EP3390635A1 - Antisense-oligomere zur behandlung eines tuberösen sklerosekomplexes - Google Patents

Antisense-oligomere zur behandlung eines tuberösen sklerosekomplexes

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Publication number
EP3390635A1
EP3390635A1 EP16876615.2A EP16876615A EP3390635A1 EP 3390635 A1 EP3390635 A1 EP 3390635A1 EP 16876615 A EP16876615 A EP 16876615A EP 3390635 A1 EP3390635 A1 EP 3390635A1
Authority
EP
European Patent Office
Prior art keywords
mrna
nucleobases
tsc2
intron
ric pre
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP16876615.2A
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English (en)
French (fr)
Other versions
EP3390635A4 (de
Inventor
Isabel AZNAREZ
Huw M. Nash
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cold Spring Harbor Laboratory
Stoke Therapeutics Inc
Original Assignee
Cold Spring Harbor Laboratory
Stoke Therapeutics Inc
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Filing date
Publication date
Application filed by Cold Spring Harbor Laboratory, Stoke Therapeutics Inc filed Critical Cold Spring Harbor Laboratory
Publication of EP3390635A1 publication Critical patent/EP3390635A1/de
Publication of EP3390635A4 publication Critical patent/EP3390635A4/de
Pending legal-status Critical Current

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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • Tuberous sclerosis complex is a disorder characterized by growth of benign tumors in multiple organ systems (Au, K., et al., J. Child Neurol., 2004, 19: 699-709).
  • Tumors of the central nervous system (CNS) are the leading cause of morbidity and mortality, followed by renal disease.
  • Patients can suffer from abnormalities of the brain that may include seizures, intellectual disability, and developmental delay, as well as abnormalities of the skin, lung, kidneys, and heart.
  • the disorder affects as many as 25,000 to 40,000 individuals in the United States and about 1 to 2 million individuals worldwide, with an estimated prevalence of one in 6,000 newborns.
  • TSC is a genetic disorder with an autosomal dominant inheritance pattern, caused by inherited defects or de novo mutations that occur on two genes, TSC1 and TSC2. Only one of the genes needs to be affected for TSC to be present.
  • 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.
  • mTOR evolutionarily conserved kinase
  • the invention provides compositions and methods for treating tuberous sclerosis complex, including antisense oligomers (ASOs) that promote constitutive splicing at intron splice sites of a TSC 2 retained-intron-containing pre-mRNA (RIC pre-mRNA).
  • ASOs antisense oligomers
  • RIC pre-mRNA TSC 2 retained-intron-containing pre-mRNA
  • the invention further provides compositions and methods for increasing production of mature TSC2 mRNA and, in turn, TSC2 protein, in cells of a subject in need thereof, for example, a subject that can benefit from increased production of TSC2 protein.
  • the described methods may be used to treat subjects having tuberous sclerosis complex caused by mutations in the TSC2 gene, including missense, splicing, frameshift and nonsense mutations, as well as whole gene deletions, that result in deficient tuberin protein production.
  • a method of increasing expression of a target protein wherein the target protein is tuberin, by cells having a retained-intron-containing pre-mRNA (RIC pre-mRNA), the RIC pre-mRNA comprising a retained intron, an exon flanking the 5' splice site of the retained intron, an exon flanking the 3' splice site of the retained intron, and wherein the RIC pre-mRNA encodes tuberin protein
  • the method comprising contacting the cells with an antisense oligomer (ASO) complementary to a targeted portion of the RIC pre-mRNA encoding tuberin protein, whereby the retained intron is constitutively spliced from the RIC pre- mRNA encoding tuberin protein, thereby increasing the level of mRNA encoding tuberin protein, and increasing the expression of tuberin protein in the cells.
  • ASO antisense oligomer
  • the target protein is tuberin.
  • the target protein or the functional RNA is a compensating protein or a compensating functional RNA that functionally augments or replaces a target protein or functional RNA that is deficient in amount or activity in the subject.
  • the cells are in or from a subject having a condition caused by a deficient amount or activity of tuberin protein.
  • the deficient amount of the target protein is caused by haploinsufficiency of the target protein, wherein the subject has a first allele encoding a functional target protein, and a second allele from which the target protein is not produced, or a second allele encoding a nonfunctional target protein, and wherein the antisense oligomer binds to a targeted portion of a RIC pre-mRNA transcribed from the first allele.
  • the subject has a condition caused by a disorder resulting from a deficiency in the amount or function of the target protein, wherein the subject has (a) a first mutant allele from which (i) the target protein is produced at a reduced level compared to production from a wild- type allele, (ii) the target protein is produced in a form having reduced function compared to an equivalent wild-type protein, or (iii) the target protein is not produced, and (b) a second mutant allele from which (i) the target protein is produced at a reduced level compared to production from a wild-type allele, (ii) the target protein is produced in a form having reduced function compared to an equivalent wild-type protein, or (iii) the target protein is not produced, and wherein when the subject has a first mutant allele a.iii., the second mutant allele is b.i.
  • the target protein is produced in a form having reduced function compared to the equivalent wild-type protein. In some embodiments, the target protein is produced in a form that is fully-functional compared to the equivalent wild-type protein.
  • the targeted portion of the RIC pre- mRNA is in the retained intron within the region +6 relative to the 5' splice site of the retained intron to -16 relative to the 3' splice site of the retained intron. In some embodiments, the targeted portion of the RIC pre-mRNA is in the retained intron within the region +500 relative to the 5' splice site of the retained intron to -500 relative to the 3' splice site of the retained intron.
  • the targeted portion of the RIC pre-mRNA is in the retained intron within: (a) the region +6 to +500, +6 to +495, or +6 to +100 relative to the 5' splice site of the retained intron; or (b) the region -16 to -500, -16 to -400, or -16 to -100 relative to the 3' splice site of the retained intron.
  • the targeted portion of the RIC pre-mRNA is within: (a) the region +2e to -4e in the exon flanking the 5' splice site of the retained intron; or (b) the region +2e to -4e in the exon flanking the 3' splice site of the retained intron.
  • the RIC pre-mRNA is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1.
  • the RIC pre-mRNA comprises a sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 2-8.
  • the antisense oligomer does not increase the amount of the target protein or the functional RNA by modulating alternative splicing of pre-mRNA transcribed from a gene encoding the functional RNA or target protein.
  • the antisense oligomer does not increase the amount of the target protein or the functional RNA by modulating aberrant splicing resulting from mutation of the gene encoding the target protein or the functional RNA.
  • the RIC pre-mRNA was produced by partial splicing of a full-length pre- mRNA or partial splicing of a wild-type pre-mRNA.
  • the mRNA encoding the target protein or functional RNA is a full-length mature mRNA, or a wild-type mature mRNA.
  • the target protein produced is full-length protein, or wild- type protein.
  • the total amount of the mRNA encoding the target protein or functional RNA produced in the cell contacted with the antisense oligomer is increased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10- fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about
  • the total amount of target protein produced by the cell contacted with the antisense oligomer is increased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5- fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1 -fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3 -fold, at least about 3.5-fold, at least about 4-fold, at least about 5-
  • the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage. In some embodiments, the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-0-methyl, a 2'-Fluoro, or a 2'-0-methoxyethyl moiety. In some embodiments, the antisense oligomer comprises at least one modified sugar moiety. In some embodiments, each sugar moiety is a modified sugar moiety. In some embodiments, the antisense oligomer consists of from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35
  • nucleobases 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40
  • nucleobases 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.
  • the antisense oligomer is at least 80%, at least 85%>, at least 90%, at least 95%, at least 98%), at least 99%, or 100%>, complementary to the targeted portion of the RIC pre-mRNA encoding the protein.
  • the targeted portion of the RIC pre-mRNA is within a sequence selected from SEQ ID NOs: 5097-5105.
  • the antisense oligomer comprises a nucleotide sequence that is at least about 80%>, 85%>, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 9-5096.
  • the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NOs: 9-5096.
  • the cell comprises a population of RIC pre- mRNAs transcribed from the gene encoding the target protein or functional RNA, wherein the population of RIC pre-mRNAs comprises two or more retained introns, and wherein the antisense oligomer binds to the most abundant retained intron in the population of RIC pre- mRNAs.
  • the binding of the antisense oligomer to the most abundant retained intron induces splicing out of the two or more retained introns from the population of RIC pre-mRNAs to produce mRNA encoding the target protein or functional RNA.
  • the cell comprises a population of RIC pre-mRNAs transcribed from the gene encoding the target protein or functional RNA, wherein the population of RIC pre-mRNAs comprises two or more retained introns, and wherein the antisense oligomer binds to the second most abundant retained intron in the population of RIC pre-mRNAs.
  • the binding of the antisense oligomer to the second most abundant retained intron induces splicing out of the two or more retained introns from the population of RIC pre-mRNAs to produce mRNA encoding the target protein or functional RNA.
  • the condition is a disease or disorder.
  • the disease or disorder is tuberous sclerosis complex.
  • the target protein and the RIC pre-mRNA are encoded by the TSC2 gene.
  • the method further comprises assessing TSC2 protein expression.
  • the antisense oligomer binds to a targeted portion of a tuberin RIC pre- mRNA, wherein the targeted portion is within a sequence selected from SEQ ID NOS: 49, 50, 51, 52, 53, 54, 55, 56, and 57.
  • the subject is a human.
  • the subject is a non-human animal. In some embodiments, the subject is a fetus, an embryo, or a child. In some embodiments, the cells are ex vivo. In some embodiments, the antisense oligomer is administered by topical application to the skin, pulmonary delivery to the lung, intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection of the subject. In some embodiments, the 9 nucleotides at -3e to -le of the exon flanking the 5' splice site and +1 to +6 of the retained intron, are identical to the corresponding wild-type sequence. In some
  • the 16 nucleotides at -15 to -1 of the retained intron and +le of the exon flanking the 3' splice site are identical to the corresponding wild-type sequence.
  • an antisense oligomer as used in a method described above.
  • 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.
  • a pharmaceutical composition comprising the antisense oligomer described above, and an excipient.
  • a method of treating a subject in need thereof by administering the pharmaceutical composition by topical application to the skin, pulmonary delivery to the lung, intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.
  • compositions comprising an antisense oligomer for use in a method of increasing expression of a target protein or a functional RNA by cells to treat tuberous sclerosis complex in a subject in need thereof, associated with a deficient protein or deficient functional RNA, wherein the deficient protein or deficient functional RNA is deficient in amount or activity in the subject, wherein the antisense oligomer enhances constitutive splicing of a retained intron-containing pre-mRNA (RIC pre-mRNA) encoding the target protein or the functional RNA, wherein the target protein is: (a) the deficient protein; or (b) a compensating protein which functionally augments or replaces the deficient protein or in the subject; and wherein the functional RNA is: (a) the deficient RNA; or (b) a compensating functional RNA which functionally augments or replaces the deficient functional RNA in the subject; wherein the RIC pre-mRNA comprises a retained in
  • compositions comprising an antisense oligomer for use in a method of treating a condition associated with tuberin protein in a subject in need thereof, the method comprising the step of increasing expression of tuberin protein by cells of the subject, wherein the cells have a retained-intron-containing pre-mRNA (RIC pre-mRNA) comprising a retained intron, an exon flanking the 5' splice site of the retained intron, an exon flanking the 3' splice site of the retained intron, and wherein the RIC pre-mRNA encodes the tuberin protein, the method comprising contacting the cells with the antisense oligomer, whereby the retained intron is constitutively spliced from the RIC pre-mRNA transcripts encoding tuberin protein, thereby increasing the level of mRNA encoding the tuberin protein, and increasing the expression of tuberin protein, in the cells of the subject.
  • RIC pre-mRNA retained-intron-containing pre-mRNA
  • the condition is a disease or disorder.
  • the disease or disorder is tuberous sclerosis complex.
  • the target protein and RIC pre-mRNA are encoded by the TSC2 gene.
  • the antisense oligomer targets a portion of the RIC pre-mRNA that is in the retained intron within the region +6 relative to the 5' splice site of the retained intron to -16 relative to the 3' splice site of the retained intron.
  • the antisense oligomer targets a portion of the RIC pre-mRNA that is in the retained intron within: (a) the region +6 to +100 relative to the 5' splice site of the retained intron; or (b) the region -16 to -100 relative to the 3' splice site of the retained intron. In some embodiments, the antisense oligomer targets a portion of the RIC pre-mRNA that is within the region about 500, 400, 300, 200, or 100 nucleotides downstream of the 5' splice site of the at least one retained intron, to about 100, 200, 300, 400, or 500 nucleotides upstream of the 3' splice site of the at least one retained intron.
  • the targeted portion of the RIC pre-mRNA is within: (a) the region +2e to - 4e in the exon flanking the 5' splice site of the retained intron; or (b) the region +2e to -4e in the ex on flanking the 3' splice site of the retained intron.
  • the RIC pre-mRNA is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. sequence identity to SEQ ID NO: 1.
  • the RIC pre-mRNA comprises a sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 2-8.
  • the antisense oligomer does not increase the amount of target protein or functional RNA by modulating alternative splicing of the pre-mRNA transcribed from a gene encoding the target protein or functional RNA.
  • the antisense oligomer does not increase the amount of the functional RNA or functional protein by modulating aberrant splicing resulting from mutation of the gene encoding the target protein or functional RNA.
  • the RIC pre- mRNA was produced by partial splicing from a full-length pre-mRNA or a wild-type pre- mRNA.
  • the mRNA encoding the target protein or functional RNA is a full-length mature mRNA, or a wild-type mature mRNA.
  • the target protein produced is full-length protein, or wild-type protein.
  • the retained intron is a rate-limiting intron. In some embodiments, said retained intron is the most abundant retained intron in said RIC pre-mRNA. In some embodiments, the retained intron is the second most abundant retained intron in said RIC pre-mRNA.
  • the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage. In some embodiments, said antisense oligomer is an antisense oligonucleotide. In some embodiments, the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-0-methyl, a 2'-Fluoro, or a 2'-0- methoxyethyl moiety. In some embodiments, the antisense oligomer comprises at least one modified sugar moiety. In some embodiments, each sugar moiety is a modified sugar moiety.
  • the antisense oligomer consists of from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50
  • nucleobases 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.
