US20210268667A1 - Antisense oligomers for treatment of non-sense mediated rna decay based conditions and diseases - Google Patents

Antisense oligomers for treatment of non-sense mediated rna decay based conditions and diseases Download PDF

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US20210268667A1
US20210268667A1 US16/758,776 US201816758776A US2021268667A1 US 20210268667 A1 US20210268667 A1 US 20210268667A1 US 201816758776 A US201816758776 A US 201816758776A US 2021268667 A1 US2021268667 A1 US 2021268667A1
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exon
grch38
mrna
intron
nucleotides
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Isabel Aznarez
Enxuan Jing
Jacob Kach
Aditya Venkatesh
Juergen Scharner
Baruch Ticho
Gene Liau
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Stoke Therapeutics Inc
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Stoke Therapeutics Inc
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    • A—HUMAN NECESSITIES
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    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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    • C12N15/09—Recombinant DNA-technology
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • B25—HAND TOOLS; PORTABLE POWER-DRIVEN TOOLS; MANIPULATORS
    • B25J—MANIPULATORS; CHAMBERS PROVIDED WITH MANIPULATION DEVICES
    • B25J15/00—Gripping heads and other end effectors
    • B25J15/0047—Gripping heads and other end effectors for internally gripping hollow or recessed objects
    • 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
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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
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    • 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
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B25—HAND TOOLS; PORTABLE POWER-DRIVEN TOOLS; MANIPULATORS
    • B25J—MANIPULATORS; CHAMBERS PROVIDED WITH MANIPULATION DEVICES
    • B25J15/00—Gripping heads and other end effectors
    • B25J15/0033—Gripping heads and other end effectors with gripping surfaces having special shapes
    • B25J15/0038—Cylindrical gripping surfaces
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/33—Alteration of splicing

Definitions

  • the targeted portion is at least about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, about 1 nucleotides downstream of 3′ end of the NMD exon.
  • the targeted portion of the mRNA comprises a sequence with at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 contiguous nucleic acids of a sequence selected from the group consisting of SEQ ID NOs: 135-191.
  • the agent is an antisense oligomer (ASO) and wherein the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complementary to at least 8 contiguous nucleic acids of a sequence selected from the group consisting of SEQ ID NOs: 135-191.
  • the targeted portion of the mRNA is upstream or downstream of the non-sense mediated RNA decay-inducing exon selected from the group consisting of: GRCh38/hg38: chr1 243564285 243564388; GRCh38/hg38: chr19 13236449 13236618; GRCh38/hg38: chr21 43059730 43060012; GRCh38/hg38: chr1 207775610 207775745; GRCh38/hg38: chr1 196675450 196675529; GRCh38/hg38: chr15 92998149 92998261; GRCh38/hg38: chr16 28479644 28479765; GRCh38/hg38: chr6 33183634 33183698; GRCh38/hg38: chr2 227296487 227296526; GRCh38/hg38: chr2 2
  • the disease or condition is associated with haploinsufficiency of a gene encoding the target protein, and 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 produced at a reduced level, or a second allele encoding a nonfunctional target protein or a partially functional target protein.
  • the disease or condition is associated with an autosomal recessive mutation of a gene encoding the target protein, wherein the subject has a first allele encoding from which: (i) the target protein is not produced or produced at a reduced level compared to a wild-type allele; or (ii) the target protein produced is nonfunctional or partially functional compared to a wild-type allele, and a second allele from which: (iii) the target protein is produced at a reduced level compared to a wild-type allele and the target protein produced is at least partially functional compared to a wild-type allele; or (iv) the target protein produced is partially functional compared to a wild-type allele.
  • the disease or condition is selected from the group consisting of: Alport syndrome; Ceroid lipofuscinosis, neuronal, 3; Galactose epimerase deficiency; Homocystinuria, B6-responsive and nonresponsive types; Methyl Malonic Aciduria; Propionic acidemia; Retinitis pigmentosa 59; Tay-Sachs disease; Insensitivity to pain, congenital; and HSAN2D, autosomal recessive.
  • exclusion of the NMD exon from the processed mRNA encoding the target protein in the cell contacted with the therapeutic agent is decreased by about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 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-
  • the therapeutic agent decreases the level of the processed mRNA encoding the target protein in the cell.
  • the level of the processed mRNA encoding the target protein in the cell contacted with the therapeutic agent is decreased by about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 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
  • the therapeutic agent inhibits exclusion of the NMD exon from the processed mRNA encoding the target protein and decreases the expression of the target protein in the cell.
  • the target protein comprises SCN8A.
  • the disease or condition comprises a central nervous system disease.
  • the disease or condition comprises epilepsy.
  • the disease or condition comprises Dravet syndrome.
  • the therapeutic agent is an antisense oligomer (ASO) and wherein the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.
  • the therapeutic agent is an antisense oligomer (ASO) and wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2′-O-methyl, a 2′-Fluoro, or a 2′-O-methoxyethyl moiety.
  • the therapeutic agent is an antisense oligomer (ASO) and wherein the antisense oligomer consists of from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30
  • the therapeutic agent is an antisense oligomer (ASO) and wherein the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, complementary to the targeted portion of the mRNA.
  • ASO antisense oligomer
  • the method further comprises assessing mRNA level or expression level of the target protein.
  • the subject is a human. In some embodiments, the subject is a non-human animal. In some embodiments, the subject is a fetus, an embryo, or a child. In some embodiments, the cells are ex vivo. In some embodiments, the therapeutic agent is administered by intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravitreal, or intravenous injection of the subject. In some embodiments, the method further comprises administering a second therapeutic agent to the subject.
  • the second therapeutic agent is a small molecule. In some embodiments, the second therapeutic agent is an antisense oligomer. In some embodiments, the second therapeutic agent corrects intron retention.
  • FIG. 2 depicts identification of an exemplary nonsense-mediated mRNA decay (NMD)-inducing exon in the CD46 gene.
  • NMD nonsense-mediated mRNA decay
  • FIG. 3 depicts identification of an exemplary nonsense-mediated mRNA decay (NMD)-inducing exon in the COL11A2 gene.
  • NMD nonsense-mediated mRNA decay
  • FIG. 6 depicts identification of an exemplary nonsense-mediated mRNA decay (NMD)-inducing exon in the DNAJC8 gene.
  • NMD nonsense-mediated mRNA decay
  • FIG. 12 depicts identification of an exemplary nonsense-mediated mRNA decay (NMD)-inducing exon in the DHDDS gene.
  • NMD nonsense-mediated mRNA decay
  • FIG. 15 depicts identification of an exemplary nonsense-mediated mRNA decay (NMD)-inducing exon in the TOPORS gene.
  • NMD nonsense-mediated mRNA decay
  • FIG. 16 depicts identification of an exemplary nonsense-mediated mRNA decay (NMD)-inducing exon in the PRPF3 gene.
  • NMD nonsense-mediated mRNA decay
  • FIG. 17 depicts identification of an exemplary nonsense-mediated mRNA decay (NMD)-inducing exon in the PRPF3 gene.
  • NMD nonsense-mediated mRNA decay
  • FIG. 18 depicts identification of an exemplary nonsense-mediated mRNA decay (NMD)-inducing exon in the NIPBL gene.
  • NMD nonsense-mediated mRNA decay
  • FIG. 19 depicts identification of an exemplary nonsense-mediated mRNA decay (NMD)-inducing exon in the CBS gene.
  • NMD nonsense-mediated mRNA decay
  • FIG. 20 depicts identification of an exemplary nonsense-mediated mRNA decay (NMD)-inducing exon in the PKP2 gene.
  • NMD nonsense-mediated mRNA decay
  • FIG. 21 depicts identification of an exemplary nonsense-mediated mRNA decay (NMD)-inducing exon in the COL4A4 gene.
  • NMD nonsense-mediated mRNA decay
  • FIG. 22 depicts identification of an exemplary nonsense-mediated mRNA decay (NMD)-inducing exon in the COL4A4 gene.
  • NMD nonsense-mediated mRNA decay
  • FIG. 23 depicts identification of an exemplary nonsense-mediated mRNA decay (NMD)-inducing exon in the CYP2J2 gene.
  • NMD nonsense-mediated mRNA decay
  • FIG. 24 depicts identification of an exemplary nonsense-mediated mRNA decay (NMD)-inducing exon in the PPARA gene.
  • NMD nonsense-mediated mRNA decay
  • FIG. 27 depicts identification of an exemplary nonsense-mediated mRNA decay (NMD)-inducing exon in the GUCY2F gene.
  • NMD nonsense-mediated mRNA decay
  • FIG. 29 depicts identification of an exemplary nonsense-mediated mRNA decay (NMD)-inducing exon in the SCN2A gene.
  • NMD nonsense-mediated mRNA decay
  • FIG. 30 depicts identification of an exemplary nonsense-mediated mRNA decay (NMD)-inducing exon in the SCN8A gene.
  • NMD nonsense-mediated mRNA decay
  • FIG. 35 depicts identification of an exemplary nonsense-mediated mRNA decay (NMD)-inducing exon in the NF1 gene.
  • NMD nonsense-mediated mRNA decay
  • FIG. 46 depicts identification of an exemplary nonsense-mediated mRNA decay (NMD)-inducing exon in the NSD1 gene.
  • NMD nonsense-mediated mRNA decay
  • FIG. 48 depicts identification of an exemplary nonsense-mediated mRNA decay (NMD)-inducing exon in the NSD1 gene.
  • NMD nonsense-mediated mRNA decay
  • FIG. 50 depicts identification of an exemplary nonsense-mediated mRNA decay (NMD)-inducing exon in the GALE gene.
  • NMD nonsense-mediated mRNA decay
  • FIG. 51 depicts identification of an exemplary nonsense-mediated mRNA decay (NMD)-inducing exon in the HEXA gene.
  • NMD nonsense-mediated mRNA decay
  • FIG. 52 depicts identification of an exemplary nonsense-mediated mRNA decay (NMD)-inducing exon in the HEXA gene.
  • NMD nonsense-mediated mRNA decay
  • FIG. 53 depicts identification of an exemplary nonsense-mediated mRNA decay (NMD)-inducing exon in the NR1H4 gene.
  • NMD nonsense-mediated mRNA decay
  • FIG. 54 depicts identification of an exemplary nonsense-mediated mRNA decay (NMD)-inducing exon in the STK11 gene.
  • NMD nonsense-mediated mRNA decay
  • FIG. 55 depicts identification of an exemplary nonsense-mediated mRNA decay (NMD)-inducing exon in the STK11 gene.
  • NMD nonsense-mediated mRNA decay
  • FIG. 60 depicts an exemplary ASO walk around AKT3 exon 8 ⁇ (GRCh38/hg38: chr1 243564285 243564388) region.
