerosis caused by whole gene deletions, resulting in a lack of tuberine protein production. It may be used to treat a subject having.

DETAILED DESCRIPTION Disclosed herein are methods for treating a subject's tuberous sclerosis in need of increasing expression of a target protein or functional RNA by the subject's cells, in certain embodiments, wherein: The cell has a retained intron-containing mRNA precursor (RIC mRNA precursor), the RIC mRNA precursor being a retained intron, an exon adjacent to the 5 ′ splice site, a 3 ′ splice site. Wherein the RIC mRNA precursor encodes a target protein or functional RNA, wherein the method comprises subjecting a subject cell to an RIC mRNA precursor encoding the target protein or functional RNA. Contacting with an antisense oligomer (ASO) complementary to the target portion of Introns are constitutively spliced from a RIC mRNA precursor that encodes a target protein or functional RNA, thereby increasing the level of mRNA encoding the target protein or functional RNA in the subject's cells, Increase the expression of protein or functional RNA. In some embodiments, also disclosed herein is a method of increasing the expression of a target protein that is tuberin by a cell having a retained intron-containing mRNA precursor (RIC mRNA precursor). The RIC mRNA precursor comprises a retained intron, an exon adjacent to the retained intron 5 ′ splice site, an exon adjacent to the retained intron 3 ′ splice site, wherein: The RIC mRNA precursor encodes a tuberin protein, and the method comprises contacting the cell with an antisense oligomer (ASO) complementary to the target portion of the RIC mRNA precursor encoding the tuberin protein; By the retained intron before the RIC mRNA encoding the tuberin protein. Constitutively spliced from the precursor, thereby increasing the level of mRNA encoding the tuberin protein and increasing the expression of the tuberin protein within the cell. In some embodiments, the target protein is tuberin. In some embodiments, the target protein or functional RNA is a compensation protein or compensation that functionally increases or replaces a target protein or functional RNA that is deficient in amount or activity in the subject. Functional RNA. In some embodiments, the cell is in or derived from a subject having a disease caused by a deficiency in the amount or activity of tuberin protein. In some embodiments, the deficiency in the amount of the target protein is caused by a haploinsufficiency of the target protein, wherein the subject is the first allele that encodes a functional target protein, the target protein is not produced. Having two alleles or a second allele encoding a non-functional target protein, the antisense oligomer binds to the target portion of the RIC mRNA precursor transcribed from the first allele. In some embodiments, the subject has a disease caused by a disorder resulting from a deficiency in the amount or function of the target protein, wherein the subject has (a) (i) the target protein is wild Produced at a reduced level compared to production from a type allele, (ii) the target protein is produced in a reduced function compared to an equivalent wild type protein, or (iii) the target protein is A first mutant allele that is not produced, and (b) (i) the target protein is produced at a reduced level compared to production from the wild-type allele, and (ii) the target protein is an equivalent wild type protein Or (iii) a second mutant allele that does not produce a target protein, wherein the subject has a reduced function compared to The first of the mutant allele a. iii. The second mutant allele is b. i. Or b. ii. Where the subject has a second mutant allele b. iii. The first mutant allele is a. i. Or a. ii. Where the RIC mRNA precursor is a. i. Or a. ii. A first mutant allele, and / or b. i. Or b. ii. Is transcribed from a second mutant allele that is In some embodiments, the target protein is produced in a reduced function compared to an equivalent wild type protein. In some embodiments, the target protein is produced in a fully functional form as compared to an equivalent wild type protein. In some embodiments, the target portion of the RIC mRNA precursor is in the region from +6 to the retained intron 5 ′ splice site to −16 to the retained intron 3 ′ splice site. In a retained intron. In some embodiments, the target portion of the RIC mRNA precursor is within the region from +500 to the retained intron 5 ′ splice site to −500 to the retained intron 3 ′ splice site. In a retained intron. In some embodiments, the target portion of the RIC mRNA precursor is in the following internal retained intron: (a) +6 to +500, +6 to +495 relative to the retained intron 5 ′ splice site Or a region from +6 to +100; or (b) a region from −16 to −500, from −16 to −400, or from −16 to −100, relative to the 3 ′ splice site of the retained intron. In some embodiments, the target portion of the RIC mRNA precursor is within the following: (a) the + 2e to −4e region in the exon adjacent to the 5 ′ splice site of the retained intron; or (b ) The region from + 2e to -4e in the exon adjacent to the 3 'splice site of the retained intron. In some embodiments, the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of SEQ ID NO: 1. It is encoded by a gene sequence having sequence identity. In some embodiments, the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% relative to any one of SEQ ID NOs: 2-8. , 99%, or 100% sequence identity. In some embodiments, the antisense oligomer does not increase the amount of the target protein or functional RNA by modulating alternative splicing of the mRNA precursor transcribed from the gene encoding the functional RNA or target protein. . In some embodiments, the antisense oligomer does not increase the amount of target protein or functional RNA by modulating aberrant splicing due to mutations in the gene encoding the target protein or functional RNA. In some embodiments, RIC mRNA precursors were generated by partial splicing of full-length mRNA precursors or partial splicing of wild-type mRNA precursors. 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 generated target protein is a full-length protein or a wild-type protein. In some embodiments, the total amount of mRNA encoding the target protein or functional RNA produced in the cell contacted with the antisense oligomer is the amount of mRNA encoding the target protein or functional RNA produced in the control cell. About 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about the total amount About 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about About 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2. Fold, at least about 3-fold, at least about 3.5 fold, at least about 4-fold, at least about 5-fold, or increased by at least about 10-fold. In some embodiments, the total amount of target protein produced by the cells contacted with the antisense oligomer is about 1.1 to about 10 times, about 1.1 to about 10 times the total amount of target protein produced by the control cells. 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1. 1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 Times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times About 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times At least about Times, or increased by at least about 10-fold. In some embodiments, the antisense oligomer comprises a backbone modification that includes a phosphorothioate linkage or a phosphorodiamidate linkage. In some embodiments, the antisense oligomer comprises a phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2′-O-methyl, 2′-fluoro, or 2′-O-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 comprises 8-50 nucleobases, 8-40 nucleobases, 8-35 nucleobases, 8-30 nucleobases, 8-25 nucleobases, 8-20 Nucleobases, 8-15 nucleobases, 9-50 nucleobases, 9-40 nucleobases, 9-35 nucleobases, 9-30 nucleobases, 9-25 nucleobases, 9-20 Nucleobase, 9-15 nucleobase, 10-50 nucleobase, 10-40 nucleobase, 10-35 nucleobase, 10-30 nucleobase, 10-25 nucleobase, 10-20 nucleic acid Bases, 10-15 nucleobases, 11-50 nucleobases, 11-40 nucleobases, 11-35 nucleobases, 11-30 nucleobases, 11-25 nucleobases, 11-20 nucleobases 11-15 nucleobases, 12-50 nucleobases, 40 nucleobases, nucleobase of 12 to 35, nucleic acid bases 12-30 consist of nucleobases 12-25, 12-20 nucleobases or 12-15 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 at the target portion of the protein-encoding RIC mRNA precursor. % Complementary. In some embodiments, the target portion of the RIC mRNA precursor is within a sequence selected from SEQ ID NO: 5097-5105. In some embodiments, the antisense oligomer is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, any one of SEQ ID NOs: 9-5096, Includes nucleotide sequences that are 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NOs: 9-5096. In some embodiments, the cell comprises a population of RIC mRNA precursors transcribed from a gene encoding a target protein or functional RNA, wherein the populations of RIC mRNA precursors are retained in two or more. Introns, where antisense oligomers bind to the most abundant retained introns in the population of RIC mRNA precursors. In some embodiments, the binding of antisense oligomers to the most abundant retained introns is retained in two or more from a population of RIC mRNA precursors that generate mRNA encoding the target protein or functional RNA. Induces intron splicing out. In some embodiments, the cell comprises a population of RIC mRNA precursors transcribed from a gene encoding a target protein or functional RNA, wherein the populations of RIC mRNA precursors are retained in two or more. Introns, where antisense oligomers bind to the second most abundant retained intron in the population of RIC mRNA precursors. In some embodiments, the binding of the antisense oligomer to the second most abundant retained intron is the retention of two or more from a population of RIC mRNA precursors that generate mRNA encoding the target protein or functional RNA. Induces splicing out of introns. In some embodiments, the disease is a disease or disorder. In some embodiments, the disease or disorder is tuberous sclerosis. In some embodiments, the target protein and RIC mRNA precursor are encoded by the TSC2 gene. In some embodiments, the method further comprises assessing TSC2 protein expression. In some embodiments, the antisense oligomers bind to the target portion of the tuberin RIC mRNA precursor, where the target portion is SEQ ID NOS: 49, 50, 51, 52, 53, 54, 55, 56, and Within the sequence selected from 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, embryo, or child. In some embodiments, the cell is ex vivo. In some embodiments, the antisense oligomer is administered locally to the skin of the subject, pulmonary delivery to the lung, intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous It is administered by internal injection. In some embodiments, the nine nucleotides at -3e to -1e of the exon adjacent to 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 in exon + 1e adjacent to the conserved intron at -15 to -1 and the 3 'splice site are identical to the corresponding wild type sequence.

  Disclosed herein are antisense oligomers used in the above methods in certain embodiments.

  In certain embodiments, comprising a sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 9-5096 Antisense oligomers are disclosed herein.

  Disclosed herein are pharmaceutical compositions comprising, in certain embodiments, the antisense oligomers described above and excipients. In some embodiments, the present specification also provides topical administration to the skin, pulmonary delivery to the lung, intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous. A method is described for treating a subject in need by administering a pharmaceutical composition by injection.

  Herein, in certain embodiments, expression of a target protein or functional RNA by a cell to treat tuberous sclerosis in a subject associated with the deficient protein or deficient functional RNA. Disclosed is a composition comprising an antisense oligomer for use in a method for increasing the amount of protein or functional RNA that is deficient in amount or activity in a subject, wherein Sense oligomers enhance constitutive splicing of a retained intron-containing mRNA precursor (RIC mRNA precursor) that encodes a target protein or functional RNA, where the target protein is: (a) deficient Protein; or (b) functionally increasing the deficient protein in the subject, It replaces the compensating protein, where the functional RNA is: (a) the deficient RNA; or (b) functionally augments or replaces the deficient functional RNA in the subject. , A functional RNA that compensates, wherein the RIC mRNA precursor was retained, including a retained intron, an exon adjacent to the 5 ′ splice site, and an exon adjacent to the 3 ′ splice site. Introns are spliced from a RIC mRNA precursor that encodes a target protein or functional RNA, thereby increasing the production or activity of the target protein or functional RNA in a subject. In some embodiments, disclosed herein is also a composition comprising an antisense oligomer for use in a method of treating a disease associated with tuberin protein in a subject, the method comprising: Increasing the expression of the tuberin protein by the cells of the cell, wherein the cells are retained in the retained intron, the exon adjacent to the retained intron 5 ′ splice site, and the retained intron 3 ′ splice site. Having a retained intron-containing mRNA precursor (RIC mRNA precursor) comprising an adjacent exon, wherein the RIC mRNA precursor encodes a tuberin protein, the method contacting the cell with an antisense oligomer So that the retained intron encodes the tuberin protein Constitutively spliced from a transcript of the RIC mRNA precursor, thereby increasing the level of mRNA encoding the tuberin protein and increasing the expression of the tuberin protein in the subject's cells. . In some embodiments, the disease is a disease or disorder. In some embodiments, the disease or disorder is tuberous sclerosis. In some embodiments, the target protein and RIC mRNA precursor are encoded by the TSC2 gene. In some embodiments, the antisense oligomer is a retained intron in a region from +6 to the retained intron 5 ′ splice site to −16 to the retained intron 3 ′ splice site. Targets a portion of the RIC mRNA precursor in In some embodiments, the antisense oligomer targets a portion of the RIC mRNA precursor, which is in the following internal retained intron: (a) to the 5 ′ splice site of the retained intron +6 to +100 region; (b) -16 to -100 region relative to the retained intron 3 'splice site. In some embodiments, the antisense oligomers are 3 ′ of at least one retained intron from about 500, 400, 300, 200, or 100 nucleotides downstream of the 5 ′ splice site of at least one retained intron. It targets a portion of the RIC mRNA precursor that is in the region up to about 100, 200, 300, 400, or 500 nucleotides upstream of the splice site. In some embodiments, the target portion of the RIC mRNA precursor is within the following: (a) the + 2e to −4e region in the exon adjacent to the 5 ′ splice site of the retained intron; or (b ) The region from + 2e to -4e in the exon adjacent to the 3 'splice site of the retained intron. In some embodiments, the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of SEQ ID NO: 1. It is encoded by a gene sequence having sequence identity. In some embodiments, the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% relative to any one of SEQ ID NOs: 2-8. , 99%, or 100% sequence identity. In some embodiments, the antisense oligomer does not increase the amount of target protein or functional RNA by modulating alternative splicing of the mRNA precursor transcribed from the gene encoding the target protein or functional RNA. In some embodiments, antisense oligomers do not increase the amount of functional RNA or functional protein by modulating aberrant splicing due to mutations in the gene encoding the target protein or functional RNA. In some embodiments, RIC mRNA precursors were generated by partial splicing from full-length mRNA precursors or wild-type mRNA precursors. 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 generated target protein is a full-length protein or a 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 of the RIC mRNA precursor. In some embodiments, the retained intron is the second most abundant retained intron in the RIC mRNA precursor. In some embodiments, the antisense oligomer comprises a backbone modification that includes 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, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-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 comprises 8-50 nucleobases, 8-40 nucleobases, 8-35 nucleobases, 8-30 nucleobases, 8-25 nucleobases, 8-20 Nucleobases, 8-15 nucleobases, 9-50 nucleobases, 9-40 nucleobases, 9-35 nucleobases, 9-30 nucleobases, 9-25 nucleobases, 9-20 Nucleobase, 9-15 nucleobase, 10-50 nucleobase, 10-40 nucleobase, 10-35 nucleobase, 10-30 nucleobase, 10-25 nucleobase, 10-20 nucleic acid Bases, 10-15 nucleobases, 11-50 nucleobases, 11-40 nucleobases, 11-35 nucleobases, 11-30 nucleobases, 11-25 nucleobases, 11-20 nucleobases 11-15 nucleobases, 12-50 nucleobases, 40 nucleobases, nucleobase of 12 to 35, nucleic acid bases 12-30 consist of nucleobases 12-25, 12-20 nucleobases or 12-15 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 at the target portion of the protein-encoding RIC mRNA precursor. % Complementary. In some embodiments, the antisense oligomer binds to a target portion of a tuberine RIC mRNA precursor, wherein the target portion is within a sequence selected from SEQ ID NO: 5097-5105. In some embodiments, the antisense oligomer is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, any one of SEQ ID NOs: 9-5096, Includes nucleotide sequences that are 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NOs: 9-5096.

  Disclosed herein are pharmaceutical compositions comprising, in certain embodiments, the antisense oligomers described above and excipients. In some embodiments, the present specification also provides topical administration to the skin, pulmonary delivery to the lung, intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous. A method is described for treating a subject in need by administering a pharmaceutical composition by injection.

  In certain embodiments, a pharmaceutical composition is disclosed herein, the composition comprising an antisense oligomer that hybridizes to a target sequence of a missing TSC2 mRNA transcript, and a pharmaceutically acceptable excipient. The TSC2 mRNA transcript, which contains the agent, where the missing TSC2 mRNA transcript contains the retained intron, and the antisense oligomer induces the retained intron splicing out from the missing TSC2 mRNA transcript. In some embodiments, the missing TSC2 mRNA transcript is a transcript of the TSC2 RIC mRNA precursor. In some embodiments, the target portion of the TSC2 RIC mRNA precursor transcript ranges from +500 to the retained intron 5 ′ splice site to −500 to the retained intron 3 ′ splice site. In the retained intron in the region of In some embodiments, the transcript of the TSC2 RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99 with respect to SEQ ID NO: 1. It is encoded by a gene sequence with% or 100% sequence identity. In some embodiments, the TSC2 RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98 for any one of SEQ ID NOs: 2-8. Includes sequences with%, 99%, or 100% sequence identity. In some embodiments, the antisense oligomer comprises a backbone modification that includes 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, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety. In some embodiments, the antisense oligomer comprises at least one modified sugar moiety. In some embodiments, the antisense oligomer comprises 8-50 nucleobases, 8-40 nucleobases, 8-35 nucleobases, 8-30 nucleobases, 8-25 nucleobases, 8-20 Nucleobases, 8-15 nucleobases, 9-50 nucleobases, 9-40 nucleobases, 9-35 nucleobases, 9-30 nucleobases, 9-25 nucleobases, 9-20 Nucleobase, 9-15 nucleobase, 10-50 nucleobase, 10-40 nucleobase, 10-35 nucleobase, 10-30 nucleobase, 10-25 nucleobase, 10-20 nucleic acid Bases, 10-15 nucleobases, 11-50 nucleobases, 11-40 nucleobases, 11-35 nucleobases, 11-30 nucleobases, 11-25 nucleobases, 11-20 nucleobases 11-15 nucleobases, 12-50 nucleobases, 40 nucleobases include nucleobases of 12 to 35, nucleic acid bases 12-30, nucleobases 12-25, 12-20 nucleobases or 12-15 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% relative to the target portion of the transcript of the TSC2 RIC mRNA precursor. Or 100% complementary. In some embodiments, the target portion of the TSC2 RIC mRNA precursor transcript is within a sequence selected from SEQ ID NO: 5097-5105. In some embodiments, the antisense oligomer is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, any one of SEQ ID NOs: 9-5096, Includes nucleotide sequences that are 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NOs: 9-5096. In some embodiments, the pharmaceutical composition is formulated for intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.

  In certain embodiments, processing of missing TSC2 mRNA transcripts to facilitate removal of retained introns to generate fully processed TSC2 mRNA transcripts encoding a functional form of the tuberin protein. Disclosed herein is a method of inducing, comprising: (a) contacting an antisense oligomer with a target cell of a subject; and (b) a transcript of TSC2 mRNA lacking the antisense oligomer. (B) a step wherein the deficient TSC2 mRNA transcript is capable of encoding a functional form of the tuberin protein and comprises at least one retained intron; To generate a fully processed TSC2 mRNA transcript that encodes a functional form of the verin protein Removing at least one retained intron from the deficient TSC2 mRNA transcript, and (d) translating the functional form of the tuberin protein from the fully processed TSC2 mRNA transcript. Including. In some embodiments, the retained intron is the entire retained intron. In some embodiments, the missing TSC2 mRNA transcript is a transcript of the TSC2 RIC mRNA precursor.

  Disclosed herein are methods for treating a subject having a disease caused by a deficiency in the amount or activity of tuberin protein, in certain embodiments, comprising SEQ ID NO: 9-5096. At least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one Administering to the subject an antisense oligomer comprising a nucleotide sequence having:

<Incorporation by quotation>
All publications, patents, and patent applications mentioned in this specification are by reference to the extent that each individual publication, patent, or patent application is specifically and individually indicated to be incorporated by reference. Incorporated herein.