  • the antisense oligomer is at least 80%, at least 85%, at least 90%), at least 95%, at least 98%>, at least 99%, or is 100% complementary to the targeted portion of the RIC pre-mRNA encoding the protein. In some embodiments, the antisense oligomer binds to a targeted portion of a tuberin RIC pre-mRNA, wherein the targeted portion is within a sequence selected from SEQ ID NOs: 5097-5105.
  • the antisense oligomer comprises a nucleotide sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 9-5096. In some embodiments, the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NOs: 9-5096.
  • a pharmaceutical composition comprising the antisense oligomer described above, and an excipient.
  • a method of treating a subject in need thereof by administering the pharmaceutical composition by topical application to the skin, pulmonary delivery to the lung, intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.
  • a pharmaceutical composition comprising: an antisense oligomer that hybridizes to a target sequence of a deficient TSC2 mRNA transcript, wherein the deficient TSC2 mRNA transcript comprises a retained intron, wherein the antisense oligomer induces splicing out of the retained intron from the deficient TSC2 mRNA transcript; and a pharmaceutical acceptable excipient.
  • the deficient TSC2 mRNA transcript is a TSC2 RIC pre-mRNA transcript.
  • the targeted portion of the TSC2 RIC pre-mRNA transcript is in the retained intron within the region +500 relative to the 5' splice site of the retained intron to -500 relative to the 3' spliced site of the retained intron.
  • the TSC2 RIC pre-mRNA transcript is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1.
  • the TSC2 RIC pre-mRNA transcript comprises a sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 2-8.
  • the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage. In some embodiments, the antisense oligomer is an antisense oligonucleotide. In some embodiments, the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-0-methyl, a 2'-Fluoro, or a 2'-0-methoxyethyl moiety. In some embodiments, the antisense oligomer comprises at least one modified sugar moiety.
  • the antisense oligomer comprises from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50
  • nucleobases 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.
  • the antisense oligomer is at least 80%>, at least 85%>, at least 90%), at least 95%, at least 98%>, at least 99%, or is 100%> complementary to a targeted portion of the TSC2 RIC pre-mRNA transcript.
  • the targeted portion of the TSC2 RIC pre-mRNA transcript is within a sequence selected from SEQ ID NOs: 5097-5105.
  • the antisense oligomer comprises a nucleotide sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 9-5096.
  • the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NOs: 9-5096.
  • the second 85%>, 90% at least 95%, at least 98%>, at least 99%, or is 100%> complementary to a targeted portion of the TSC2 RIC pre-mRNA transcript.
  • composition is formulated for intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.
  • a method of inducing processing of a deficient TSC2 mRNA transcript to facilitate removal of a retained intron to produce a fully processed TSC2 mRNA transcript that encodes a functional form of a tuberin protein comprising: (a) contacting an antisense oligomer to a target cell of a subject; (b) hybridizing the antisense oligomer to the deficient TSC2 mRNA transcript, wherein the deficient TSC2 mRNA transcript is capable of encoding the functional form of tuberin protein and comprises at least one retained intron; (c) removing the at least one retained intron from the deficient TSC2 mRNA transcript to produce the fully processed TSC2 mRNA transcript that encodes the functional form of tuberin protein; and (d) translating the functional form of tuberin protein from the fully processed TSC2 mRNA transcript.
  • the retained intron is an entire retained intron.
  • the deficient TSC2 mRNA transcript is an entire retained intron.
  • a method of treating a subject having a condition caused by a deficient amount or activity of tuberin protein comprising: administering to the subject an antisense oligomer comprising a nucleotide sequence with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 9-5096.
  • FIG. 1 shows a schematic representation of an exemplary retained-intron-containing (RIC) pre-mRNA transcript.
  • the 5' splice site consensus sequence is indicated with underlined letters (letters are nucleotides; upper case: exonic portion and lower case: intronic portion) from -3e to -le and +1 to +6 (numbers labeled "e” are exonic and unlabeled numbers are intronic).
  • the 3' splice site consensus sequence is indicated with underlined letters (letters are nucleotides; upper case: exonic portion and lower case: intronic portion) from -15 to -1 and +le (numbers labeled "e” are exonic and unlabeled numbers are intronic).
  • Intronic target regions for ASO screening comprise nucleotides +6 relative to the 5' splice site of the retained intron (arrow at left) to -16 relative to the 3' splice site of the retained intron (arrow at right).
  • intronic target regions for ASO screening comprise nucleotides +6 to +100 relative to the 5' splice site of the retained intron and -16 to -100 relative to the 3' splice site of the retained intron.
  • Exonic target regions comprise nucleotides +2e to -4e in the ex on flanking the 5' splice site of the retained intron and +2e to -4e in the ex on flanking the 3' splice site of the retained intron.
  • n or N denote any nucleotide
  • y denotes pyrimidine.
  • the sequences shown represent consensus sequences for mammalian splice sites and individual introns and exons need not match the consensus sequences at every position.
  • FIG. 2A-FIG. 2B illustrate schematic representations of the Targeted Augmentation of Nuclear Gene Output (TANGO) approach.
  • FIG. 2A shows a cell divided into nuclear and cytoplasmic compartments.
  • a pre-mRNA transcript of a target gene consisting of exons (rectangles) and introns (connecting lines) undergoes splicing to generate an mRNA, and this mRNA is exported to the cytoplasm and translated into target protein.
  • the splicing of intron 1 is inefficient and a retained intron-containing (RIC) pre-mRNA accumulates primarily in the nucleus, and if exported to the cytoplasm, is degraded, leading to no target protein production.
  • FIG. 2B shows an example of the same cell divided into nuclear and cytoplasmic compartments.
  • Treatment with an antisense oligomer (ASO) promotes the splicing of intron 1 and results in an increase in mRNA, which is in turn translated into higher levels of target protein
  • FIG. 3 shows intron-retention in the TSC2 gene with intron 4 detail.
  • the identification of intron-retention events in the TSC2 gene using RNA sequencing (RNAseq) is shown, visualized in the UCSC genome browser.
  • the upper panel shows the read density
  • the TSC2 transcript expressed in HCN human cortical neurons
  • HCN human cortical neurons
  • nuclear fraction nuclear fraction
  • a graphic representation of the TSC2 gene is shown to scale.
  • the read density is shown as peaks.
  • the highest read density corresponds to exons (black boxes), while no reads are observed for the majority of the introns (lines with arrow heads) in either cellular fraction.
  • Higher read density is detected for introns 4, 25/26, and 31/32 (indicated by the arrows) in the nuclear fraction compared to the cytoplasmic fraction indicating that splicing efficiency of introns 4, 25/26, and 31/32 is low, resulting in intron retention.
  • the retained-intron containing pre-mRNA transcripts are retained in the nucleus and are not exported out to the cytoplasm.
  • the read density for intron 4 in HCN is shown in detail in the lower panel.
  • FIG. 4 shows TSC2 gene IVS 4 ASO walk.
  • FIG. 5 shows intron-retention in the TSC2 gene with introns 25 and 26 detail.
  • Intron retention in the TSC2 gene was identified by RNA sequencing (RNAseq), visualized in the UCSC genome browser, as described herein in the Examples.
  • RNAseq RNA sequencing
  • the read density for introns 25 and 26 in HCN is shown in detail in the lower panel.
  • Introns 25 and 26 flank exon 26, an alternatively spliced exon.
  • FIG. 6 shows TSC2 gene IVS 25 and 26 ASO walk.
  • the splice site intronic regions flanking alternative exon 26 are not targeted to avoid affecting the inclusion level of exon 26.
  • ASOs were designed to cover these regions by shifting 5 nucleotides at a time.
  • the TSC2 exon-intron structure is drawn to scale.
  • FIG. 7 shows intron-retention in the TSC2 gene with introns 31 and 32 detail.
  • Intron retention in the TSC2 gene was identified by RNA sequencing (RNAseq), visualized in the UCSC genome browser, as described herein in the Examples.
  • the read density for introns 31 and 32 is shown in detail in the lower panel. Introns 31 and 32 flank exon 32, an alternatively spliced exon. This is evidenced in the graphic representations of the TSC2 transcripts and the RNAseq data, such that the rectangle depicting exon 32 is present in some transcripts while absent in others, and the read density corresponding to exon 32 in the cytoplasmic fraction is significantly lower than that of the constitutively spliced exons in TSC2.
  • the read density for intron 31 is shown in detail in the lower panel indicating 43% intron retention as calculated by bioinformatic analysis.
  • FIG. 8 shows TSC2 gene IVS 31 and 32 ASO walk.
  • the splice site intronic regions flanking alternative exon 32 are not targeted to avoid affecting the inclusion level of exon 32.
  • ASOs were designed to cover these regions by shifting 5 nucleotides at a time with the exception of TSC2-IVS32-33 and TSC2- IVS32-51.
  • the TSC2 exon-intron structure is drawn to scale.
  • FIG. 9 depicts a schematic of the ReSeq Genes for TSC2 intron 4 corresponding to NM 000548.
  • the Percent Intron Retention (PIR) of the circled intron is shown.
  • FIG. 11 depicts a schematic of the ReSeq Genes for TSC2 intron 25 corresponding to NM 000548.
  • the Percent Intron Retention (PIR) of the circled intron is shown.
  • FIG. 13 depicts a schematic of the ReSeq Genes for TSC2 intron 26 corresponding to NM 000548.
  • the Percent Intron Retention (PIR) of the circled intron is shown.
  • FIG. 14 depicts a schematic of the ReSeq Genes for TSC2 intron 31 corresponding to NM 000548.
  • the Percent Intron Retention (PIR) of the circled intron is shown.
  • FIG. 16 depicts a schematic of the ReSeq Genes for TSC2 intron 32 corresponding to NM 000548.
  • the Percent Intron Retention (PIR) of the circled intron is shown.
  • introns in primary transcripts of protein-coding genes having more than one intron are spliced from the primary transcript with different efficiencies. In most cases only the fully spliced mRNA is exported through nuclear pores for subsequent translation in the cytoplasm. Unspliced and partially spliced transcripts are detectable in the nucleus. It is generally thought that nuclear accumulation of transcripts that are not fully spliced is a mechanism to prevent the accumulation of potentially deleterious mRNAs in the cytoplasm that may be translated to protein. For some genes, splicing of the least efficient intron is a rate- limiting post-transcriptional step in gene expression, prior to translation in the cytoplasm.
  • the present invention provides compositions and methods for upregulating splicing of one or more retained TSC2 introns that are rate-limiting for the nuclear stages of gene expression to increase steady-state production of fully-spliced, mature mRNA, and thus, translated tuberin protein levels.
  • These compositions and methods utilize antisense oligomers (ASOs) that promote constitutive splicing at an intron splice sites of a retained-intron-containing TSC2 pre- mRNA that accumulates in the nucleus.
  • ASOs antisense oligomers
  • TSC2 protein is increased using the methods of the invention to treat a condition caused by TSC2 deficiency.
  • the methods of the invention are used to increase TSC2 production to treat a condition in a subject in need thereof.
  • the subject has condition in which TSC2 is not necessarily deficient relative to wild-type, but where an increase in TSC2 mitigates the condition nonetheless.
  • the condition is a caused by a TSC2 haploinsufficiency.
  • the condition is an autosomal dominant disorder.
  • the condition is an autosomal recessive disorder.
  • Tuberous sclerosis complex is a disease characterized by tumor growth in multiple organ systems (Au et al., J. Child Neurol. 2004, 19, 699-709). Tumors are usually benign but are occasionally malignant. Approximately 90% of tuberous sclerosis complex cases display cortical tuber; facial angiofibroma and renal angiomyolipoma occur in more than 80% of cases. In addition, approximately 80% of cases display subependymal nodule, approximately 50% of cases display cardiac rhabdomyoma, and 51% to approximately 88% of cases display ungual/ subungual fibroma. Tumors of the central nervous system (CNS) are the leading cause of morbidity and mortality, followed by renal disease (Au et al., J. Child Neurol. 2004, 19, 699- 709).
  • CNS central nervous system
  • Tuberous sclerosis complex is a genetic disorder with an autosomal dominant inheritance pattern and a high mutation rate (Au et al., J. Child Neurol. 2004, 19, 699-709).
  • Linkage of tuberous sclerosis complex to chromosomal regions 9q34.3 and 16pl3.3 led to the identification of the TSC1 and TSC2 genes, respectively.
  • More than 69% of tuberous sclerosis complex cases result from haploinsufficency of TSC2, and disease in approximately two thirds of patients results from de novo mutation or deletion (Au et al., J. Child Neurol. 2004, 19, 699-709).
  • Severe disease is thought to require a "second hit" reduction of the other allele.
  • the prevalence of tuberous sclerosis complex is as high as one in 5,800 live births, or approximately 300 births per year in the United States, with 200 cases per year resulting from mutation of TSC2.
  • the disease is described, e.g., by OMTM #613254 (Online Mendelian Inheritance in Man, Johns Hopkins University, 1966-2015), incorporated by reference herein.
  • the TSC2 gene codes for a protein named tuberin.
  • the TSC2 gene contains 41 exons, two of which are alternatively spliced, and spans 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 the TSC1 gene product hamartin.
  • the tuberin-hamartin complex plays a role in the regulation of cell growth, translation, cell size, and cell adhesion and migration through a variety of signal cascades.
  • the tuberin-hamartin complex functions in the phosphatidyl-inositol-3- kinase and protein B pathway (PI3K/PKB) that regulates translation.
  • PI3K/PKB phosphatidyl-inositol-3- kinase and protein B pathway
  • the tuberin- hamartin complex suppresses cell growth and translation by suppressing mTOR kinase activity, resulting in suppression of the p70 ribosomal protein S6 kinase (S6K) 1 and activation of eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1).
  • S6K ribosomal protein S6 kinase
  • 4E-BP1 eukaryotic translation initiation factor 4E binding protein 1
  • the GAP domain of tuberin hydrolyzes guanosine triphosphate (GTP) bound to the small G protein Rheb, a Ras homolog enriched in brain, thus preventing mTOR activation.
  • tuberin and hamartin regulate cell growth and cell adhesion through the MAP kinase (MAPK) and E-cadherin-beta-catenin pathways, respectively.
  • MAPK MAP kinase
  • E-cadherin-beta-catenin pathways respectively.