  • a graphic representation of an ASO walk performed for around AKT3 exon 8 ⁇ (GRCh38/hg38: chr1 243564285 243564388) region targeting sequences upstream of the 3′ splice site, across the 3′splice site, exon 8 ⁇ , across the 5′ splice site, and downstream of the 5′ splice site is shown.
  • ASOs were designed to cover these regions by shifting 5 nucleotides at a time.
  • FIG. 62 depicts confirmation of NMD-inducing exon via cycloheximide treatment in various cell lines.
  • RT-PCR analysis using total RNA from DMSO-treated or cycloheximide-treated cells confirmed the presence of a band corresponding to the NMD-inducing exon 14 ⁇ (GRCh38/hg38: chr13 100305751 100305834) of PCCA gene
  • FIG. 65 depicts confirmation of NMD-inducing exon via puromycin or cycloheximide treatment in various cell lines, as well as the confirmation of NMD-inducing exon in brain and retina samples.
  • RT-PCR analysis using total RNA from water-treated, DMSO-treated, puromycin-treated, or cycloheximide-treated cells confirmed the presence of a band corresponding to the NMD-inducing exon 7 ⁇ (GRCh38/hg38: chr3 193628509 193628616) of OPA1 gene
  • FIG. 66 depicts an exemplary ASO walk around OPA1 exon 7 ⁇ (GRCh38/hg38: chr3 193628509 193628616) region.
  • a graphic representation of an ASO walk performed for around OPA1 exon 7 ⁇ (GRCh38/hg38: chr3 193628509 193628616) region targeting sequences upstream of the 3′ splice site, across the 3′splice site, exon 7 ⁇ , across the 5′ splice site, and downstream of the 5′ splice site is shown.
  • ASOs were designed to cover these regions by shifting 5 nucleotides at a time or 3 nucleotides across the splice site regions.
  • FIG. 69 depicts confirmation of NMD-inducing exon via cycloheximide treatment in ReNCell VM and existence of NMD-inducing exon mRNA (NF1) in both human and monkey cortices.
  • RT-PCR analysis using total RNA from DMSO-treated or cycloheximide-treated cells confirmed the presence of a band corresponding to the NMD-inducing exon 31 ⁇ (GRCh38/hg38: chr17 31249955 31250125) of NF1 gene
  • FIG. 70 depicts an exemplary ASO walk around NF1 exon 31 ⁇ (GRCh38/hg38: chr17 31249955 31250125) region.
  • a graphic representation of an ASO walk performed for around NF1 exon 31 ⁇ (GRCh38/hg38: chr17 31249955 31250125) region targeting sequences upstream of the 3′ splice site, across the 3′splice site, exon 31 ⁇ , across the 5′ splice site, and downstream of the 5′ splice site is shown.
  • ASOs were designed to cover these regions by shifting 5 nucleotides at a time.
  • FIG. 71 depicts NF1 exon 31 ⁇ (GRCh38/hg38: chr17 31249955 31250125) region ASO walk evaluated by RT-PCR (top) and RT-Taqman-qPCR (bottom). RT-PCR results indicating a decrease in exon 31 ⁇ and a graph of fold-change of the NF1 productive mRNA product relative to Sham are shown.
  • FIG. 73 depicts an exemplary ASO walk around SYNGAP1 exon 18 ⁇ (GRCh38/hg38: chr6 33448789 33448868) region.
  • a graphic representation of an ASO walk performed for around SYNGAP1 exon 18 ⁇ (GRCh38/hg38: chr6 33448789 33448868) region targeting sequences upstream of the 3′ splice site, across the 3′splice site, exon 18 ⁇ , across the 5′ splice site, and downstream of the 5′ splice site is shown.
  • ASOs were designed to cover these regions by shifting 5 nucleotides at a time.
  • FIG. 74 depicts SYNGAP1 exon 18 ⁇ (GRCh38/hg38: chr6 33448789 33448868) region ASO walk evaluated by RT-PCR (top) and Taqman-qPCR (bottom). Graphs of % exon 18 ⁇ inclusion and fold-change of the SYNGAP1 productive mRNA product relative to Sham are plotted (top and bottom, respectively).
  • FIG. 75 depicts confirmation of NMD-inducing exon via cycloheximide treatment.
  • RT-PCR analysis using total RNA from DMSO-treated or cycloheximide-treated cells confirmed the presence of a band corresponding to the NMD-inducing exon 30 ⁇ (GRCh38/hg38: chr15 92998149 92998261) of CHD2 gene. Also shown is the RT-PCR analysis demonstrating the presence of mRNA containing NMD-inducing exon 30 ⁇ in cortex samples from mouse, non-human primate and human.
  • FIG. 76 depicts an exemplary ASO walk around CHD2 exon 30 ⁇ (GRCh38/hg38: chr15 92998149 92998261) region.
  • a graphic representation of an ASO walk performed for around CHD2 exon 30 ⁇ (GRCh38/hg38: chr15 92998149 92998261) region targeting sequences upstream of the 3′ splice site, across the 3′ splice site, exon 30 ⁇ , across the 5′ splice site, and downstream of the 5′ splice site is shown.
  • ASOs were designed to cover these regions by shifting 5 nucleotides at a time.
  • FIG. 77 depicts CHD2 exon 30 ⁇ (GRCh38/hg38: chr15 92998149 92998261) region ASO walk evaluated by RT-PCR. RT-PCR results are shown demonstrating the changes in amount of mRNA containing NMD-inducing exon 30 ⁇ .
  • FIG. 78 depicts changes induced by different ASOs in levels of CHD2 non-productive exon (exon 30 ⁇ (GRCh38/hg38: chr15 92998149 92998261)) and CHD2 productive mRNA.
  • Such therapeutic agents can be used to treat a condition caused by ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2, CR1, CR
  • Intervening sequences or introns are removed by a large and highly dynamic RNA-protein complex termed the spliceosome, which orchestrates complex interactions between primary transcripts, small nuclear RNAs (snRNAs) and a large number of proteins.
  • Spliceosomes assemble ad hoc on each intron in an ordered manner, starting with recognition of the 5′ splice site (5′ss) by U1 snRNA or the 3′splice site (3′ss) by the U2 pathway, which involves binding of the U2 auxiliary factor (U2AF) to the 3′ss region to facilitate U2 binding to the branch point sequence (BPS).
  • 5′ss 5′ splice site
  • U1 snRNA small nuclear RNAs
  • 3′ss 3′splice site
  • U2AF is a stable heterodimer composed of a U2AF2-encoded 65-kD subunit (U2AF65), which binds the polypyrimidine tract (PPT), and a U2AF1-encoded 35-kD subunit (U2AF35), which interacts with highly conserved AG dinucleotides at 3′ ss and stabilizes U2AF65 binding.
  • U2AF65 U2AF2-encoded 65-kD subunit
  • PPT polypyrimidine tract
  • U2AF35 U2AF1-encoded 35-kD subunit
  • accurate splicing requires auxiliary sequences or structures that activate or repress splice site recognition, known as intronic or exonic splicing enhancers or silencers.
  • ESSs can be mediated by members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family and can alter recruitment of core splicing factors to adjacent splice sites.
  • hnRNP nuclear ribonucleoprotein
  • silencer elements are suggested to have a role in repression of pseudo-exons, sets of decoy intronic splice sites with the typical spacing of an exon but without a functional open reading frame.
  • ESEs and ESSs in cooperation with their cognate trans-acting RBPs, represent important components in a set of splicing controls that specify how, where and when mRNAs are assembled from their precursors.
  • sequences marking the exon-intron boundaries are degenerate signals of varying strengths that can occur at high frequency within human genes.
  • different pairs of splice sites can be linked together in many different combinations, creating a diverse array of transcripts from a single gene. This is commonly referred to as alternative pre-mRNA splicing.
  • alternative pre-mRNA splicing Although most mRNA isoforms produced by alternative splicing can be exported from the nucleus and translated into functional polypeptides, different mRNA isoforms from a single gene can vary greatly in their translation efficiency.
  • mRNA isoforms with premature termination codons (PTCs) at least 50 bp upstream of an exon junction complex are likely to be targeted for degradation by the nonsense-mediated mRNA decay (NMD) pathway.
  • Mutations in traditional (BPS/PPT/3′ss/5′ss) and auxiliary splicing motifs can cause aberrant splicing, such as exon skipping or cryptic (or pseudo-) exon inclusion or splice-site activation, and contribute significantly to human morbidity and mortality. Both aberrant and alternative splicing patterns can be influenced by natural DNA variants in exons and introns.
  • NMD is a translation-coupled mechanism that eliminates mRNAs containing PTCs. NMD can function as a surveillance pathway that exists in all eukaryotes.
  • NMD can reduce errors in gene expression by eliminating mRNA transcripts that contain premature stop codons. Translation of these aberrant mRNAs could, in some cases, lead to deleterious gain-of-function or dominant-negative activity of the resulting proteins. NMD targets not only transcripts with PTCs but also a broad array of mRNA isoforms expressed from many endogenous genes, suggesting that NMD is a master regulator that drives both fine and coarse adjustments in steady-state RNA levels in the cell.
  • NMD-inducing exon is an exon or a pseudo-exon that is a region within an intron and can activate the NMD pathway if included in a mature RNA transcript.
  • the intron containing an NIE is usually spliced out, but the intron or a portion thereof (e.g. NIE) may be retained during alternative or aberrant splicing events.
  • Mature mRNA transcripts containing such an NIE may be non-productive due to frame shifts which induce the NMD pathway. Inclusion of a NIE in mature RNA transcripts may downregulate gene expression.
  • mRNA transcripts containing an NIE may be referred to as “NIE containing mRNA” or “NMD exon mRNA” in the current disclosure.
  • Cryptic (or pseudo-splice sites) have the same splicing recognition sequences as genuine splice sites but are not used in splicing reactions. They outnumber genuine splice sites in the human genome by an order of a magnitude and are normally repressed by thus far poorly understood molecular mechanisms.
  • Cryptic 5′ splice sites have the consensus NNN/GUNNNN or NNN/GCNNNN where N is any nucleotide and/is the exon-intron boundary.
  • Cryptic 3′ splice sites have the consensus NAG/N.
  • the cryptic splice sites or splicing regulatory sequences may compete for RNA-binding proteins, such as U2AF, with a splice site of the NIE.
  • an agent may bind to a cryptic splice site or splicing regulatory sequence to prevent binding of RNA-binding proteins and thereby favor binding of RNA-binding proteins to the NIE splice sites.
  • the cryptic splice site may not comprise the 5′ or 3′ splice site of the NIE.
  • the cryptic splice site may be at least 10 nucleotides, at least 20 nucleotides, at least 50 nucleotides, at least 100 nucleotides or at least 200 nucleotides upstream of the NIE 5′ splice site.