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 of which: .
Schematic representation of a typical retained intron-containing (RIC) mRNA precursor transcript. The 5 'splice site consensus sequence is the letters (letters) underlined to -3e to -1e and +1 to +6 (numbers labeled "e" are exons and numbers not labeled are introns). Are nucleotides; upper case: exon part, and lower case: intron part). The 3 'splice site consensus sequence is a letter underlined from -15 to -1 and + 1e (numbers labeled "e" are exons and unlabeled numbers are introns) (letters are nucleotides Upper case: exon part, and lower case: intron part). The intron target region for ASO screening ranges from nucleotide +6 for the retained intron 5 'splice site (left arrow) to nucleotide -16 for the retained intron 3' splice site (right arrow). including. In embodiments, the intron target region for ASO screening comprises nucleotides +6 to +100 to the retained intron 5 'splice site and nucleotides -16 to -100 to the retained intron 3' splice site. . Exon target sites include nucleotides from + 2e to -4e in the exon adjacent to the 5 'splice site of the retained intron and + 2e to -4e in the exon adjacent to the 3' splice site of the retained intron. Including. “N” or “N” represents a nucleotide and “y” represents a pyrimidine. The depicted sequence represents a consensus sequence for the mammalian splice site, and individual introns and exons need not match the consensus sequence at every position. FIG. 2 illustrates a schematic diagram of a targeted enhancement (TANGO) method of nuclear gene output. FIG. 2A shows a cell divided into a nucleus and a cytoplasmic compartment. In the nucleus, transcripts of target gene mRNA precursors, consisting of exons (rectangles) and introns (connecting lines), are spliced to produce mRNAs that are transported into the cytoplasm and translated into target proteins. The For this target gene, intron 1 splicing is inefficient, and retained intron-containing (RIC) mRNA precursors accumulate primarily in the nucleus and degrade when transported to the cytoplasm. It does not lead to production. FIG. 2 illustrates a schematic diagram of a targeted enhancement (TANGO) method of nuclear gene output. FIG. 2B shows an example of a similar cell divided into a nucleus and a cytoplasmic compartment. Treatment with antisense oligomers (ASO) promotes intron 1 splicing and leads to increased mRNA, which in turn is translated into high levels of the target protein. Intron retention in the TSC2 gene with intron 4 is shown in detail. Identification of intron retention events in the TSC2 gene using RNA sequencing (RNAseq) is visualized and shown in the UCSC genome browser. The upper panel shows the read density corresponding to the TSC2 transcript expressed in HCN (human cortical neurons) and localized in either the cytoplasm (top) or nuclear fraction (bottom). At the bottom of this panel, a graphical representation of the TSC2 gene is shown to scale. The read density is shown as a peak. The highest reading density corresponds to an exon (black box), while no reading is observed for the majority of introns (lines with arrow heads) in any cell fraction. A higher reading density compared to the cytoplasmic fraction was detected in introns 4, 25/26, and 31/32 (indicated by arrows) in the nuclear fraction, which were introns 4, 25/26, and 31 / 32 shows that splicing efficiency is low, resulting in intron retention. The transcript of the retained intron-containing mRNA precursor is retained in the nucleus and not transported to the cytoplasm. The read density of intron 4 in HCN is shown in detail in the lower panel. The TSC2 gene IVS 4 ASO walk is shown. Illustrated is an ASO walk performed on TSC2 IVS 4 using 2'-OMe ASO, PS backbone, targeting sequences immediately downstream of the 5 'splice site or upstream of the 3' splice site. ASO was designed to cover these regions by changing the position of 5 nucleotides at a time. The TSC2 exon-intron structure is shown to scale. Intron retention in the TSC2 gene with introns 25 and 26 is shown in detail. Intron retention in the TSC2 gene was identified by RNA sequencing (RNAseq) and visualized in the UCSC genome browser, as described in the Examples herein. The read density of introns 25 and 26 in HCN is shown in detail in the lower panel. Introns 25 and 26 are adjacent to exon 26, which is an alternatively spliced exon. This is demonstrated in the graphical representation of the TSC2 transcript and RNAseq data, so that the rectangle representing exon 26 is present in some transcripts but not in other transcripts, and in the cytoplasmic fraction. The read density corresponding to exon 26 is significantly lower than the constitutively spliced exon in TSC2. The read density for intron 26 is shown in detail in the lower panel showing 51% intron retention as calculated by bioinformatic analysis. ASO walk of TSC2 gene IVS 25 and 26 is shown. Performed for TSC2 IVS 25 and 26 targeting sequences immediately downstream of the 5 'splice site of intron 25 or upstream of the 3' splice site of intron 26 using 2'-OMe ASO, PS backbone. A graphical representation of the ASO walk is shown. The intron region of the splice site adjacent to alternative exon 26 is not targeted to avoid affecting the inclusion level of exon 26. ASO was designed to cover these regions by changing the position of 5 nucleotides at a time. The TSC2 exon-intron structure is shown to scale. Intron retention in the TSC2 gene with introns 31 and 32 is shown in detail. Intron retention in the TSC2 gene was identified by RNA sequencing (RNAseq) and visualized in the UCSC genome browser, as described in the Examples herein. The read density of introns 31 and 32 is shown in detail in the lower panel. Introns 31 and 32 are adjacent to exon 32, which is an alternatively spliced exon. This is demonstrated in the schematic representation of the TSC2 transcript and RNAseq data, so that a rectangle representing exon 32 is present in some transcripts but not in other transcripts, and in the cytoplasmic fraction. The read density corresponding to exon 32 is significantly lower than the constitutively spliced exon in TSC2. The read density for intron 31 is shown in detail in the lower panel showing 43% intron retention as calculated by bioinformatic analysis. The ASO walk of TSC2 gene IVS 31 and 32 is shown. 2'-OMe ASO, performed on TSC2 IVS 31 and 32 targeting sequences immediately downstream of the 5 'splice site of intron 31 or upstream of the 3' splice site of intron 32 using the PS backbone. A graphical representation of the ASO walk is shown. The intron region of the splice site adjacent to alternative exon 32 is not targeted to avoid affecting the inclusion level of exon 32. ASO was designed to cover these regions by changing the position of 5 nucleotides at a time, with the exception of TSC2-IVS32-33 and TSC2-IVS32-51. The TSC2 exon-intron structure is shown to scale. The schematic diagram of the ReSeq gene of TSC2 intron 4 corresponding to NM_000548 is represented. The intron retention (PIR) of the circled intron is shown. FIG. 4 represents a typical graph showing the mean (n = 3) fold change in expression level of TSC2 mRNA without intron 4 in ARPE-19 cells treated with 80 nM of indicated ASO for 24 hours on mock-treated cells . Data are normalized to RPL32 expression. The schematic diagram of the ReSeq gene of TSC2 intron 25 corresponding to NM_000548 is represented. The intron retention (PIR) of the circled intron is shown. FIG. 6 represents a typical graph showing the mean (n = 3) fold change in expression level of TSC2 mRNA without intron 25 in ARPE-19 cells treated with 80 nM of indicated ASO for 24 hours on mock-treated cells . Data are normalized to RPL32 expression. The schematic diagram of the ReSeq gene of TSC2 intron 26 corresponding to NM_000548 is represented. The intron retention (PIR) of the circled intron is shown. The schematic diagram of the ReSeq gene of TSC2 intron 31 corresponding to NM_000548 is shown. The intron retention (PIR) of the circled intron is shown. FIG. 6 represents a typical graph showing the mean (n = 3) fold change in expression levels of TSC2 mRNA without intron 31 in ARPE-19 cells treated with 80 nM of indicated ASO for 24 hours on mock-treated cells . Data are normalized to RPL32 expression. The schematic diagram of the ReSeq gene of TSC2 intron 32 corresponding to NM_000548 is represented. The intron retention (PIR) of the circled intron is shown.

  Individual introns in the primary transcript of a protein-encoding gene having two or more introns are spliced from primary transcripts with different efficiencies. In most cases, only fully spliced mRNA is transported through the nuclear pore for subsequent cytoplasmic translation. Unspliced or partially spliced transcripts can be detected in the nucleus. Nuclear accumulation of transcripts that are not completely spliced is generally thought to be a mechanism that prevents the accumulation of potentially harmful mRNAs in the cytoplasm that can be translated into proteins. For some genes, the least efficient intron splicing is the rate-limiting post-transcriptional step in gene expression prior to translation in the cytoplasm.

  The present invention is one that is rate-limiting to the nuclear stage of gene expression in order to increase the steady state production of fully spliced mature mRNA and therefore increase translated protein levels. Compositions and methods for upregulating splicing of the above retained TSC2 intron are provided. These compositions and methods take advantage of antisense oligomers (ASO) that promote constitutive splicing at the intron splice sites of retained intron-containing TSC2 mRNA precursors that accumulate in the nucleus. Thus, in an embodiment, the method of the invention is used to increase TSC2 protein to treat a disease resulting from TSC2 deficiency.

  In other embodiments, the methods of the invention are used to increase TSC2 production to treat a disease in a subject in need thereof. In embodiments, the subject has a disease in which TSC2 is not necessarily deficient relative to the wild type, but nevertheless an increase in TSC2 alleviates the disease. In an embodiment, the disease is caused by TSC2 haploinsufficiency. In an embodiment, the disease is an autosomal dominant disorder. In an embodiment, the disease is an autosomal recessive disorder.

<Nodular sclerosis>
Tuberous sclerosis is a disease characterized by tumor growth in a multi-organ system (Au et al., J. Child Neurol. 2004, 19, 699-709). Tumors are usually benign, but sometimes malignant. Approximately 90% of tuberous sclerosis cases represent cortical nodules; facial fibroangioma and renal angiomyolipoma occur in more than 80% of cases. In addition, approximately 80% of cases represent epiphyseal nodules, approximately 50% of cases represent rhabdomyomas of the heart, and 51% to approximately 88% of cases represent nail / subungual fibromas. Central nervous system (CNS) tumors are a major cause of morbidity and mortality followed by renal disease (Au et al., J. Child Neurol. 2004, 19, 699-709).

  Tuberous sclerosis is a genetic disease with an autosomal dominant inheritance pattern and a high mutation rate (Au et al., J. Child Neurol. 2004, 19, 699-709). Binding of tuberous sclerosis to chromosomal regions 9q34.3 and 16p13.3 led to the identification of the TSC1 and TSC2 genes, respectively. More than 69% of tuberous sclerosis cases result from haploinsufficiency of TSC2, and approximately two thirds of patients result from de novo mutations or deletions (Au et al., J. et al. Child Neurol.2004, 19, 699-709). Severe disease is thought to require a reduction in the “second hit” of other alleles. The prevalence of tuberous sclerosis is about 1,800 out of 5,800 live births per year or about 300 births in the United States, with 200 cases per year resulting from TSC2 mutations. Arise. The disease is described, for example, by OMIM # 613254 (Online Mendellian Inheritance in Man, Johns Hopkins University, 1966-2015), incorporated herein by reference.

  The TSC2 gene encodes a protein called tuberin. The TSC2 gene contains 41 exons, two of which are alternatively spliced, spanning approximately 40 kb on chromosome 16 (Au et al., J. Child Neurol. 2004, 19, 699-709). Tuberin is a 200 kDa protein that forms a complex with hamartin, a TSC1 gene product. The tuberin-hamartin complex plays a role in regulating cell proliferation, transport, cell size, cell adhesion, and migration through various signal cascades. For example, the tuberin-hamartin complex functions in the phosphatidylinositol-3-kinase and protein B pathway (PI3K / PKB) that regulate translation. Specifically, the combined value of tuberin-hamartin suppresses cell growth and translation by suppressing mTOR kinase activity, resulting in inhibition of p70 ribosomal protein S6 kinase (S6K) 1 and eukaryotic translation initiation factor 4E binding protein. 1 (4E-BP1) activation. Furthermore, tuberin's GAP domain hydrolyzes guanosine triphosphate (GTP) bound to the small G protein Rheb, which is an abundant Ras homolog in the brain, thus preventing mTOR activation.

  In addition, tuberin and hamartin regulate cell proliferation and cell adhesion, respectively, via the MAP kinase (MAPK) and E-cadherin-beta-catenin pathways. A number of mutations were described for TSC2 (Au et al., J. Child Neurol. 2004, 19, 699-709), first discovered by identifying a loss of heterozygosity in the portion of chromosome 16p13. Mutations include frameshifts and protein shortening, nonsense mutations, mutations affecting splicing, in-frame mutations, deletions, insertions, duplications, and large deletions and translocations. Mutations in TSC2 that lead to a shortened protein product are unable to form the tuberin-hamalin complex, thus resulting in loss of cell growth regulation. The role of the tuberin-hamartin complex as a key regulator of multiple signaling pathways involves multiple organ systems, including neurological and neuroreactive abnormalities such as seizures, intellectual disability, and developmental delay Consistent with the broad spectrum of clinical findings among patients with tuberous sclerosis (Au et al., J. Child Neurol. 2004, 19, 699-709).

<Retained intron-containing mRNA precursor (RIC mRNA precursor)>
In embodiments, the methods herein take advantage of the presence of a retained intron-containing mRNA precursor (RIC mRNA precursor) that is transcribed from the TSC2 gene and encodes a tuberin protein in the nucleus of the cell. Splicing of specific TSC2 RIC mRNA precursor species to yield mature, fully spliced TSC2 mRNA is induced using ASO that stimulates splicing out of retained introns. The resulting mature TSC2 mRNA is transported to the cytoplasm and translated thereby increasing the amount of tuberin protein in the patient's cells and alleviating the symptoms of tuberous sclerosis. This method is known as Targeted Enhancement of Nuclear Gene Output (TANGO), described further below.

<TSC2 nuclear transcript>
As described in the Examples herein, the TSC2 gene was analyzed for intron retention events and retention of introns 4, 25, 26, 31, and 32 was observed. RNA sequencing (RNAseq) visualized by the UCSC genome browser showed TSC2 transcripts expressed in HCN (human cortical neuron) cells, localized to either the cytoplasmic or nuclear fraction. No reading was observed for the majority of introns in either fraction. However, a higher reading density compared to the cytoplasmic fraction was detected in the introns 4, 25, 26, 31, and 32 of the nuclear fraction, which is the splicing efficiency of introns 4, 25, 26, 31, and 32 Is low, resulting in intron retention. The transcript of the retained intron-containing mRNA precursor is mainly accumulated in the nucleus and is not translated into tuberin protein. The read density for AST intron 26 is shown in detail in the lower panel showing 51% intron retention as calculated by bioinformatic analysis. Intron 26 intron retention (PIR) values were obtained by averaging four values (87, 83, 20 and 12). Each value uses one of four different algorithms and the read density for AST intron 31 determined in renal epithelial cells is detailed in the lower panel showing 43% intron retention. . Intron retention (PIR) values for intron 31 were obtained by averaging four values (78, 71, 16 and 8). Each value was determined in renal epithelial cells using one of four different algorithms. Introns 4 and 32 were not mapped. Analysis of ENCODE data to identify intron retention events (e.g., Tigner, et al., 2012, “Deep sequencing of subcellular RNA fractions sequencial transcriptional beneficient co-transienti- 22 (9): 1616-25) did not identify introns 25, 26, or 31 as retained and did not map introns 4 and 32.

  Table 1 shows the target sequences of TSC2 RIC mRNA precursor transcripts per sequence ID, and sequence IDs useful for increasing the production of tuberine protein by targeting regions of TSC2 RIC mRNA precursor Provides a non-limiting list of each ASO. In embodiments, other ASOs useful for these purposes are identified using, for example, the methods described herein.

  In some embodiments, the ASO disclosed herein targets RIC mRNA precursors transcribed from the TSC2 genomic sequence. In some embodiments, ASO targets RIC mRNA precursor transcripts from TSC2 genomic sequences that contain retained introns. In some embodiments, the ASO targets the transcript of the SEQ ID NO: 1 RIC mRNA precursor. In some embodiments, the ASO targets the transcript of the SEQ ID NO: 1 RIC mRNA precursor containing a retained intron. In some embodiments, the ASO disclosed herein targets the sequence of the TSC2 RIC mRNA precursor. In some embodiments, the ASO targets a transcript of a TSC2 RIC mRNA precursor that includes an intron retained at 3, 24, 29, or a combination thereof, where the intron numbering is mRNA in NM_001318829. Corresponds to an array. In some embodiments, the ASO targets TSC2 RIC mRNA precursor transcripts containing introns retained at 4, 25, 26, 31, 32, or combinations thereof, where the intron numbering is , Corresponds to the mRNA sequence in NM_000548. In some embodiments, the ASO targets a transcript of a TSC2 RIC mRNA precursor that contains an intron retained at 4, 25, 30, or a combination thereof, where the intron numbering is mRNA in NM_001077183. Corresponds to an array. In some embodiments, the ASO targets a transcript of a TSC2 RIC mRNA precursor that includes an intron retained at 4, 25, 26, 31, or a combination thereof, where the intron numbering is NM_001114382. Corresponds to the mRNA sequence. In some embodiments, the ASO targets a transcript of a TSC2 RIC mRNA precursor that contains an intron retained at 22, 27, 28, or a combination thereof, where the intron numbering is mRNA in NM_001318831 Corresponds to an array. In some embodiments, the ASO targets a transcript of a TSC2 RIC mRNA precursor that contains an intron retained at 4, 25, 30, or a combination thereof, wherein the intron numbering is mRNA in NM_001318832. Corresponds to an array. In some embodiments, the ASO targets a transcript of a TSC2 RIC mRNA precursor that includes an intron retained at 3, 24, 29, or a combination thereof, where the intron numbering is mRNA in NM_001318827. Corresponds to an array.

  In some embodiments, the ASO targets the TSC2 RIC mRNA precursor sequence according to SEQ ID NO: 2. In some embodiments, the ASO comprises a sequence of a TSC2 RIC mRNA precursor according to SEQ ID NO: 2, comprising retained intron 24, retained intron 3, retained intron 29, or combinations thereof. Target. In some embodiments, the ASO targets the TSC2 RIC mRNA precursor sequence according to SEQ ID NO: 3. In some embodiments, the ASO includes a retained intron 25, a retained intron 26, a retained intron 4, a retained intron 31, or a combination thereof, TSC2 RIC according to SEQ ID NO: 3 Target the sequence of the mRNA precursor. In some embodiments, the ASO targets the TSC2 RIC mRNA precursor sequence according to SEQ ID NO: 4. In some embodiments, the ASO comprises a sequence of a TSC2 RIC mRNA precursor according to SEQ ID NO: 4, comprising retained intron 25, retained intron 4, retained intron 30, or combinations thereof. Target. In some embodiments, the ASO targets the TSC2 RIC mRNA precursor sequence according to SEQ ID NO: 5. In some embodiments, the ASO includes a retained intron 25, a retained intron 26, a retained intron 4, a retained intron 31, or a combination thereof, TSC2 RIC according to SEQ ID NO: 5 Target the sequence of the mRNA precursor. In some embodiments, the ASO targets the TSC2 RIC mRNA precursor sequence according to SEQ ID NO: 6. In some embodiments, the ASO comprises a sequence of a TSC2 RIC mRNA precursor according to SEQ ID NO: 6, comprising retained intron 27, retained intron 28, retained intron 22, or combinations thereof. Target. In some embodiments, the ASO targets the TSC2 RIC mRNA precursor sequence according to SEQ ID NO: 7. In some embodiments, the ASO comprises a sequence of a TSC2 RIC mRNA precursor according to SEQ ID NO: 7, comprising retained intron 25, retained intron 4, retained intron 30, or combinations thereof. Target. In some embodiments, the ASO targets the sequence of the TSC2 RIC mRNA precursor according to SEQ ID NO: 8. In some embodiments, the ASO comprises a sequence of a TSC2 RIC mRNA precursor according to SEQ ID NO: 8, comprising retained intron 24, retained intron 3, retained intron 29, or combinations thereof. Target. In some embodiments, the ASO disclosed herein targets SEQ ID NO: 5097-5105. In some embodiments, the ASO has a sequence according to any one of SEQ ID NOs: 9-5096.