  • Mutations include frameshift and protein truncations, nonsense mutations, mutations that affect splicing, in-frame mutations, deletions, insertions, duplications, and large deletions and rearrangement. Mutations of TSC2 that yield truncated protein products fail to form the tuberin- hamartin complex, thus resulting in loss of cell growth regulation.
  • the role of the tuberin- hamartin complex as a key regulator of multiple signaling pathways is consistent with the wide spectrum of clinical findings among patients with tuberous sclerosis complex that involve multiple organ systems, including neurological and neurobehavioral abnormalities such as seizures, intellectual disability, and developmental delay (Au et al., J. Child Neurol. 2004, 19, 699-709).
  • RIC Pre -mRNA Retained Intron Containing Pre-mRNA
  • the methods of the present invention exploit the presence of retained- intron-containing pre-mRNA (RIC pre-mRNA) transcribed from the TSC2 gene and encoding tuberin protein, in the cell nucleus.
  • Splicing of the identified TSC2 RIC pre-mRNA species to produce mature, fully-spliced, TSC2 mRNA is induced using ASOs that stimulate splicing out of the retained introns.
  • the resulting mature TSC2 mRNA can be exported to the cytoplasm and translated, thereby increasing the amount of tuberin protein in the patient's cells and alleviating symptoms of tuberous sclerosis complex.
  • This method described further below, is known as Targeted Augmentation of Nuclear Gene Output (TANGO).
  • RNA sequencing visualized in the UCSC genome browser, showed TSC2 transcripts expressed in HCN (human cortical neurons) cells and localized in either the cytoplasmic or nuclear fraction. In both fractions, reads were not observed for the majority of the introns. However, higher read density was detected for introns 4, 25, 26, 31, and 32 in the nuclear fraction compared to the cytoplasmic fraction indicating that splicing efficiency of introns 4, 25, 26, 31, and 32 is low, resulting in intron retention.
  • the retained-intron containing pre-mRNA transcripts accumulate primarily in the nucleus and not translated into the tuberin protein.
  • the read density for intron 26 in AST is shown in detail in the lower panel indicating 51% intron retention as calculated by bioinformatic analysis.
  • the percent intron retention (PIR) value for intron 26 was obtained by averaging four values (87, 83, 20, and 12), each determined in renal epithelial cells using one of four different algorithms.
  • the read density for intron 31 in AST is shown in detail in the lower panel indicating 43% intron retention.
  • the percent intron retention (PIR) value for intron 31 was obtained by averaging four values (78, 71, 16, and 8), each determined in renal epithelial cells using one of four different algorithms.
  • Introns 4 and 32 were not mapped.
  • Analysis of the ENCODE data (described by, e.g., Tilgner, et al., 2012, "Deep sequencing of subcellular RNA fractions shows splicing to be predominantly co-transcriptional in the human genome but inefficient for IncRNAs," Genome Research 22(9): 1616-25) to identify intron retention events did not identify intron 25, 26, or 31 as retained, and did not map introns 4 and 32.
  • Table 1 provides a non-limiting list of target sequences of a TSC2 RIC pre-mRNA transcript by sequence ID, and ASOs by sequence ID, useful for increasing production of tuberin protein by targeting a region of a TSC2 RIC pre-mRNA.
  • ASOs useful for these purposes are identified, using, e.g., methods described herein.
  • TSC2 NM 001318831 SEQ ID NOs: 3346-
  • TSC2 NM 001318827 SEQ ID NOs: 4646-
  • the ASOs disclosed herein target a RIC pre-mRNA transcribed from a TSC2 genomic sequence. In some embodiments, the ASO targets a RIC pre-mRNA transcript from a TSC2 genomic sequence comprising a retained intron. In some embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ ID NO: 1. In some embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ ID NO: 1 comprising a retained intron. In some embodiments, the ASOs disclosed herein target a TSC2 RIC pre-mRNA sequence.
  • the ASO targets a TSC2 RIC pre-mRNA transcript comprising a retained intron at 3, 24, 29 or a combination thereof, wherein the intron numbering correspond to the mRNA sequence at NM_001318829. In some embodiments, the ASO targets a TSC2 RIC pre-mRNA transcript comprising a retained intron at 4, 25, 26, 31, 32 or a combination thereof, wherein the intron numbering correspond to the mRNA sequence at NM 000548. In some embodiments, the ASO targets a TSC2 RIC pre-mRNA transcript comprising a retained intron at 4, 25, 30 or a combination thereof, wherein the intron numbering correspond to the mRNA sequence at NM_001077183.
  • the ASO targets a TSC2 RIC pre-mRNA transcript comprising a retained intron at 4, 25, 26, 31, or a combination thereof, wherein the intron numbering correspond to the mRNA sequence at NM OOl 114382. In some embodiments, the ASO targets a TSC2 RIC pre-mRNA transcript comprising a retained intron at 22, 27, 28 or a combination thereof, wherein the intron numbering correspond to the mRNA sequence at NM_001318831. In some embodiments, the ASO targets a TSC2 RIC pre-mRNA transcript comprising a retained intron at 4, 25, 30 or a combination thereof, wherein the intron numbering correspond to the mRNA sequence at NM 001318832. In some embodiments, the ASO targets a TSC2 RIC pre-mRNA transcript comprising a retained intron at 3, 24, 29 or a combination thereof, wherein the intron numbering correspond to the mRNA sequence at NM_001318827.
  • the ASO targets a TSC2 RIC pre-mRNA sequence according to SEQ ID NO: 2. In some embodiments, the ASO targets a TSC2 RIC pre-mRNA sequence according to SEQ ID NO: 2 comprising a retained intron 24, a retained intron 3, a retained intron 29, or a combination thereof. In some embodiments, the ASO targets a TSC2 RIC pre-mRNA sequence according to SEQ ID NO: 3. In some embodiments, the ASO targets a TSC2 RIC pre- mRNA sequence according to SEQ ID NO: 3 comprising a retained intron 32, a retained intron 25, a retained intron 26, a retained intron 4, a retained intron 31 or a combination thereof.
  • the ASO targets a TSC2 RIC pre-mRNA sequence according to SEQ ID NO: 4. In some embodiments, the ASO targets a TSC2 RIC pre-mRNA sequence according to SEQ ID NO: 4 comprising a retained intron 25, a retained intron 4, a retained intron 30 or a combination thereof. In some embodiments, the ASO targets a TSC2 RIC pre-mRNA sequence according to SEQ ID NO: 5. In some embodiments, the ASO targets a TSC2 RIC pre-mRNA sequence according to SEQ ID NO: 5 comprising a retained intron 25, a retained intron 26, a retained intron 4, a retained intron 31 or a combination thereof.
  • the ASO targets a TSC2 RIC pre-mRNA sequence according to SEQ ID NO: 6. In some embodiments, the ASO targets a TSC2 RIC pre-mRNA sequence according to SEQ ID NO: 6 comprising a retained intron 27, a retained intron 28, a retained intron 22 or a combination thereof. In some embodiments, the ASO targets a TSC2 RIC pre-mRNA sequence according to SEQ ID NO: 7. In some embodiments, the ASO targets a TSC2 RIC pre-mRNA sequence according to SEQ ID NO: 7 comprising a retained intron 25, a retained intron 4, a retained intron 30 or a combination thereof.
  • the ASO targets a TSC2 RIC pre-mRNA sequence according to SEQ ID NO: 8 . In some embodiments, the ASO targets a TSC2 RIC pre-mRNA sequence according to SEQ ID NO: 8 comprising a retained intron 24, a retained intron 3, a retained intron 29 or a combination thereof. In some embodiments, the ASOs disclosed herein target SEQ ID NOs: 5097-5105. In some embodiments, the ASO has a sequence according to any one of SEQ ID NOs: 9-5096.
  • the ASO targets exon 24 or exon 25 of a TSC2 RIC pre-mRNA comprising a retained intron 24, wherein the intron numbering correspond to the mRNA sequence at M 001318829.
  • the ASO targets an exon 24 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 24.
  • the ASO targets an exon 24 sequence about 2 to about 75 or about 4 to about 74 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 24.
  • the ASO targets an exon 25 sequence downstream (or 3 ') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 24. In some embodiments, the ASO targets an exon 25 sequence about 2 to about 150 or about 2 to about 147 nucleotides downstream (or 3') from the 3' splice site of a TSC2 RIC pre- mRNA comprising the retained intron 24.
  • the ASO targets intron 24 in a TSC2 RIC pre-mRNA comprising a retained intron 24, wherein the intron numbering correspond to the mRNA sequence at NM 001318829.
  • the ASO targets an intron 24 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 24.
  • 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 pre-mRNA comprising the retained intron 24.
  • the ASO targets an intron 24 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising 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 pre-mRNA comprising the retained intron 24.
  • the ASO targets exon 3 or exon 4 of a TSC2 RIC pre-mRNA comprising a retained intron 3, wherein the intron numbering correspond to the mRNA sequence at NM 001318829.
  • the ASO targets an exon 3 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 3.
  • 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 pre-mRNA comprising the retained intron 3.
  • the ASO targets an exon 4 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 3. In some embodiments, the ASO targets an exon 4 sequence about 2 to about 127 nucleotides downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 3.
  • the ASO targets intron 3 in a TSC2 RIC pre-mRNA comprising a retained intron 3, wherein the intron numbering correspond to the mRNA sequence at
  • the ASO targets an intron 3 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the 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 pre-mRNA comprising the 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 pre-mRNA comprising the 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 pre-mRNA comprising the retained intron 3.
  • the ASO targets exon 29 or exon 30 of a TSC2 RIC pre-mRNA comprising a retained intron 29, wherein the intron numbering correspond to the mRNA sequence at NM 001318829.
  • the ASO targets an exon 29 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 29.
  • 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 pre-mRNA comprising the retained intron 29.
  • the ASO targets an exon 30 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the 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 pre-mRNA comprising the retained intron 29.
  • the ASO targets intron 29 in a TSC2 RIC pre-mRNA comprising a retained intron 29, wherein the intron numbering correspond to the mRNA sequence at NM 001318829.
  • the ASO targets an intron 29 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 29.
  • 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 pre-mRNA comprising the retained intron 29.
  • the ASO targets an intron 29 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 29. In some embodiments, the ASO targets an intron 29 sequence about 16 to about 500 or about 36 to about 500 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 29.
  • the ASO targets exon 32 or exon 33 of a TSC2 RIC pre-mRNA comprising a retained intron 32, wherein the intron numbering correspond to the mRNA sequence at NM_000548.
  • the ASO targets an exon 32 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 32.
  • 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 pre-mRNA comprising the retained intron 32.
  • the ASO targets an exon 33 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the 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 a TSC2 RIC pre-mRNA comprising the retained intron 32.
  • the ASO targets intron 32 in a TSC2 RIC pre-mRNA comprising a retained intron 32, wherein the intron numbering correspond to the mRNA sequence at NM_000548.
  • the ASO targets an intron 32 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 32.
  • the ASO targets an intron 32 sequence about 6 to about 495 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 32.
  • the ASO targets an intron 32 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 32. In some embodiments, the ASO targets an intron 32 sequence about 16 to about 500 or about 36 to about 500 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 32.
  • the ASO targets exon 25 or exon 26 of a TSC2 RIC pre-mRNA comprising a retained intron 25, wherein the intron numbering correspond to the mRNA sequence at NM_000548.
  • the ASO targets an exon 25 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
  • the ASO targets an exon 25 sequence about 4 to about 74 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
  • the ASO targets an exon 26 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising 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 a TSC2 RIC pre-mRNA comprising the retained intron 25.
  • the ASO targets intron 25 in a TSC2 RIC pre-mRNA comprising a retained intron 25, wherein the intron numbering correspond to the mRNA sequence at NM_000548.
  • the ASO targets an intron 25 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
  • the ASO targets an intron 25 sequence about 6 to about 497 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
  • the ASO targets an intron 25 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising 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 a TSC2 RIC pre-mRNA comprising the retained intron 25.
  • the ASO targets exon 26 or exon 27 of a TSC2 RIC pre-mRNA comprising a retained intron 26, wherein the intron numbering correspond to the mRNA sequence at NM_000548.
  • the ASO targets an exon 26 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 26.
  • the ASO targets an exon 26 sequence about 4 to about 111 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 26.
  • the ASO targets an exon 27 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising 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 a TSC2 RIC pre-mRNA comprising the retained intron 26.
  • the ASO targets intron 26 in a TSC2 RIC pre-mRNA comprising a retained intron 26, wherein the intron numbering correspond to the mRNA sequence at NM 000548.
  • the ASO targets an intron 26 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 26.
  • the ASO targets an intron 26 sequence about 6 to about 500 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 26.
  • the ASO targets an intron 26 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising 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 a TSC2 RIC pre-mRNA comprising the retained intron 26.
  • the ASO targets exon 4 or exon 5 of a TSC2 RIC pre-mRNA comprising a retained intron 4, wherein the intron numbering correspond to the mRNA sequence at NM_000548.
  • the ASO targets an exon 4 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
  • the ASO targets an exon 4 sequence about 4 to about 94 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
  • the ASO targets an exon 5 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4. In some embodiments, the ASO targets an exon 5 sequence about 2 to about 127 nucleotides downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
  • the ASO targets intron 4 in a TSC2 RIC pre-mRNA comprising a retained intron 4, wherein the intron numbering correspond to the mRNA sequence at M 000548.
  • the ASO targets an intron 4 sequence downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
  • the ASO targets an intron 4 sequence about 6 to about 435 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
  • the ASO targets an intron 4 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
  • the ASO targets an intron 4 sequence about 16 to about 432 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
  • the ASO targets exon 31 or exon 32 of a TSC2 RIC pre-mRNA comprising a retained intron 31, wherein the intron numbering correspond to the mRNA sequence at NM_000548.
  • the ASO targets an exon 31 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 31.
  • the ASO targets an exon 31 sequence about 4 to about 184 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 31.
  • the ASO targets an exon 32 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the 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 a TSC2 RIC pre-mRNA comprising the retained intron 31.
  • the ASO targets intron 31 in a TSC2 RIC pre-mRNA comprising a retained intron 31, wherein the intron numbering correspond to the mRNA sequence at NM 000548.
  • the ASO targets an intron 31 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 31.