  • the cryptic splice site may be at least 10 nucleotides, at least 20 nucleotides, at least 50 nucleotides, at least 100 nucleotides, at least 200 nucleotides downstream of the NIE 3′ splice site.
  • Induction of exon skipping may result in inhibition of an NMD pathway.
  • the diseases or conditions that can be treated or ameliorated using the method or composition disclosed herein are not directly associated with the target protein (gene) that the therapeutic agent targets.
  • a therapeutic agent provided herein can target a protein (gene) that is not directly associated with a disease or condition, but the modulation of expression of the target protein (gene) can treat or ameliorate the disease or condition. For instance, targeting genes like CD46, CFH, CR1, DNAJC8, EIF2AK3, ERN1, GUCY2F, GUCY2F, SEMA3C, SEMA3D, SIRT3, or AKT3 by a therapeutic agent provided herein can treat or ameliorate eye diseases or conditions.
  • targeting genes CD46, CFH, CR1, DNAJC8, EIF2AK3, ERN1, GUCY2F, GUCY2F, SEMA3C, SEMA3D, SIRT3, or AKT3 are said to be indicated for Pathway (eye).
  • targeting gene like SCN8A can treat or ameliorate central nervous system diseases, e.g., epilepsy, e.g., Dravet syndrome.
  • target genes like SCN8A are said to be indicated for Pathway (central nervous system) or Pathway (central nervous system, epilepsy).
  • the ASO targets a sequence downstream (or 3′) from the 3′ end of an NIE (5′ss) of a ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3
  • the ASO targets a sequence that is within an intron flanking the 3′ end of the NIE of a ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN
  • the ASO targets a sequence comprising an NIE-intron boundary of a ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46,
  • the ASO targets a sequence within an intron of a ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2,
  • the ASO targets a sequence comprising both a portion of an intron and a portion of an exon of a ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A,
  • the ASO targets a sequence about 4 to about 300 nucleotides upstream (or 5′) from the 5′ end of the NIE. In some embodiments, the ASO targets a sequence about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, or about 250 to about 300 nucleotides upstream (or 5′) from the 5′ end of the NIE region. In some embodiments, the ASO may target a sequence more than 300 nucleotides upstream from the 5′ end of the NIE.
  • the targeted portion of the NMD exon mRNA comprises a sequence with at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 contiguous nucleic acids of any one of SEQ ID NOs: 60-191.
  • the ASO targets exon 8 ⁇ of a ABCB4 NIE containing pre-mRNA comprising NIE exon 8, exon 9 ⁇ of a ASS1 NIE containing pre-mRNA comprising NIE exon 9, exon 16 ⁇ of a ATP8B1 NIE containing pre-mRNA comprising NIE exon 16, exon 1 ⁇ of a BAG3 NIE containing pre-mRNA comprising NIE exon 1, exon 31 ⁇ of a CACNA1A NIE containing pre-mRNA comprising NIE exon 31, exon 36 ⁇ of a CACNA1A NIE containing pre-mRNA comprising NIE exon 36, exon 37 ⁇ of a CACNA1A NIE containing pre-mRNA comprising NIE exon 37, exon 3 ⁇ of a CBS NIE containing pre-mRNA comprising NIE exon 3, exon 12 ⁇ of a CBS NIE containing pre-mRNA comprising NIE exon 12, exon 1 ⁇ of a CD55 NIE containing pre-
  • the ASO targets exon (GRCh38/hg38: chr1 243564285 243564388) of AKT3; exon (GRCh38/hg38: chr19 13236449 13236618) of CACNA1A; exon (GRCh38/hg38: chr21 43059730 43060012) of CBS; exon (GRCh38/hg38: chr1 207775610 207775745) of CD46; exon (GRCh38/hg38: chr1 196675450 196675529) of CFH; exon (GRCh38/hg38: chr15 92998149 92998261) of CHD2; exon (GRCh38/hg38: chr16 28479644 28479765) of CLN3; exon (GRCh38/hg38: chr6 33183634 33183698) of COL11A2; exon (GRCh38/hg38: chr6 331836
  • the ASO targets a sequence about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides upstream (or 5′) from the 5′ end of exon 8 ⁇ of ABCB4, exon 9 ⁇ of ASS1, exon 16 ⁇ of ATP8B1, exon 1 ⁇ of BAG3, exon 31 ⁇ of CACNA1A, exon 36 ⁇ of CACNA1A, exon 37 ⁇ of CACNA1A, exon 3 ⁇ of CBS, exon 12 ⁇ of CBS, exon 1 ⁇ of CD55, exon 16 ⁇ of CDKL5, exon 3 ⁇ of CFH, exon 30 ⁇ of CHD2, exon 4 ⁇
  • the ASO targets a sequence about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides upstream (or 5′) from GRCh38/hg38: chr1 243564388 of AKT3; GRCh38/hg38: chr19 13236618 of CACNA1A; GRCh38/hg38: chr21 43060012 of CBS; GRCh38/hg38: chr1 207775610 of CD46; GRCh38/hg38: chr1 196675450 of CFH; GRCh38/hg38: chr15 929
  • the ASO targets a sequence at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides upstream (or 5′) from the 5′ end of exon 8 ⁇ of ABCB4, exon 9 ⁇ of ASS1, exon 16 ⁇ of ATP8B1, exon 1 ⁇ of BAG3, exon 31 ⁇ of CACNA1A, exon 36 ⁇ of CACNA1A, exon 37 ⁇ of CACNA1A, exon 3 ⁇ of CBS, exon 12 ⁇ of CBS, exon 1 ⁇ of CD55, exon 16 ⁇ of CDKL5, exon 3 ⁇ of CFH, exon 30 ⁇ of CHD2, exon
  • the ASO targets a sequence at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides upstream (or 5′) from GRCh38/hg38: chr1 243564388 of AKT3; GRCh38/hg38: chr19 13236618 of CACNA1A; GRCh38/hg38: chr21 43060012 of CBS; GRCh38/hg38: chr1 207775610 of CD46; GRCh38/hg38: chr1 196675450 of CFH; GRCh38/hg38: chr15
  • the ASO targets a sequence about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream (or 3′) from the 3′ end of exon 8 ⁇ of ABCB4, exon 9 ⁇ of ASS1, exon 16 ⁇ of ATP8B1, exon 1 ⁇ of BAG3, exon 31 ⁇ of CACNA1A, exon 36 ⁇ of CACNA1A, exon 37 ⁇ of CACNA1A, exon 3 ⁇ of CBS, exon 12 ⁇ of CBS, exon 1 ⁇ of CD55, exon 16 ⁇ of CDKL5, exon 3 ⁇ of CFH, exon 30 ⁇ of CHD2, exon 4 ⁇ of
  • the ASO targets a sequence about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream (or 3′) from GRCh38/hg38: chr1 243564285 of AKT3; GRCh38/hg38: chr19 13236449 of CACNA1A; GRCh38/hg38: chr21 43059730 of CBS; GRCh38/hg38: chr1 207775745 of CD46; GRCh38/hg38: chr1 196675529 of CFH; GRCh38/hg38: chr15 92
  • the ASO targets a sequence at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream (or 3′) from the 3′ end of exon 8 ⁇ of ABCB4, exon 9 ⁇ of ASS1, exon 16 ⁇ of ATP8B1, exon 1 ⁇ of BAG3, exon 31 ⁇ of CACNA1A, exon 36 ⁇ of CACNA1A, exon 37 ⁇ of CACNA1A, exon 3 ⁇ of CBS, exon 12 ⁇ of CBS, exon 1 ⁇ of CD55, exon 16 ⁇ of CDKL5, exon 3 ⁇ of CFH, exon 30 ⁇ of CHD2, exon 4
  • the ASO targets a sequence at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream (or 3′) from GRCh38/hg38: chr1 243564285 of AKT3; GRCh38/hg38: chr19 13236449 of CACNA1A; GRCh38/hg38: chr21 43059730 of CBS; GRCh38/hg38: chr1 207775745 of CD46; GRCh38/hg38: chr1 196675529 of CFH; GRCh38/hg38: chr15
  • the ASO targets a sequence upstream from the 5′ end of an NIE.
  • ASOs targeting a sequence upstream from the 5′ end of an NIE e.g. exon 8 ⁇ of ABCB4, exon 9 ⁇ of ASS1, exon 16 ⁇ of ATP8B1, exon 1 ⁇ of BAG3, exon 31 ⁇ of CACNA1A, exon 36 ⁇ of CACNA1A, exon 37 ⁇ of CACNA1A, exon 3 ⁇ of CBS, exon 12 ⁇ of CBS, exon 1 ⁇ of CD55, exon 16 ⁇ of CDKL5, exon 3 ⁇ of CFH, exon 30 ⁇ of CHD2, exon 4 ⁇ of CHRNA7, exon 1 ⁇ of CISD2, exon 15 ⁇ of CLN3, exon 11 ⁇ of COL4A3, exon 41 ⁇ of COL4A3, exon 22 ⁇ of COL4A4, exon 44 ⁇ of COL4A4, exon 20 ⁇ of DEPDC5, exon 2 ⁇ of DHDDS, exon 3 ⁇ of
  • ASOs targeting a sequence upstream from the 5′ end of an NIE e.g., exon (GRCh38/hg38: chr1 243564285 243564388) of AKT3; exon (GRCh38/hg38: chr19 13236449 13236618) of CACNA1A; exon (GRCh38/hg38: chr21 43059730 43060012) of CBS; exon (GRCh38/hg38: chr1 207775610 207775745) of CD46; exon (GRCh38/hg38: chr1 196675450 196675529) of CFH; exon (GRCh38/hg38: chr15 92998149 92998261) of CHD2; exon (GRCh38/hg38: chr16 28479644 28479765) of CLN3; exon (GRCh38/hg38: chr6 33183634 33183698) of COL
  • the ASOs target a sequence containing an exon-intron boundary (or junction).
  • ASOs targeting a sequence containing an exon-intron boundary can comprise a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complimentary to at least 8 contiguous nucleic acids of any one of SEQ ID NOs: 60-191.
  • the ASOs target a sequence downstream from the 3′ end of an NIE.
  • ASOs targeting a sequence downstream from the 3′ end of an NIE e.g.