  In some embodiments, the ASO targets TSC2 RIC mRNA precursor exon 24 or exon 25 containing a retained intron 24, where the intron numbering corresponds to the mRNA sequence in NM_001318829. In some embodiments, the ASO targets the exon 24 sequence upstream (or 5 ') from the 5' splice site of the TSC2 RIC mRNA precursor containing the retained intron 24. In some embodiments, the ASO is an exon 24 about 2 to about 75 or about 4 to about 74 nucleotides upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor comprising a retained intron 24. Target the sequence. In some embodiments, the ASO targets an exon 25 sequence downstream (or 3 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 24. In some embodiments, the ASO is an exon about 2 to about 150 or about 2 to about 147 nucleotides downstream (or 3 ′) from the 3 ′ splice site of a TSC2 RIC mRNA precursor comprising a retained intron 24. Target 25 sequences.

  In some embodiments, the ASO targets the intron 24 of a TSC2 RIC mRNA precursor that contains a retained intron 24, where the intron numbering corresponds to the mRNA sequence in NM_001318829. In some embodiments, the ASO targets an intron 24 sequence downstream (or 3 ') from the 5' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 24. In some embodiments, the ASO targets an intron 24 sequence about 6 to about 497 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 24. . In some embodiments, the ASO targets an intron 24 sequence upstream (or 5 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 24. In some embodiments, the ASO targets an intron 24 sequence about 16 to about 496 nucleotides upstream (or 5 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 24. .

  In some embodiments, the ASO targets exon 3 or exon 4 of the TSC2 RIC mRNA precursor containing the retained intron 3, where the intron numbering corresponds to the mRNA sequence in NM_001318829. In some embodiments, ASO targets an exon 3 sequence upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor containing a retained intron 3. In some embodiments, the ASO targets an exon 3 sequence about 4 to about 94 nucleotides upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 3. . In some embodiments, ASO targets an exon 4 sequence downstream (or 3 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 3. In some embodiments, the ASO targets an exon 4 sequence from about 2 to about 127 nucleotides downstream (or 3 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 3. .

  In some embodiments, ASO targets the intron 3 of the TSC2 RIC mRNA precursor that contains the retained intron 3, where the intron numbering corresponds to the mRNA sequence in NM_001318829. In some embodiments, ASO targets an intron 3 sequence downstream (or 3 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 3. In some embodiments, the ASO targets an intron 3 sequence about 6 to about 435 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 3. . In some embodiments, the ASO targets an intron 3 sequence upstream (or 5 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 3. In some embodiments, the ASO targets an intron 3 sequence about 16 to about 432 nucleotides upstream (or 5 ′) from the 3 ′ splice site of a TSC2 RIC mRNA precursor that contains a retained intron 3. .

  In some embodiments, the ASO targets TSC2 RIC mRNA precursor exon 29 or exon 30 containing a retained intron 29, where the intron numbering corresponds to the mRNA sequence in NM — 001318829. In some embodiments, the ASO targets an exon 29 sequence upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 29. In some embodiments, the ASO targets an exon 29 sequence about 4 to about 184 nucleotides upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 29. . In some embodiments, the ASO targets an exon 30 sequence downstream (or 3 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 29. In some embodiments, the ASO targets an exon 30 sequence about 2 to about 102 nucleotides downstream (or 3 ') from the 3' splice site of a TSC2 RIC mRNA precursor that includes a retained intron 29. .

  In some embodiments, the ASO targets the intron 29 of the TSC2 RIC mRNA precursor that contains the retained intron 29, where the intron numbering corresponds to the mRNA sequence in NM_001318829. In some embodiments, the ASO targets an intron 29 sequence downstream (or 3 ') from the 5' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 29. In some embodiments, the ASO targets an intron 29 sequence about 6 to about 492 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 29. . In some embodiments, the ASO targets an intron 29 sequence upstream (or 5 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 29. In some embodiments, the ASO is an intron about 16 to about 500, or about 36 to about 500 nucleotides upstream (or 5 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 29. Targets 29 sequences.

  In some embodiments, the ASO targets the exon 32 or exon 33 of the TSC2 RIC mRNA precursor containing the retained intron 32, where the intron numbering corresponds to the mRNA sequence in NM_000548. In some embodiments, the ASO targets an exon 32 sequence upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 32. In some embodiments, the ASO targets an exon 32 sequence about 4 to about 49 nucleotides upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 32. In some embodiments, the ASO targets an exon 33 sequence downstream (or 3 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 32. In some embodiments, the ASO targets an exon 33 sequence about 2 to about 102 nucleotides downstream (or 3 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 32.

  In some embodiments, ASO targets intron 32 in a TSC2 RIC mRNA precursor that contains a retained intron 32, where the intron numbering corresponds to the mRNA sequence in NM_000548. In some embodiments, the ASO targets an intron 32 sequence downstream (or 3 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 32. In some embodiments, the ASO targets an intron 32 sequence about 6 to about 495 nucleotides downstream (or 3 ') from the 5' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 32. In some embodiments, the ASO targets an intron 32 sequence upstream (or 5 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 32. In some embodiments, the ASO is about 16 to about 500 or about 36 to about 500 nucleotides upstream (or 5 ') intron 32 from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 32. Target the sequence.

  In some embodiments, the ASO targets the exon 25 or exon 26 of the TSC2 RIC mRNA precursor containing the retained intron 25, where the intron numbering corresponds to the mRNA sequence in NM_000548. In some embodiments, ASO targets an exon 25 sequence upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 25. In some embodiments, the ASO targets an exon 25 sequence from about 4 to about 74 nucleotides upstream (or 5 ') from the 5' splice site of the TSC2 RIC mRNA precursor containing the retained intron 25. In some embodiments, the ASO targets the exon 26 sequence downstream (or 3 ') from the 3' splice site of the TSC2 RIC mRNA precursor containing the retained intron 25. In some embodiments, the ASO targets an exon 26 sequence about 2 to about 112 nucleotides downstream (or 3 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 25.

  In some embodiments, ASO targets intron 25 in a TSC2 RIC mRNA precursor containing retained intron 25, where the intron numbering corresponds to the mRNA sequence in NM_000548. In some embodiments, ASO targets an intron 25 sequence downstream (or 3 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 25. In some embodiments, the ASO targets an intron 25 sequence about 6 to about 497 nucleotides downstream (or 3 ') from the 5' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 25. In some embodiments, the ASO targets an intron 25 sequence upstream (or 5 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 25. In some embodiments, the ASO targets an intron 25 sequence about 16 to about 498 nucleotides upstream (or 5 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 25.

  In some embodiments, the ASO targets the TSC2 RIC mRNA precursor exon 26 or exon 27 containing the retained intron 26, where the intron numbering corresponds to the mRNA sequence in NM_000548. In some embodiments, the ASO targets an exon 26 sequence upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 26. In some embodiments, the ASO targets an exon 26 sequence from about 4 to about 111 nucleotides upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor containing a retained intron 26. In some embodiments, ASO targets the exon 27 sequence downstream (or 3 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 26. In some embodiments, the ASO targets an exon 27 sequence about 2 to about 147 nucleotides downstream (or 3 ') from the 3' splice site of the TSC2 RIC mRNA precursor containing the retained intron 26.

  In some embodiments, ASO targets intron 26 in a TSC2 RIC mRNA precursor that contains a retained intron 26, where the intron numbering corresponds to the mRNA sequence in NM_000548. In some embodiments, the ASO targets an intron 26 sequence downstream (or 3 ') from the 5' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 26. In some embodiments, the ASO targets an intron 26 sequence from about 6 to about 500 nucleotides downstream (or 3 ') from the 5' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 26. In some embodiments, the ASO targets an intron 26 sequence upstream (or 5 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 26. In some embodiments, the ASO targets an intron 26 sequence about 16 to about 496 nucleotides upstream (or 5 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 26.

  In some embodiments, ASO targets exon 4 or exon 5 of the TSC2 RIC mRNA precursor containing retained intron 4, where the intron numbering corresponds to the mRNA sequence in NM_000548. In some embodiments, the ASO targets an exon 4 sequence upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 4. In some embodiments, the ASO targets an exon 4 sequence from about 4 to about 94 nucleotides upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor containing a retained intron 4. In some embodiments, ASO targets an exon 5 sequence downstream (or 3 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 4. In some embodiments, ASO targets exon 5 sequences from about 2 to about 127 nucleotides downstream (or 3 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 4.

  In some embodiments, ASO targets intron 4 in a TSC2 RIC mRNA precursor containing retained intron 4, where the intron numbering corresponds to the mRNA sequence in NM_000548. In some embodiments, ASO targets an intron 4 sequence downstream (or 3 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 4. In some embodiments, the ASO targets an intron 4 sequence from about 6 to about 435 nucleotides downstream (or 3 ') from the 5' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 4. In some embodiments, the ASO targets an intron 4 sequence upstream (or 5 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 4. In some embodiments, the ASO targets an intron 4 sequence from about 16 to about 432 nucleotides upstream (or 5 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 4.

  In some embodiments, the ASO targets TSC2 RIC mRNA precursor exon 31 or exon 32 containing a retained intron 31, where the intron numbering corresponds to the mRNA sequence in NM_000548. In some embodiments, the ASO targets the exon 31 sequence upstream (or 5 ') from the 5' splice site of the TSC2 RIC mRNA precursor containing the retained intron 31. In some embodiments, the ASO targets an exon 31 sequence from about 4 to about 184 nucleotides upstream (or 5 ') from the 5' splice site of the TSC2 RIC mRNA precursor containing the retained intron 31. In some embodiments, ASO targets an exon 32 sequence downstream (or 3 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 31. In some embodiments, the ASO targets an exon 32 sequence about 2 to about 52 nucleotides downstream (or 3 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 31.

  In some embodiments, ASO targets intron 31 in a TSC2 RIC mRNA precursor that contains a retained intron 31, where the intron numbering corresponds to the mRNA sequence in NM_000548. In some embodiments, ASO targets an intron 31 sequence downstream (or 3 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 31. In some embodiments, the ASO targets an intron 31 sequence from about 6 to about 331 nucleotides downstream (or 3 ') from the 5' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 31. In some embodiments, the ASO targets an intron 31 sequence upstream (or 5 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 31. In some embodiments, the ASO targets an intron 31 sequence about 16 to about 333 nucleotides upstream (or 5 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 31.

  In some embodiments, the ASO targets the exon 25 or exon 26 of the TSC2 RIC mRNA precursor that contains the retained intron 25, where the intron numbering corresponds to the mRNA sequence in NM_001077183. In some embodiments, ASO targets an exon 25 sequence upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 25. In some embodiments, the ASO targets an exon 25 sequence from about 4 to about 74 nucleotides upstream (or 5 ') from the 5' splice site of the TSC2 RIC mRNA precursor containing the retained intron 25. In some embodiments, the ASO targets the exon 26 sequence downstream (or 3 ') from the 3' splice site of the TSC2 RIC mRNA precursor containing the retained intron 25. In some embodiments, the ASO targets an exon 26 sequence about 2 to about 142 nucleotides downstream (or 3 ') from the 3' splice site of the TSC2 RIC mRNA precursor containing the retained intron 25.

  In some embodiments, ASO targets intron 25 in a TSC2 RIC mRNA precursor that contains a retained intron 25, where the intron numbering corresponds to the mRNA sequence in NM_001077183. In some embodiments, ASO targets an intron 25 sequence downstream (or 3 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 25. In some embodiments, the ASO targets an intron 25 sequence about 6 to about 497 nucleotides downstream (or 3 ') from the 5' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 25. In some embodiments, the ASO targets an intron 25 sequence upstream (or 5 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 25. In some embodiments, the ASO targets an intron 25 sequence about 16 to about 499 nucleotides upstream (or 5 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 25.

  In some embodiments, ASO targets exon 4 or exon 5 of the TSC2 RIC mRNA precursor containing retained intron 4, where the intron numbering matches the mRNA sequence in NM_001077183. In some embodiments, the ASO targets an exon 4 sequence upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 4. In some embodiments, the ASO targets an exon 4 sequence from about 4 to about 94 nucleotides upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor containing a retained intron 4. In some embodiments, ASO targets an exon 5 sequence downstream (or 3 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 4. In some embodiments, ASO targets exon 5 sequences from about 2 to about 127 nucleotides downstream (or 3 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 4.

  In some embodiments, ASO targets intron 4 in a TSC2 RIC mRNA precursor that contains a retained intron 4, where the intron numbering corresponds to the mRNA sequence in NM_001077183. In some embodiments, ASO targets an intron 4 sequence downstream (or 3 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 4. In some embodiments, the ASO targets an intron 4 sequence from about 6 to about 435 nucleotides downstream (or 3 ') from the 5' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 4. In some embodiments, the ASO targets an intron 4 sequence upstream (or 5 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 4. In some embodiments, the ASO targets an intron 4 sequence from about 16 to about 432 nucleotides upstream (or 5 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 4.

  In some embodiments, the ASO targets the TSC2 RIC mRNA precursor exon 30 or exon 31 containing the retained intron 30, where the intron numbering matches the mRNA sequence in NM_001077183. In some embodiments, the ASO targets an exon 30 sequence upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor containing a retained intron 30. In some embodiments, the ASO targets an exon 30 sequence from about 4 to about 184 nucleotides upstream (or 5 ') from the 5' splice site of the TSC2 RIC mRNA precursor containing the retained intron 30. In some embodiments, the ASO targets an exon 31 sequence downstream (or 3 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 30. In some embodiments, the ASO targets an exon 31 sequence about 2 to about 102 nucleotides downstream (or 3 ') from the 3' splice site of the TSC2 RIC mRNA precursor containing the retained intron 30.

  In some embodiments, ASO targets intron 30 in a TSC2 RIC mRNA precursor that contains a retained intron 30, where the intron numbering corresponds to the mRNA sequence in NM_001077183. In some embodiments, the ASO targets an intron 30 sequence downstream (or 3 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 30. In some embodiments, the ASO targets an intron 30 sequence from about 6 to about 492 nucleotides downstream (or 3 ') from the 5' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 30. In some embodiments, the ASO targets an intron 30 sequence upstream (or 5 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 30. In some embodiments, the ASO is about 16 to about 500 or about 36 to about 500 nucleotides upstream (or 5 ′) intron 30 from the 3 ′ splice site of the TSC2 RIC mRNA precursor that contains the retained intron 30. Target the sequence.

  In some embodiments, the ASO targets the exon 25 or exon 26 of the TSC2 RIC mRNA precursor containing the retained intron 25, where the intron numbering corresponds to the mRNA sequence in NM_001114382. In some embodiments, ASO targets an exon 25 sequence upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 25. In some embodiments, the ASO targets an exon 25 sequence from about 4 to about 74 nucleotides upstream (or 5 ') from the 5' splice site of the TSC2 RIC mRNA precursor containing the retained intron 25. In some embodiments, the ASO targets the exon 26 sequence downstream (or 3 ') from the 3' splice site of the TSC2 RIC mRNA precursor containing the retained intron 25. In some embodiments, the ASO targets an exon 26 sequence about 2 to about 112 nucleotides downstream (or 3 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 25.

  In some embodiments, ASO targets intron 25 in a TSC2 RIC mRNA precursor that contains retained intron 25, where the intron numbering corresponds to the mRNA sequence in NM_001114382. In some embodiments, ASO targets an intron 25 sequence downstream (or 3 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 25. In some embodiments, the ASO targets an intron 25 sequence about 6 to about 497 nucleotides downstream (or 3 ') from the 5' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 25. In some embodiments, the ASO targets an intron 25 sequence upstream (or 5 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 25. In some embodiments, the ASO targets an intron 25 sequence about 16 to about 498 nucleotides upstream (or 5 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 25.

  In some embodiments, the ASO targets the TSC2 RIC mRNA precursor exon 26 or exon 27 that contains the retained intron 26, where the intron numbering corresponds to the mRNA sequence in NM_001114382. In some embodiments, the ASO targets an exon 26 sequence upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 26. In some embodiments, the ASO targets an exon 26 sequence from about 4 to about 111 nucleotides upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor containing a retained intron 26. In some embodiments, ASO targets the exon 27 sequence downstream (or 3 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 26. In some embodiments, the ASO targets an exon 27 sequence about 2 to about 147 nucleotides downstream (or 3 ') from the 3' splice site of the TSC2 RIC mRNA precursor containing the retained intron 26.

  In some embodiments, ASO targets intron 26 in a TSC2 RIC mRNA precursor that contains a retained intron 26, where the intron numbering corresponds to the mRNA sequence in NM_001114382. In some embodiments, the ASO targets an intron 26 sequence downstream (or 3 ') from the 5' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 26. In some embodiments, the ASO targets an intron 26 sequence from about 6 to about 500 nucleotides downstream (or 3 ') from the 5' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 26. In some embodiments, the ASO targets an intron 26 sequence upstream (or 5 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 26. In some embodiments, the ASO targets an intron 26 sequence about 16 to about 496 nucleotides upstream (or 5 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 26.

  In some embodiments, the ASO targets exon 4 or exon 5 of a TSC2 RIC mRNA precursor that contains a retained intron 4, where the intron numbering corresponds to the mRNA sequence in NM_001114382. In some embodiments, the ASO targets an exon 4 sequence upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 4. In some embodiments, the ASO targets an exon 4 sequence from about 4 to about 94 nucleotides upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor containing a retained intron 4. In some embodiments, ASO targets an exon 5 sequence downstream (or 3 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 4. In some embodiments, ASO targets exon 5 sequences from about 2 to about 127 nucleotides downstream (or 3 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 4.

  In some embodiments, ASO targets intron 4 in a TSC2 RIC mRNA precursor that contains retained intron 4, where the intron numbering corresponds to the mRNA sequence in NM_001114382. In some embodiments, ASO targets an intron 4 sequence downstream (or 3 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 4. In some embodiments, the ASO targets an intron 4 sequence from about 6 to about 435 nucleotides downstream (or 3 ') from the 5' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 4. In some embodiments, the ASO targets an intron 4 sequence upstream (or 5 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 4. In some embodiments, the ASO targets an intron 4 sequence from about 16 to about 432 nucleotides upstream (or 5 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 4.

  In some embodiments, the ASO targets the exon 31 or exon 32 of the TSC2 RIC mRNA precursor containing the retained intron 31, where the intron numbering corresponds to the mRNA sequence in NM_001114382. In some embodiments, the ASO targets the exon 31 sequence upstream (or 5 ') from the 5' splice site of the TSC2 RIC mRNA precursor containing the retained intron 31. In some embodiments, the ASO targets an exon 31 sequence from about 4 to about 184 nucleotides upstream (or 5 ') from the 5' splice site of the TSC2 RIC mRNA precursor containing the retained intron 31. In some embodiments, ASO targets an exon 32 sequence downstream (or 3 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 31. In some embodiments, the ASO targets an exon 32 sequence about 2 to about 102 nucleotides downstream (or 3 ') from the 3' splice site of the TSC2 RIC mRNA precursor containing the retained intron 31.

  In some embodiments, ASO targets intron 31 in a TSC2 RIC mRNA precursor that contains a retained intron 31, where the intron numbering corresponds to the mRNA sequence in NM_001114382. In some embodiments, ASO targets an intron 31 sequence downstream (or 3 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 31. In some embodiments, the ASO targets an intron 31 sequence about 6 to about 492 nucleotides downstream (or 3 ') from the 5' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 31. In some embodiments, the ASO targets an intron 31 sequence upstream (or 5 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 31. In some embodiments, the ASO is about 16 to about 500 or about 36 to about 500 nucleotides upstream (or 5 ′) intron 31 from the 3 ′ splice site of the TSC2 RIC mRNA precursor that contains the retained intron 31. Target the sequence.