  • the ASO targets an intron 31 sequence about 6 to about 331 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 31.
  • the ASO targets an intron 31 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the 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 a TSC2 RIC pre-mRNA comprising the retained intron 31.
  • the ASO targets exon 25 or exon 26 of a TSC2 RIC pre-mRNA comprising a retained intron 25, wherein the intron numbering correspond to the mRNA sequence at NM_001077183.
  • the ASO targets an exon 25 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
  • the ASO targets an exon 25 sequence about 4 to about 74 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
  • the ASO targets an ex on 26 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25. In some embodiments, the ASO targets an ex on 26 sequence about 2 to about 142 nucleotides downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
  • the ASO targets intron 25 in a TSC2 RIC pre-mRNA comprising a retained intron 25, wherein the intron numbering correspond to the mRNA sequence at NM OO 1077183.
  • the ASO targets an intron 25 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
  • the ASO targets an intron 25 sequence about 6 to about 497 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
  • the ASO targets an intron 25 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising 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 a TSC2 RIC pre-mRNA comprising the retained intron 25.
  • the ASO targets exon 4 or exon 5 of a TSC2 RIC pre-mRNA comprising a retained intron 4, wherein the intron numbering correspond to the mRNA sequence at NM_001077183.
  • the ASO targets an exon 4 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
  • the ASO targets an exon 4 sequence about 4 to about 94 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
  • the ASO targets an exon 5 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4. In some embodiments, the ASO targets an exon 5 sequence about 2 to about 127 nucleotides downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
  • the ASO targets intron 4 in a TSC2 RIC pre-mRNA comprising a retained intron 4, wherein the intron numbering correspond to the mRNA sequence at
  • the ASO targets an intron 4 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4. In some embodiments, the ASO targets an intron 4 sequence about 6 to about 435 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising 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 pre-mRNA comprising the retained intron 4. In some
  • the ASO targets an intron 4 sequence about 16 to about 432 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
  • the ASO targets exon 30 or exon 31 of a TSC2 RIC pre-mRNA comprising a retained intron 30, wherein the intron numbering correspond to the mRNA sequence at NM_001077183.
  • the ASO targets an exon 30 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 30.
  • the ASO targets an exon 30 sequence about 4 to about 184 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising 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 pre-mRNA comprising the 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 a TSC2 RIC pre-mRNA comprising the retained intron 30.
  • the ASO targets intron 30 in a TSC2 RIC pre-mRNA comprising a retained intron 30, wherein the intron numbering correspond to the mRNA sequence at NM OO 1077183.
  • the ASO targets an intron 30 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 30.
  • 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 pre-mRNA comprising the retained intron 30.
  • the ASO targets an intron 30 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 30. In some embodiments, the ASO targets an intron 30 sequence about 16 to about 500 or about 36 to about 500 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 30.
  • the ASO targets exon 25 or exon 26 of a TSC2 RIC pre-mRNA comprising a retained intron 25, wherein the intron numbering correspond to the mRNA sequence at NM_001114382.
  • the ASO targets an exon 25 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
  • the ASO targets an exon 25 sequence about 4 to about 74 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
  • the ASO targets an exon 26 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising 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 a TSC2 RIC pre-mRNA comprising the retained intron 25.
  • the ASO targets intron 25 in a TSC2 RIC pre-mRNA comprising a retained intron 25, wherein the intron numbering correspond to the mRNA sequence at NM OOl 114382.
  • the ASO targets an intron 25 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
  • the ASO targets an intron 25 sequence about 6 to about 497 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
  • the ASO targets an intron 25 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising 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 a TSC2 RIC pre-mRNA comprising the retained intron 25.
  • the ASO targets exon 26 or exon 27 of a TSC2 RIC pre-mRNA comprising a retained intron 26, wherein the intron numbering correspond to the mRNA sequence at NM OOl 114382.
  • the ASO targets an exon 26 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 26.
  • the ASO targets an exon 26 sequence about 4 to about 111 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 26.
  • the ASO targets an exon 27 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising 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 a TSC2 RIC pre-mRNA comprising the retained intron 26.
  • the ASO targets intron 26 in a TSC2 RIC pre-mRNA comprising a retained intron 26, wherein the intron numbering correspond to the mRNA sequence at NM OOl 114382.
  • the ASO targets an intron 26 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 26.
  • the ASO targets an intron 26 sequence about 6 to about 500 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 26.
  • the ASO targets an intron 26 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising 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 a TSC2 RIC pre-mRNA comprising the retained intron 26.
  • the ASO targets exon 4 or exon 5 of a TSC2 RIC pre-mRNA comprising a retained intron 4, wherein the intron numbering correspond to the mRNA sequence at NM OOl 114382.
  • the ASO targets an exon 4 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
  • the ASO targets an exon 4 sequence about 4 to about 94 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
  • the ASO targets an exon 5 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4. In some embodiments, the ASO targets an exon 5 sequence about 2 to about 127 nucleotides downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
  • the ASO targets intron 4 in a TSC2 RIC pre-mRNA comprising a retained intron 4, wherein the intron numbering correspond to the mRNA sequence at
  • the ASO targets an intron 4 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4. In some embodiments, the ASO targets an intron 4 sequence about 6 to about 435 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising 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 pre-mRNA comprising the retained intron 4. In some
  • the ASO targets an intron 4 sequence about 16 to about 432 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
  • the ASO targets exon 31 or exon 32 of a TSC2 RIC pre-mRNA comprising a retained intron 31, wherein the intron numbering correspond to the mRNA sequence at NM OOl 114382.
  • the ASO targets an exon 31 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 31.
  • the ASO targets an exon 31 sequence about 4 to about 184 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 31.
  • the ASO targets an exon 32 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the 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 a TSC2 RIC pre-mRNA comprising the retained intron 31.
  • the ASO targets intron 31 in a TSC2 RIC pre-mRNA comprising a retained intron 31, wherein the intron numbering correspond to the mRNA sequence at NM OOl 114382.
  • the ASO targets an intron 31 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 31.
  • the ASO targets an intron 31 sequence about 6 to about 492 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 31.
  • the ASO targets an intron 31 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 31. In some embodiments, the ASO targets an intron 31 sequence about 16 to about 500 or about 36 to about 500 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 31.
  • the ASO targets exon 27 or exon 28 of a TSC2 RIC pre-mRNA comprising a retained intron 27, wherein the intron numbering correspond to the mRNA sequence at NM 001318831.
  • the ASO targets an exon 27 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron
  • 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 pre-mRNA comprising the retained intron 27. In some embodiments, the ASO targets an exon 28 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the 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 a TSC2 RIC pre-mRNA comprising the retained intron 27.
  • the ASO targets intron 27 in a TSC2 RIC pre-mRNA comprising a retained intron 27, wherein the intron numbering correspond to the mRNA sequence at
  • the ASO targets an intron 27 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the 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 a TSC2 RIC pre-mRNA comprising 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 pre-mRNA comprising the 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 a TSC2 RIC pre-mRNA comprising the retained intron 27.
  • the ASO targets exon 28 or exon 29 of a TSC2 RIC pre-mRNA comprising a retained intron 28, wherein the intron numbering correspond to the mRNA sequence at NM 001318831.
  • the ASO targets an exon 28 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron
  • 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 pre-mRNA comprising the 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 pre-mRNA comprising the 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 pre-mRNA comprising the retained intron 28.
  • the ASO targets intron 28 in a TSC2 RIC pre-mRNA comprising a retained intron 28, wherein the intron numbering correspond to the mRNA sequence at M 001318831.
  • the ASO targets an intron 28 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 28.
  • the ASO targets an intron 28 sequence about 6 to about 495 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 28.
  • the ASO targets an intron 28 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 28. In some embodiments, the ASO targets an intron 28 sequence about 16 to about 500 or about 36 to about 500 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 28.
  • the ASO targets exon 22 or exon 23 of a TSC2 RIC pre-mRNA comprising a retained intron 22, wherein the intron numbering correspond to the mRNA sequence at NM 001318831.
  • the ASO targets an exon 22 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 22.
  • the ASO targets an exon 22 sequence about 4 to about 74 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 22.
  • the ASO targets an exon 23 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising 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 a TSC2 RIC pre-mRNA comprising the retained intron 22.
  • the ASO targets intron 22 in a TSC2 RIC pre-mRNA comprising a retained intron 22, wherein the intron numbering correspond to the mRNA sequence at NM 001318831.
  • the ASO targets an intron 22 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 22.
  • the ASO targets an intron 22 sequence about 6 to about 497 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 22.
  • the ASO targets an intron 22 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising 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 a TSC2 RIC pre-mRNA comprising the retained intron 22.
  • the ASO targets exon 25 or exon 26 of a TSC2 RIC pre-mRNA comprising a retained intron 25, wherein the intron numbering correspond to the mRNA sequence at NM 001318832.
  • the ASO targets an exon 25 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
  • the ASO targets an exon 25 sequence about 4 to about 74 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
  • the ASO targets an ex on 26 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising 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 a TSC2 RIC pre-mRNA comprising the retained intron 25.
  • the ASO targets intron 25 in a TSC2 RIC pre-mRNA comprising a retained intron 25, wherein the intron numbering correspond to the mRNA sequence at NM 001318832.
  • the ASO targets an intron 25 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
  • the ASO targets an intron 25 sequence about 6 to about 497 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 25.
  • the ASO targets an intron 25 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising 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 a TSC2 RIC pre-mRNA comprising the retained intron 25.
  • the ASO targets exon 4 or exon 5 of a TSC2 RIC pre-mRNA comprising a retained intron 4, wherein the intron numbering correspond to the mRNA sequence at NM 001318832.
  • the ASO targets an exon 4 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
  • the ASO targets an exon 4 sequence about 4 to about 94 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
  • the ASO targets an exon 5 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4. In some embodiments, the ASO targets an exon 5 sequence about 2 to about 127 nucleotides downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
  • the ASO targets intron 4 in a TSC2 RIC pre-mRNA comprising a retained intron 4, wherein the intron numbering correspond to the mRNA sequence at
  • the ASO targets an intron 4 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4. In some embodiments, the ASO targets an intron 4 sequence about 6 to about 435 nucleotides downstream (or 3 ') from the 5' splice site of a TSC2 RIC pre-mRNA comprising 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 pre-mRNA comprising the retained intron 4. In some embodiments, the ASO targets an intron 4 sequence about 16 to about 432 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 4.
  • the ASO targets exon 30 or exon 31 of a TSC2 RIC pre-mRNA comprising a retained intron 30, wherein the intron numbering correspond to the mRNA sequence at NM 001318832.
  • the ASO targets an exon 30 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 30.
  • the ASO targets an exon 30 sequence about 4 to about 184 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 30.
  • the ASO targets an exon 31 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the 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 a TSC2 RIC pre-mRNA comprising the retained intron 30.
  • the ASO targets intron 30 in a TSC2 RIC pre-mRNA comprising a retained intron 30, wherein the intron numbering correspond to the mRNA sequence at NM 001318832.
  • the ASO targets an intron 30 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 30.
  • 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 pre-mRNA comprising the retained intron 30.
  • the ASO targets an intron 30 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 30. In some embodiments, the ASO targets an intron 30 sequence about 16 to about 500 or about 36 to about 500 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 30.
  • the ASO targets exon 24 or exon 25 of a TSC2 RIC pre-mRNA comprising a retained intron 24, wherein the intron numbering correspond to the mRNA sequence at NM_001318827.
  • the ASO targets an exon 24 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 24.
  • 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 pre-mRNA comprising the retained intron 24.
  • the ASO targets an exon 25 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the 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 pre-mRNA comprising the retained intron 24. [0088] In some embodiments, the ASO targets intron 24 in a TSC2 RIC pre-mRNA comprising a retained intron 24, wherein the intron numbering correspond to the mRNA sequence at NM 001318827.
  • the ASO targets an intron 24 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising 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 pre-mRNA comprising the retained intron 24. In some embodiments, the ASO targets an intron 24 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising 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 pre-mRNA comprising the retained intron 24.
  • the ASO targets exon 3 or exon 4 of a TSC2 RIC pre-mRNA comprising a retained intron 3, wherein the intron numbering correspond to the mRNA sequence at NM 001318827.
  • the ASO targets an exon 3 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 3.
  • 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 pre-mRNA comprising the retained intron 3.
  • the ASO targets an exon 4 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 3. In some embodiments, the ASO targets an exon 4 sequence about 2 to about 127 nucleotides downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 3.
  • the ASO targets intron 3 in a TSC2 RIC pre-mRNA comprising a retained intron 3, wherein the intron numbering correspond to the mRNA sequence at
  • the ASO targets an intron 3 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the 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 pre-mRNA comprising the 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 pre-mRNA comprising the 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 pre-mRNA comprising the retained intron 3.
  • the ASO targets exon 29 or exon 30 of a TSC2 RIC pre-mRNA comprising a retained intron 29, wherein the intron numbering correspond to the mRNA sequence at NM 001318827.
  • the ASO targets an exon 29 sequence upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 29.
  • the ASO targets an ex on 29 sequence about 4 to about 184 nucleotides upstream (or 5') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 29.
  • the ASO targets an exon 30 sequence downstream (or 3') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the 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 pre-mRNA comprising the retained intron 29.
  • the ASO targets intron 29 in a TSC2 RIC pre-mRNA comprising a retained intron 29, wherein the intron numbering correspond to the mRNA sequence at NM 001318827.
  • the ASO targets an intron 29 sequence downstream (or 3') from the 5' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 29.
  • 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 pre-mRNA comprising the retained intron 29.
  • the ASO targets an intron 29 sequence upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 29. In some embodiments, the ASO targets an intron 29 sequence about 16 to about 500 or about 36 to about 500 nucleotides upstream (or 5') from the 3' splice site of a TSC2 RIC pre-mRNA comprising the retained intron 29.
  • the degree of intron retention can be represented using the metric percent intron retention (PIR), the percentage of transcripts in which a given intron is retained.
  • PIR metric percent intron retention
  • PIR can be calculated as the percentage of the average number of reads mapping to the exon-intron junctions, over the sum of the average of the exon-intron junction reads plus the exon-exon junction reads.
  • the methods described herein are used to increase the production of a functional tuberin protein.