  • ASOs targeting a sequence downstream from the 3′ end of an NIE e.g., exon (GRCh38/hg38: chr1 243564285 243564388) of AKT3; exon (GRCh38/hg38: chr19 13236449 13236618) of CACNA1A; exon (GRCh38/hg38: chr21 43059730 43060012) of CBS; exon (GRCh38/hg38: chr1 207775610 207775745) of CD46; exon (GRCh38/hg38: chr1 196675450 196675529) of CFH; exon (GRCh38/hg38: chr15 92998149 92998261) of CHD2; exon (GRCh38/hg38: chr16 28479644 28479765) of CLN3; exon (GRCh38/hg38: chr6 33183634 33183698) of COL11
  • the ASO targets exon 8 ⁇ of a ABCB4 NIE containing pre-mRNA comprising NIE exon 8, exon 9 ⁇ of a ASS1 NIE containing pre-mRNA comprising NIE exon 9, exon 16 ⁇ of a ATP8B1 NIE containing pre-mRNA comprising NIE exon 16, exon 1 ⁇ of a BAG3 NIE containing pre-mRNA comprising NIE exon 1, exon 31 ⁇ of a CACNA1A NIE containing pre-mRNA comprising NIE exon 31, exon 36 ⁇ of a CACNA1A NIE containing pre-mRNA comprising NIE exon 36, exon 37 ⁇ of a CACNA1A NIE containing pre-mRNA comprising NIE exon 37, exon 3 ⁇ of a CBS NIE containing pre-mRNA comprising NIE exon 3, exon 12 ⁇ of a CBS NIE containing pre-mRNA comprising NIE exon 12, exon 1 ⁇ of a CD55 NIE containing pre-
  • the methods are used to increase the production of a partially functional ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2,
  • a partially functional protein or RNA will have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% less activity relative to the fully functional protein or RNA.
  • the method is a method of increasing the expression of the ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAIL RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2, CR1,
  • the subject has a first allele encoding a functional ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2,
  • the subject has a first allele encoding a functional ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAIL RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2,
  • the subject has a first allele encoding a functional ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2,
  • the antisense oligomer binds to a targeted portion of the NIE containing pre-mRNA transcribed from the second allele, thereby inducing exon skipping of the pseudo-exon from the pre-mRNA, and causing an increase in the level of mature mRNA encoding functional ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A,
  • the method is a method of increasing the expression of the ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAIL RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2, CR1,
  • the method is a method of increasing the expression of the ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAIL RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2, CR1,
  • the method is a method of increasing the expression of the ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAIL RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2, CR1,
  • the subject has:
  • a subject treated using the methods of the present disclosure expresses a partially functional ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46,
  • a subject treated using the methods of the disclosure has a ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2,
  • an antisense oligomer targeted to the most abundant pseudo-exon in the population of NIE containing pre-mRNAs encoding the target protein induces exon skipping of one or two or more pseudo-exons in the population, including the pseudo-exon to which the antisense oligomer is targeted or binds.
  • the degree of exon inclusion can be expressed as percent exon inclusion, e.g., the percentage of transcripts in which a given pseudo-exon is included.
  • percent exon inclusion can be calculated as the percentage of the amount of RNA transcripts with the exon inclusion, over the sum of the average of the amount of RNA transcripts with exon inclusion plus the average of the amount of RNA transcripts with exon exclusion.
  • a control compound can be, for example, an oligonucleotide that is not complementary to a targeted portion of the ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN,
  • the NIE can be in any length. In some embodiments, the NIE comprises a full sequence of an intron, in which case, it can be referred to as intron retention. In some embodiments, the NIE can be a portion of the intron. In some embodiments, the NIE can be a 5′ end portion of an intron including a 5′ss sequence. In some embodiments, the NIE can be a 3′ end portion of an intron including a 3′ss sequence. In some embodiments, the NIE can be a portion within an intron without inclusion of a 5′ss sequence. In some embodiments, the NIE can be a portion within an intron without inclusion of a 3′ss sequence.
  • the NIE can be a portion within an intron without inclusion of either a 5′ss or a 3′ss sequence.
  • the NIE can be from 5 nucleotides to 10 nucleotides in length, from 10 nucleotides to 15 nucleotides in length, from 15 nucleotides to 20 nucleotides in length, from 20 nucleotides to 25 nucleotides in length, from 25 nucleotides to 30 nucleotides in length, from 30 nucleotides to 35 nucleotides in length, from 35 nucleotides to 40 nucleotides in length, from 40 nucleotides to 45 nucleotides in length, from 45 nucleotides to 50 nucleotides in length, from 50 nucleotides to 55 nucleotides in length, from 55 nucleotides to 60 nucleotides in length, from 60 nucleotides to 65 nucleotides in length, from 65 nucleo
  • the NIE can be at least 10 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleoids, at least 70 nucleotides, at least 80 nucleotides in length, at least 90 nucleotides, or at least 100 nucleotides in length.
  • the NIE can be from 100 to 200 nucleotides in length, from 200 to 300 nucleotides in length, from 300 to 400 nucleotides in length, from 400 to 500 nucleotides in length, from 500 to 600 nucleotides in length, from 600 to 700 nucleotides in length, from 700 to 800 nucleotides in length, from 800 to 900 nucleotides in length, from 900 to 1,000 nucleotides in length. In some embodiments, the NIE may be longer than 1,000 nucleotides in length.
  • compositions and methods comprising a therapeutic agent are provided to modulate protein expression level of ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT
  • a therapeutic agent disclosed herein can be a NIE repressor agent.
  • a therapeutic agent may comprise a polynucleic acid polymer.
  • the reduction may be complete, e.g., 100%, or may be partial.
  • the reduction may be clinically significant.
  • the reduction/correction may be relative to the level of NIE inclusion in the subject without treatment, or relative to the amount of NIE inclusion in a population of similar subjects.
  • the reduction/correction may be at least 10% less NIE inclusion relative to the average subject, or the subject prior to treatment.
  • the reduction may be at least 20% less NIE inclusion relative to an average subject, or the subject prior to treatment.
  • the reduction may be at least 40% less NIE inclusion relative to an average subject, or the subject prior to treatment.
  • the reduction may be at least 50% less NIE inclusion relative to an average subject, or the subject prior to treatment.
  • the reduction may be at least 60% less NIE inclusion relative to an average subject, or the subject prior to treatment.
  • the reduction may be at least 80% less NIE inclusion relative to an average subject, or the subject prior to treatment.
  • the reduction may be at least 90% less NIE inclusion relative to an average subject, or the subject prior to treatment.
  • the increase may be relative to the level of active ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2, CR1, CRX, DNA
  • the increase may be at least 10% more active ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2, CR1, CRX, DNAJC
  • the increase may be at least 20% more active ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2, CR1, CRX, DNAJC
  • the increase may be at least 40% more active ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2, CR1, CRX, DNAJC
  • the increase may be at least 80% more active ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2, CR1, CRX, DNA
  • the polynucleic acid polymer may be about 50 nucleotides in length.
  • the polynucleic acid polymer may be about 45 nucleotides in length.
  • the polynucleic acid polymer may be about 40 nucleotides in length.
  • the polynucleic acid polymer may be about 35 nucleotides in length.
  • the polynucleic acid polymer may be about 30 nucleotides in length.
  • the polynucleic acid polymer may be about 24 nucleotides in length.
  • the polynucleic acid polymer may be about 25 nucleotides in length.
  • the sequence of the polynucleic acid polymer may be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% complementary to a target sequence of an mRNA transcript, e.g., a partially processed mRNA transcript.
  • the sequence of the polynucleic acid polymer may be 100% complementary to a target sequence of a pre-mRNA transcript.
  • the sequence of the polynucleic acid polymer may have 4 or fewer mismatches to a target sequence of the pre-mRNA transcript.
  • the sequence of the polynucleic acid polymer may have 3 or fewer mismatches to a target sequence of the pre-mRNA transcript.
  • the sequence of the polynucleic acid polymer may have 2 or fewer mismatches to a target sequence of the pre-mRNA transcript.
  • the sequence of the polynucleic acid polymer may have 1 or fewer mismatches to a target sequence of the pre-mRNA transcript.
  • the sequence of the polynucleic acid polymer may have no mismatches to a target sequence of the pre-mRNA transcript.
  • the polynucleic acid polymer may specifically hybridize to a target sequence of the pre-mRNA transcript.
  • the polynucleic acid polymer may have 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence complementarity to a target sequence of the pre-mRNA transcript.
  • the hybridization may be under high stringent hybridization conditions.
  • the polynucleic acid polymer comprising a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 60-191.
  • the polynucleic acid polymer may comprise a sequence with 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 60-191.
  • sequence identity may be determined by BLAST sequence alignment using standard/default parameters. For example, the sequence may have 99% identity and still function according to the present disclosure. In other embodiments, the sequence may have 98% identity and still function according to the present disclosure. In another embodiment, the sequence may have 95% identity and still function according to the present disclosure. In another embodiment, the sequence may have 90% identity and still function according to the present disclosure.
  • composition comprising an antisense oligomer that induces exon skipping by binding to a targeted portion of a ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3
  • 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,” incorporated by reference herein, 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 NIE containing pre-mRNA.
  • hybridization occurs with a T m substantially greater than 37° C., preferably at least 50° C., and typically between 60° C. to approximately 90° C.
  • T m 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 polynucleotides.
  • a double-stranded polynucleotide can be “complementary” to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second.
  • Complementarity (the degree to which one polynucleotide is complementary with another) is quantifiable in terms of the proportion (e.g., the percentage) of bases in opposing strands that are expected to form hydrogen bonds with each other, according to generally accepted base-pairing rules.
  • ASO antisense oligomer
  • 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 complementarity.
  • 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.
  • the ASOs described herein comprise nucleobases that are complementary to nucleobases present in a target portion of a NIE containing pre-mRNA.
  • the term ASO embodies oligonucleotides and any other oligomeric molecule that comprises nucleobases capable of hybridizing to a complementary nucleobase on a target mRNA but does not comprise a sugar moiety, such as a peptide nucleic acid (PNA).
  • 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.
  • the term “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. Pat. No. 8,258,109 B2, U.S. Pat. No. 5,656,612, U.S. Patent Publication No. 2012/0190728, and Dias and Stein, Mol. Cancer Ther. 2002, 347-355, herein incorporated by reference in their entirety.
  • One or more nucleobases 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-methylcytosine, and 5-hydroxymethoylcytosine.
  • 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.
  • an ASO used in the methods of the disclosure comprises an ASO having phosphorus internucleotide linkages that are not random.
  • a composition used in the methods of the disclosure comprises a pure diastereomeric ASO.
  • 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 disclosure comprising, but not limited to, any of the ASOs set forth herein comprise a sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 contiguous nucleic acids of any one of SEQ ID NOs: 60-191, 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
  • an ASO used in the methods of the disclosure comprising a sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 contiguous nucleic acids of any one of SEQ ID NOs: 60-191, 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
  • 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′-O-methyl (2′-O-Me), 2′-O-methoxyethyl (2′MOE), 2′-O-aminoethyl, 2′F; N3′->P5′ phosphoramidate, 2′dimethylaminooxyethoxy, 2′dimethylaminoethoxyethoxy, 2′-guanidinidium, 2′-O-guanidinium ethyl, carbamate modified sugars, and bicyclic modified sugars.