  In some embodiments, the ASO targets TSC2 RIC mRNA precursor exon 27 or exon 28 containing a retained intron 27, where the intron numbering corresponds to the mRNA sequence in NM_001318831. In some embodiments, ASO targets an exon 27 sequence upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 27. In some embodiments, the ASO targets an exon 27 sequence about 4 to about 184 nucleotides upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 27. In some embodiments, ASO targets an exon 28 sequence downstream (or 3 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 27. In some embodiments, the ASO targets an exon 28 sequence about 2 to about 52 nucleotides downstream (or 3 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 27.

  In some embodiments, ASO targets intron 27 in a TSC2 RIC mRNA precursor that contains retained intron 27, where the intron numbering corresponds to the mRNA sequence in NM_001318831. In some embodiments, ASO targets an intron 27 sequence downstream (or 3 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 27. In some embodiments, the ASO targets an intron 27 sequence about 6 to about 331 nucleotides downstream (or 3 ') from the 5' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 27. In some embodiments, the ASO targets an intron 27 sequence upstream (or 5 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 27. In some embodiments, the ASO targets an intron 27 sequence about 16 to about 333 nucleotides upstream (or 5 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 27.

  In some embodiments, the ASO targets the TSC2 RIC mRNA precursor exon 28 or exon 29 containing the retained intron 28, where the intron numbering corresponds to the mRNA sequence in NM_001318831. In some embodiments, the ASO targets an exon 28 sequence upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 28. In some embodiments, the ASO targets an exon 28 sequence about 4 to about 49 nucleotides upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 28. In some embodiments, the ASO targets an exon 29 sequence downstream (or 3 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 28. In some embodiments, the ASO targets an exon 29 sequence about 2 to about 102 nucleotides downstream (or 3 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 28.

  In some embodiments, ASO targets intron 28 in a TSC2 RIC mRNA precursor that contains retained intron 28, where the intron numbering corresponds to the mRNA sequence in NM_001318831. In some embodiments, the ASO targets an intron 28 sequence downstream (or 3 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 28. In some embodiments, the ASO targets an intron 28 sequence about 6 to about 495 nucleotides downstream (or 3 ') from the 5' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 28. In some embodiments, the ASO targets an intron 28 sequence upstream (or 5 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 28. In some embodiments, the ASO has an intron 28 that is about 16 to about 500 or about 36 to about 500 nucleotides upstream (or 5 ′) from the 3 ′ splice site of a TSC2 RIC mRNA precursor that contains a retained intron 28. Target the sequence.

  In some embodiments, the ASO targets TSC2 RIC mRNA precursor exon 22 or exon 23 containing a retained intron 22, where the intron numbering corresponds to the mRNA sequence in NM_001318831. In some embodiments, the ASO targets the exon 22 sequence upstream (or 5 ') from the 5' splice site of the TSC2 RIC mRNA precursor containing the retained intron 22. In some embodiments, the ASO targets an exon 22 sequence from about 4 to about 74 nucleotides upstream (or 5 ') from the 5' splice site of the TSC2 RIC mRNA precursor containing the retained intron 22. In some embodiments, the ASO targets the exon 23 sequence downstream (or 3 ') from the 3' splice site of the TSC2 RIC mRNA precursor containing the retained intron 22. In some embodiments, the ASO targets an exon 23 sequence about 2 to about 142 nucleotides downstream (or 3 ') from the 3' splice site of the TSC2 RIC mRNA precursor containing the retained intron 22.

  In some embodiments, ASO targets intron 22 in a TSC2 RIC mRNA precursor that contains a retained intron 22, where the intron numbering corresponds to the mRNA sequence in NM_001318831. In some embodiments, the ASO targets an intron 22 sequence downstream (or 3 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 22. In some embodiments, the ASO targets an intron 22 sequence about 6 to about 497 nucleotides downstream (or 3 ') from the 5' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 22. In some embodiments, the ASO targets an intron 22 sequence upstream (or 5 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 22. In some embodiments, the ASO targets an intron 22 sequence about 16 to about 499 nucleotides upstream (or 5 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 22.

  In some embodiments, the ASO targets the exon 25 or exon 26 of the TSC2 RIC mRNA precursor containing the retained intron 25, where the intron numbering corresponds to the mRNA sequence in NM_001318832. In some embodiments, ASO targets an exon 25 sequence upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 25. In some embodiments, the ASO targets an exon 25 sequence from about 4 to about 74 nucleotides upstream (or 5 ') from the 5' splice site of the TSC2 RIC mRNA precursor containing the retained intron 25. In some embodiments, the ASO targets the exon 26 sequence downstream (or 3 ') from the 3' splice site of the TSC2 RIC mRNA precursor containing the retained intron 25. In some embodiments, the ASO targets an exon 26 sequence about 2 to about 142 nucleotides downstream (or 3 ') from the 3' splice site of the TSC2 RIC mRNA precursor containing the retained intron 25.

  In some embodiments, ASO targets intron 25 in a TSC2 RIC mRNA precursor containing retained intron 25, where the intron numbering corresponds to the mRNA sequence in NM_001318832. In some embodiments, ASO targets an intron 25 sequence downstream (or 3 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 25. In some embodiments, the ASO targets an intron 25 sequence about 6 to about 497 nucleotides downstream (or 3 ') from the 5' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 25. In some embodiments, the ASO targets an intron 25 sequence upstream (or 5 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 25. In some embodiments, the ASO targets an intron 25 sequence about 16 to about 499 nucleotides upstream (or 5 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 25.

  In some embodiments, ASO targets exon 4 or exon 5 of the TSC2 RIC mRNA precursor containing retained intron 4, where the intron numbering corresponds to the mRNA sequence in NM_001318832. In some embodiments, the ASO targets an exon 4 sequence upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 4. In some embodiments, the ASO targets an exon 4 sequence from about 4 to about 94 nucleotides upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor containing a retained intron 4. In some embodiments, ASO targets an exon 5 sequence downstream (or 3 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 4. In some embodiments, ASO targets exon 5 sequences from about 2 to about 127 nucleotides downstream (or 3 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 4.

  In some embodiments, ASO targets intron 4 in a TSC2 RIC mRNA precursor that contains retained intron 4, where the intron numbering corresponds to the mRNA sequence in NM_001318832. In some embodiments, ASO targets an intron 4 sequence downstream (or 3 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 4. In some embodiments, the ASO targets an intron 4 sequence from about 6 to about 435 nucleotides downstream (or 3 ') from the 5' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 4. In some embodiments, the ASO targets an intron 4 sequence upstream (or 5 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 4. In some embodiments, the ASO targets an intron 4 sequence from about 16 to about 432 nucleotides upstream (or 5 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 4.

  In some embodiments, the ASO targets the exon 30 or exon 31 of the TSC2 RIC mRNA precursor containing the retained intron 30, where the intron numbering corresponds to the mRNA sequence in NM_001318832. In some embodiments, the ASO targets an exon 30 sequence upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor containing a retained intron 30. In some embodiments, the ASO targets an exon 30 sequence from about 4 to about 184 nucleotides upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor comprising a retained intron 30. . In some embodiments, the ASO targets an exon 31 sequence downstream (or 3 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 30. In some embodiments, the ASO targets an exon 31 sequence from about 2 to about 102 nucleotides downstream (or 3 ') from the 3' splice site of a TSC2 RIC mRNA precursor comprising a retained intron 30. .

  In some embodiments, ASO targets intron 30 in a TSC2 RIC mRNA precursor that contains a retained intron 30, where the intron numbering corresponds to the mRNA sequence in NM_001318832. In some embodiments, the ASO targets an intron 30 sequence downstream (or 3 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 30. In some embodiments, the ASO targets an intron 30 sequence about 6 to about 492 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 30. . In some embodiments, the ASO targets an intron 30 sequence upstream (or 5 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 30. In some embodiments, the ASO is about 16 to about 500, or about 36 to about 500 nucleotides upstream (or 5 ′) from the 3 ′ splice site of a TSC2 RIC mRNA precursor comprising a retained intron 30. Target the intron 30 sequence.

  In some embodiments, the ASO targets the TSC2 RIC mRNA precursor exon 24 or exon 25 containing the retained intron 24, where the intron numbering corresponds to the mRNA sequence in NM_001318827. In some embodiments, the ASO targets the exon 24 sequence upstream (or 5 ') from the 5' splice site of the TSC2 RIC mRNA precursor containing the retained intron 24. In some embodiments, the ASO targets an exon 24 sequence about 4 to about 74 nucleotides upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor containing a retained intron 24. . In some embodiments, the ASO targets an exon 25 sequence downstream (or 3 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 24. In some embodiments, the ASO targets an exon 25 sequence about 2 to about 147 nucleotides downstream (or 3 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 24. .

  In some embodiments, ASO targets intron 24 in a TSC2 RIC mRNA precursor that contains a retained intron 24, where the intron numbering corresponds to the mRNA sequence in NM_001318827. In some embodiments, the ASO targets an intron 24 sequence downstream (or 3 ') from the 5' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 24. In some embodiments, the ASO targets an intron 24 sequence about 6 to about 497 nucleotides downstream (or 3 ') from the 5' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 24. . In some embodiments, the ASO targets an intron 24 sequence upstream (or 5 ') from the 3' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 24. In some embodiments, the ASO targets an intron 24 sequence about 16 to about 496 nucleotides upstream (or 5 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 24. .

  In some embodiments, the ASO targets TSC2 RIC mRNA precursor exon 3 or exon 4 containing a retained intron 3, where the intron numbering corresponds to the mRNA sequence in NM_001318827. In some embodiments, ASO targets an exon 3 sequence upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor containing a retained intron 3. In some embodiments, the ASO targets an exon 3 sequence about 4 to about 69 nucleotides upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 3. . In some embodiments, ASO targets an exon 4 sequence downstream (or 3 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 3. In some embodiments, the ASO targets an exon 4 sequence that is about 2 to about 127 nucleotides downstream (or 3 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 3. .

  In some embodiments, the ASO targets intron 3 in a TSC2 RIC mRNA precursor that contains a retained intron 3, where the intron numbering corresponds to the mRNA sequence in NM_001318827. In some embodiments, ASO targets an intron 3 sequence downstream (or 3 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 3. In some embodiments, the ASO targets an intron 3 sequence about 6 to about 499 nucleotides downstream (or 3 ′) from the 5 ′ splice site of a TSC2 RIC mRNA precursor that contains a retained intron 3. . In some embodiments, the ASO targets an intron 3 sequence upstream (or 5 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 3. In some embodiments, the ASO targets an intron 3 sequence about 16 to about 497 nucleotides upstream (or 5 ′) from the 3 ′ splice site of a TSC2 RIC mRNA precursor that contains a retained intron 3. .

  In some embodiments, the ASO targets the TSC2 RIC mRNA precursor exon 29 or exon 30 containing the retained intron 29, where the intron numbering corresponds to the mRNA sequence in NM_001318827. In some embodiments, the ASO targets an exon 29 sequence upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 29. In some embodiments, the ASO targets an exon 29 sequence about 4 to about 184 nucleotides upstream (or 5 ') from the 5' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 29. . In some embodiments, the ASO targets an exon 30 sequence downstream (or 3 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 29. In some embodiments, the ASO targets an exon 30 sequence from about 2 to about 102 nucleotides downstream (or 3 ') from the 3' splice site of a TSC2 RIC mRNA precursor that includes a retained intron 29. .

  In some embodiments, ASO targets intron 29 in a TSC2 RIC mRNA precursor that contains a retained intron 29, where the intron numbering corresponds to the mRNA sequence in NM_001318827. In some embodiments, the ASO targets an intron 29 sequence downstream (or 3 ') from the 5' splice site of the TSC2 RIC mRNA precursor that contains the retained intron 29. In some embodiments, the ASO targets an intron 29 sequence about 6 to about 492 nucleotides downstream (or 3 ′) from the 5 ′ splice site of the TSC2 RIC mRNA precursor that contains the retained intron 29. . In some embodiments, the ASO targets an intron 29 sequence upstream (or 5 ') from the 3' splice site of a TSC2 RIC mRNA precursor that contains a retained intron 29. In some embodiments, the ASO is about 16 to about 500, or about 36 to about 500 nucleotides upstream (or 5 ') from the 3' splice site of the TSC2 RIC mRNA precursor comprising the retained intron 29. Target the intron 29 sequence.

  The degree of intron retention can be expressed as percent intron retention (PIR), the percentage of transcript that a given intron is retained. Briefly, the PIR can be calculated as a percentage of the average number of reads that map to the exon-intron linkage relative to the sum of the exon-intron linkage readings and the average of the exon-exon linkage readings.

<Tuberin protein expression>
Over 69% of tuberous sclerosis cases result from haploinsufficiency of TSC2, and approximately two-thirds of patients' disease results from a de novo mutation or deletion (Au, K. et al. , J. Child Neurol., 2004, 19: 699-709).

  In embodiments, the methods described herein are used to increase production of functional tuberin protein. The term “functional” as used herein refers to the amount of tuberin protein activity or function required to eliminate one or more symptoms of tuberous sclerosis. In an embodiment, the method is used to increase production of partially functional tuberin protein. As used herein, the term “partial functional” refers to an activity or function of a tuberin protein that is less than the amount of activity or function required to eliminate or prevent one or more symptoms of the disease or condition. Refers to the quantity. In some embodiments, the partially functional protein or RNA is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60 relative to the fully functional protein or RNA. %, At least 70%, at least 75%, at least 80%, 85%, at least 90%, or at least 95% less active.

  In an embodiment, the method is a method of increasing the expression of tuberin protein by a cell of a subject having a RIC mRNA precursor encoding a tuberin protein, wherein the subject has an amount of tuberin protein activity. Having tuberous sclerosis due to deficiency and the deficiency in the amount of tuberin protein is due to haploinsufficiency of tuberin protein. In such embodiments, the subject has a first allele that encodes a functional tuberin protein and a second allele that does not produce a tuberin protein. In another such embodiment, the subject has a first allele that encodes a functional tuberin protein and a second allele that encodes a non-functional tuberin protein. In another such embodiment, the subject has a first allele that encodes a functional tuberin protein and a second allele that encodes a partially functional tuberin protein. In any of these embodiments, the antisense oligomer binds to the target portion of the RIC mRNA precursor transcribed from the first allele (encoding a functional tuberin protein), thereby causing RIC mRNA. It induces constitutive splicing of retained introns from the precursor, resulting in increased levels of mature mRNA encoding functional tuberin protein and increased expression of tuberin protein in the subject's cells.

  In an embodiment, the subject has a first allele that encodes a functional tuberin protein and a second allele that encodes a partially functional tuberin protein, wherein the antisense oligomer comprises a first allele The target portion of the RIC mRNA precursor transcribed from the allele (encoding a functional tuberin protein) or the RIC mRNA precursor transcribed from the second allele (encoding a partially functional tuberin protein) And thereby induces constitutive splicing of the retained intron from the RIC mRNA precursor, increasing the level of mature mRNA encoding the tuberin protein, and functional or This results in increased partial functional tuberin protein expression.

  In a related embodiment, the method is a method that uses ASO to increase the expression of a protein or functional RNA. In an embodiment, ASO is used to increase tuberin protein expression in cells of a subject having a RIC mRNA precursor encoding a tuberin protein, said subject being in an amount or function of tuberin protein. Having a deficiency, for example tuberous sclerosis.

  In embodiments, a transcript of a RIC mRNA precursor encoding a protein that is responsible for a disease or condition is targeted by ASO as described herein. In some embodiments, transcripts of RIC mRNA precursors encoding proteins that are not causative of the disease are targeted by ASO. For example, a disease caused by a mutation or deletion of a first protein in a particular pathway is improved by targeting a RIC mRNA precursor that encodes a second protein, thereby increasing production of the second protein. Yes. In some embodiments, the activity of the second protein can compensate for a mutation or deficiency in the first protein that is responsible for the disease or condition.

In embodiments, the subject is
a. A first mutant allele,
i) Tuberin protein is produced at a reduced level compared to production from the wild type allele,
ii) a first mutant allele in which the tuberin protein is produced in a reduced function compared to an equivalent wild-type protein, or iii) no tuberin protein or functional RNA is produced;
And b. A second mutant allele,
i) Tuberin protein is produced at a reduced level compared to production from the wild type allele,
ii) the tuberin protein is produced in a reduced function compared to an equivalent wild type protein, or iii) has a second mutant allele that does not produce the tuberin protein, wherein
The RIC mRNA precursor is transcribed from the first allele and / or the second allele. In these embodiments, the ASO binds to the target portion of the RIC mRNA precursor transcribed from the first or second allele, thereby constructing a retained intron from the RIC mRNA precursor. Splicing is induced, causing an increase in the level of mRNA encoding the tuberin protein, resulting in increased expression of the target protein or functional RNA in the subject's cells. In these embodiments, target proteins or functional RNAs with increased expression levels resulting from constitutive splicing of retained introns from RIC mRNA precursors are functional compared to equivalent wild-type proteins. Can be either of a low form (partially functional) or fully functional (fully functional) compared to an equivalent wild type protein.

  In embodiments, the level of mRNA encoding the tuberin protein is produced in a control cell, eg, not treated with an antisense oligomer or treated with an antisense oligomer that does not bind to the target portion of the TSC2 RIC mRNA precursor. When compared with the amount of mRNA encoding TSC2, it increases from 1.1 times to 10 times.

  In an embodiment, the disease resulting from a deficiency in the amount or activity of a tuberin protein is not a disease resulting from alternative splicing or aberrant splicing of a retained intron targeted to ASO. In an embodiment, the disease due to a lack of tuberin protein amount or activity is not a disease due to alternative or abnormal splicing of retained introns in the RIC mRNA precursor encoding the tuberin protein. In embodiments, alternative splicing or aberrant splicing can occur in an mRNA precursor transcribed from a gene. However, the compositions and methods of the present invention do not prevent or modify this alternative or abnormal splicing.

  In an embodiment, a subject treated using the methods of the present invention expresses a partially functional tuberin protein from one allele, the partially functional tuberin protein comprising a frameshift mutation, nonsense. Due to mutations, missense mutations, or partial gene deletions. In an embodiment, a subject treated using the methods of the invention expresses a non-functional tuberin protein from one allele, the non-functional tuberin protein being a frame in one allele. Caused by shift mutations, nonsense mutations, missense mutations, or partial gene deletions. In an embodiment, a subject treated using the methods of the invention has a TSC2 whole gene deletion in one allele.

  In embodiments, the subject has a TSC2 missense mutation selected from R611Q / W and R905W. In embodiments, the subject has a TSC2 deletion mutation of at least 6 amino acids in exon 38. In embodiments, the subject has a TSC2 substitution mutation of arginine 611 for tryptophan or glutamine. In embodiments, those known in the art and described in documents such as those previously referenced (Au, K. et al., J. Child Neurol., 2004, 19: 699-709), A subject with any TSC2 mutation is treated using the methods and compositions of the invention.

<Use of TANGO to increase tuberin protein expression>
As described above, in embodiments, targeted enhancement of nuclear gene output (TANGO) is used in the methods of the invention to increase the expression of tuberin protein. In these embodiments, the retained intron-containing mRNA precursor (RIC mRNA precursor) encoding the tuberin protein is in the nucleus of the cell. Cells having a retained intron encoding a tuberin protein, a TSC2 RIC mRNA precursor containing an exon adjacent to the 5 ′ splice site, and an exon adjacent to the 3 ′ splice site are included in the target portion of the RIC mRNA precursor. It is contacted with a complementary antisense oligomer (ASO). Hybridization of ASO to the target portion of the RIC mRNA precursor enhances splicing of the retained intron splice site (5 ′ splice site or 3 ′ splice site) and increases subsequent target protein production.