  • the term “functional” refers to the amount of activity or function of a tuberin protein that is necessary to eliminate any one or more symptoms of tuberous sclerosis complex.
  • the methods are used to increase the production of a partially functional tuberin protein.
  • the term “partially functional” refers to any amount of activity or function of the tuberin protein that is less than the amount of activity or function that is necessary to eliminate or prevent any one or more symptoms of a disease or condition.
  • a partially functional protein or RNA will have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%), 85%, at least 90%, or at least 95% less activity relative to the fully functional protein or RNA.
  • the method is a method of increasing the expression of the tuberin protein by cells of a subject having a RIC pre-mRNA encoding the tuberin protein, wherein the subject has tuberous sclerosis complex caused by a deficient amount of activity of tuberin protein, and wherein the deficient amount of the tuberin protein is caused by haploinsufficiency of the tuberin protein.
  • the subject has a first allele encoding a functional tuberin protein, and a second allele from which the tuberin protein is not produced.
  • the subject has a first allele encoding a functional tuberin protein, and a second allele encoding a nonfunctional tuberin protein.
  • the subject has a first allele encoding a functional tuberin protein, and a second allele encoding a partially functional tuberin protein.
  • the antisense oligomer binds to a targeted portion of the RIC pre-mRNA transcribed from the first allele (encoding functional tuberin protein), thereby inducing constitutive splicing of the retained intron from the RIC pre- mRNA, and causing an increase in the level of mature mRNA encoding functional tuberin protein, and an increase in the expression of the tuberin protein in the cells of the subject.
  • the subject has a first allele encoding a functional tuberin protein, and a second allele encoding a partially functional tuberin protein, and the antisense oligomer binds to a targeted portion of the RIC pre-mRNA transcribed from the first allele (encoding functional tuberin protein) or a targeted portion of the RIC pre-mRNA transcribed from the second allele (encoding partially functional tuberin protein), thereby inducing constitutive splicing of the retained intron from the RIC pre-mRNA, and causing an increase in the level of mature mRNA encoding the tuberin protein, and an increase in the expression of functional or partially functional tuberin protein in the cells of the subject.
  • the method is a method of using an ASO to increase the expression of a protein or functional RNA.
  • an ASO is used to increase the expression of tuberin protein in cells of a subject having a RIC pre-mRNA encoding tuberin protein, wherein the subject has a deficiency, e.g., tuberous sclerosis complex, in the amount or function of a tuberin protein.
  • the RIC pre-mRNA transcript that encodes the protein that is causative of the disease or condition is targeted by the ASOs described herein.
  • a RIC pre-mRNA transcript that encodes a protein that is not causative of the disease is targeted by the ASOs.
  • a disease or condition that is the result of a mutation or deficiency of a first protein in a particular pathway may be ameliorated by targeting a RIC pre-mRNA that encodes a second protein, thereby increasing production of the second protein.
  • the function of the second protein is able to compensate for the mutation or deficiency of the first protein (which is causative of the disease or condition).
  • the subject has:
  • the tuberin protein is produced at a reduced level compared to production from a wild-type allele
  • tuberin protein is produced in a form having reduced function
  • the tuberin protein is produced at a reduced level compared to production from a wild-type allele
  • tuberin protein is produced in a form having reduced function
  • the RIC pre-mRNA is transcribed from the first allele and/or the second allele.
  • the ASO binds to a targeted portion of the RIC pre-mRNA transcribed from the first allele or the second allele, thereby inducing constitutive splicing of the retained intron from the RIC pre-mRNA, and causing an increase in the level of mRNA encoding tuberin protein and an increase in the expression of the target protein or functional RNA in the cells of the subject.
  • the target protein or functional RNA having an increase in expression level resulting from the constitutive splicing of the retained intron from the RIC pre- mRNA is either in a form having reduced function compared to the equivalent wild-type protein (partially-functional), or having full function compared to the equivalent wild-type protein (fully-functional).
  • the level of mRNA encoding tuberin protein is increased 1.1 to 10-fold, when compared to the amount of mRNA encoding TSC2 that is produced in a control cell, e.g., one that is not treated with the anti sense oligomer or one that is treated with an anti sense oligomer that does not bind to the targeted portion of the TSC2 RIC pre-mRNA.
  • condition caused by a deficient amount or activity of tuberin protein is not a condition caused by alternative or aberrant splicing of the retained intron to which the
  • the condition caused by a deficient amount or activity of the tuberin protein is not a condition caused by alternative or aberrant splicing of any retained intron in a RIC pre-mRNA encoding the tuberin protein.
  • alternative or aberrant splicing may occur in a pre-mRNA transcribed from the gene, however the compositions and methods of the invention do not prevent or correct this alternative or aberrant splicing.
  • a subject treated using the methods of the invention expresses a partially functional tuberin protein from one allele, wherein the partially functional tuberin protein is caused by a frameshift mutation, a nonsense mutation, a missense mutation, or a partial gene deletion.
  • a subject treated using the methods of the invention expresses a nonfunctional tuberin protein from one allele, wherein the nonfunctional tuberin protein is caused by a frameshift mutation, a nonsense mutation, a missense mutation, a partial gene deletion, in one allele.
  • a subject treated using the methods of the invention has a TSC2 whole gene deletion, in one allele.
  • the subject has a TSC2 missense mutation selected from R611Q/W and R905W.
  • the subject has TSC2 deletion mutation of at least 6 amino acids in exon 38.
  • the subject has TSC2 substitution mutation of arginine 611 for tryptophan or glutamine.
  • Targeted Augmentation of Nuclear Gene Output is used in the methods of the invention to increase expression of a tuberin protein.
  • a retained-intron-containing pre-mRNA (RIC pre-mRNA) encoding tuberin protein is present in the nucleus of a cell.
  • Cells having a TSC2 RIC pre-mRNA that comprises a retained intron, an exon flanking the 5' splice site, and an exon flanking the 3' splice site, encoding the tuberin protein are contacted with antisense oligomers (ASOs) that are
  • Hybridization of the ASOs to the targeted portion of the RIC pre-mRNA results in enhanced splicing at the splice site (5' splice site or 3' splice site) of the retained intron and subsequently increases target protein production.
  • pre-mRNA and “pre-mRNA transcript” may be used interchangeably and refer to any pre-mRNA species that contains at least one intron.
  • pre-mRNA or pre-mRNA transcripts comprise a 5'-7-methylguanosine cap and/or a poly-A tail.
  • pre-mRNA or pre-mRNA transcripts comprise both a 5'-7-methylguanosine cap and a poly-A tail. In some embodiments, the pre-mRNA transcript does not comprise a 5 '-7- methylguanosine cap and/or a poly-A tail.
  • a pre-mRNA transcript is a non-productive messenger RNA (mRNA) molecule if it is not translated into a protein (or transported into the cytoplasm from the nucleus).
  • mRNA non-productive messenger RNA
  • RIC pre-mRNA is a pre- mRNA transcript that contains at least one retained intron.
  • the RIC pre-mRNA contains a retained intron, an ex on flanking the 5' splice site of the retained intron, an exon flanking the 3 ' splice site of the retained intron, and encodes the target protein.
  • An "RIC pre-mRNA encoding a target protein” is understood to encode the target protein when fully spliced.
  • a "retained intron” is any intron that is present in a pre-mRNA transcript when one or more other introns, such as an adjacent intron, encoded by the same gene have been spliced out of the same pre- mRNA transcript.
  • the retained intron is the most abundant intron in RIC pre-mRNA encoding the target protein.
  • the retained intron is the most abundant intron in a population of RIC pre-mRNAs transcribed from the gene encoding the target protein in a cell, wherein the population of RIC pre-mRNAs comprises two or more retained introns.
  • an antisense oligomer targeted to the most abundant intron in the population of RIC pre-mRNAs encoding the target protein induces splicing out of two or more retained introns in the population, including the retained intron to which the antisense oligomer is targeted or binds.
  • a mature mRNA encoding the target protein is thereby produced.
  • the terms "mature mRNA,” and "fully-spliced mRNA,” are used
  • the targeted region is in a retained intron that is the most abundant intron in a RIC pre-mRNA encoding the tuberin protein.
  • the most abundant retained intron in a RIC pre-mRNA encoding the tuberin protein is intron 4, 25, 26, 31, 32, 25 and 26, or 31 and 32.
  • the most abundant retained intron in a RIC pre-mRNA encoding the tuberin protein is intron 26. In embodiments, the most abundant retained intron in a RIC pre-mRNA encoding the tuberin protein is intron 31. In some embodiments, the most abundant retained intron in a RIC pre- mRNA encoding the tuberin protein is intron 26, and the second most abundant retained intron in a RIC pre-mRNA encoding the tuberin protein is intron 31.
  • a retained intron is an intron that is identified as a retained intron based on a determination of at least about 5%, at least about 10%, at least about 15%, at least about 20%), at least about 25%, at least about 30%>, at least about 35%, at least about 40%, at least about 45%), or at least about 50%, retention.
  • a retained intron is an intron that is identified as a retained intron based on a determination of about 5% to about 100%, about 5% to about 95%), about 5% to about 90%, about 5% to about 85%, about 5% to about 80%, about 5% to about 75%, about 5% to about 70%, about 5% to about 65%, about 5% to about 60%, about 5% to about 65%, about 5% to about 60%, about 5% to about 55%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 10% to about 100%, about 10% to about 95%, about 10% to about 90%, about 10% to about 85%, about 10% to about 80%, about 10% to about 75%, about 10% to about 70%, about 10% to about 65%, about 10% to about 60%, about 10% to about 65%, about 10% to about 60%, about 10% to about 5
  • the term “comprise” or variations thereof such as “comprises” or “comprising” are to be read to indicate the inclusion of any recited feature (e.g. in the case of an antisense oligomer, a defined nucleobase sequence) but not the exclusion of any other features.
  • the term “comprising” is inclusive and does not exclude additional, unrecited features (e.g. in the case of an antisense oligomer, the presence of additional, unrecited nucleobases).
  • compositions and methods comprising
  • comprising may be replaced with “consisting essentially of " or “consisting of.”
  • the phrase “consisting essentially of is used herein to require the specified feature(s) (e.g. nucleobase sequence) as well as those which do not materially affect the character or function of the claimed invention.
  • the term “consisting” is used to indicate the presence of the recited feature (e.g. nucleobase sequence) alone (so that in the case of an antisense oligomer consisting of a specified nucleobase sequence, the presence of additional, unrecited nucleobases is excluded).
  • the targeted region is in a retained intron that is the second most abundant intron in a RIC pre-mRNA encoding the tuberin protein.
  • the second most abundant retained intron may be targeted rather than the most abundant retained intron due to the uniqueness of the nucleotide sequence of the second most abundant retained intron, ease of ASO design to target a particular nucleotide sequence, and/or amount of increase in protein production resulting from targeting the intron with an ASO.
  • the retained intron is the second most abundant intron in a population of RIC pre-mRNAs transcribed from the gene encoding the target protein in a cell, wherein the population of RIC pre-mRNAs comprises two or more retained introns.
  • an antisense oligomer targeted to the second most abundant intron in the population of RIC pre-mRNAs encoding the target protein induces splicing out of two or more retained introns in the population, including the retained intron to which the antisense oligomer is targeted or binds.
  • fully-spliced (mature) RNA encoding the target protein is thereby produced.
  • the second most retained intron in a RIC pre-mRNA encoding the tuberin protein is intron 4, 25, 26, 31, 32, 25 and 26, or 31 and 32.
  • the second most abundant retained intron in a RIC pre- mRNA encoding the tuberin protein is intron 31.
  • an ASO is complementary to a targeted region that is within a non- retained intron in a RIC pre-mRNA.
  • the targeted portion of the RIC pre- mRNA is within: the region +6 to +100 relative to the 5' splice site of the non-retained intron; or the region -16 to -100 relative to the 3' splice site of the non-retained intron.
  • the targeted portion of the RIC pre-mRNA is within the region +100 relative to the 5' splice site of the non-retained intron to -100 relative to the 3' splice site of the non-retained intron.
  • RNA encoding the target protein is thereby produced.
  • the retained intron of the RIC pre-mRNA is an inefficiently spliced intron.
  • "inefficiently spliced” may refer to a relatively low frequency of splicing at a splice site adjacent to the retained intron (5' splice site or 3' splice site) as compared to the frequency of splicing at another splice site in the RIC pre-mRNA.
  • inefficiently spliced may also refer to the relative rate or kinetics of splicing at a splice site, in which an "inefficiently spliced" intron may be spliced or removed at a slower rate as compared to another intron in a RIC pre-mRNA.
  • the 9-nucleotide sequence at -3e to -le of the exon flanking the 5' splice site and +1 to +6 of the retained intron is identical to the corresponding wild-type sequence.
  • the 16 nucleotide sequence at -15 to -1 of the retained intron and +le of the exon flanking the 3' splice site is identical to the corresponding wild-type sequence.
  • 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.
  • wild-type sequence refers to the canonical sequence for the TSC2 gene found at NCBI Gene ID 7249.
  • nucleotide position denoted with an "e” indicates the nucleotide is present in the sequence of an exon (e.g., the exon flanking the 5' splice site or the exon flanking the 3' splice site).
  • the methods involve contacting cells with an ASO that is complementary to a portion of a pre-mRNA encoding tuberin protein, resulting in increased expression of TSC2.
  • contacting or administering to cells refers to any method of providing an ASO in immediate proximity with the cells such that the ASO and the cells interact.
  • a cell that is contacted with an ASO will take up or transport the ASO into the cell.
  • the method involves contacting a condition or disease-associated or condition or disease-relevant cell with any of the ASOs described herein.
  • the ASO may be further modified or attached (e.g., covalently attached) to another molecule to target the ASO to a cell type, enhance contact between the ASO and the condition or disease-associated or condition or disease-relevant cell, or enhance uptake of the ASO.
  • the term “increasing protein production” or “increasing expression of a target protein” means enhancing the amount of protein that is translated from an mRNA in a cell.
  • a “target protein” may be any protein for which increased expression/production is desired.
  • contacting a cell that expresses a TSC2 RIC pre-mRNA with an ASO that is complementary to a targeted portion of the TSC2 RIC pre-mRNA transcript results in a measurable increase in the amount of the tuberin protein (e.g., a target protein) encoded by the pre-mRNA.