  • the sugar moiety modification is selected from 2′-O-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′O-methyloxyethyl (cMOE) modifications.
  • 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 modifications. In some embodiments, the ASO comprises one or more sugar moiety modification. In some embodiments, the ASO comprises one or more backbone modifications and one or more sugar moiety modifications. In some embodiments, the ASO comprises a 2′MOE modification and a phosphorothioate backbone. In some embodiments, the ASO comprises a phosphorodiamidate morpholino (PMO). In some embodiments, the ASO comprises a peptide nucleic acid (PNA).
  • any of the ASOs or any component of an ASO 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 components 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/or modulate the half-life of the ASO.
  • 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-exon junction in mRNA
  • a nucleotide that is directly adjacent and upstream of the reference point is designated “minus one,” e.g., “ ⁇ 1”
  • a nucleotide that is directly adjacent and downstream of the reference point is designated “plus one,” e.g., “+1.”
  • the ASOs are complementary to a targeted portion of the ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2,
  • the ASOs may be complementary to a targeted portion of a ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A
  • the ASOs are complementary to a targeted portion that is within the region about +1 to about +40,000, about +1 to about +30,000, about +1 to about +20,000, about +1 to about +15,000, about +1 to about +10,000, about +1 to about +5,000, about +1 to about +4,000, about +1 to about +3,000, about +1 to about +2,000, about +1 to about +1,000, about +1 to about +500, about +1 to about +490, about +1 to about +480, about +1 to about +470, about +1 to about +460, about +1 to about +450, about +1 to about +440, about +1 to about +430, about +1 to about +420, about +1 to about +410, about +1 to about +400, about +1 to about +390, about +1 to about +380, about +1 to about +370, about +1 to about +360, about +1 to about +350, about +1 to about +340, about +1 to about +330, about +1 to about +320,
  • the ASOs are complementary to a targeted portion that is within the region from about +1 to about +100, from about +100 to about +200, from about +200 to about +300, from about +300 to about +400, or from about +400 to about +500 relative to 5′ splice site (or 3′ end) of the included exon.
  • the ASOs are complementary to (and bind to) a targeted portion of a ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46
  • the ASOs are complementary to a targeted portion of the ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2,
  • the ASOs may be complementary to a targeted portion of a ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A
  • the ASOs are complementary to a targeted portion that is within the region about ⁇ 1 to about ⁇ 40,000, about ⁇ 1 to about ⁇ 30,000, about ⁇ 1 to about ⁇ 20,000, about ⁇ 1 to about ⁇ 15,000, about ⁇ 1 to about ⁇ 10,000, about ⁇ 1 to about ⁇ 5,000, about ⁇ 1 to about ⁇ 4,000, about ⁇ 1 to about ⁇ 3,000, about ⁇ 1 to about ⁇ 2,000, about ⁇ 1 to about ⁇ 1,000, about ⁇ 1 to about ⁇ 500, about ⁇ 1 to about ⁇ 490, about ⁇ 1 to about ⁇ 480, about ⁇ 1 to about ⁇ 470, about ⁇ 1 to about ⁇ 460, about ⁇ 1 to about ⁇ 450, about ⁇ 1 to about ⁇ 440, about ⁇ 1 to about ⁇ 430, about ⁇ 1 to about ⁇ 420, about ⁇ 1 to about ⁇ 410, about ⁇ 1 to about ⁇ 400, about ⁇ 1 to about ⁇ 390, about ⁇ 1 to about ⁇ 380, about
  • the ASOs are complementary to a targeted region of a ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2,
  • the ASOs are complementary to a targeted portion of the ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2,
  • the ASOs are complementary to a targeted portion of the ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2,
  • the ASOs are complementary to a targeted portion that is within the region about ⁇ 1 to about ⁇ 40,000, about ⁇ 1 to about ⁇ 30,000, ⁇ 1 to about ⁇ 20,000, about ⁇ 1 to about ⁇ 15,000, about ⁇ 1 to about ⁇ 10,000, about ⁇ 1 to about ⁇ 5,000, about ⁇ 1 to about ⁇ 4,000, about ⁇ 1 to about ⁇ 3,000, about ⁇ 1 to about ⁇ 2,000, about ⁇ 1 to about ⁇ 1,000, about ⁇ 1 to about ⁇ 500, about ⁇ 1 to about ⁇ 490, about ⁇ 1 to about ⁇ 480, about ⁇ 1 to about ⁇ 470, about ⁇ 1 to about ⁇ 460, about ⁇ 1 to about ⁇ 450, about ⁇ 1 to about ⁇ 440, about ⁇ 1 to about ⁇ 430, about ⁇ 1 to about ⁇ 420, about ⁇ 1 to about ⁇ 410, about ⁇ 1 to about ⁇ 400, about ⁇ 1 to about ⁇ 390, about ⁇ 1 to about ⁇ 380, about ⁇
  • the ASOs are complementary to a targeted portion that is within the region from about ⁇ 1 to about ⁇ 100, from about ⁇ 100 to about ⁇ 200, from about ⁇ 200 to about ⁇ 300, from about ⁇ 300 to about ⁇ 400, or from about ⁇ 400 to about ⁇ 500 relative to 3′ splice site of the included exon.
  • the ASOs are complementary to a targeted region of a ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2,
  • the ASOs are complementary to a targeted portion of the ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2,
  • the ASOs may be of any length suitable for specific binding and effective enhancement of splicing.
  • 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, 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
  • two or more ASOs with different chemistries but complementary to the same targeted portion of the NIE containing pre-mRNA are used. In some embodiments, two or more ASOs that are complementary to different targeted portions of the NIE containing pre-mRNA are used.
  • the antisense oligonucleotides of the disclosure 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.
  • Oligonucleotides comprising lipophilic moieties and preparation methods have been described in the published literature.
  • the antisense oligonucleotide is conjugated with a moiety including, but not limited to, an abasic nucleotide, a polyether, a polyamine, a polyamide, a peptides, a carbohydrate, e.g., N-acetylgalactosamine (GalNAc), N—Ac-Glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate), a lipid, or a polyhydrocarbon compound.
  • a moiety including, but not limited to, an abasic nucleotide, a polyether, a polyamine, a polyamide, a peptides, a carbohydrate, e.g., N-acetylgalactosamine (GalNAc), N—Ac-Glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate), a lipid, or a polyhydrocarbon compound.
  • Conjugates can be linked to one or more of any nucleotides comprising the antisense oligonucleotide at any of several positions on the sugar, base or phosphate group, as understood in the art and described in the literature, e.g., using a linker.
  • Linkers can include a bivalent or trivalent branched linker.
  • the conjugate is attached to the 3′ end of the antisense oligonucleotide.
  • the term “cell” may refer to a population of cells.
  • the cell is in a subject.
  • the cell is isolated from a subject.
  • the cell is ex vivo.
  • the cell is a condition or disease-relevant cell or a cell line.
  • the cell is in vitro (e.g., in cell culture).
  • salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate,
  • alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.
  • the compositions are formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas.
  • the compositions are formulated as suspensions in aqueous, non-aqueous or mixed media.
  • Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • a pharmaceutical formulation or composition of the present disclosure includes, but is not limited to, a solution, emulsion, microemulsion, foam or liposome-containing formulation (e.g., cationic or noncationic liposomes).
  • liposomes may comprise one or more penetration enhancers, carriers, 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.
  • PEG polyethylene glycol
  • a surfactant is included in the pharmaceutical formulation or compositions.
  • the present disclosure 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 nonsurfactant.
  • the pharmaceutical formulation comprises multiple antisense oligonucleotides.
  • the antisense oligonucleotide is administered in combination with another drug or therapeutic agent.
  • the ASOs disclosed in the present disclosure can be used in combination with one or more additional therapeutic agents.
  • the one or more additional therapeutic agents can comprise a small molecule.
  • the one or more additional therapeutic agents can comprise a small molecule described in WO2016128343A1, WO2017053982A1, WO2016196386A1, WO201428459A1, WO201524876A2, WO2013119916A2, and WO2014209841A2, which are incorporated by reference herein in their entirety.
  • the one or more additional therapeutic agents comprise an ASO that can be used to correct intron retention.
  • 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 a 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. For example, an individual may be at an increased risk of having such a disease or disorder because of family history of the disease.
  • a fetus is treated in utero, e.g., by administering the ASO composition to the fetus directly or indirectly (e.g., via the mother).
  • Suitable routes for administration of ASOs of the present disclosure may vary depending on cell type to which delivery of the ASOs is desired. Multiple tissues and organs are affected by Dravet syndrome, with the brain being the most significantly affected tissue.
  • the ASOs of the present disclosure may be administered to patients parenterally, for example, by intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.
  • 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.
  • agents capable of promoting penetration of the subject antisense oligonucleotide across the blood-brain barrier by any method known in the art.
  • delivery of agents by administration of an adenovirus vector to motor neurons in muscle tissue is described in U.S. Pat. No. 6,632,427, “Adenoviral-vector-mediated gene transfer into medullary motor neurons,” incorporated herein by reference.
  • Delivery of vectors directly to the brain e.g., the striatum, the thalamus, the hippocampus, or the substantia nigra, is described, e.g., in U.S. Pat. No. 6,756,523, “Adenovirus vectors for the transfer of foreign genes into cells of the central nervous system particularly in brain,” incorporated herein by reference.
  • 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.
  • an ASO of the disclosure is coupled to a dopamine reuptake inhibitor (DRI), a selective serotonin reuptake inhibitor (S SRI), a noradrenaline reuptake inhibitor (NRI), a norepinephrine-dopamine reuptake inhibitor (NDRI), and a serotonin-norepinephrine-dopamine reuptake inhibitor (SNDRI), using methods described in, e.g., U.S. Pat. No. 9,193,969, incorporated herein by reference.
  • DRI dopamine reuptake inhibitor
  • S SRI selective serotonin reuptake inhibitor
  • NRI noradrenaline reuptake inhibitor
  • NDRI norepinephrine-dopamine reuptake inhibitor
  • SNDRI serotonin-norepinephrine-dopamine reuptake inhibitor
  • subjects treated using the methods and compositions are evaluated for improvement in condition using any methods known and described in the art.
  • a method can comprise identifying or determining ASOs that induce pseudo-exon skipping of a ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAIL RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD
  • a round of screening 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 3′ splice site of the included exon (e.g., a portion of sequence of the exon located upstream of the target/included exon) to approximately 100 nucleotides downstream of the 3′ splice site of the target/included exon and/or from approximately 100 nucleotides upstream of the 5′ splice site of the included exon to approximately 100 nucleotides downstream of the 5′ splice site of the target/included exon (e.g., a portion of sequence of the exon located downstream of the target/included exon).