  The terms “mRNA precursor” and “mRNA precursor transcript” are used interchangeably and may refer to an mRNA precursor species comprising at least one intron. In embodiments, the mRNA precursor or mRNA precursor transcript comprises a 5'-7-methylguanosine cap, and / or a poly A terminus. In embodiments, the mRNA precursor or transcript of the mRNA precursor comprises both a 5'-7-methylguanosine cap and a poly A terminus. In some embodiments, the transcript of the mRNA precursor does not include a 5'-7-methylguanosine cap and / or a poly A terminus. When not translated into protein (or transported from the nucleus into the cytoplasm), the mRNA precursor transcript is a non-productive messenger RNA (mRNA) molecule.

  As used herein, a “retained intron-containing mRNA precursor” (“RIC mRNA precursor”) is a transcript of an mRNA precursor that contains at least one retained intron. The RIC mRNA precursor contains a retained intron, an exon adjacent to the 5 'splice site of the retained intron, an exon adjacent to the 3' splice site of the retained intron, and encodes the target protein. A “RIC mRNA precursor encoding a target protein” is understood to encode a target protein when fully spliced. A “retained intron” is an mRNA precursor transcript that is encoded by the same gene when one or more other introns, such as adjacent introns, are spliced out of the transcript of the same mRNA precursor. Is an intron. In some embodiments, the retained intron is the most abundant intron in the RIC mRNA precursor that encodes the target protein. In embodiments, the retained intron is the most abundant intron in the population of RIC mRNA precursors transcribed from the gene encoding the target protein in the cell, and the population of RIC mRNA precursors is more than one retained. Containing introns. In embodiments, antisense oligomers targeted to the most abundant introns in a population of RIC mRNA precursors encoding a target protein are within a population that contains retained introns to which the antisense oligomer is targeted or bound. Inducing splicing out of two or more retained introns. In embodiments, mature mRNA encoding the target protein is thereby produced. The terms “mature mRNA” and “fully spliced mRNA” refer to fully processed mRNA that encodes a target protein (eg, mRNA that is transported from the nucleus to the cytoplasm and translated into the target protein), or completely Are used interchangeably herein to describe the functional RNA processed. The term “productive mRNA” can also be used as described for a fully processed mRNA encoding the target protein. In embodiments, the target region is in a retained intron that is most abundant in the RIC mRNA precursor population encoding the tuberin protein. In embodiments, the most abundant retained intron in the RIC mRNA precursor encoding the tuberin protein is intron 4, 25, 26, 31, 32, 25 and 26, or 31 and 32. In an embodiment, the most abundant intron in the RIC mRNA precursor encoding the tuberin protein is intron 26. In an embodiment, the most abundant retained intron in the RIC mRNA precursor encoding the tuberin protein is intron 31. In some embodiments, the most abundant retained intron in the RIC mRNA precursor encoding tuberin protein is intron 26 and the second most abundant retained in the RIC mRNA precursor encoding tuberin protein. The resulting intron is intron 31.

  In embodiments, the retained intron is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, An intron identified as a retained intron based on a retention rate of retention of at least about 45%, or at least about 50%. In embodiments, the retained intron is 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% 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% 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 %, From about 25% to about 55%, from about 25% to about 50%, from about 25% to about 45%, from about 25% to about 40%, or from about 25% to about 35%, It is an intron identified as a retained intron.

  As used herein, the term “comprise” or variations such as “comprises” or “comprising” are used to describe the listed features (eg, for antisense oligomers). The defined nucleobase sequence), but should not be construed as excluding any other features. Thus, as used herein, the term “comprising” is inclusive and refers to additional unlisted features (eg, the presence of additional unlisted nucleobases in the case of antisense oligomers). ) Is not excluded.

  In any embodiment of the compositions and methods provided herein, “comprising” can be replaced with “consisting essentially” or “consisting of”. is there. The phrase “consisting essentially” is intended to require a feature that does not substantially affect the nature or function of the claimed invention, as well as the specified feature (eg, nucleobase sequence). Used in the description. As used herein, the term “consisting” is used to indicate the presence of a listed feature (eg, nucleobase sequence) alone (as a result of an antisense consisting of a specified nucleobase sequence). In the case of oligomers, the presence of additional unlisted nucleobases is excluded).

  In embodiments, the target region is in a retained intron that is the second most abundant intron in the RIC mRNA precursor encoding the tuberin protein. For example, the second most abundant retained intron can be targeted rather than the most abundant retained intron. The factors are the uniqueness of the nucleotide sequence of the second most abundant retained intron, the ease of ASO design targeting a specific nucleotide sequence, and / or the amount of protein produced from targeting the intron by ASO Is an increase. In embodiments, the retained intron is the second most abundant intron in the RIC mRNA precursor population transcribed from the gene encoding the target protein in the cell, and the RIC mRNA precursor population is more than one Contains retained introns. In an embodiment, an antisense oligomer that targets the second most abundant intron in a RIC mRNA precursor population encoding a target protein is in a population that contains a retained intron to which the antisense oligomer is targeted or bound. Induces splicing out of two or more retained introns. Thereby, in an embodiment, a fully spliced (mature) RNA encoding the target protein is produced. In embodiments, the second most retained intron in the RIC mRNA precursor encoding the tuberin protein is intron 4, 25, 26, 31, 32, 25 and 26, or 31 and 32. In some embodiments, the second most abundant intron in the RIC mRNA precursor encoding tuberin protein is intron 31.

  In an embodiment, the ASO is complementary to a target region that is within the unretained intron of the RIC mRNA precursor. In embodiments, the target portion of the RIC mRNA precursor is: within the region from +6 to +100 to the unretained intron 5 ′ splice site; or from −16 to −100 to the unretained intron 3 ′ splice site It is in. In embodiments, the target portion of the RIC mRNA precursor is in the region from +100 to the unretained intron 5 'splice site to -100 to the unretained intron 3' splice site. As used to identify a region or sequence location, “within” is understood to include residues at the recited positions. For example, the region from +6 to +100 includes residues at positions +6 and +100. Thereby, in an embodiment, a fully spliced (mature) RNA encoding the target protein is produced.

  In embodiments, the retained intron of the RIC mRNA precursor is an inefficiently spliced intron. As used herein, “inefficiently spliced” refers to a splice site adjacent to a retained intron (as compared to the frequency of splicing at another splice site in the RIC mRNA precursor. It may refer to a relatively low frequency of splicing at the 5 ′ splice site or 3 ′ splice site). The term “inefficiently spliced” can also refer to the relative rate or kinetics of splicing at the splice site. This “inefficiently spliced” intron can be spliced or removed at a slower rate compared to another intron in the RIC mRNA precursor.

  In an embodiment, the exon -3e to -1e adjacent to the 5 'splice site, and the +1 to +6 9-nucleotide sequence of the retained intron are identical to the corresponding wild-type sequence. In an embodiment, the 16 nucleotide sequence of exon + 1e adjacent to the -15 to -1 and 3 'splice sites of the retained intron is identical to the corresponding wild type sequence. As used herein, “wild type sequence” refers to the nucleotide sequence for the TSC2 gene in the published reference genome deposited in the NCBI repository of biological and scientific information. As used herein, “wild type sequence” refers to the standard sequence for the TSC2 gene found in NCBI Gene ID 7249. As further used herein, the nucleotide position indicated by “e” is a sequence in which the nucleotide is an exon (eg, an exon adjacent to a 5 ′ splice site, or an exon adjacent to a 3 ′ splice site). Is present.

  In the method, cells are contacted with ASO that is complementary to a portion of the mRNA precursor encoding the tuberin protein, resulting in increased expression of TSC2. As used herein, “contacting” or administering a cell refers to a method of bringing ASO into immediate proximity to the cell so that the cell interacts with ASO. Cells that are contacted with ASO take up or transport ASO into the cell. The method includes contacting a cell associated with or associated with a disease or disorder with any of the ASOs described herein. In some embodiments, ASO targets ASO to a cell type, enhances contact between ASO and a cell associated with or associated with a disease or disorder, or uptake of ASO Can be further modified or conjugated to another molecule (eg, covalently linked).

  As used herein, the term “increasing protein production” or “increasing target protein expression” means increasing the amount of protein translated from mRNA in a cell. A “target protein” can be a protein for which increased expression / production is desired.

  In an embodiment, the ASO that is complementary to the target site of the transcript of the TSC2 RIC mRNA precursor is contacted with a cell that expresses the TSC2 RIC mRNA precursor to produce a tuberin protein encoded by the mRNA precursor (eg, Resulting in a measurable increase in the amount of target protein). Methods for measuring or detecting protein production will be apparent to those of skill in the art and include any known method such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA.

  In an embodiment, contacting the cell with an ASO that is complementary to the target site of the TSC2 RIC mRNA transcript, as compared to the amount of protein produced by the cell in the absence / treatment of ASO, This results in an increase in the amount of produced tuberin protein of at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000%. In an embodiment, the total amount of tuberin protein produced by the cells contacted with the antisense oligomer is about 1.1 to about 10 times compared to the amount of target protein produced by the control compound, From about 1.5 times to about 10 times, from about 2 times to about 10 times, from about 3 times to about 10 times, from about 4 times to about 10 times, from about 1.1 times to about 5 times, from about 1.1 times About 6 times, about 1.1 times to about 7 times, about 1.1 times to about 8 times, about 1.1 times to about 9 times, about 2 times to about 5 times, about 2 times to about 6 times, About 2 times to about 7 times, about 2 times to about 8 times, about 2 times to about 9 times, about 3 times to about 6 times, about 3 times to about 7 times, about 3 times to about 8 times, about 3 times Times to about 9 times, about 4 times to about 7 times, about 4 times to about 8 times, about 4 times to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, less When About 2.5-fold, at least about 3-fold, at least about 3.5 fold, at least 4 fold, at least about 5-fold, or is increased at least about 10-fold. The control compound may be, for example, an oligonucleotide that is not complementary to the target site of the RIC mRNA precursor.

  In some embodiments, contacting the cell with ASO that is complementary to the target site of the TSC2 RIC mRNA precursor results in an increase in the amount of mRNA encoding TSC2, including mature mRNA encoding the target protein. . In some embodiments, the amount of mRNA encoding the tuberin protein, or mature mRNA encoding the tuberin protein is compared to the amount of protein produced by the cell in the absence of ASO / treatment. Increase by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000%. In an embodiment, the total amount of mRNA encoding the tuberin protein or mature mRNA encoding the tuberin protein produced in the cell contacted with the antisense oligomer is untreated cells, eg, untreated cells. Or about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times the amount of mature RNA produced in cells treated with the control compound. About 3 times to about 10 times, about 4 times to about 10 times, about 1.1 times to about 5 times, about 1.1 times to about 6 times, about 1.1 times to about 7 times, about 1. 1 to 8 times, 1.1 to 9 times, 2 to 5 times, 2 to 6 times, 2 to 7 times, 2 to 8 times, 2 to 9 times, 3 to 6 times, 3 to 7 times, 3 to 8 times, 3 to 9 times, 4 times About 7 times, about 4 times to about 8 times, about 4 times to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times Fold, at least about 3.5 times, at least 4 times, at least about 5 times, or at least about 10 times. The control compound may be, for example, an oligonucleotide that is not complementary to the target site of the TSC2 RIC mRNA precursor.

<Constitutive splicing of retained introns from RIC mRNA precursor>
The methods and antisense oligonucleotide compositions provided herein may comprise, for example, an mRNA encoding tuberin protein, or a tuberculosis in a subject having tuberous sclerosis resulting from a lack of tuberin protein amount or activity. It is useful to increase the expression of tuberin protein in the cell by increasing the level of mature mRNA encoding the verin protein. In particular, the methods and compositions as described herein induce constitutive splicing of retained introns from transcripts of TSC2 RIC mRNA precursors encoding tuberin protein, thereby producing tuberin. The level of mRNA encoding the protein or mature mRNA encoding the tuberin protein increases and the expression of the tuberin protein increases.

  Constitutive splicing of the retained intron from the RIC mRNA precursor correctly removes the retained intron from the RIC mRNA precursor. This retained intron has a wild type splice sequence. Constitutive splicing, as used herein, is RIC mRNA transcribed from a gene or allele having a mutation that causes alternative or aberrant splicing of the mRNA precursor transcribed from the gene or allele. Does not include splicing of retained introns from precursors. For example, constitutive splicing of retained introns induced using the methods and antisense oligonucleotides provided herein does not correct aberrant splicing in the mRNA precursor, or is selective for mRNA precursor. Splicing is not affected, resulting in increased expression of the target protein or functional RNA.

  In embodiments, constitutive splicing correctly removes retained introns from the TSC2 RIC mRNA precursor. This TSC2 RIC mRNA precursor is transcribed from a wild-type gene or allele, or a polymorphic gene or allele, and encodes a fully functional target protein or functional RNA. The gene or allele has no mutation that causes alternative or aberrant splicing of the retained intron.

  In some embodiments, constitutive splicing of the retained intron from the TSC2 RIC mRNA precursor encoding the TSC2 protein correctly removes the retained intron from the TSC2 RIC mRNA precursor encoding the tuberin protein. . The TSC2 RIC mRNA precursor is transcribed from a gene or allele that produces a target gene or functional RNA at a reduced level compared to production from a wild type allele. And the gene or allele has no mutation that causes alternative or abnormal splicing of the retained intron. In these embodiments, correct removal of the constitutively spliced retained intron results in the production of a target protein or functional RNA that is functional when compared to an equivalent wild type protein or functional RNA. .

  In other embodiments, constitutive splicing correctly removes retained introns from the TSC2 RIC mRNA precursor. This TSC2 RIC mRNA precursor is transcribed from a gene or allele that encodes a target protein or functional RNA produced in a reduced function compared to an equivalent wild type protein or functional RNA. And the gene or allele has no mutation that causes alternative or abnormal splicing of the retained intron. In these embodiments, correct removal of the constitutively spliced retained intron is partially functional when compared to a partially functional target protein, or equivalent wild-type protein or functional RNA. Resulting in the production of functional RNA that is functional.

  “Correct removal” of retained introns by constitutive splicing refers to removal of all introns without removing any part of the exon.

  In embodiments, an antisense oligomer as described herein or used in the methods described herein modulates alternative splicing or aberrant splicing of an mRNA precursor transcribed from the TSC2 gene. By doing so, the amount of mRNA encoding the tuberin protein or the amount of tuberin protein is not increased. Alternative splicing or aberrant splicing modulation can be performed using any known method for analyzing the sequence and length of RNA species, eg, by RT-PCR, and as described elsewhere in the specification and literature. Can be measured using. In embodiments, alternative splicing or aberrant splicing modulation is determined based on an increase or decrease in the amount of the species of interest spliced at least 10% or 1.1 fold. In embodiments, the modulation is at least 10% to 100% or 1.1 to 10 times as described herein in connection with determining the increase in mRNA encoding the tuberin protein in the methods of the invention. Determined based on an increase or decrease in double level.

  In an embodiment, the method is a method wherein the TSC2 RIC mRNA precursor was produced by partial splicing of the wild type TSC2 mRNA precursor. In an embodiment, the method is a method wherein the TSC2 RIC mRNA precursor was produced by partial splicing of the full-length wild type TSC2 mRNA precursor. In embodiments, the TSC2 RIC mRNA precursor was produced by partial splicing of the full-length TSC2 mRNA precursor. In these embodiments, the full-length TSC2 mRNA precursor is in a retained intron splice site that does not impair the correct splicing of the retained intron compared to the spliced intron retained with the wild-type splice site sequence. Can have polymorphism.

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

<Antisense oligomer>
One aspect of the present disclosure is a composition comprising an antisense oligomer that enhances splicing by binding to a targeted portion of a TSC2 RIC mRNA precursor. As used herein, the terms “ASO” and “antisense oligomer” are used interchangeably and refer to a target nucleic acid (eg, TSC2 RIC) by Watson-Crick base pair or wobble base pair (GU). mRNA precursor) refers to an oligomer, such as a polynucleotide, containing a nucleobase that hybridizes to a sequence. The ASO can have a precise sequence that is complementary or nearly complementary to the target sequence (eg, sufficient complementarity to bind to the target sequence and enhance splicing at the splice site). ASOs are designed to bind (hybridize) to a target nucleic acid (eg, a targeted portion of a transcript of an mRNA precursor) and remain hybridized under physiological conditions. Typically, ASO hybridizes to a limited number of sequences that are not the target nucleic acid (in a few sites other than the target nucleic acid) when hybridizing to sites other than the intended (targeted) nucleic acid sequence. The design of the ASO allows for the generation of a nucleic acid sequence of the targeted portion of the mRNA precursor transcript, or a sufficiently similar nucleic acid sequence elsewhere in the genomic or cellular mRNA precursor or transcriptome. As a result, the potential for ASO to bind to other sites and cause “off-target” effects is limited. Antisense oligomers known to those skilled in the art are described herein, such as, for example, in PCT International Application No. PCT / US2014 / 054151, published as WO2015 / 035091 of the title “Reducing Nonsense-Mediated mRNA Decay”. Can be used to implement the method.

  In some embodiments, the ASO “specifically hybridizes” or “specific” to the targeted portion of the target nucleic acid or RIC mRNA precursor. Typically, such hybridization occurs at a melting temperature (Tm) substantially higher than 37 ° C, preferably at least 50 ° C, and typically between 60 ° C and approximately 90 ° C. Such hybridization preferably corresponds to stringent hybridization conditions. For a given ionic strength and pH, Tm is the temperature at which 50% of the target sequence hybridizes to a complementary oligonucleotide.

  Oligomers, such as oligonucleotides, are “complementary” to each other when hybridization occurs in an antiparallel configuration between two single stranded polynucleotides. A double-stranded polynucleotide can be “complementary” to another polynucleotide when hybridization occurs between one of the strands of the first polynucleotide and one of the strands of the second polynucleotide. Complementarity (the degree to which one polynucleotide is complementary to another polynucleotide) is the proportion of bases in opposing strands (eg, percentages) that are expected to form hydrogen bonds with each other according to generally accepted base-pairing rules. It can be quantified from the point of. The sequence of the antisense oligomer (ASO) need not be 100% complementary to the sequence of its target nucleic acid to hybridize. In certain embodiments, the ASO is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, relative to the target site within the targeted target nucleic acid sequence, It can comprise at least 97%, at least 98%, or at least 99% sequence complementarity. For example, 18 of the 20 nucleobases of the oligomeric compound are complementary to the target site, thus specifically hybridizing ASO represents 90 percent complementarity. In this example, the remaining non-complementary nucleobases can be clustered together or interspersed with complementary nucleobases and need not be adjacent to each other or to complementary nucleobases. The percent complementarity of the target nucleic acid region of ASO is determined by the BLAST program (basic local alignment search tool) and the PowerBLAST program known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

  ASO does not need to hybridize to every nucleobase in the target sequence, and the nucleobase to which it hybridizes may or may not be adjacent. The ASO may hybridize over one or more segments of the mRNA precursor transcript so that intervening or adjacent segments are not involved in the hybridization event (eg, a loop structure or hairpin structure may be formed). ). In certain embodiments, the ASO hybridizes to non-contiguous nucleobases in the target mRNA precursor transcript. For example, ASO can hybridize to a nucleobase in a transcript of an mRNA precursor that is separated by one or more nucleobases to which ASO does not hybridize.