  • Methods of measuring or detecting production of a protein will be evident to one of skill in the art and include any known method, for example, Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA.
  • contacting cells with an ASO that is complementary to a targeted portion of a TSC2 RIC pre-mRNA transcript results in an increase in the amount of tuberin protein produced by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000%, compared to the amount of the protein produced by a cell in the absence of the ASO/absence of treatment.
  • the total amount of tuberin protein produced by the cell to which the antisense oligomer was contacted is increased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5 -fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 1.1 to
  • contacting cells with an ASO that is complementary to a targeted portion of a TSC2 RIC pre-mRNA transcript results in an increase in the amount of mRNA encoding TSC2, including the mature mRNA encoding the target protein.
  • the amount of mRNA encoding tuberin protein, or the mature mRNA encoding the tuberin protein is increased by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000%, compared to the amount of the protein produced by a cell in the absence of the ASO/absence of treatment.
  • the total amount of the mRNA encoding tuberin protein, or the mature mRNA encoding tuberin protein produced in the cell to which the antisense oligomer was contacted is increased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8- fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 6-fold, about 3 to about
  • a control compound can be, for example, an oligonucleotide that is not complementary to the targeted portion of the TSC2 RIC pre-mRNA.
  • the methods and antisense oligonucleotide compositions provided herein are useful for increasing the expression of tuberin protein in cells, for example, in a subject having Tuberous
  • Sclerosis Complex caused by a deficiency in the amount or activity of tuberin protein, by increasing the level of mRNA encoding tuberin protein, or the mature mRNA encoding tuberin protein.
  • the methods and compositions as described herein induce the constitutive splicing of a retained intron from a TSC2 RIC pre-mRNA transcript encoding tuberin protein, thereby increasing the level of mRNA encoding tuberin protein, or the mature mRNA encoding tuberin protein and increasing the expression of tuberin protein.
  • Constitutive splicing of a retained intron from a RIC pre-mRNA correctly removes the retained intron from the RIC pre-mRNA, wherein the retained intron has wild-type splice sequences.
  • Constitutive splicing does not encompass splicing of a retained intron from a RIC pre-mRNA transcribed from a gene or allele having a mutation that causes alternative splicing or aberrant splicing of a pre-mRNA transcribed from the gene or allele.
  • constitutive splicing of a retained intron does not correct aberrant splicing in or influence alternative splicing of a pre-mRNA to result in an increased expression of a target protein or functional RNA.
  • constitutive splicing correctly removes a retained intron from a TSC2 RIC pre-mRNA, wherein the TSC2 RIC pre-mRNA is transcribed from a wild-type gene or allele, or a polymorphic gene or allele, that encodes a fully-functional target protein or functional RNA, and wherein the gene or allele does not have a mutation that causes alternative splicing or aberrant splicing of the retained intron.
  • constitutive splicing of a retained intron from a TSC2 RIC pre- mRNA encoding tuberin protein correctly removes a retained intron from a TSC2 RIC pre- mRNA encoding tuberin protein, wherein the TSC2 RIC pre-mRNA is transcribed from a gene or allele from which the target gene or functional RNA is produced at a reduced level compared to production from a wild-type allele, and wherein the gene or allele does not have a mutation that causes alternative splicing or aberrant splicing of the retained intron.
  • the correct removal of the constitutively spliced retained intron results in production of target protein or functional RNA that is functional when compared to an equivalent wild-type protein or functional RNA.
  • constitutive splicing correctly removes a retained intron from a TSC2 RIC pre-mRNA, wherein the TSC2 RIC pre-mRNA is transcribed from a gene or allele that encodes a target protein or functional RNA produced in a form having reduced function compared to an equivalent wild-type protein or functional RNA, and wherein the gene or allele does not have a mutation that causes alternative splicing or aberrant splicing of the retained intron.
  • the correct removal of the constitutively spliced retained intron results in production of partially functional target protein, or functional RNA that is partially functional when compared to an equivalent wild-type protein or functional RNA.
  • an antisense oligomer as described herein or used in any method described herein does not increase the amount of mRNA encoding tuberin protein or the amount of tuberin protein by modulating alternative splicing or aberrant splicing of a pre-mRNA transcribed from the TSC2 gene.
  • Modulation of alternative splicing or aberrant splicing can be measured using any known method for analyzing the sequence and length of RNA species, e.g., by RT-PCR and using methods described elsewhere herein and in the literature.
  • modulation of alternative or aberrant splicing is determined based on an increase or decrease in the amount of the spliced species of interest of at least 10% or 1.1-fold. In embodiments, modulation is determined based on an increase or decrease at a level that is at least 10% to 100% or 1.1 to 10-fold, as described herein regarding determining an increase in mRNA encoding tuberin protein in the methods of the invention.
  • the method is a method wherein the TSC2 RIC pre-mRNA was produced by partial splicing of a wild-type TSC2 pre-mRNA. In embodiments, the method is a method wherein the TSC2 RIC pre-mRNA was produced by partial splicing of a full-length wild-type TSC2 pre-mRNA. In embodiments, the TSC2 RIC pre-mRNA was produced by partial splicing of a full-length TSC2 pre-mRNA.
  • a full-length TSC2 pre- mRNA may have a polymorphism in a splice site of the retained intron that does not impair correct splicing of the retained intron as compared to splicing of the retained intron having the wild-type splice site sequence.
  • the mRNA encoding tuberin protein is a full-length mature mRNA, or a wild-type mature mRNA.
  • a full-length mature mRNA may have a polymorphism that does not affect the activity of the target protein or the functional RNA encoded by the mature mRNA, as compared to the activity of tuberin protein encoded by the wild-type mature mRNA.
  • compositions comprising antisense oligomers that enhances splicing by binding to a targeted portion of a TSC2 RIC pre-mRNA.
  • ASO antisense oligomer
  • the terms "ASO” and "antisense oligomer” are used interchangeably and refer to an oligomer such as a polynucleotide, comprising nucleobases that hybridizes to a target nucleic acid (e.g., a TSC2 RIC pre-mRNA) sequence by Watson-Crick base pairing or wobble base pairing (G-U).
  • the ASO may have exact sequence complementary to the target sequence or near complementarity (e.g., sufficient complementarity to bind the target sequence and enhancing splicing at a splice site).
  • ASOs are designed so that they bind (hybridize) to a target nucleic acid (e.g., a targeted portion of a pre-mRNA transcript) and remain hybridized under physiological conditions. Typically, if they hybridize to a site other than the intended (targeted) nucleic acid sequence, they hybridize to a limited number of sequences that are not a target nucleic acid (to a few sites other than a target nucleic acid).
  • Design of an ASO can take into consideration the occurrence of the nucleic acid sequence of the targeted portion of the pre- mRNA transcript or a sufficiently similar nucleic acid sequence in other locations in the genome or cellular pre-mRNA or transcriptome, such that the likelihood the ASO will bind other sites and cause "off-target” effects is limited.
  • Any antisense oligomers known in the art for example in PCT Application No. PCT/US2014/054151, published as WO 2015/035091, titled "Reducing Nonsense-Mediated mRNA Decay,” can be used to practice the methods described herein.
  • ASOs "specifically hybridize” to or are “specific” to a target nucleic acid or a targeted portion of a RIC pre-mRNA.
  • hybridization occurs with a Tm substantially greater than 37°C, preferably at least 50°C, and typically between 60°C to approximately 90°C.
  • Tm substantially greater than 37°C, preferably at least 50°C, and typically between 60°C to approximately 90°C.
  • Such hybridization preferably corresponds to stringent hybridization conditions.
  • the Tm is the temperature at which 50% of a target sequence hybridizes to a complementary oligonucleotide.
  • Oligomers such as oligonucleotides, are "complementary" to one another when hybridization occurs in an antiparallel configuration between two single-stranded
  • a double-stranded polynucleotide can be "complementary" to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second.
  • Complementarity (the degree to which one polynucleotide is complementary with another) is quantifiable in terms of the proportion (e.g., the percentage) of bases in opposing strands that are expected to form hydrogen bonds with each other, according to generally accepted base-pairing rules.
  • the sequence of an antisense oligomer (ASO) need not be 100%) complementary to that of its target nucleic acid to hybridize.
  • ASOs can comprise at least 70%>, at least 75%>, at least 80%>, at least 85%>, at least 90%>, at least 95%, at least 96%>, at least 97%>, at least 98%>, or at least 99%> sequence complementarity to a target region within the target nucleic acid sequence to which they are targeted.
  • an ASO in which 18 of 20 nucleobases of the oligomeric compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent
  • the remaining non-complementary nucleobases may be clustered together or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases.
  • Percent complementarity of an ASO with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).
  • An ASO need not hybridize to all nucleobases in a target sequence and the nucleobases to which it does hybridize may be contiguous or noncontiguous. ASOs may hybridize over one or more segments of a pre-mRNA transcript, such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure may be formed). In certain embodiments, an ASO hybridizes to noncontiguous nucleobases in a target pre-mRNA transcript. For example, an ASO can hybridize to nucleobases in a pre-mRNA transcript that are separated by one or more nucleobase(s) to which the ASO does not hybridize.
  • ASOs described herein comprise nucleobases that are complementary to
  • ASO embodies oligonucleotides and any other oligomeric molecule that comprises nucleobases capable of hybridizing to a complementary nucleobase on a target mRNA but does not comprise a sugar moiety, such as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the ASOs may comprise naturally-occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination of two or three of the preceding.
  • naturally occurring nucleotides includes deoxyribonucleotides and ribonucleotides.
  • modified nucleotides includes nucleotides with modified or substituted sugar groups and/or having a modified backbone. In some embodiments, all of the nucleotides of the ASO are modified nucleotides. Chemical modifications of ASOs or components of ASOs that are compatible with the methods and compositions described herein will be evident to one of skill in the art and can be found, for example, in U. S. Patent No.
  • the nucleobase of an ASO may be any naturally occurring, unmodified nucleobase such as adenine, guanine, cytosine, thymine and uracil, or any synthetic or modified nucleobase that is sufficiently similar to an unmodified nucleobase such that it is capable of hydrogen bonding with a nucleobase present on a target pre-mRNA.
  • modified nucleobases include, without limitation, hypoxanthine, xanthine, 7-methylguanine, 5, 6-dihydrouracil, 5- m ethyl cytosine, and 5 -hydroxymethoyl cytosine.
  • the ASOs described herein also comprise a backbone structure that connects the components of an oligomer.
  • backbone structure and “oligomer linkages” may be used interchangeably and refer to the connection between monomers of the ASO.
  • the backbone comprises a 3 '-5 ' phosphodiester linkage connecting sugar moieties of the oligomer.
  • the backbone structure or oligomer linkages of the ASOs described herein may include (but are not limited to) phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoramidate, and the like. See e.g., LaPlanche et al., Nucleic Acids Res. 14:9081 (1986); Stec et al., J. Am. Chem. Soc. 106:6077 (1984), Stein et al., Nucleic Acids Res.
  • the backbone structure of the ASO does not contain phosphorous but rather contains peptide bonds, for example in a peptide nucleic acid (PNA), or linking groups including carbamate, amides, and linear and cyclic hydrocarbon groups.
  • PNA peptide nucleic acid
  • the backbone modification is a phosphorothioate linkage. In some embodiments, the backbone modification is a phosphoramidate linkage.
  • the stereochemistry at each of the phosphorus internucleotide linkages of the ASO backbone is random. In embodiments, the stereochemistry at each of the
  • an ASO used in the methods of the invention comprises an ASO having phosphorus internucleotide linkages that are not random.
  • a composition used in the methods of the invention comprises a pure diastereomeric ASO.
  • a composition used in the methods of the invention comprises an ASO that has diastereomeric purity of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%), at least about 96%>, at least about 97%>, at least about 98%>, at least about 99%>, about 100%, about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100% , or about 99% to about 100%.
  • the ASO has a nonrandom mixture of Rp and Sp configurations at its phosphorus internucleotide linkages.
  • Rp and Sp are required in antisense oligonucleotides to achieve a balance between good activity and nuclease stability.
  • an ASO used in the methods of the invention comprises about 5-100% Rp, at least about 5% Rp, at least about 10%) Rp, at least about 15% Rp, at least about 20% Rp, at least about 25% Rp, at least about 30%) Rp, at least about 35% Rp, at least about 40% Rp, at least about 45% Rp, at least about 50% Rp, at least about 55% Rp, at least about 60% Rp, at least about 65% Rp, at least about 70%) Rp, at least about 75% Rp, at least about 80% Rp, at least about 85% Rp, at least about 90%) Rp, or at least about 95% Rp, with the remainder Sp, or about 100% Rp.
  • an ASO used in the methods of the invention comprises about 10% to about 100% Rp, about 15% to about 100% Rp, about 20% to about 100% Rp, about 25% to about 100% Rp, about 30% to about 100% Rp, about 35% to about 100% Rp, about 40% to about 100% Rp, about 45% to about 100% Rp, about 50% to about 100% Rp, about 55% to about 100% Rp, about 60% to about 100% Rp, about 65% to about 100% Rp, about 70% to about 100% Rp, about 75% to about 100% Rp, about 80% to about 100% Rp, about 85% to about 100% Rp, about 90% to about 100% Rp, or about 95% to about 100% Rp, about 20% to about 80% Rp, about 25% to about 75% Rp, about 30% to about 70% Rp, about 40% to about 60% Rp, or about 45% to about 55%) Rp, with the remainder Sp.
  • an ASO used in the methods of the invention comprises about 5-100%> Sp, at least about 5% Sp, at least about 10% Sp, at least about 15% Sp, at least about 20% Sp, at least about 25% Sp, at least about 30% Sp, at least about 35% Sp, at least about 40% Sp, at least about 45% Sp, at least about 50% Sp, at least about 55% Sp, at least about 60% Sp, at least about 65% Sp, at least about 70%) Sp, at least about 75% Sp, at least about 80% Sp, at least about 85% Sp, at least about 90%) Sp, or at least about 95% Sp, with the remainder Rp, or about 100% Sp.