  • a first ASO of 15 nucleotides in length may be designed to specifically hybridize to nucleotides +6 to +20 relative to the 3′ splice site of the target/included exon.
  • a second ASO may be designed to specifically hybridize to nucleotides +11 to +25 relative to the 3′ splice site of the target/included exon.
  • ASOs are designed as such spanning the target region of the pre-mRNA. In embodiments, the ASOs can be tiled more closely, e.g., every 1, 2, 3, or 4 nucleotides.
  • 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., a NIE containing pre-mRNA described herein).
  • a disease-relevant cell line that expresses the target pre-mRNA (e.g., a NIE containing pre-mRNA described herein).
  • the exon skipping 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 in Example 4.
  • RT reverse transcriptase
  • the exon skipping efficiency (or the splicing efficiency to splice the intron containing the NIE), 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 functional 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 exon skipping (or enhanced splicing of NIE).
  • 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 NIE, as described herein (see, e.g., Example 4).
  • a reduction or absence of a longer RT-PCR product produced using the primers spanning the NIE in ASO-treated cells as compared to in control ASO-treated cells indicates that exon skipping (or splicing of the target intron containing an NIE) has been enhanced.
  • the exon skipping efficiency (or the splicing efficiency to splice the intron containing the NIE), 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 functional 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 exon skipping (or enhanced splicing of the intron containing a NIE) and increased protein production may be tested in vivo using animal models, for example transgenic mouse models in which the full-length human gene has been knocked-in or in humanized mouse models of disease.
  • Suitable routes for administration of ASOs may vary depending on the disease and/or the cell types to which delivery of the ASOs is desired.
  • ASOs may be administered, for example, by intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.
  • 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 (e.g., 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 2 Also within the scope of the present disclosure is a method to identify or validate an NMD-inducing exon in the presence of an NMD inhibitor, for example, cycloheximide.
  • an NMD inhibitor for example, cycloheximide.
  • Embodiment A1 A method of treating Alport syndrome; Amyotrophic lateral sclerosis (ALS); Angelman syndrome; Aphasia, primary progressive; Arrhythmogenic right ventricular dysplasia 9; Autism spectrum disorder; Cardiomyopathy, dilated, 1HH; Myopathy, myofibrillar 6; Ceriod lipofuscinosis, neuronal, 3; Cholestasis, intrahepatic, of pregnancy, 3; Cholestasis, progressive familial intrahepatic 1; Citrullinemia Type II; Citrullinemia, Type 1; Cognitive impairment with or without cerebral ataxia; Cornelia de Lange; Early-onset epileptic encephalopathy; Epilepsy-aphasia spectrum; Epilepsy, generalized, with febrile seizures plus, type 7; Epileptic encephalopathy, childhood-onset; Epileptic encephalopathy, early infantile, 11; Epileptic encephalopathy, early infantile, 12; Epileptic encephalopathy, early infantile, 13; Epileptic ence
  • Embodiment A2 The method of embodiment A1, wherein the target protein is ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAIL RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, or VCAN.
  • Embodiment A3 A method of increasing expression of ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, or VCAN protein by a cell having an mRNA that contains a non-s
  • Embodiment A4 The method of any one of embodiments A1 to A3, wherein the non-sense mediated RNA decay-inducing exon is spliced out from the NMD exon mRNA encoding the target protein or functional RNA.
  • Embodiment A5 The method of any one of embodiments A1 to A4, wherein the target protein does not comprise an amino acid sequence encoded by the non-sense mediated RNA decay-inducing exon.
  • Embodiment A6 The method of any one of embodiments A1 to A5, wherein the target protein is a full-length target protein.
  • Embodiment A7 The method of any one of embodiments A1 to A6, wherein the agent is an antisense oligomer (ASO) complementary to the targeted portion of the NMD exon mRNA.
  • ASO antisense oligomer
  • Embodiment A8 The method of any one of embodiments A1 to A7, wherein the mRNA is pre-mRNA.
  • Embodiment A9 The method of any one of embodiments A1 to A8, wherein the contacting comprises contacting the therapeutic agent to the mRNA, wherein the mRNA is in a nucleus of the cell.
  • Embodiment A10 The method of any one of embodiments A1 to A9, wherein the target protein or the functional RNA corrects a deficiency in the target protein or functional RNA in the subject.
  • Embodiment A11 The method of any one of embodiments A1 to A10, wherein the cells are in or from a subject with a condition caused by a deficient amount or activity of ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAIL RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK
  • Embodiment A12 The method of any one of embodiments A1 to A11, wherein the deficient amount of the target protein is caused by haploinsufficiency of the target protein, wherein the subject has a first allele encoding a functional target protein, and a second allele from which the target protein is not produced, or a second allele encoding a nonfunctional target protein, and wherein the antisense oligomer binds to a targeted portion of a NMD exon mRNA transcribed from the first allele.
  • Embodiment A13 The method of any one of embodiments A1 to A11, wherein the subject has a condition caused by a disorder resulting from a deficiency in the amount or function of the target protein, wherein the subject has
  • Embodiment A14 The method of embodiment A13, wherein the target protein is produced in a form having reduced function compared to the equivalent wild-type protein.
  • Embodiment A15 The method of embodiment A13, wherein the target protein is produced in a form that is fully-functional compared to the equivalent wild-type protein.
  • Embodiment A16 The method of any one of embodiments A1 to A15, wherein the targeted portion of the NMD exon mRNA is within the non-sense mediated RNA decay-inducing exon.
  • Embodiment A17 The method of any one of embodiments A1 to A15, wherein the targeted portion of the NMD exon mRNA is either upstream or downstream of the non-sense mediated RNA decay-inducing exon.
  • Embodiment A19 The method of any one of embodiments A1 to A17, wherein the NMD exon mRNA is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NOs: 1-59.
  • Embodiment A20 The method of any one of embodiments A1 to A17, wherein the targeted portion of the NMD exon mRNA comprises a sequence with at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 contiguous nucleic acids of SEQ ID NO: SEQ ID NOs: 60-134.
  • Embodiment A21 The method of any one of embodiments A1 to A20, wherein the agent is an antisense oligomer (ASO) and wherein the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complementary to at least 8 contiguous nucleic acids of SEQ ID NOs: 60-134.
  • ASO antisense oligomer
  • Embodiment A22 The method of any one of embodiments A1 to A15, wherein the targeted portion of the NMD exon mRNA is within the non-sense mediated RNA decay-inducing exon 8 ⁇ of ABCB4, exon 9 ⁇ of ASS1, exon 16 ⁇ of ATP8B1, exon 1 ⁇ of BAG3, exon 31 ⁇ of CACNA1A, exon 36 ⁇ of CACNA1A, exon 37 ⁇ of CACNA1A, exon 3 ⁇ of CBS, exon 12 ⁇ of CBS, exon 1 ⁇ of CD55, exon 16 ⁇ of CDKL5, exon 3 ⁇ of CFH, exon 30 ⁇ of CHD2, exon 4 ⁇ of CHRNA7, exon 1 ⁇ of CISD2, exon 15 ⁇ of CLN3, exon 11 ⁇ of COL4A3, exon 41 ⁇ of COL4A3, exon 22 ⁇ of COL4A4, exon 44 ⁇ of COL4A4, exon 20 ⁇ of DEPDC5, exon 2 ⁇ of DHDDS, exon 3 ⁇ of
  • Embodiment A23 The method of any one of embodiments A1 to A15, wherein the targeted portion of the NMD exon mRNA is upstream or downstream of the non-sense mediated RNA decay-inducing exon 8 ⁇ of ABCB4, exon 9 ⁇ of ASS1, exon 16 ⁇ of ATP8B1, exon 1 ⁇ of BAG3, exon 31 ⁇ of CACNA1A, exon 36 ⁇ of CACNA1A, exon 37 ⁇ of CACNA1A, exon 3 ⁇ of CBS, exon 12 ⁇ of CBS, exon 1 ⁇ of CD55, exon 16 ⁇ of CDKL5, exon 3 ⁇ of CFH, exon 30 ⁇ of CHD2, exon 4 ⁇ of CHRNA7, exon 1 ⁇ of CISD2, exon 15 ⁇ of CLN3, exon 11 ⁇ of COL4A3, exon 41 ⁇ of COL4A3, exon 22 ⁇ of COL4A4, exon 44 ⁇ of COL4A4, exon 20 ⁇ of DEPDC5, exon 2 ⁇ of DHDDS, ex
  • Embodiment A24 The method of any one of embodiments A1 to A15, wherein the targeted portion of the NMD exon mRNA comprises an exon-intron junction exon 8 ⁇ of ABCB4, exon 9 ⁇ of ASS1, exon 16 ⁇ of ATP8B1, exon 1 ⁇ of BAG3, exon 31 ⁇ of CACNA1A, exon 36 ⁇ of CACNA1A, exon 37 ⁇ of CACNA1A, exon 3 ⁇ of CBS, exon 12 ⁇ of CBS, exon 1 ⁇ of CD55, exon 16 ⁇ of CDKL5, exon 3 ⁇ of CFH, exon 30 ⁇ of CHD2, exon 4 ⁇ of CHRNA7, exon 1 ⁇ of CISD2, exon 15 ⁇ of CLN3, exon 11 ⁇ of COL4A3, exon 41 ⁇ of COL4A3, exon 22 ⁇ of COL4A4, exon 44 ⁇ of COL4A4, exon 20 ⁇ of DEPDC5, exon 2 ⁇ of DHDDS, exon 3 ⁇ of ELOVL4, exon 5
  • Embodiment A25 The method of any one of embodiments A1 to A24, wherein the target protein produced is full-length protein, or wild-type protein.
  • Embodiment A26 The method of any one of embodiments A1 to A25, wherein the total amount of the mRNA encoding the target protein or functional RNA produced in the cell contacted with the antisense oligomer is increased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5
  • Embodiment A27 The method of any one of embodiments A1 to A25, wherein the total amount of the mRNA encoding the target protein or functional RNA produced in the cell contacted with the antisense oligomer is increased about 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 10%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%, compared to the total amount of the mRNA encoding the target protein or
  • Embodiment A28 The method of one any of embodiments A1 to A25, wherein the total amount of target protein produced by the cell contacted with the antisense oligomer is increased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about
  • Embodiment A29 The method of one any of embodiments A1 to A25, wherein the total amount of target protein produced by the cell contacted with the antisense oligomer is increased about 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 10%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%, compared to the total amount of target protein produced by a control cell.
  • Embodiment A30 The method of any one of embodiments A1 to 29, wherein the agent is an antisense oligomer (ASO) and wherein the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.