  The ASO described herein includes nucleobases that are complementary to nucleobases present in the target portion of the RIC mRNA precursor. The term “ASO” refers to oligonucleotides and other oligomeric molecules that contain nucleobases that can hybridize to complementary nucleobases on the target mRNA, but do not include sugar moieties such as peptide nucleic acids (PNA). Make it concrete. ASOs can include naturally occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination of two or three thereof. The term “naturally occurring nucleotides” includes deoxyribonucleotides and ribonucleotides. The term “modified nucleotide” includes a nucleotide having a modified or substituted saccharide and / or a modified backbone. In some embodiments, all of the ASO nucleotides are modified nucleotides. Chemical modifications of ASO and components of ASO that are compatible with the methods and compositions described herein will be apparent to those skilled in the art, eg, US Pat. No. 8,258,109 B2, US Pat. , 656,612, US Patent Publication No. 2012/0190728, and Diaz and Stein, Mol. Cancer Ther. 2002, 347-355, which are incorporated herein by reference in their entirety.

  The nucleobases of ASO are unmodified so that they can hydrogen bond with naturally occurring unmodified nucleobases such as adenine, guanine, cytosine, thymine and uracil, or with nucleobases present on the target mRNA precursor. It can be a synthetic or modified nucleobase that is sufficiently similar to the nucleobase. Examples of modified nucleobases include, but are not limited to, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine.

  The ASO described herein also includes a backbone structure that binds to the components of the oligomer. The terms “backbone structure” and “oligomer linkage” are used interchangeably and may refer to the linkage between monomers of ASO. In naturally occurring oligonucleotides, the backbone contains a 3'-5 'phosphodiester linkage that connects the sugar moieties of the oligomer. The backbone structures or oligomeric linkages of ASO described herein include (but are not limited to) phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilate. ), Phosphoramidate and the like. See, for example, LaPlanche et al. , Nucleic Acids Res. 14: 9081 (1986); Stec et al. , J .; Am. Chem. Soc. 106: 6077 (1984), Stein et al. , Nucleic Acids Res. 16: 3209 (1988), Zon et al. , Anti Cancer Drug Design 6: 539 1991; Zon et al. , Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90: 543 (1990). In some embodiments, the backbone structure of ASO does not contain phosphorous acid, but includes, for example, peptide nucleic acids (PNA), or carbamates, amides, and linear and cyclic hydrocarbon groups The linking group contains a peptide bond. In some embodiments, the backbone modification is a phosphorothioate linkage. In some embodiments, the backbone modification is a phosphoramidate linkage.

  In embodiments, the stereochemistry at each of the linkages between the phosphonucleotides of the ASO backbone is random. In embodiments, the stereochemistry at each of the bonds between the phosphonucleotides of the ASO backbone is controlled and not random. For example, US Patent Application Publication No. 2014/0194610, “Methods for the Synthesis of Functionalized Nucleic Acids”, which is incorporated herein by reference, independently determines the handedness of chirality at each phosphorus atom in a nucleic acid oligomer. The method of selecting is described. In embodiments, ASOs used in the methods of the invention, including but not limited to any of the ASOs specified in Table 1 herein, include ASOs that have non-random linkages between phosphonucleotides. In an embodiment, the composition used in the method of the invention comprises pure diastereomeric ASO. In embodiments, the composition used in the methods of the invention comprises 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% ASO having a diastereomeric purity of

  In embodiments, the ASO has a non-random mixture of Rp and Sp configurations at the linkage between its phosphonucleotides. For example, it has been suggested that a mixture of Rp and Sp is required for antisense oligonucleotides in order to achieve a good balance between activity and nuclease stability (Wan, et al., 2014 “Synthesis, biophysical properties”. and biologic activity of second generation antisense oligonucleotides containing chiral phosphorothioate links, ”Nucleic Acids Research 42 (134). In embodiments, the ASO used in the methods of the invention, including but not limited to any of the ASOs specified herein in Table 1, is 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 and the remaining sp, or about 100% Including the Rp. In embodiments, ASOs used in the methods of the invention, including but not limited to any of the ASOs specified herein in Table 1, are about 10% to about 100% Rp, about 15% 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% 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% 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% about 100% Rp, or about 95% to about 100% Rp, about 20% 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, and the rest Sp is included.

  In embodiments, the ASO used in the methods of the invention, including but not limited to any of the ASOs specified herein in Table 1, is 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 and the remaining Rp, or about 100% Including the Sp. In embodiments, the ASO used in the methods of the invention, including but not limited to any of the ASOs specified herein in Table 1, is from about 10% to about 100% Sp, from about 15%. 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% 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% 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% 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, and the rest Of Rp.

  Any of the ASOs described herein contain modified sugar moieties or sugar analogs that contain ribose or deoxyribose, or a morpholine ring, such as those present in naturally occurring nucleotides. obtain. Non-limiting examples of modified sugar moieties are 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'F N3 ′-> P5 ′ phosphoramidate, 2′dimethylaminooxyethoxy, 2′dimethylaminoethoxyethoxy, 2′-guanidinium, 2′-O-guanidinium ethyl, carbamate modified sugars; And 2 ′ substituents such as bicyclic modified sugars. In some embodiments, the sugar modification is selected from 2'-O-Me, 2'F, and 2'MOE. In some embodiments, the sugar modification is an additional crosslink as in locked nucleic acids (LNA). In some embodiments, the sugar analog contains a morpholine ring such as phosphorodiamidate morpholino (PMO). In some embodiments, the sugar moiety comprises a modification of ribofuranosyl or 2'deoxyribofuranosyl. In some embodiments, the sugar moiety comprises a 2'4'-constrained 2'O-methyloxyethyl (cMOE) modification. In some embodiments, the sugar moiety comprises a cEt 2'4'-inhibited 2'-O ethyl BNA modification. In some embodiments, the sugar moiety comprises a tricyclo DNA (tcDNA) modification. In some embodiments, the sugar moiety comprises an ethylene nucleic acid (ENA) modification. In some embodiments, the sugar moiety comprises an MCE modification. Modifications are known in the art and are described in the literature, for example, Jarver, et al. , 2014, “A Chemical View of Oligonucleotides for Exon Skipping and Related Drug Applications”, which is incorporated by reference in this specification, incorporated herein by reference, Nucleic Acid Therapeutics 24 (1): 37-47.

  In some examples, each monomer of ASO is modified in the same manner, for example, each bond of the backbone of ASO contains a phosphorothioate bond, or each ribose sugar moiety contains a 2'O-methyl modification. Such modifications that are present on each of the monomeric components of ASO are referred to as “homogeneous modifications”. In some examples, combinations of different modifications may be desired, for example, ASO may include a combination of a phosphorodiamidate linkage and a sugar moiety (morpholino) that contains a morpholine ring. The combination of different modifications to ASO is called “mixed modification” or “mixed chemistry”.

  In some embodiments, the ASO includes one or more backbone modifications. In some embodiments, the ASO includes one or more sugar modification. In some embodiments, the ASO includes one or more backbone modifications and one or more sugar moiety modifications. In some embodiments, the ASO comprises a 2'MOE modification and a phosphorothioate backbone. In some embodiments, the ASO comprises phosphorodiamidate morpholino (PMO). In some embodiments, the ASO comprises a peptide nucleic acid (PNA). Any of the ASOs or components of ASO described herein (eg, nucleobases, sugar moieties, backbones) to achieve the desired properties or activities of ASO or reduce the undesirable properties or activities of ASO May be modified for For example, ASO or one or more components of ASO enhances the binding affinity of the mRNA precursor to the target sequence on the transcript; reduces binding to non-target sequences; cellular nucleases (ie, RNase H To improve ASO uptake into cells and / or cell nuclei; alter pharmacokinetics or pharmacodynamics of ASO; and to modulate the half-life of ASO Can be modified.

  In some embodiments, the ASO is composed of 2'-O- (2-methoxyethyl) (MOE) phosphorothioate modified nucleotides. ASOs composed of such nucleotides are particularly well suited for the methods disclosed herein, and oligomers having such modifications have significantly enhanced resistance to nuclease degradation and increased It has been shown to have bioavailability, making it suitable for oral delivery, for example, in some embodiments described herein. For example, Gary et al. , J Pharmacol Exp Ther. 2001; 296 (3): 890-7; Geary et al. , J Pharmacol Exp Ther. 2001; 296 (3): 898-904.

  Methods for synthesizing ASO are known to those skilled in the art. Alternatively or additionally, ASO may be obtained from commercial sources.

  Unless specified otherwise, the left-hand end of single-stranded nucleic acid (eg, mRNA precursor transcripts, oligonucleotides, ASO, etc.) sequences is the 5 ′ end, and the left-hand direction of single- or double-stranded nucleic acid sequences Is called the 5 'direction. Similarly, the right end or right direction of a nucleic acid sequence (single stranded or double stranded) is the 3 'end or 3' direction. In general, a region or sequence 5 'to a reference point in a nucleic acid is referred to as "upstream" and a region or sequence 3' to the reference point in a nucleic acid is referred to as "downstream". In general, the 5 'direction or 5' end of an mRNA is where the initiation or start codon is located, while the 3 'end or 3' direction is where the stop codon is located. In some aspects, nucleotides upstream of a reference point in a nucleic acid can be designated by negative numbers and nucleotides downstream of the reference point can be designated by positive numbers. For example, a reference point (eg, exon-exon linkage in an mRNA) can be designated as a “zero” site, and nucleotides immediately adjacent to and upstream from the reference point are “minus 1”, eg, “−1”. A nucleotide that is designated, while immediately adjacent to and downstream of the reference point is designated “plus 1”, eg, “+1”.

  In other embodiments, the ASO is against the target portion of the TSC2 RIC mRNA precursor (eg, 5 ′ splice site) that is downstream (3 ′ direction) of the 5 ′ splice site of the intron retained in the TSC2 RIC mRNA precursor. In the direction indicated by a positive number) (and bind to the target moiety) (FIG. 1). In some embodiments, the ASO is a TSC2 RIC mRNA precursor that is within the region of +6 to +500, +6 to +400, +6 to +300, +6 to +200, or +6 to +100 to the 5 ′ splice site of the retained intron. Is complementary to the target portion of In some embodiments, the ASO is not complementary to the 5 'splice site from +1 to +5 nucleotides (the first 5 nucleotides located downstream of the 5' splice site). In some embodiments, the ASO may be complementary to the target portion of the TSC2 RIC mRNA precursor that is within the region of +6 to +50 nucleotides relative to the retained intron 5 'splice site. In some embodiments, the ASO is from +6 to +90, +6 to +80, +6 to +70, +6 to +60, +6 to +50, +6 to +40, +6 to +30, or +6 to the 5 ′ splice site of the retained intron. Complementary to the target portion within the +20 region.

  In some embodiments, the ASO is a target region (eg, negative number) of the TSC2 RIC mRNA precursor upstream (5 ′ direction) of the 3 ′ splice site of the intron retained in the TSC2 RIC mRNA precursor. Complementary to the specified direction (FIG. 1). In some embodiments, the ASO is from −16 to −500, −16 to −400, −16 to −300, −16 to −200, −16 to −100 to the retained intron 3 ′ splice site. Complementary to the target portion of the TSC2 RIC mRNA precursor within the region. In some embodiments, the ASO is not complementary to the 3 'splice site from -1 to -15 nucleotides (the first 15 nucleotides located upstream of the 3' splice site). In some embodiments, the ASO is complementary to the target portion of the TSC2 RIC mRNA precursor that is within the region from -16 to -50 to the retained intron 3 'splice site. In some embodiments, the ASO is from −16 to −90, −16 to −80, −16 to −70, −16 to −60, −16 to −50, − to the 3 ′ splice site of the retained intron. Complementary to the target moiety in the region of 16 to -40, or -16 to -30.

  In embodiments, the target portion of the TSC2 RIC mRNA precursor is in the region from +100 to the retained intron 5 'splice site to -100 to the retained intron 3' splice site.

  In some embodiments, the ASO is complementary to the target portion of the TSC2 RIC mRNA precursor located within (upstream) the exon adjacent to the retained intron 5 'splice site (Figure 1). In some embodiments, the ASO is complementary to the target portion of the TSC2 RIC mRNA precursor within the + 2e to -4e region in the exon adjacent to the retained intron 5 'splice site. In some embodiments, the ASO is not complementary to the -1e to -3e nucleotides for the retained intron 5 'splice site. In some embodiments, the ASO is -4e to -100e, -4e to -90e, -4e to -80e, -4e to -70e, -4e to -60e, relative to the retained intron 5 'splice site, It is complementary to the target portion of the TSC2 RIC mRNA precursor within the region of -4e to -50e, -4e to -40e, -4e to -30e, or -4e to -20e.

  In some embodiments, the ASO is complementary to the target portion of the TSC2 RIC mRNA precursor within the exon (downstream) adjacent to the retained intron 3 'splice site (Figure 1). In some embodiments, the ASO is complementary to the target portion of the TSC2 RIC mRNA precursor within the + 2e to -4e region in the exon adjacent to the retained intron 3 'splice site. In some embodiments, the ASO is not complementary to nucleotide + 1e to the retained intron 3 'splice site. In some embodiments, ASO is from + 2e to + 100e, + 2e to + 90e, + 2e to + 80e, + 2e to + 70e, + 2e to + 60e, + 2e to + 50e, + 2e to + 40e, + 2e to the 3 ′ splice site of the retained intron. It is complementary to the target portion of the TSC2 RIC mRNA precursor, which is within the region of + 30e, or + 2e to + 20e. The ASO may be of any length suitable for splicing specific binding and effective potentiation. In some embodiments, the ASO consists of 8 to 50 nucleobases. For example, ASO has a length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, It may be 29, 30, 31, 32, 33, 34, 35, 45 or 50 nucleobases. In some embodiments, the ASO consists of more than 50 nucleobases. In some embodiments, the ASO is 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases. Bases, 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 From 5 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 nucleic acids Bases, 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 Length of 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 It is. In some embodiments, the ASO is 30 nucleotides in length. In some embodiments, the ASO is 29 nucleotides in length. In some embodiments, the ASO is 28 nucleotides in length. In some embodiments, the ASO is 27 nucleotides in length. In some embodiments, the ASO is 26 nucleotides in length. In some embodiments, the ASO is 25 nucleotides in length. In some embodiments, the ASO is 24 nucleotides in length. In some embodiments, the ASO is 23 nucleotides in length. In some embodiments, the ASO is 22 nucleotides in length. In some embodiments, the ASO is 21 nucleotides in length. In some embodiments, the ASO is 20 nucleotides in length. In some embodiments, the ASO is 19 nucleotides in length. In some embodiments, the ASO is 18 nucleotides in length. In some embodiments, the ASO is 17 nucleotides in length. In some embodiments, the ASO is 16 nucleotides in length. In some embodiments, the ASO is 15 nucleotides in length. In some embodiments, the ASO is 14 nucleotides in length. In some embodiments, the ASO is 13 nucleotides in length. In some embodiments, the ASO is 12 nucleotides in length. In some embodiments, the ASO is 11 nucleotides in length. In some embodiments, the ASO is 10 nucleotides in length.

  In some embodiments, two or more ASOs with different chemistries but complementary to the same target portion of the RIC mRNA precursor are used. In some embodiments, two or more ASOs that are complementary to different target portions of the RIC mRNA precursor are used.

  In embodiments, the antisense oligonucleotides of the invention are chemically linked to one or more moieties or conjugates, eg, targeting moieties or other conjugates that enhance oligonucleotide activity or cellular uptake. . Such moieties include, but are not limited to, for example, cholesterol moieties, lipid moieties such as cholesteryl moieties, fatty chains such as dodecanediol or undecyl residues, polyamine chains or polyethylene glycol chains, or adamantane acetic acid. Oligonucleotides containing lipophilic moieties and methods of preparation are described in the published literature. In embodiments, antisense oligonucleotides include, but are not limited to, abasic nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates such as N-acetylgalactosamine (GalNAc), N-Ac-glucosamine (GulNAc), or mannose. (Eg, mannose-6-phosphate), lipid, or conjugated with a moiety that includes a polyhydrocarbon compound. The conjugate includes an antisense oligonucleotide at any of several positions on the sugar, base or phosphate group, as understood in the art and described in the literature, for example using a linker. It can be linked to one or more of the nucleotides. The linker can comprise a divalent or trivalent branched linker. In embodiments, the conjugate is attached to the 3 'end of the antisense oligonucleotide. Methods for preparing oligonucleotide conjugates are described, for example, in US Pat. No. 8,450,467 “Carbohydrate conjugates as delivery agents for oligonucleotides”, which is incorporated herein by reference.

  In some embodiments, the nucleic acid targeted by ASO is a TSC2 RIC mRNA precursor 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 present in the subject. In some embodiments, the cell is isolated from the subject. In some embodiments, the cell is ex vivo. In some embodiments, the cell is a disease or disease-related cell or cell line. In some embodiments, the cell is in vitro (eg, in a cell culture medium).

<Pharmaceutical composition>
Pharmaceutical compositions or formulations for use in any of the described methods comprising antisense oligonucleotides of the described compositions are in accordance with conventional techniques well known in the pharmaceutical industry and described in published literature. Can be prepared. In embodiments, a pharmaceutical composition or formulation for treating a subject comprises an effective amount of an antisense oligomer, as described above, or a pharmaceutically acceptable salt, solvate, hydrate, or ester thereof. And a pharmaceutically acceptable diluent. The antisense oligomer of the pharmaceutical formulation can further comprise a pharmaceutically acceptable excipient, diluent or carrier.

  Pharmaceutically acceptable salts are suitable for use in contact with human and lower animal tissues that are free of excessive toxicity, irritation, allergic reactions, etc. and correspond to a reasonable benefit / risk ratio ( See, for example, S. M. Berge, et al., J. Pharmaceutical Sciences, 66: 1-19 (1977), which is incorporated herein by reference for this purpose). The salts can be prepared in situ during the final separation and purification of the compound or alternatively by reacting the free base functionality with a suitable organic acid. Examples of pharmaceutically acceptable non-toxic acid addition salts include inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, An organic acid such as succinic acid or malonic acid, or a salt of an amino group formed by using other documented methods such as ion exchange. Other pharmaceutically acceptable salts are adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, Camphor sulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethane sulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate , Heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate , Methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinic acid , Persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartaric acid, thiocyanate, p-toluenesulfonic acid Salt, undecanoate, valerate, etc. Typical alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In addition, pharmaceutically acceptable salts may have counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates where appropriate. Includes non-toxic ammonium, quaternary ammonium, and amine cations formed using.

  In embodiments, the composition is formulated into any of a number of possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. In embodiments, the composition is formulated as a suspension in an aqueous, non-aqueous, or mixed medium. The aqueous suspension may further comprise certain 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, the pharmaceutical formulation or composition of the present invention includes, but is not limited to, a solution, emulsion, microemulsion, foam or liposome-containing formulation (eg, cationic liposomes or non-cationic liposomes).

  The pharmaceutical composition or formulation of the present invention may contain one or more penetration enhancers, carriers, excipients, or other active or non-active agents as needed and described in the literature well known or published to those skilled in the art. It may contain active ingredients. In embodiments, liposomes also include sterically stable liposomes, eg, liposomes that include one or more specific lipids. These specific lipids consequently increase the circulation life of the liposomes. In embodiments, sterically stable liposomes contain one or more glycolipids or are derivatized with one or more lipophilic polymers, such as polyethylene glycol (PEG) moieties. In embodiments, the surfactant is included in a pharmaceutical formulation or composition. The use of surfactants in drug products, formulations and emulsions is well known in the art. In embodiments, the present invention utilizes penetration enhancers to achieve efficient delivery of antisense oligonucleotides, eg, to aid diffusion across cell membranes and / or enhance the permeability of lipophilic drugs. In embodiments, the penetration enhancer is a surfactant, fatty acid, bile salt, chelator, or non-chelating non-surfactant.