  • an ASO used in the methods of the invention comprises about 10% to about 100% Sp, about 15% to about 100% Sp, about 20% to about 100% Sp, about 25% to about 100% Sp, about 30% to about 100% Sp, about 35% to about 100% Sp, about 40% to about 100% Sp, about 45% to about 100% Sp, about 50% to about 100% Sp, about 55% to about 100% Sp, about 60% to about 100% Sp, about 65% to about 100% Sp, about 70% to about 100% Sp, about 75% to about 100% Sp, about 80% to about 100% Sp, about 85% to about 100% Sp, about 90% to about 100% Sp, or about 95% to about 100% Sp, about 20% to about 80% Sp, about 25% to about 75% Sp, about 30% to about 70% Sp, about 40% to about 60% Sp, or about 45% to about 55% Sp, with the remainder Rp.
  • Any of the ASOs described herein may contain a sugar moiety that comprises ribose or deoxyribose, as present in naturally occurring nucleotides, or a modified sugar moiety or sugar analog, including a morpholine ring.
  • modified sugar moieties include 2' substitutions such as 2'-0-methyl (2'-0-Me), 2'-0-methoxyethyl (2'MOE), 2'-0-aminoethyl, 2'F; N3'->P5' phosphoramidate, 2'dimethylaminooxyethoxy, 2'dimethylaminoethoxyethoxy, 2'-guanidinidium, 2'-0-guanidinium ethyl, carbamate modified sugars, and bicyclic modified sugars.
  • the sugar moiety modification is selected from 2'-0-Me, 2'F, and 2'MOE.
  • the sugar moiety modification is an extra bridge bond, such as in a locked nucleic acid (LNA).
  • the sugar analog contains a morpholine ring, such as phosphorodiamidate morpholino (PMO).
  • the sugar moiety comprises a ribofuransyl or 2'deoxyribofuransyl modification.
  • the sugar moiety comprises 2' 4' -constrained 2'0-methyloxyethyl (cMOE) modifications.
  • the sugar moiety comprises cEt 2', 4' constrained 2'-0 ethyl BNA modifications.
  • the sugar moiety comprises tricycloDNA (tcDNA) modifications.
  • the sugar moiety comprises ethylene nucleic acid (ENA) modifications.
  • the sugar moiety comprises MCE modifications. Modifications are known in the art and described in the literature, e.g., by Jarver, et al., 2014, "A Chemical View of Oligonucleotides for Exon Skipping and Related Drug Applications,” Nucleic Acid Therapeutics 24(1): 37-47, incorporated by reference for this purpose herein.
  • each monomer of the ASO is modified in the same way, for example each linkage of the backbone of the ASO comprises a phosphorothioate linkage or each ribose sugar moiety comprises a 2'O-methyl modification.
  • Such modifications that are present on each of the monomer components of an ASO are referred to as “uniform modifications.”
  • a combination of different modifications may be desired, for example, an ASO may comprise a combination of phosphorodiamidate linkages and sugar moieties comprising morpholine rings (morpholinos).
  • Combinations of different modifications to an ASO are referred to as “mixed modifications” or “mixed chemistries.”
  • the ASO comprises one or more backbone modification. In some embodiments, the ASO comprises one or more sugar moiety modification. In some
  • the ASO comprises one or more backbone modification and one or more sugar moiety modification.
  • the ASO comprises 2'MOE modifications and a phosphorothioate backbone.
  • the ASO comprises a phosphorodiamidate morpholino (PMO).
  • the ASO comprises a peptide nucleic acid (PNA). Any of the ASOs or any component of an ASO (e.g., a nucleobase, sugar moiety, backbone) described herein may be modified in order to achieve desired properties or activities of the ASO or reduce undesired properties or activities of the ASO.
  • an ASO or one or more component of any ASO may be modified to enhance binding affinity to a target sequence on a pre-mRNA transcript; reduce binding to any non-target sequence; reduce degradation by cellular nucleases (i.e., RNase H); improve uptake of the ASO into a cell and/or into the nucleus of a cell; alter the pharmacokinetics or pharmacodynamics of the ASO; and modulate the half-life of the ASO.
  • RNase H cellular nucleases
  • the ASOs are comprised of 2'-0-(2-methoxyethyl) (MOE) phosphorothioate-modified nucleotides.
  • MOE 2-methoxyethyl
  • ASOs comprised of such nucleotides are especially well-suited to the methods disclosed herein; oligomers having such modifications have been shown to have significantly enhanced resistance to nuclease degradation and increased bioavailability, making them suitable, for example, for oral delivery in some embodiments described herein. See e.g., Geary et al., J Pharmacol Exp Ther. 2001; 296(3):890-7; Geary et al., J Pharmacol Exp Ther. 2001; 296(3):898-904.
  • ASOs may be obtained from a commercial source.
  • the left-hand end of single-stranded nucleic acid e.g., pre- mRNA transcript, oligonucleotide, ASO, etc.
  • sequences is the 5' end and the left-hand direction of single or double-stranded nucleic acid sequences is referred to as the 5' direction.
  • the right-hand end or direction of a nucleic acid sequence is the 3' end or direction.
  • nucleotides that are upstream of a reference point in a nucleic acid may be designated by a negative number, while nucleotides that are downstream of a reference point may be designated by a positive number.
  • a reference point e.g., an exon- ex on junction in mRNA
  • a nucleotide that is directly adjacent and upstream of the reference point is designated “minus one,” e.g., while a nucleotide that is directly adjacent and downstream of the reference point is designated “plus one,” e.g., "+1.”
  • the ASOs are complementary to (and bind to) a targeted portion of a TSC2 RIC pre-mRNA that is downstream (in the 3' direction) of the 5' splice site of the retained intron in a TSC2 RIC pre-mRNA (e.g., the direction designated by positive numbers relative to the 5' splice site) (FIG. 1).
  • the ASOs are complementary to a targeted portion of the TSC2 RIC pre-mRNA that is within the region +6 to +500, +6 to +400, +6 to +300, +6 to +200, or +6 to +100 relative to the 5' splice site of the retained intron.
  • the ASO is not complementary to nucleotides +1 to +5 relative to the 5' splice site (the first five nucleotides located downstream of the 5' splice site).
  • the ASOs may be complementary to a targeted portion of a TSC2 RIC pre-mRNA that is within the region between nucleotides +6 and +50 relative to the 5' splice site of the retained intron.
  • the ASOs are complementary to a targeted portion that is within the region +6 to +90, +6 to +80, +6 to +70, +6 to +60, +6 to +50, +6 to +40, +6 to +30, or +6 to +20 relative to 5' splice site of the retained intron.
  • the ASOs are complementary to a targeted region of a TSC2 RIC pre-mRNA that is upstream (5' relative) of the 3 ' splice site of the retained intron in a TSC2 RIC pre-mRNA (e.g., in the direction designated by negative numbers) (FIG. 1).
  • a TSC2 RIC pre-mRNA that is upstream (5' relative) of the 3 ' splice site of the retained intron in a TSC2 RIC pre-mRNA (e.g., in the direction designated by negative numbers) (FIG. 1).
  • the ASOs are complementary to a targeted portion of the TSC2 RIC pre-mRNA that is within the region -16 to -500, -16 to -400, -16 to -300, -6 to -200, -16 to -100, relative to the 3' splice site of the retained intron.
  • the ASO is not complementary to nucleotides -1 to -15 relative to the 3' splice site (the first 15 nucleotides located upstream of the 3' splice site).
  • the ASOs are complementary to a targeted portion of the TSC2 RIC pre-mRNA that is within the region -16 to -50 relative to the 3' splice site of the retained intron.
  • the ASOs are complementary to a targeted portion that is within the region -16 to -90, -16 to -80, -16 to -70, -16 to -60, -16 to -50, -16 to -40, or -16 to -30 relative to 3' splice site of the retained intron.
  • the targeted portion of the TSC2 RIC pre-mRNA is within the region +100 relative to the 5' splice site of the retained intron to -100 relative to the 3' splice site of the retained intron.
  • the ASOs are complementary to a targeted portion of a TSC2 RIC pre-mRNA that is within the ex on flanking the 5' splice site (upstream) of the retained intron (FIG. 1). In some embodiments, the ASOs are complementary to a targeted portion of the TSC2 RIC pre-mRNA that is within the region +2e to -4e in the exon flanking the 5' splice site of the retained intron. In some embodiments, the ASOs are not complementary to nucleotides -le to - 3e relative to the 5' splice site of the retained intron.
  • the ASOs are complementary to a targeted portion of the TSC2 RIC pre-mRNA that is within the region -4e to-lOOe, -4e to -90e, -4e to -80e, -4e to -70e, -4e to -60e, -4e to -50e, -4 to -40e, -4e to -30e, or - 4e to -20e relative to the 5' splice site of the retained intron.
  • the ASOs are complementary to a targeted portion of a TSC2 RIC pre-mRNA that is within the exon flanking the 3' splice site (downstream) of the retained intron (FIG. 1). In some embodiments, the ASOs are complementary to a targeted portion to the TSC2 RIC pre-mRNA that is within the region +2e to -4e in the exon flanking the 3' splice site of the retained intron. In some embodiments, the ASOs are not complementary to nucleotide +le relative to the 3' splice site of the retained intron.
  • the ASOs are complementary to a targeted portion of the TSC2 RIC pre-mRNA that is within the region+2e to +100e, +2e to +90e, +2e to +80e, +2e to +70e, +2e to +60e, +2e to +50e, +2e to +40e, +2e to +30e, or +2 to +20e relative to the 3' splice site of the retained intron.
  • the ASOs may be of any length suitable for specific binding and effective enhancement of splicing. In some
  • the ASOs consist of 8 to 50 nucleobases.
  • the ASO may be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, or 50 nucleobases in length.
  • the ASOs consist of more than 50 nucleobases.
  • the ASO is from 8 to 50 nucleobases, 8 to 40 nucleobases,
  • nucleobases 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, 12 to 15 nucleobases, 13 to 50 nucleobases, 13 to 40 nucleobases, 13 to 35 nucleobases, 13 to 30 nucleo
  • ASOs are 30 nucleotides in length. In some embodiments, the ASOs are 29 nucleotides in length. In some embodiments, the ASOs are 28 nucleotides in length. In some embodiments, the ASOs are 27 nucleotides in length. In some embodiments, the ASOs are 26 nucleotides in length. In some embodiments, the ASOs are 25 nucleotides in length. In some embodiments, the ASOs are 24 nucleotides in length. In some embodiments, the ASOs are 23 nucleotides in length. In some embodiments, the ASOs are 22 nucleotides in length. In some embodiments, the ASOs are 21 nucleotides in length.
  • two or more ASOs with different chemistries but complementary to the same targeted portion of the RIC pre-mRNA are used. In some embodiments, two or more ASOs that are complementary to different targeted portions of the RIC pre-mRNA are used.
  • the antisense oligonucleotides of the invention are chemically linked to one or more moieties or conjugates, e.g., a targeting moiety or other conjugate that enhances the activity or cellular uptake of the oligonucleotide.
  • moieties include, but are not limited to, a lipid moiety, e.g., as a cholesterol moiety, a cholesteryl moiety, an aliphatic chain, e.g., dodecandiol or undecyl residues, a polyamine or a polyethylene glycol chain, or adamantane acetic acid.
  • the antisense oligonucleotide is conjugated with a moiety including, but not limited to, an abasic nucleotide, a polyether, a polyamine, a polyamide, a peptides, a carbohydrate, e.g., N-acetylgalactosamine (GalNAc), N- Ac-Glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate), a lipid, or a
  • Conjugates can be linked to one or more of any nucleotides comprising the antisense oligonucleotide at any of several positions on the sugar, base or phosphate group, as understood in the art and described in the literature, e.g., using a linker.
  • Linkers can include a bivalent or trivalent branched linker.
  • the conjugate is attached to the 3' end of the antisense oligonucleotide.
  • the nucleic acid to be targeted by an ASO is a TSC2 RIC pre- mRNA expressed in a cell, such as a eukaryotic cell.
  • a cell may refer to a population of cells.
  • the cell is in a subject.
  • the cell is isolated from a subject. In some embodiments, the cell is ex vivo. In some embodiments, the cell is a condition or disease-relevant cell or a cell line. In some embodiments, the cell is in vitro ⁇ e.g., in cell culture).
  • compositions or formulations comprising the antisense oligonucleotide of the described compositions and for use in any of the described methods can be prepared according to conventional techniques well known in the pharmaceutical industry and described in the published literature.
  • a pharmaceutical composition or formulation for treating a subject comprises an effective amount of any antisense oligomer as described above, or a pharmaceutically acceptable salt, solvate, hydrate or ester thereof, and a pharmaceutically acceptable diluent.
  • the antisense oligomer of a pharmaceutical formulation may further comprise a pharmaceutically acceptable excipient, diluent or carrier.
  • salts can be prepared in situ during the final isolation and purification of the compounds, or separately by reacting the free base function with a suitable organic acid.
  • suitable organic acid examples include salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other documented methodologies such as ion exchange.
  • salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphor sulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemi sulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, peroxine sodium
  • pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.
  • compositions are formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas.
  • the compositions are formulated as suspensions in aqueous, non-aqueous or mixed media.
  • Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium
  • a pharmaceutical formulation or composition of the present invention includes, but is not limited to, a solution, emulsion, microemulsion, foam or liposome-containing formulation (e.g., cationic or noncationic liposomes).
  • the pharmaceutical composition or formulation of the present invention may comprise one or more penetration enhancer, carrier, excipients or other active or inactive ingredients as appropriate and well known to those of skill in the art or described in the published literature.
  • liposomes also include sterically stabilized liposomes, e.g., liposomes comprising one or more specialized lipids. These specialized lipids result in liposomes with enhanced circulation lifetimes.
  • a sterically stabilized liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety.
  • a surfactant is included in the pharmaceutical formulation or compositions.
  • the present invention employs a penetration enhancer to effect the efficient delivery of the antisense oligonucleotide, e.g., to aid diffusion across cell membranes and /or enhance the permeability of a lipophilic drug.
  • the penetration enhancers are a surfactant, fatty acid, bile salt, chelating agent, or non-chelating non-surfactant.
  • the pharmaceutical formulation comprises multiple antisense oligonucleotides.
  • the antisense oligonucleotide is administered in combination with another drug or therapeutic agent.