  • ASO antisense oligomer
  • Embodiment A31 The method of any one of embodiments A1 to A30, wherein the agent is an antisense oligomer (ASO) and wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2′-O-methyl, a 2′-Fluoro, or a 2′-O-methoxyethyl moiety.
  • ASO antisense oligomer
  • Embodiment A32 The method of any one of embodiments A1 to A31, wherein the agent is an antisense oligomer (ASO) and wherein the antisense oligomer comprises at least one modified sugar moiety.
  • ASO antisense oligomer
  • Embodiment A33 The method of embodiment A32, wherein each sugar moiety is a modified sugar moiety.
  • Embodiment A34 The method of any one of embodiments A1 to A33, wherein the agent is an antisense oligomer (ASO) and wherein the antisense oligomer consists of from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nu
  • Embodiment A35 The method of any one of embodiments A1 to A34, wherein the agent is an antisense oligomer (ASO) and wherein the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, complementary to the targeted portion of the NMD exon mRNA encoding the protein.
  • ASO antisense oligomer
  • Embodiment A36 The method of any one of embodiments A1 to A35, wherein the method further comprises assessing ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAH, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, or VCAN
  • Embodiment A37 The method of any one of embodiments A1 to A36, wherein Alport syndrome; Amyotrophic lateral sclerosis (ALS); Angelman syndrome; Aphasia, primary progressive; Arrhythmogenic right ventricular dysplasia 9; Autism spectrum disorder; Cardiomyopathy, dilated, 1HH; Myopathy, myofibrillar 6; Ceriod lipofuscinosis, neuronal, 3; Cholestasis, intrahepatic, of pregnancy, 3; Cholestasis, progressive familial intrahepatic 1; Citrullinemia Type II; Citrullinemia, Type 1; Cognitive impairment with or without cerebral ataxia; Cornelia de Lange; Early-onset epileptic encephalopathy; Epilepsy-aphasia spectrum; Epilepsy, generalized, with febrile seizures plus, type 7; Epileptic encephalopathy, childhood-onset; Epileptic encephalopathy, early infantile, 11; Epileptic encephalopathy, early infantile, 12; Epileptic encephalopathy
  • Embodiment A38 The method of any one of embodiments A1 to A37, wherein the subject is a human.
  • Embodiment A39 The method of any one of embodiments A1 to A38, wherein the subject is a non-human animal.
  • Embodiment A40 The method of any one of embodiments A1 to A39, wherein the subject is a fetus, an embryo, or a child.
  • Embodiment A41 The method of any one of embodiments A1 to A40, wherein the cells are ex vivo.
  • Embodiment A42 The method of any one of embodiments A1 to A41, wherein the therapeutic agent is administered by intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection of the subject.
  • Embodiment A43 The method of any of embodiments A1 to A42, wherein the method further comprises administering a second therapeutic agent to the subject.
  • Embodiment A44 The method of embodiment A43, wherein the second therapeutic agent is a small molecule.
  • Embodiment A45 The method of embodiment A43, wherein the second therapeutic agent is an ASO.
  • Embodiment A47 An antisense oligomer as used in a method of any of embodiments A1 to A46.
  • Embodiment A50 A method of treating a subject in need thereof, comprising administering the pharmaceutical composition of embodiment A49 to the subject, wherein the administering is by intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.
  • Embodiment A51 A composition comprising a therapeutic agent for use in a method of increasing expression of a target protein or a functional RNA by cells to treat Alport syndrome; Amyotrophic lateral sclerosis (ALS); Angelman syndrome; Aphasia, primary progressive; Arrhythmogenic right ventricular dysplasia 9; Autism spectrum disorder; Cardiomyopathy, dilated, 1HH; Myopathy, myofibrillar 6; Ceriod lipofuscinosis, neuronal, 3; Cholestasis, intrahepatic, of pregnancy, 3; Cholestasis, progressive familial intrahepatic 1; Citrullinemia Type II; Citrullinemia, Type 1; Cognitive impairment with or without cerebral ataxia; Cornelia de Lange; Early-onset epileptic encephalopathy; Epilepsy-aphasia spectrum; Epilepsy, generalized, with febrile seizures plus, type 7; Epileptic encephalopathy, childhood-onset; Epileptic encephalopathy, early infantile, 11; Epileptic ence
  • Embodiment A52 A composition comprising a therapeutic agent for use in a method of treating a condition associated with ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, or VCAN protein in
  • Embodiment A54 The composition of embodiment A53, wherein the disease or disorder is Alport syndrome; Amyotrophic lateral sclerosis (ALS); Angelman syndrome; Aphasia, primary progressive; Arrhythmogenic right ventricular dysplasia 9; Autism spectrum disorder; Cardiomyopathy, dilated, 1HH; Myopathy, myofibrillar 6; Ceriod lipofuscinosis, neuronal, 3; Cholestasis, intrahepatic, of pregnancy, 3; Cholestasis, progressive familial intrahepatic 1; Citrullinemia Type II; Citrullinemia, Type 1; Cognitive impairment with or without cerebral ataxia; Cornelia de Lange; Early-onset epileptic encephalopathy; Epilepsy-aphasia spectrum; Epilepsy, generalized, with febrile seizures plus, type 7; Epileptic encephalopathy, childhood-onset; Epileptic encephalopathy, early infantile, 11; Epileptic encephalopathy, early infantile, 12; Epileptic encephalopathy, early
  • Embodiment A55 The composition of any one of embodiments A52 to 54, wherein the ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, or VCAN protein and NMD exon
  • Embodiment A56 The composition of any one of embodiments A51 to A55, wherein the non-sense mediated RNA decay-inducing exon is spliced out from the NMD exon mRNA encoding the ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25
  • Embodiment A58 The composition of any one of embodiments A51 to A57, wherein the ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, or VCAN protein is a full-
  • Embodiment A59 The composition of any one of embodiments A51 to A58, wherein the therapeutic agent is an antisense oligomer (ASO) complementary to the targeted portion of the NMD exon mRNA.
  • ASO antisense oligomer
  • Embodiment A60 The composition of any of embodiments A51 to A59, wherein the therapeutic agent is an antisense oligomer (ASO) and wherein the antisense oligomer targets a portion of the NMD exon mRNA that is within the non-sense mediated RNA decay-inducing exon.
  • ASO antisense oligomer
  • Embodiment A61 The composition of any of embodiments A51 to A59, wherein the therapeutic agent is an antisense oligomer (ASO) and wherein the antisense oligomer targets a portion of the NMD exon mRNA that is upstream or downstream of the non-sense mediated RNA decay-inducing exon.
  • ASO antisense oligomer
  • Embodiment A62 The composition of any one of embodiments A51 to A61, wherein the target protein is ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, or VCAN.
  • Embodiment A63 The composition of embodiment A62, wherein the NMD exon mRNA comprises a sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 60-134.
  • Embodiment A64 The composition of embodiment A62, wherein the NMD exon mRNA is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 1-59.
  • Embodiment A66 The composition of any one of embodiments A62 to A65, wherein the targeted portion of the NMD exon mRNA is within the non-sense mediated RNA decay-inducing exon 8 ⁇ of ABCB4, exon 9 ⁇ of ASS1, exon 16 ⁇ of ATP8B1, exon 1 ⁇ of BAG3, exon 31 ⁇ of CACNA1A, exon 36 ⁇ of CACNA1A, exon 37 ⁇ of CACNA1A, exon 3 ⁇ of CBS, exon 12 ⁇ of CBS, exon 1 ⁇ of CD55, exon 16 ⁇ of CDKL5, exon 3 ⁇ of CFH, exon 30 ⁇ of CHD2, exon 4 ⁇ of CHRNA7, exon 1 ⁇ of CISD2, exon 15 ⁇ of CLN3, exon 11 ⁇ of COL4A3, exon 41 ⁇ of COL4A3, exon 22 ⁇ of COL4A4, exon 44 ⁇ of COL4A4, exon 20 ⁇ of DEPDC5, exon 2 ⁇ of DHDDS, exon 3 ⁇ of
  • Embodiment A67 The composition of any one of embodiments A62 to A65, wherein the targeted portion of the NMD exon mRNA is upstream or downstream of the non-sense mediated RNA decay-inducing exon 8 ⁇ of ABCB4, exon 9 ⁇ of ASS1, exon 16 ⁇ of ATP8B1, exon 1 ⁇ of BAG3, exon 31 ⁇ of CACNA1A, exon 36 ⁇ of CACNA1A, exon 37 ⁇ of CACNA1A, exon 3 ⁇ of CBS, exon 12 ⁇ of CBS, exon 1 ⁇ of CD55, exon 16 ⁇ of CDKL5, exon 3 ⁇ of CFH, exon 30 ⁇ of CHD2, exon 4 ⁇ of CHRNA7, exon 1 ⁇ of CISD2, exon 15 ⁇ of CLN3, exon 11 ⁇ of COL4A3, exon 41 ⁇ of COL4A3, exon 22 ⁇ of COL4A4, exon 44 ⁇ of COL4A4, exon 20 ⁇ of DEPDC5, exon 2 ⁇ of DHDDS, ex
  • Embodiment A68 The composition of any one of embodiments A62 to A65, wherein the targeted portion of the NMD exon mRNA comprises an exon-intron junction of exon 8 ⁇ of ABCB4, exon 9 ⁇ of ASS1, exon 16 ⁇ of ATP8B1, exon 1 ⁇ of BAG3, exon 31 ⁇ of CACNA1A, exon 36 ⁇ of CACNA1A, exon 37 ⁇ of CACNA1A, exon 3 ⁇ of CBS, exon 12 ⁇ of CBS, exon 1 ⁇ of CD55, exon 16 ⁇ of CDKL5, exon 3 ⁇ of CFH, exon 30 ⁇ of CHD2, exon 4 ⁇ of CHRNA7, exon lx of CISD2, exon 15 ⁇ of CLN3, exon 11 ⁇ of COL4A3, exon 41 ⁇ of COL4A3, exon 22 ⁇ of COL4A4, exon 44 ⁇ of COL4A4, exon 20 ⁇ of DEPDC5, exon 2 ⁇ of DHDDS, exon 3 ⁇ of ELOVL4, ex
  • Embodiment A69 The composition of any one of embodiments A62 to A68, wherein the therapeutic agent is an antisense oligomer (ASO) and wherein the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complementary to a region comprising at least 8 contiguous nucleic acids of SEQ ID NOs: 60-134.
  • ASO antisense oligomer
  • Embodiment A70 The composition of any one of embodiments A51 to A69, wherein the mRNA encoding the target protein or functional RNA is a full-length mature mRNA, or a wild-type mature mRNA.
  • Embodiment A71 The composition of any one of embodiments A51 to A70, wherein the target protein produced is full-length protein, or wild-type protein.