  In embodiments, the pharmaceutical formulation comprises a plurality of 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 that can facilitate penetration of the subject antisense oligonucleotide across the blood brain barrier by methods known in the art. For example, the delivery of drugs by administration of adenoviral vectors to motor neurons in muscle tissue is described in US Pat. No. 6,632,427 “Adenoviral-vector-generated gene transfer into motorized neurones”, which is incorporated by reference. Incorporated herein. Delivery of vectors directly to the brain, eg, striatum, thalamus, hippocampus, or substantia nigra, is described, for example, in US Pat. “system partly in brain”, which is incorporated herein by reference.

  In embodiments, the antisense oligonucleotide is linked or conjugated with an agent that provides the desired pharmaceutical or pharmacodynamic properties. In embodiments, antisense oligonucleotides are conjugated to substances known in the art, such as antibodies to transferrin receptors, to facilitate penetration or transport across the blood brain barrier. In embodiments, the antisense oligonucleotide is linked to a viral vector, eg, to make the antisense composition more effective or to increase transport across the blood brain barrier. In embodiments, the disruption of the permeable blood brain barrier is a sugar such as meso-erythritol, xylitol, D (+) galactose, D (+) lactose, D (+) xylose, dulcitol, myo-inositol, L (−. ) Fructose, D (-) mannitol, D (+) glucose, D (+) arabinose, D (-) arabinose, cellobiose, D (+) maltose, D (+) raffinose, L (+) rhamnose, D (+) ) Melibiose, D (-) ribose, adonitol, D (+) arabitol, L (-) arabitol, D (+) fucose, L (-) fucose, D (-) lyxose, L (+) lyxose, and L ( -) Lyxose or amino acids such as glutamine, lysine, arginine, asparagine, aspartic acid, cysteine Glutamic acid, glycine, histidine, leucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, assisted by injection of valine, and taurine. Methods and materials for enhancing blood brain barrier penetration are described, for example, in US Pat. No. 4,866,042, “Method for the delivery of genetic material the brain brain”, US Pat. No. 6,294,520. “Material for passage through the blood-brain barrier” and US Pat. No. 6,936,589 “Parental delivery systems”, each of which is incorporated herein by reference.

  In embodiments, the antisense oligonucleotides of the invention are chemically linked to one or more moieties or conjugates, eg, targeting moieties or other conjugates that enhance oligonucleotide activity or cellular uptake. . Such moieties include, but are not limited to, for example, cholesterol moieties, lipid moieties such as cholesteryl moieties, fatty chains such as dodecanediol or undecyl residues, polyamine chains or polyethylene glycol chains, or adamantane acetic acid. Oligonucleotides containing lipophilic moieties and methods of preparation are described in the published literature. In other embodiments, antisense oligonucleotides include, but are not limited to, abasic nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates such as N-acetylgalactosamine (GalNAc), N-Ac-glucosamine (GulNAc), Or conjugated with a moiety comprising mannose (eg, mannose-6-phosphate), lipid, or polyhydrocarbon compound. The conjugate includes an antisense oligonucleotide at any of several positions on the sugar, base or phosphate group, as understood in the art and described in the literature, for example using a linker. It can be linked to one or more of the nucleotides. The linker can comprise a divalent or trivalent branched linker. In embodiments, the conjugate is attached to the 3 'end of the antisense oligonucleotide. Methods for preparing oligonucleotides are described, for example, in US Pat. No. 8,450,467 “Carbohydrate conjugates as delivery agents for oligonucleotides”, which is incorporated herein by reference.

<Treatment of subject>
Any of the compositions provided herein can be administered to an individual. “Individual” may be used interchangeably with “subject” or “patient”. The individual may be a mammal, such as a human or non-human primate, rodent, rabbit, rat, mouse, horse, donkey, goat, cat, dog, cow, pig, or sheep. Good. In embodiments, the individual is a human. In embodiments, the individual is a fetus, embryo, or child. In other embodiments, the individual may be another eukaryote such as a plant. In some embodiments, the compounds provided herein are administered to cells ex vivo.

  In some embodiments, the compositions provided herein are administered to an individual as a method of treating a disease or disorder. In some embodiments, the individual has a genetic disease, such as any of the diseases described herein. In some embodiments, the individual is at risk of having a disease, such as any of the diseases described herein. In some embodiments, the individual is at increased risk of having a disease or disorder caused by a lack of protein or a lack of protein activity. Where an individual is at increased risk of having a disease or disorder caused by a lack of protein or a lack of protein activity, the method includes preventive or prophylactic treatment. For example, an individual may have an increased risk of having such a disease or disorder due to a family history of the disease. Typically, individuals with an increased risk of having such a disease or disorder benefit from prophylactic treatment (eg, by preventing or delaying the onset or progression of the disease or disorder).

  The appropriate route for administration of the ASO of the invention may vary depending on the cell type for which delivery of ASO is desired. Several tissues and organs are affected by tuberous sclerosis, and the brain, kidney, skin, lung and heart are the most severely affected tissues. The ASO of the present invention can be administered topically to the patient's skin or to the lung by pulmonary delivery. The ASO of the present invention may be administered parenterally to a patient, for example, by intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. In embodiments, delivery to the brain, kidney, skin, lung or heart is performed. In embodiments, the fetus is treated in utero, for example, by administering the ASO composition directly or indirectly to the fetus (eg, via the mother).

<Method of identifying additional ASOs that enhance splicing>
Also within the scope of the present invention are methods of identifying (determining) additional ASOs that enhance splicing of the TSC2 RIC mRNA precursor, particularly in the target intron. ASOs that specifically hybridize various nucleotides within the target region of the mRNA precursor 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 splicing inhibitor / silencer binding site. Methods known in the art to identify (determine) ASOs that result in desirable effects (eg, splicing, enhanced protein or functional RNA production) when hybridized to a target region of an intron May be used. These methods can also be used to identify ASOs that enhance splicing of retained introns by binding to target regions in exons adjacent to retained introns or in unretained introns. it can. An example of a method that can be used is provided below.

  A round of screening, called an ASO “walk”, can be performed using ASO that is designed to hybridize to the target site of the mRNA precursor. For example, the ASO used for the ASO walk is approximately 100 nucleotides upstream of the 5 ′ splice site of the retained intron (eg, part of the sequence of the exon located upstream of the targeted / retained intron). To / from approximately 100 nucleotides downstream of the 5 'splice site of the targeted / retained intron and / or from approximately 100 nucleotides upstream of the 3' splice site of the retained intron. Tiled every 5 nucleotides to approximately 100 nucleotides downstream of the 3 ′ splice site of the intron (eg, part of the sequence of the exon located downstream of the targeted / retained intron) obtain. For example, a first ASO of 15 nucleotides in length can be designed to specifically hybridize from nucleotide +6 to +20 to the 5 'splice site of the targeted / retained intron. The second ASO is designed to hybridize specifically from nucleotide +11 to +25 to the 5 'splice site of the targeted / retained intron. ASO is designed to span the target region of the mRNA precursor. In embodiments, ASO can be more densely laid down, eg, every 1, 2, 3, or 4 nucleotides. Furthermore, ASO can be laid from 100 nucleotides downstream of the 5 'splice site to 100 nucleotides upstream of the 3' splice site.

  One or more ASOs, or one control ASO (ASO with a scrambled sequence, which is a sequence not expected to hybridize to the target site) is a target mRNA precursor (eg, as described herein), eg, by transfection. To a disease-related cell line that expresses a RIC mRNA precursor as described elsewhere in (1). The effect of each ASO on splicing is assessed by methods known in the art, eg, reverse transcriptase (RT) -PCR using primers spanning splice junctions, as described herein. (See “Identifying Intron Retention Events”). Reduced or lack of RT-PCR products generated using primers spanning splice junctions in ASO-treated cells compared to control ASO-treated cells enhanced target intron splicing It is shown that. In some embodiments, splicing efficiency, ratio of spliced mRNA precursor to unspliced mRNA precursor, rate of splicing, or degree of splicing is improved using ASO as described herein. Can be done. The amount of protein or functional RNA encoded by the target mRNA precursor can also be evaluated to determine whether each ASO has achieved the desired effect (eg, enhanced protein production). Methods known in the art for assessing and / or quantifying protein production, such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA can also be used.

  A second round of screening, called ASO “micro-walk”, can be performed using ASO designed to hybridize to the target site of the mRNA precursor. The ASO used in the ASO microwalk is laid out one nucleotide at a time to further refine the nucleotide acid sequence of the mRNA precursor that, when hybridized with ASO, results in enhanced splicing.

  Regions defined by ASO that facilitate splicing of target introns include longer ASOs, typically 18-25 nt, in addition to ASOs spaced in 1-nt steps , More detailed by the ASO “Microwalk”.

  As described above for ASO walks, ASO microwalks can include one or more ASOs, or a control ASO (an ASO with a scrambled sequence that is a sequence that is not expected to hybridize to a target site), eg, Performed by transfection by delivering to a disease-related cell line that expresses the target mRNA precursor. The effect of each ASO on splicing is assessed by methods known in the art, eg, reverse transcriptase (RT) -PCR using primers spanning splice junctions, as described herein. (See “Identifying Intron Retention Events”). Reduced or lack of RT-PCR products generated using primers spanning splice junctions in ASO-treated cells compared to control ASO-treated cells enhanced target intron splicing It is shown that. In some embodiments, splicing efficiency, ratio of spliced mRNA precursor to unspliced mRNA precursor, rate of splicing, or degree of splicing is improved using ASO as described herein. Can be done. The amount of protein or functional RNA encoded by the target mRNA precursor can also be assessed to determine whether each ASO has achieved the desired effect (eg, enhanced protein production). Methods known in the art for assessing and / or quantifying protein production, such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA can also be used.

  ASO, which when hybridized to a region of the mRNA precursor, results in enhanced splicing and increased protein production is an animal model, eg, a transgenic mouse model in which a full-length human gene is knocked in or humanized disease It can be tested in vivo using a mouse model. Suitable routes for administration of ASO may vary depending on the disease and / or cell type for which delivery of ASO is desired. ASO can be administered, for example, by topical application to the skin, pulmonary delivery to the lung, intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. After administration, the cells, tissues, and / or organs of the model animal are evaluated, for example, for splicing (efficiency, speed, degree) and protein production by methods known in the art and described herein. And can be evaluated to determine the effect of ASO treatment. An animal model can also be a phenotype or behavioral sign of disease or disease severity.

  While preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many modifications, changes and substitutions will 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 can be utilized in the practice of the invention. The following claims are intended to define the scope of the invention, and the methods and structures within the scope of these claims and their equivalents are intended to be encompassed thereby.

  The invention is more specifically illustrated by the following examples. However, it should be understood that the invention is not limited in any way by these examples.

Example 1: Identification of intron retention events of TSC2 transcripts by RNAseq using next generation sequencing Next generation sequencing to reveal a snapshot of transcripts produced by the TSC2 gene to identify intron retention events The determination was used to perform shotgun sequencing of the entire transcriptome. To this end, polyA + RNA from the nuclear and cytoplasmic fractions of HCN (human cortical neurons), renal epithelial cells, bronchial epithelial cells, and THLE-3 (human hepatic epithelial) cells were isolated and Illumina TruSeq Stranded mRNA. Library A cDNA library was constructed using Prep Kit. The library was pair-ended sequence and, as a result, 100 nucleotide readings were mapped to the human genome (February 2009, GRCh37 / hg19 assembly). The results of sequencing for TSC2 are shown in FIG. Briefly, FIG. 3 (UCSC Genome Informatics Group (Center for Biomolecular Science, Engineering, CA, et al., H 2015, “The UCSC Genome Browser database: 2015 update” Nucleic Acids Research 43, Database Issue, doi: 10.1093 / nar / gku1177 read and mapped using the genome browser of UCSC The coverage and number of readings can be inferred by the peak signal. The peak height indicates the level of expression given by the density of readings in a particular area. A schematic of TSC2 (drawn to scale) is provided by the UCSC genome browser (under the reading signal) so that the peaks are matched to the TSC2 exon and intron regions. Based on this representation, HCN (as shown for intron 4 in the lower diagram of FIG. 3, for introns 25 and 26 in the lower diagram of FIG. 5 and for introns 31 and 32 in the lower diagram of FIG. 7). 5 introns (4, 25 indicated by arrows) that have a high reading density in the nuclear fraction, but have very low or no readings in the cytoplasmic fraction of these cells. , 26, 31, and 32). This indicates that these introns are retained and that transcripts containing intron-4, intron-25, intron-26, intron-31, and intron-32 remain in the cell nucleus. This suggests that these retained TSC2 RIC mRNA precursors are non-productive because they are not transported into the cytoplasm.

Example 2: ASO-walk design targeting intron 4 of TSC2 An ASO walk was designed to target intron 4 using the methods described herein (Figure 4; Table 2). . The region immediately downstream of the 5 ′ splice site of intron 4 spanning nucleotides +6 to +58 and the region immediately upstream of the 3 ′ splice site of intron 4 spanning nucleotides −16 to −68 are expressed as 2′-O-Me RNA, PS Targeting with the backbone, 18-mer ASO was shifted by 5-nucleotide intervals.

Example 3: ASO-walk design targeting introns 25 and 26 of TSC2 An ASO walk was designed to target introns 25 and 26 using the methods described herein (Figure 6). Table 2). The region immediately downstream of the 5 ′ splice site of intron 25 spanning nucleotides +6 to +58 and the region immediately upstream of the 3 ′ splice site of intron 26 spanning nucleotides −16 to −68 are expressed as 2′-O-Me RNA, PS Targeting with the backbone, 18-mer ASO was shifted by 5-nucleotide intervals. The intron region of the splice site adjacent to the alternative exon 26 is not targeted to avoid affecting the inclusion level of exon 26.

Example 4: ASO-walk design targeting introns 31 and 32 of TSC2 An ASO walk was designed to target introns 31 and 32 using the methods described herein (Figure 8). Table 2). The region immediately downstream of the 5 ′ splice site of intron 31 spanning nucleotides +6 to +58 and the region immediately upstream of the 3 ′ splice site of intron 32 spanning nucleotides −18 to −68 are expressed as 2′-O-Me RNA, PS Targeted with the backbone, 18-mer ASO was shifted by 5-nucleotide intervals (with the exception of two ASOs, TSC2-IVS32-33 and TSC2-IVS32-51). The intron region of the splice site adjacent to the alternative exon 32 is not targeted to avoid affecting the inclusion level of exon 32.

Example 5: Improved splicing efficiency through ASO targets of TSC2 introns 4, 25, 26, 31 or 32 increases transcript levels. Using ASO, increased TSC2 expression is associated with TSC2 introns 4, 25, To determine whether it could be achieved by improving the splicing efficiency of 26, 31, or 32, RT-PCR products were evaluated using radioactive RT-PCR and RT-qPCR. ARPE-19 cell (American Type Culture Collection (ATCC), USA), which is a human retinal epithelial cell line, or Huh-7 (NIBIOHN, Japan), which is a human hepatocellular carcinoma cell line, or a human neuroblastoma cell line SK-N-AS (ATCC), which was mock transfected or transfected with the target ASO described in FIGS. 10, 12, 15 and Table 1-2. Cells were transfected with Lipofectamine RNAiMax transfection reagent (Thermo Fisher) according to the supplier's specifications. Briefly, ASO was plated in 96 well tissue culture plates and combined with RNAiMax diluted in Opti-MEM. Cells were detached with trypsin, resuspended in complete medium, and approximately 25,000 cells were added to the ASO transfection mixture. Transfection experiments were performed in triplicate plate replications. The final ASO concentration was 80 nM. According to the supplier's specifications, the medium was changed 6 hours after transfection and the cells were harvested at 24 hours using Cells-to-Ct RT reagent supplemented with DNAse (Thermo Fisher). CDNA was generated with Cells-to-Ct RT reagent (Thermo Fisher) according to the supplier's specifications. To quantify the amount of splicing at the intron of interest, quantitative PCR was performed using the Taqman assay with probes spanning the corresponding exon-exon linkages (Thermo Fisher) listed in Table 1-2. . The Taqman assay was performed on a QuantStudio 7Flex Real-Time PCR system (Thermo Fisher) according to the supplier's specifications. Target gene assay values are normalized to RPL32 (deltaCt) and plate-matched mock transfection samples (delta-delta Ct), resulting in a fold change over pseudo-quantification (2 ^-(delta-delta Ct)). The average fold change over the mock of replication was plotted (Figures 10, 12 and 15) In Figures 10, 12 and 15, some ASOs were identified as increasing target gene expression, This suggests increased splicing at each target intron.