  • the antisense oligonucleotide is administered with one or more agents capable of promoting penetration of the subject antisense oligonucleotide across the blood-brain barrier by any method known in the art. For example, delivery of agents by administration of an adenovirus vector to motor neurons in muscle tissue is described in U.S. Pat. No. 6,632,427, "Adenoviral -vector-mediated gene transfer into medullary motor neurons,” incorporated herein by reference.
  • vectors directly to the brain e.g., the striatum, the thalamus, the hippocampus, or the substantia nigra
  • Delivery of vectors directly to the brain e.g., the striatum, the thalamus, the hippocampus, or the substantia nigra, is described, e.g., in U.S. Pat. No. 6,756,523, "Adenovirus vectors for the transfer of foreign genes into cells of the central nervous system particularly in brain," incorporated herein by reference.
  • the antisense oligonucleotides are linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties.
  • the antisense oligonucleotide is coupled to a substance, known in the art to promote penetration or transport across the blood-brain barrier, e.g., an antibody to the transferrin receptor.
  • the antisense oligonucleotide is linked with a viral vector, e.g., to render the antisense compound more effective or increase transport across the blood-brain barrier.
  • osmotic blood brain barrier disruption is assisted by infusion of sugars, e.g.,, meso erythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose, dulcitol, myo-inositol, L(-) fructose, D(-) mannitol, D(+) glucose, D(+) arabinose, D(-) arabinose, cellobiose, D(+) maltose, D(+) raffinose, L(+) rhamnose, D(+) melibiose, D(-) ribose, adonitol, D(+) arabitol, L(-) arabitol, D(+) fucose, L(-) fucose, D(-) lyxose, L(+) lyxose, and L(-
  • the antisense oligonucleotides of the invention are chemically linked to one or more moieties or conjugates, e.g., a targeting moiety or other conjugate that enhances the activity or cellular uptake of the oligonucleotide.
  • moieties include, but are not limited to, a lipid moiety, e.g., as a cholesterol moiety, a cholesteryl moiety, an aliphatic chain, e.g., dodecandiol or undecyl residues, a polyamine or a polyethylene glycol chain, or adamantane acetic acid.
  • the antisense oligonucleotide is conjugated with a moiety including, but not limited to, an abasic nucleotide, a polyether, a polyamine, a polyamide, a peptides, a carbohydrate, e.g., N-acetylgalactosamine (GalNAc), N- Ac-Glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate), a lipid, or a
  • Conjugates can be linked to one or more of any nucleotides comprising the antisense oligonucleotide at any of several positions on the sugar, base or phosphate group, as understood in the art and described in the literature, e.g., using a linker.
  • Linkers can include a bivalent or trivalent branched linker.
  • the conjugate is attached to the 3' end of the antisense oligonucleotide.
  • compositions provided herein may be administered to an individual.
  • “Individual” may be used interchangeably with “subject” or "patient.”
  • An individual may be a mammal, for example a human or animal such as a non-human primate, a rodent, a rabbit, a rat, a mouse, a horse, a donkey, a goat, a cat, a dog, a cow, a pig, or a sheep.
  • the individual is a human.
  • the individual is a fetus, an embryo, or a child.
  • the individual may be another eukaryotic organism, such as a plant.
  • the compositions provided herein are administered to a cell ex vivo.
  • the compositions provided herein are administered to an individual as a method of treating a disease or disorder.
  • the individual has a genetic disease, such as any of the diseases described herein.
  • the individual is at risk of having the disease, such as any of the diseases described herein.
  • the individual is at increased risk of having a disease or disorder caused by insufficient amount of a protein or insufficient activity of a protein. If an individual is "at an increased risk" of having a disease or disorder caused insufficient amount of a protein or insufficient activity of a protein, the method involves preventative or prophylactic treatment.
  • an individual may be at an increased risk of having such a disease or disorder because of family history of the disease.
  • individuals at an increased risk of having such a disease or disorder benefit from prophylactic treatment (e.g., by preventing or delaying the onset or progression of the disease or disorder).
  • Suitable routes for administration of ASOs of the present invention may vary depending on cell type to which delivery of the ASOs is desired. Multiple tissues and organs are affected by tuberous sclerosis complex, with the brain, kidney, skin, lung and heart being the most significantly affected tissues.
  • the ASOs of the present invention may be administered to patients topically to the skin or by pulmonary delivery to the lung.
  • the ASOs of the present invention may be administered to patients parenterally, for example, by intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.
  • delivery is to the brain, kidney, skin, lung or heart.
  • a fetus is treated in utero, e.g., by administering the ASO composition to the fetus directly or indirectly (e.g., via the mother).
  • ASOs that specifically hybridize to different nucleotides within the target region of the pre-mRNA may be screened to identify (determine) ASOs that improve the rate and/or extent of splicing of the target intron.
  • the ASO may block or interfere with the binding site(s) of a splicing repressor(s)/silencer.
  • Any method known in the art may be used to identify (determine) an ASO that when hybridized to the target region of the intron results in the desired effect (e.g., enhanced splicing, protein or functional RNA production). These methods also can be used for identifying ASOs that enhance splicing of the retained intron by binding to a targeted region in an exon flanking the retained intron, or in a non-retained intron. An example of a method that may be used is provided below. [0166] A round of screening, referred to as an ASO "walk” may be performed using ASOs that have been designed to hybridize to a target region of a pre-mRNA.
  • the ASOs used in the ASO walk can be tiled every 5 nucleotides from approximately 100 nucleotides upstream of the 5' splice site of the retained intron (e.g., a portion of sequence of the ex on located upstream of the target/retained intron) to approximately 100 nucleotides downstream of the 5' splice site of the target/retained intron and/or from approximately 100 nucleotides upstream of the 3 ' splice site of the retained intron to approximately 100 nucleotides downstream of the 3 ' splice site of the target/retained intron (e.g., a portion of sequence of the ex on located
  • a first ASO of 15 nucleotides in length may be designed to specifically hybridize to nucleotides +6 to +20 relative to the 5' splice site of the target/retained intron.
  • a second ASO is designed to specifically hybridize to nucleotides +1 1 to +25 relative to the 5' splice site of the target/retained intron.
  • ASOs are designed as such spanning the target region of the pre-mRNA.
  • the ASOs can be tiled more closely, e.g., every 1, 2, 3, or 4 nucleotides. Further, the ASOs can be tiled from 100 nucleotides downstream of the 5 ' splice site, to 100 nucleotides upstream of the 3 ' splice site.
  • One or more ASOs, or a control ASO are delivered, for example by transfection, into a disease-relevant cell line that expresses the target pre-mRNA (e.g., the RIC pre-mRNA described elsewhere herein).
  • the splicing-inducing effects of each of the ASOs may be assessed by any method known in the art, for example by reverse transcriptase (RT)-PCR using primers that span the splice junction, as described herein (see “Identification of intron-retention events").
  • a reduction or absence of the RT-PCR product produced using the primers spanning the splice junction in ASO-treated cells as compared to in control ASO-treated cells indicates that splicing of the target intron has been enhanced.
  • the splicing efficiency, the ratio of spliced to unspliced pre-mRNA, the rate of splicing, or the extent of splicing may be improved using the ASOs described herein.
  • the amount of protein or functional RNA that is encoded by the target pre-mRNA can also be assessed to determine whether each ASO achieved the desired effect (e.g., enhanced protein production). Any method known in the art for assessing and/or quantifying protein production, such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA, can be used.
  • a second round of screening referred to as an ASO "micro-walk” may be performed using ASOs that have been designed to hybridize to a target region of a pre-mRNA.
  • the ASOs used in the ASO micro-walk are tiled every 1 nucleotide to further refine the nucleotide acid sequence of the pre-mRNA that when hybridized with an ASO results in enhanced splicing.
  • Regions defined by ASOs that promote splicing of the target intron are explored in greater detail by means of an ASO "micro-walk", involving ASOs spaced in 1-nt steps, as well as longer ASOs, typically 18-25 nt.
  • the ASO micro-walk is performed by delivering one or more ASOs, or a control ASO (an ASO with a scrambled sequence, sequence that is not expected to hybridize to the target region), for example by transfection, into a disease-relevant cell line that expresses the target pre-mRNA.
  • the splicing-inducing effects of each of the ASOs may be assessed by any method known in the art, for example by reverse transcriptase (RT)- PCR using primers that span the splice junction, as described herein (see “Identification of intron-retention events").
  • a reduction or absence of the RT-PCR product produced using the primers spanning the splice junction in ASO-treated cells as compared to in control ASO-treated cells indicates that splicing of the target intron has been enhanced.
  • the splicing efficiency, the ratio of spliced to unspliced pre-mRNA, the rate of splicing, or the extent of splicing may be improved using the ASOs described herein.
  • the amount of protein or functional RNA that is encoded by the target pre-mRNA can also be assessed to determine whether each ASO achieved the desired effect (e.g., enhanced protein production). Any method known in the art for assessing and/or quantifying protein production, such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA, can be used.
  • ASOs that when hybridized to a region of a pre-mRNA result in enhanced splicing and increased protein production may be tested in vivo using animal models, for example transgenic mouse models in which the full-length human gene has been knocked-in or in humanized mouse models of disease. Suitable routes for administration of ASOs may vary depending on the disease and/or the cell types to which delivery of the ASOs is desired. ASOs may be
  • the cells, tissues, and/or organs of the model animals may be assessed to determine the effect of the ASO treatment by for example evaluating splicing (efficiency, rate, extent) and protein production by methods known in the art and described herein.
  • the animal models may also be any phenotypic or behavioral indication of the disease or disease severity.
  • Example 1 Identification of intron retention events in TSC2 transcripts by RNAseq using next generation sequencing
  • FIG. 3 shows the mapped reads visualized using the UCSC genome browser (operated by the UCSC Genome Informatics Group (Center for Biomolecular Science & Engineering, University of California, Santa Cruz, 1156 High Street, Santa Cruz, CA 95064) and described by, e.g., Rosenbloom, et al., 2015, "The UCSC Genome Browser database: 2015 update," Nucleic Acids Research 43, Database Issue, doi: 10.1093/nar/gkul 177) and the coverage and number of reads can be inferred by the peak signals.
  • the height of the peaks indicates the level of expression given by the density of the reads in a particular region.
  • TSC2 drawn to scale
  • UCSC genome browser below the read signals
  • An ASO walk was designed to target intron 4 using the method described herein (FIG. 4; Table 2).
  • a region immediately downstream of the 5' splice site of intron 4 spanning nucleotides +6 to +58 and a region immediately upstream of the 3' splice site of intron 4 spanning nucleotides -16 to -68 of the intron were targeted with 2'-0-Me RNA, PS backbone, 18-mer ASOs shifted by 5-nucleotide intervals.
  • An ASO walk was designed to target introns 25 and 26 using the method described herein (FIG. 6; Table 2).
  • a region immediately downstream of the intron 25 5' splice site spanning nucleotides +6 to +58 and a region immediately upstream of intron 26 3' splice site spanning nucleotides -16 to -68 of the intron were targeted with 2'-0-Me RNA, PS backbone, 18-mer ASOs shifted by 5-nucleotide intervals.
  • the splice site intronic regions flanking alternative exon 26 are not targeted to avoid affecting the inclusion level of exon 26.
  • An ASO walk was designed to target introns 31 and 32 using the method described herein (FIG. 8; Table 2).
  • a region immediately downstream of the 5' splice site of intron 31 spanning nucleotides +6 to +58 and a region immediately upstream 3' splice site of intron 32 spanning nucleotides -18 to -68 were targeted with 2'-0-Me RNA, PS backbone, 18-mer ASOs shifted by 5-nucleotide intervals (with the exception of 2 ASOs, TSC2-IVS32-33 and TSC2- IVS32-51).
  • the splice site intronic regions flanking alternative exon 32 are not targeted to avoid affecting the inclusion level of exon 32.
  • Example 5 Improved splicing efficiency via ASO-targeting of TSC2 intron 4, 25, 26, 31 or 32 increases transcript levels
  • RT-PCR products were evaluated using radioactive RT-PCR and RT-qPCR.
  • ARPE-19 cells a human retinal epithelium cell line (American Type Culture Collection (ATCC), USA), or Huh-7, a human hepatoma cell line (NIBIOHN, Japan), or SK-N-AS, a human neuroblastoma cell line (ATCC) were mock- transfected, or transfected with the targeting ASOs described in FIG. 10, FIG. 12, FIG. 15 and Tables 1-2.
  • RNAiMax transfection reagent (Thermo Fisher) according to vendor's specifications. Briefly, ASOs were plated in 96-well tissue culture plates and combined with RNAiMax diluted in Opti-MEM. Cells were detached using trypsin and resuspended in full medium, and approximately 25,000 cells were added the ASO- transfection mixture. Transfection experiments were carried out in triplicate plate
  • Taqman assays were carried out according to vendor's specifications, on a QuantStudio 7 Flex Real-Time PCR system (Thermo Fisher).
  • Target gene assay values were normalized to RPL32 (deltaCt) and plate-matched mock transfected samples (delta-delta Ct), generating fold- change over mock quantitation (2 A -(delta-deltaCt). Average fold-change over mock of the three plate replicates was plotted (FIG. 10, FIG. 12 and FIG. 15). In FIG. 10, FIG. 12 and FIG. 15, several ASOs were identified to increase the target gene expression, indicating an increase in splicing at the respective target intron.
  • TSC2-IVS31+26 cagugggagcagagcccg 31 4900
  • TSC2-IVS31+31 caggccagugggagcaga 31 4901

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US11096956B2 (en) 2015-12-14 2021-08-24 Stoke Therapeutics, Inc. Antisense oligomers and uses thereof
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JP2020516283A (ja) 2017-04-11 2020-06-11 リジェネロン・ファーマシューティカルズ・インコーポレイテッドＲｅｇｅｎｅｒｏｎ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ， Ｉｎｃ． ヒドロキシステロイド（１７−ベータ）デヒドロゲナーゼ（ｈｓｄ１７ｂ）ファミリーのメンバーのモジュレーターの活性をスクリーニングするためのアッセイ
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US10961583B2 (en) 2017-10-11 2021-03-30 Regeneron Phramaceuticals, Inc. Inhibition of HSD17B13 in the treatment of liver disease in patients expressing the PNPLA3 I148M variation
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