Claims (101)

A method for treating tuberous sclerosis in a subject by increasing the expression of a target protein or functional RNA by the cells of the subject, comprising:
Here, the cell has a retained intron-containing mRNA precursor (RIC mRNA precursor), and the RIC mRNA precursor is a retained intron, an exon adjacent to a 5 ′ splice site, a 3 ′ splice. Including an exon adjacent to the site, the RIC mRNA precursor encodes a target protein or functional RNA;
The method
Contacting the subject's cells with an antisense oligomer (ASO) complementary to the target portion of a RIC mRNA precursor encoding a target protein or functional RNA, whereby the retained intron is Or constitutively spliced from a RIC mRNA precursor encoding functional RNA, thereby increasing the level of mRNA encoding the target protein or functional RNA in the subject's cells, and the target protein or functional RNA A method of increasing the expression of A method of increasing expression of a target protein that is tuberin by a cell having a retained intron-containing mRNA precursor (RIC mRNA precursor) comprising:
The RIC mRNA precursor comprises a retained intron, an exon adjacent to the retained intron 5 ′ splice site, an exon adjacent to the retained 3 ′ splice site of the retained intron, and the RIC mRNA precursor is Encoding the tuberin protein,
The method
Contacting the cell with an antisense oligomer (ASO) complementary to the target portion of the RIC mRNA precursor encoding the tuberin protein, whereby the retained intron is the RIC mRNA encoding the tuberin protein. A method that is constitutively spliced from a precursor, thereby increasing the level of mRNA encoding the tuberin protein and increasing the expression of the tuberin protein in the cell.   The method of claim 1, wherein the target protein is tuberin.   The target protein or functional RNA is a compensation protein or functional compensation RNA that functionally increases or replaces a target protein or functional RNA that is deficient in amount or activity in a subject. Item 2. The method according to Item 1.   3. The method of claim 2, wherein the cell is in or derived from a subject having a disease caused by a deficiency in the amount or activity of tuberin protein.   The deficiency in the amount of target protein is caused by haploinsufficiency of the target protein, where the subject has a first allele that encodes a functional target protein, a second allele that does not produce a target protein, or 6. Any of claims 1-5, having a second allele encoding a non-functional target protein, wherein the antisense oligomer binds to the target portion of the RIC mRNA precursor transcribed from the first allele. The method according to any one of the above. The subject has a disease caused by a disorder resulting from a deficiency in the amount or function of the target protein,
Where the subject is
a.
i. The target protein is produced at a reduced level compared to production from the wild type allele,
ii. The target protein is produced in a reduced function compared to an equivalent wild type protein, or
iii. No target protein is produced,
A first mutant allele;
b.
i. The target protein is produced at a reduced level compared to production from the wild type allele,
ii. The target protein is produced in a reduced function compared to an equivalent wild type protein, or
iii. No target protein is produced,
A second mutant allele,
Wherein the subject has the first mutant allele a. iii. The second mutant allele is b. i. Or b. ii. Where the subject has a second mutant allele b. iii. The first mutant allele is a. i. Or a. ii. Where the RIC mRNA precursor is a. i. Or a. ii. A first mutant allele, and / or b. i. Or b. ii. 6. The method of any one of claims 1-5, wherein the method is transcribed from a second mutant allele that is   8. The method of claim 7, wherein the target protein is produced in a reduced function compared to an equivalent wild type protein.   8. The method of claim 7, wherein the target protein is produced in a fully functional form compared to an equivalent wild type protein.   The target portion of the RIC mRNA precursor is in the retained intron within the region from +6 to the retained intron 5 ′ splice site to −16 to the retained intron 3 ′ splice site, 10. A method according to any one of claims 1-9.   The target portion of the RIC mRNA precursor is in the retained intron in the region from +500 to the retained intron 5 ′ splice site to −500 to the retained intron 3 ′ splice site, 10. A method according to any one of claims 1-9. The target portion of the RIC mRNA precursor is
(A) a region from +6 to +500, +6 to +495, or +6 to +100, relative to the retained intron 5 ′ splice site, or
(B) a region of −16 to −500, −16 to −400, or −16 to −100, relative to the retained 3 ′ splice site of the intron,
10. The method according to any one of claims 1-9, wherein the method is in a retained intron inside. The target portion of the RIC mRNA precursor is
(A) a region from + 2e to −4e in the exon adjacent to the 5 ′ splice site of the retained intron, or
(B) a region from + 2e to -4e in the exon adjacent to the 3 ′ splice site of the retained intron,
10. The method according to any one of claims 1-9, wherein the method is within.   The RIC mRNA precursor has a gene sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 14. A method according to any one of claims 1-13, encoded by:   RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% relative to any one of SEQ ID NOs: 2-8 15. The method of any one of claims 1-14, comprising a sequence with the sequence identity of:   16. The antisense oligomer of claim 1-15, wherein the antisense oligomer does not increase the amount of target protein or functional RNA by modulating alternative splicing of an mRNA precursor transcribed from a gene encoding functional RNA or target protein. The method according to any one of the above.   16. The antisense oligomer of any of claims 1-15, wherein the antisense oligomer does not increase the amount of target protein or functional RNA by modulating aberrant splicing due to mutation of a gene encoding the target protein or functional RNA. The method according to one.   18. The method of any one of claims 1-17, wherein the RIC mRNA precursor was generated by partial splicing of a full-length mRNA precursor or partial splicing of a wild-type mRNA precursor.   The method according to any one of claims 1 to 18, wherein the mRNA encoding the target protein or functional RNA is a full-length mature mRNA or a wild-type mature mRNA.   The method according to any one of claims 1 to 19, wherein the generated target protein is a full-length protein or a wild-type protein.   The total amount of mRNA encoding the target protein or functional RNA produced in the cells contacted with the antisense oligomer is approximately compared to the total amount of mRNA encoding the target protein or functional RNA produced in the control cells. 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1. 1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about About 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, less Least about 3.5 times, at least about 4-fold, at least about 5-fold, or increased by at least about 10-fold, The method according to any one of claims 1-20.   The total amount of target protein produced by the cells contacted with the antisense oligomer is about 1.1 to about 10 times, about 1.5 to about 10 times compared to the total amount of target protein produced by the control cells. About 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 Times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times At least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or At least about 1 Fold increase, method according to any one of claims 1-21.   23. The method of any one of claims 1-22, wherein the antisense oligomer comprises a backbone modification that includes a phosphorothioate linkage or a phosphorodiamidate linkage.   24. The antisense oligomer of any one of claims 1-23, wherein the antisense oligomer comprises a phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety. The method described in 1.   25. A method according to any one of claims 1-24, wherein the antisense oligomer comprises at least one modified sugar moiety.   26. The method of claim 25, wherein each sugar moiety is a modified sugar moiety.   Antisense oligomers are 8-50 nucleobases, 8-40 nucleobases, 8-35 nucleobases, 8-30 nucleobases, 8-25 nucleobases, 8-20 nucleobases, 8-15 Nucleobases, 9-50 nucleobases, 9-40 nucleobases, 9-35 nucleobases, 9-30 nucleobases, 9-25 nucleobases, 9-20 nucleobases, 9-15 Nucleobase, 10-50 nucleobase, 10-40 nucleobase, 10-35 nucleobase, 10-30 nucleobase, 10-25 nucleobase, 10-20 nucleobase, 10-15 nucleic acid Base, 11-50 nucleobase, 11-40 nucleobase, 11-35 nucleobase, 11-30 nucleobase, 11-25 nucleobase, 11-20 nucleobase, 11-15 nucleobase , 12-50 nucleobases, 12-40 nucleobases, 12-3 Nucleobase, a nucleobase of 12 to 30, nucleic acid bases 12-25, consisting of a nucleic acid base or 12-15 nucleobases, 12 to 20, the method according to any one of claims 1-26.   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 target portion of the protein-encoding RIC mRNA precursor The method according to any one of Items 1-27.   29. The method of any one of claims 1-28, wherein the target portion of the RIC mRNA precursor is within a sequence selected from SEQ ID NO: 5097-5105.   The antisense oligomer is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 for any one of SEQ ID NOs: 9-5096. 30. The method of any one of claims 1-29, comprising a nucleotide sequence that is%, 98%, or 99% sequence identity.   30. The method of any one of claims 1-29, wherein the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NOs: 9-5096.   The cell comprises a population of RIC mRNA precursors transcribed from a gene encoding a target protein or functional RNA, wherein the population of RIC mRNA precursors comprises two or more retained introns, and wherein 32. The method of any one of claims 1-31, wherein the antisense oligomer binds to the most abundant retained intron in the population of RIC mRNA precursors.   Binding of antisense oligomers to the most abundant retained introns triggers splicing out of two or more retained introns from a population of RIC mRNA precursors that generate mRNAs that encode target proteins or functional RNAs 35. The method of claim 32.   The cell comprises a population of RIC mRNA precursors transcribed from a gene encoding a target protein or functional RNA, wherein the population of RIC mRNA precursors comprises two or more retained introns, and wherein 32. The method of any one of claims 1-31, wherein the antisense oligomer binds to the second most abundant retained intron in a population of RIC mRNA precursors.   Antisense oligomer binding to the second most abundant retained intron results in the splicing out of two or more retained introns from a population of RIC mRNA precursors that generate mRNA encoding the target protein or functional RNA. 35. The method of claim 34, wherein the method is triggered.   36. The method of any one of claims 5-35, wherein the disease is a disease or disorder.   40. The method of claim 36, wherein the disease or disorder is tuberous sclerosis.   38. The method of claim 37, wherein the target protein and RIC mRNA precursor are encoded by the TSC2 gene.   40. The method of any one of claims 1-38, wherein the method further comprises assessing TSC2 protein expression.   Antisense oligomers bind to the target portion of the tuberine RIC mRNA precursor, where the target portion is within a sequence selected from SEQ ID NOs: 49, 50, 51, 52, 53, 54, 55, 56, and 57. 40. A method according to any one of claims 1-39.   41. The method of any one of claims 1-40, wherein the subject is a human.   41. The method of any one of claims 1-40, wherein the subject is a non-human animal.   42. The method according to any one of claims 1-41, wherein the subject is a fetus, embryo, or child.   43. The method of any one of claims 1-42, wherein the cell is ex vivo.   The antisense oligomer is administered by topical administration to the skin of the subject, pulmonary delivery to the lung, intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. 43. A method according to any one of claims 1-42.   46. The exon -3e to -1e adjacent to the 5 'splice site and the nine nucleotides +1 to +6 of the retained intron are identical to the corresponding wild type sequence. The method described in one.   47. The 16 nucleotides in + 1e of exon adjacent to the conserved intron -15 to -1 and 3 'splice sites are identical to the corresponding wild type sequence. the method of.   40. An antisense oligomer used in the method of any one of claims 1-39.   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: 9-5096.   50. A pharmaceutical composition comprising the antisense oligomer of claim 48 or 49 and an excipient.   51. The pharmaceutical composition of claim 50 administered by topical administration to the skin, pulmonary delivery to the lung, intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. A method of treating a subject by: Antisense oligomers for use in a method of increasing expression of a target protein or functional RNA by a cell to treat tuberous sclerosis in a subject associated with the deficient protein or deficient functional RNA A composition comprising:
Here, the deficient protein or functional RNA is deficient in amount or activity in the subject, wherein the antisense oligomer is a retained intron-containing mRNA that encodes the target protein or functional RNA. Enhance constitutive splicing of the precursor (RIC mRNA precursor);
Here, the target protein is
(A) missing protein, or
(B) a compensating protein that functionally increases or replaces a deficient protein in a subject;
Here, functional RNA is
(A) missing RNA, or
(B) functional RNA that functionally enhances or replaces functional RNA that is deficient in the subject, and
Here, the RIC mRNA precursor comprises a retained intron, an exon adjacent to the 5 ′ splice site, and an exon adjacent to the 3 ′ splice site, the retained intron being a target protein or functional A composition that is spliced from a RIC mRNA precursor encoding RNA, thereby increasing the production or activity of a target protein or functional RNA in a subject. A composition comprising an antisense oligomer for use in a method of treating a disease associated with tuberin protein in a subject comprising:
The method increases the expression of tuberin protein by a subject's cells, wherein the cell retains an intron, an exon adjacent to the retained intron 5 ′ splice site, and a retained intron. Having a retained intron-containing mRNA precursor (RIC mRNA precursor) containing an exon adjacent to the 3 ′ splice site of RIC mRNA precursor, wherein the RIC mRNA precursor encodes a tuberin protein; Contacting the sense oligomer, whereby the retained intron is constitutively spliced from the transcript of the RIC mRNA precursor encoding the tuberin protein, thereby allowing the tube to grow in the subject's cells. Increases the level of mRNA encoding verin protein, Increase expression of quality, comprising the step, composition.   54. The composition of claim 53, wherein the disease is a disease or disorder.   55. The composition of claim 54, wherein the disease or disorder is tuberous sclerosis.   56. The composition of claim 55, wherein the target protein and RIC mRNA precursor are encoded by the TSC2 gene.   Antisense oligomers are those of the RIC mRNA precursor in the retained intron in the region from +6 to the retained intron 5 ′ splice site to −16 to the retained intron 3 ′ splice site. 57. The composition of any one of claims 52-56, wherein the composition targets a portion. Antisense oligomers target a portion of the RIC mRNA precursor,
(A) a region from +6 to +100 to the 5 ′ splice site of the retained intron,
(B) a region from -16 to -100 to the retained intron 3 'splice site;
58. The composition of any one of claims 52-57, wherein the composition is in a retained intron inside.   Antisense oligomers are about 500, 400, 300, 200, or 100 nucleotides downstream of the 5 ′ splice site of at least one retained intron and about 100, 200 of the 3 ′ splice site of at least one retained intron. 57. The composition of any one of claims 52-56, wherein the composition targets a portion of a RIC mRNA precursor within a region up to 300, 400, or 500 nucleotides upstream. The target portion of the RIC mRNA precursor is
(A) a region from + 2e to −4e in the exon adjacent to the 5 ′ splice site of the retained intron, or
(B) a region from + 2e to -4e in the exon adjacent to the 3 ′ splice site of the retained intron,
57. The composition of any one of claims 52-56, wherein   The RIC mRNA precursor has a gene sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 61. A composition according to any one of claims 52-60, encoded by:   RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% relative to any one of SEQ ID NOs: 2-8 62. The composition of any one of claims 52-61, comprising a sequence with the sequence identity of:   63. Any of claims 52-62, wherein the antisense oligomer does not increase the amount of target protein or functional RNA by modulating alternative splicing of the mRNA precursor transcribed from the gene encoding the target protein or functional RNA. The composition according to any one of the above.   63. Any of claims 52-62, wherein the antisense oligomer does not increase the amount of functional RNA or functional protein by modulating aberrant splicing due to mutations in a gene encoding the target protein or functional RNA The composition according to any one of the above.   66. The composition of any one of claims 52-64, wherein the RIC mRNA precursor was generated by partial splicing from a full-length mRNA precursor or a wild-type mRNA precursor.   66. The composition of any one of claims 52-65, wherein the mRNA encoding the target protein or functional RNA is a full length mature mRNA or a wild type mature mRNA.   67. The composition of any one of claims 52-66, wherein the generated target protein is a full length protein or a wild type protein.   68. The composition of any one of claims 52-67, wherein the retained intron is a rate limiting intron.   69. The composition of any one of claims 52-68, wherein the retained intron is the most abundant retained intron in the RIC mRNA precursor.   69. The composition of any one of claims 52-68, wherein a retained intron is a retained intron that is the second most abundant in the RIC mRNA precursor.   71. The composition of any one of claims 52-70, wherein the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.   72. The composition of any one of claims 52-71, wherein the antisense oligomer is an antisense oligonucleotide.   73. Any one of claims 52-72, wherein the antisense oligomer comprises a phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety. A composition according to 1.   74. The composition of any one of claims 52-73, wherein the antisense oligomer comprises at least one modified sugar moiety.   75. The composition of claim 74, wherein each sugar moiety is a modified sugar moiety.   Antisense oligomers are 8-50 nucleobases, 8-40 nucleobases, 8-35 nucleobases, 8-30 nucleobases, 8-25 nucleobases, 8-20 nucleobases, 8-15 Nucleobases, 9-50 nucleobases, 9-40 nucleobases, 9-35 nucleobases, 9-30 nucleobases, 9-25 nucleobases, 9-20 nucleobases, 9-15 Nucleobase, 10-50 nucleobase, 10-40 nucleobase, 10-35 nucleobase, 10-30 nucleobase, 10-25 nucleobase, 10-20 nucleobase, 10-15 nucleic acid Base, 11-50 nucleobase, 11-40 nucleobase, 11-35 nucleobase, 11-30 nucleobase, 11-25 nucleobase, 11-20 nucleobase, 11-15 nucleobase , 12-50 nucleobases, 12-40 nucleobases, 12-3 76. The composition of any one of claims 52-75, consisting of: 12-30 nucleobases, 12-25 nucleobases, 12-20 nucleobases, or 12-15 nucleobases. .   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 target portion of the protein-encoding RIC mRNA precursor Item 76. The composition according to any one of Items 52-76.   78. The antisense oligomer binds to a target portion of a tuberine RIC mRNA precursor, wherein the target portion is within a sequence selected from SEQ ID NO: 5097-5105. The composition as described.   The antisense oligomer is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 for any one of SEQ ID NOs: 9-5096. 79. The composition of any one of claims 52-78, comprising a nucleotide sequence that is%, 98%, or 99% sequence identity.   79. The composition of any one of claims 52-78, wherein the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NOs: 9-5096.   81. A pharmaceutical composition comprising the antisense oligomer of any of the compositions of claims 52-80 and an excipient.   82. Administration of the pharmaceutical composition of claim 81 by topical administration to the skin, pulmonary delivery to the lung, intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. A method of treating a subject by: An antisense oligomer that hybridizes to a target sequence of a missing TSC2 mRNA transcript, comprising an intron that retains the missing TSC2 mRNA transcript, and lacking an antisense oligomer An antisense oligomer that induces splicing out of the retained intron from the transcript of
A pharmaceutically acceptable excipient,
Pharmaceutical composition.   84. The pharmaceutical composition of claim 83, wherein the missing TSC2 mRNA transcript is a TSC2 RIC mRNA precursor transcript.   The target portion of the TSC2 RIC mRNA precursor transcript was retained in the region from +500 to the retained intron 5 ′ splice site to −500 to the retained intron 3 ′ splice site. 85. A pharmaceutical composition according to claim 83 or 84, which is in an intron.   TSC2 RIC mRNA precursor transcript is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identical to SEQ ID NO: 1 The pharmaceutical composition according to any one of claims 83 to 85, which is encoded by a gene sequence having sex.   The TSC2 RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 for any one of SEQ ID NOs: 2-8. 87. The pharmaceutical composition according to any one of claims 83-86, comprising a sequence with% sequence identity.   88. The pharmaceutical composition according to any one of claims 83-87, wherein the antisense oligomer comprises a backbone modification comprising a phosphorothioate bond or a phosphorodiamidate bond.   89. The pharmaceutical composition according to any one of claims 83-88, wherein the antisense oligomer is an antisense oligonucleotide.   90. The antisense oligomer of any one of claims 83-89, wherein the antisense oligomer comprises a phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety. A pharmaceutical composition according to 1.   95. The pharmaceutical composition according to any one of claims 83-90, wherein the antisense oligomer comprises at least one modified sugar moiety.   Antisense oligomers are 8-50 nucleobases, 8-40 nucleobases, 8-35 nucleobases, 8-30 nucleobases, 8-25 nucleobases, 8-20 nucleobases, 8-15 Nucleobases, 9-50 nucleobases, 9-40 nucleobases, 9-35 nucleobases, 9-30 nucleobases, 9-25 nucleobases, 9-20 nucleobases, 9-15 Nucleobase, 10-50 nucleobase, 10-40 nucleobase, 10-35 nucleobase, 10-30 nucleobase, 10-25 nucleobase, 10-20 nucleobase, 10-15 nucleic acid Base, 11-50 nucleobase, 11-40 nucleobase, 11-35 nucleobase, 11-30 nucleobase, 11-25 nucleobase, 11-20 nucleobase, 11-15 nucleobase , 12-50 nucleobases, 12-40 nucleobases, 12-3 92. The pharmaceutical composition of any one of claims 83-91, comprising: 12-30 nucleobases, 12-25 nucleobases, 12-20 nucleobases, or 12-15 nucleobases. object.   Antisense oligomers are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to the target portion of the TSC2 RIC mRNA precursor transcript. 94. The pharmaceutical composition according to any one of claims 83-92.   94. The pharmaceutical composition according to any one of claims 83-93, wherein the target portion of the TSC2 RIC mRNA precursor transcript is within a sequence selected from SEQ ID NO: 5097-5105.   The antisense oligomer is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 for any one of SEQ ID NOs: 9-5096. 95. The pharmaceutical composition of any one of claims 83-94, comprising a nucleotide sequence that is%, 98%, or 99% sequence identity.   95. The pharmaceutical composition according to any one of claims 83 to 94, wherein the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NO: 9-5096.   96. The medicament of any one of claims 83-95, wherein the pharmaceutical composition is formulated for intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. Composition. In a method that induces the processing of missing TSC2 mRNA transcripts to facilitate removal of retained introns to generate fully processed TSC2 mRNA transcripts that encode a functional form of the tuberin protein. There,
The method
(A) contacting an antisense oligomer with a target cell of a subject;
(B) a step of hybridizing to an antisense oligomer-deficient TSC2 mRNA transcript, wherein the deficient TSC2 mRNA transcript can encode a functional form of tuberin protein, Including one retained intron;
(C) removing at least one retained intron from a TSC2 mRNA transcript that is deficient to produce a fully processed TSC2 mRNA transcript that encodes a functional form of the tuberin protein;
(D) translating the functional form of the tuberin protein from a fully processed TSC2 mRNA transcript.   99. The method of claim 98, wherein the retained intron is the entire retained intron.   100. The method of claim 98 or 99, wherein the missing TSC2 mRNA transcript is a TSC2 RIC mRNA precursor transcript. A method of treating a subject having a disease caused by a deficiency in the amount or activity of tuberin protein comprising:
The method
SEQ ID NO: At least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% for any one of 9-5096 Or administering to the subject an antisense oligomer comprising a nucleotide sequence having 99% sequence identity.