  • Embodiment A72 The composition of any one of embodiments A51 to A71, wherein the therapeutic agent is an antisense oligomer (ASO) and wherein the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.
  • ASO antisense oligomer
  • Embodiment A73 The composition of any of embodiments A51 to A72, wherein the therapeutic agent is an antisense oligomer (ASO) and wherein said antisense oligomer is an antisense oligonucleotide.
  • the therapeutic agent is an antisense oligomer (ASO) and wherein said antisense oligomer is an antisense oligonucleotide.
  • Embodiment A74 The composition of any of embodiments A51 to A73, wherein the therapeutic agent is an antisense oligomer (ASO) and wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2′-O-methyl, a 2′-Fluoro, or a 2′-O-methoxyethyl moiety.
  • ASO antisense oligomer
  • Embodiment A75 The composition of any of embodiments A51 to A74, wherein the therapeutic agent is an antisense oligomer (ASO) and wherein the antisense oligomer comprises at least one modified sugar moiety.
  • ASO antisense oligomer
  • Embodiment A76 The composition of embodiment A75, wherein each sugar moiety is a modified sugar moiety.
  • Embodiment A77 The composition of any of embodiments A51 to A76, wherein the therapeutic agent is an antisense oligomer (ASO) and wherein the antisense oligomer consists of from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nu
  • Embodiment A78 A pharmaceutical composition comprising the therapeutic agent of any of the compositions of embodiments A51 to A77, and an excipient.
  • Embodiment A79 A method of treating a subject in need thereof, comprising administering the pharmaceutical composition of embodiment A78 to the subject, wherein the administering is by intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.
  • Embodiment A80 The method of any of embodiments A51 to A79, wherein the method further comprises administering a second therapeutic agent to the subject.
  • Embodiment A81 The method of embodiment A80, wherein the second therapeutic agent is a small molecule.
  • Embodiment A82 The method of embodiment A80, wherein the second therapeutic agent is an ASO.
  • Embodiment A83 The method of any one of embodiments A80 to A82, wherein the second therapeutic agent corrects intron retention.
  • Embodiment A84 A pharmaceutical composition comprising: an antisense oligomer that hybridizes to a target sequence of a ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A,
  • Embodiment A86 The pharmaceutical composition of embodiment A84 or A85, wherein the targeted portion of the NMD exon mRNA is within the non-sense mediated RNA decay-inducing exon 8 ⁇ of ABCB4, exon 9 ⁇ of ASS1, exon 16 ⁇ of ATP8B1, exon 1 ⁇ of BAG3, exon 31 ⁇ of CACNA1A, exon 36 ⁇ of CACNA1A, exon 37 ⁇ of CACNA1A, exon 3 ⁇ of CBS, exon 12 ⁇ of CBS, exon 1 ⁇ of CD55, exon 16 ⁇ of CDKL5, exon 3 ⁇ of CFH, exon 30 ⁇ of CHD2, exon 4 ⁇ of CHRNA7, exon 1 ⁇ of CISD2, exon 15 ⁇ of CLN3, exon 11 ⁇ of COL4A3, exon 41 ⁇ of COL4A3, exon 22 ⁇ of COL4A4, exon 44 ⁇ of COL4A4, exon 20 ⁇ of DEPDC5, exon 2 ⁇ of DHDDS, exon 3 ⁇ of ELOV
  • Embodiment A88 The pharmaceutical composition of embodiment A84 or A85, wherein the targeted portion of the NMD exon mRNA comprises an exon-intron junction exon 8 ⁇ of ABCB4, exon 9 ⁇ of ASS1, exon 16 ⁇ of ATP8B1, exon 1 ⁇ of BAG3, exon 31 ⁇ of CACNA1A, exon 36 ⁇ of CACNA1A, exon 37 ⁇ of CACNA1A, exon 3 ⁇ of CBS, exon 12 ⁇ of CBS, exon 1 ⁇ of CD55, exon 16 ⁇ of CDKL5, exon 3 ⁇ of CFH, exon 30 ⁇ of CHD2, exon 4 ⁇ of CHRNA7, exon 1 ⁇ of CISD2, exon 15 ⁇ of CLN3, exon 11 ⁇ of COL4A3, exon 41 ⁇ of COL4A3, exon 22 ⁇ of COL4A4, exon 44 ⁇ of COL4A4, exon 20 ⁇ of DEPDC5, exon 2 ⁇ of DHDDS, exon 3 ⁇ of ELOVL4, exon 5 ⁇ of FA
  • Embodiment A89 The pharmaceutical composition of any one of embodiments A84 to A88, wherein the ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, or VCAN NMD exon
  • Embodiment A93 The pharmaceutical composition of embodiment A84, wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2′-O-methyl, a 2′-Fluoro, or a 2′-O-methoxyethyl moiety.
  • Embodiment A94 The pharmaceutical composition of embodiment A84, wherein the antisense oligomer comprises at least one modified sugar moiety.
  • Embodiment A96 The pharmaceutical composition of embodiment A84 or A85, wherein the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or is 100% complementary to a targeted portion of the ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN
  • Embodiment A98 The pharmaceutical composition of embodiment A84, wherein the antisense oligomer comprises a nucleotide sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a region comprising at least 8 contiguous nucleic acids of SEQ ID NOs: 60-134.
  • Embodiment A99 The pharmaceutical composition of embodiment A84, wherein the antisense oligomer comprises a nucleotide sequence that is identical a region comprising at least 8 contiguous nucleic acids of SEQ ID NOs: 60-134.
  • Embodiment A100 The pharmaceutical composition of any one of the embodiments A84 to A99, wherein the pharmaceutical composition is formulated for intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.
  • Embodiment A101 The method of any of embodiments A84 to A100, wherein the method further comprises administering a second therapeutic agent to the subject.
  • Embodiment A102 The method of embodiment A101, wherein the second therapeutic agent is a small molecule.
  • Embodiment A103 The method of embodiment A101, wherein the second therapeutic agent is an ASO.
  • Embodiment A104 The method of any one of embodiments A101 to A103, wherein the second therapeutic agent corrects intron retention.
  • Embodiment A105 A method of inducing processing of a deficient ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, or VCAN mRNA transcript to facilitate removal of a non
  • Embodiment A106 A method of treating a subject having a condition caused by a deficient amount or activity of ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, or VCAN protein compris
  • Embodiment A107 A method of treating Alport syndrome; Amyotrophic lateral sclerosis (ALS); Angelman syndrome; Aphasia, primary progressive; Arrhythmogenic right ventricular dysplasia 9; Autism spectrum disorder; Cardiomyopathy, dilated, 1HH; Myopathy, myofibrillar 6; Ceriod lipofuscinosis, neuronal, 3; Cholestasis, intrahepatic, of pregnancy, 3; Cholestasis, progressive familial intrahepatic 1; Citrullinemia Type II; Citrullinemia, Type 1; Cognitive impairment with or without cerebral ataxia; Cornelia de Lange; Early-onset epileptic encephalopathy; Epilepsy-aphasia spectrum; Epilepsy, generalized, with febrile seizures plus, type 7; Epileptic encephalopathy, childhood-onset; Epileptic encephalopathy, early infantile, 11; Epileptic encephalopathy, early infantile, 12; Epileptic encephalopathy, early infantile, 13; Epileptic ence
  • Embodiment A108 A method of increasing expression of ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, or VCAN protein by a cell having an mRNA that contains a non-s
  • Embodiment 1 A method of modulating expression of a target protein, by a cell having an mRNA that comprises a non-sense mediated RNA decay-inducing exon (NMD exon) and encodes the target protein, the method comprising contacting a therapeutic agent to the cell, whereby the therapeutic agent modulates splicing of the NMD exon from the mRNA, thereby modulating level of processed mRNA encoding the target protein, and modulating the expression of the target protein in the cell, wherein the target protein is selected from the group consisting of: AKT3, CACNA1A, CBS, CD46, CFH, CHD2, CLN3, COL11A2, COL4A3, COL4A4, COL4A4, CR1, CRX, CYP2J2, DHDDS, DNAJC8, EIF2AK3, ERN1, GALE, GUCY2F, GUCY2F, HEXA, HEXA, MAPK3, MBD5, MBD5, MBD5, MUT, MYH
  • Embodiment 2 A method of treating a disease or condition in a subject in need thereof by modulating expression of a target protein in a cell of the subject, comprising: contacting the cell of the subject with a therapeutic agent that modulates splicing of a non-sense mediated mRNA decay-inducing exon (NMD exon) from an mRNA in the cell, wherein the mRNA comprises the NMD exon and encodes the target protein, thereby modulating level of processed mRNA encoding the target protein, and modulating expression of the target protein in the cell of the subject, wherein the target protein is selected from the group consisting of: AKT3, CACNA1A, CBS, CD46, CFH, CHD2, CLN3, COL11A2, COL4A3, COL4A4, COL4A4, CR1, CRX, CYP2J2, DHDDS, DNAJC8, EIF2AK3, ERN1, GALE, GUCY2F, GUCY2F, HEXA, HEX
  • Embodiment 3 The method of embodiment 1 or 2, wherein the therapeutic agent
  • Embodiment 4 The method of embodiment 3, wherein the therapeutic agent interferes with binding of the factor involved in splicing of the NMD exon to a region of the targeted portion.
  • Embodiment 5 The method of embodiment 3 or 4, wherein the targeted portion is proximal to the NMD exon.
  • Embodiment 7 The method of any one of embodiments 3 to 6, wherein the targeted portion is at least about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, about 1 nucleotides upstream of 5′ end of the NMD exon.
  • Embodiment 8 The method of any one of embodiments 3 to 5, wherein the targeted portion is at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream of 3′ end of the NMD exon.
  • Embodiment 9 The method of any one of embodiments 3 to 5 or 8, wherein the targeted portion is at least about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, about 1 nucleotides downstream of 3′ end of the NMD exon.
  • Embodiment 11 The method of any one of embodiments 3 to 5 or 10, wherein the targeted portion is about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides upstream of genomic site selected from the group consisting of: GRCh38/hg38: chr1 243564388; GRCh38/hg38: chr19 13236618; GRCh38/hg38: chr21 43060012; GRCh38/hg38: chr1 207775610; GRCh38/hg38: chr1 196675450; GRCh38/hg38: chr15 92998
  • Embodiment 12 The method of any one of embodiments 3 to 5, wherein the targeted portion is at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream of genomic site selected from the group consisting of: GRCh38/hg38: chr1 243564285; GRCh38/hg38: chr19 13236449; GRCh38/hg38: chr21 43059730; GRCh38/hg38: chr1 207775745; GRCh38/hg38: chr1 196675529; GRCh38/hg38: chr15 9299
  • Embodiment 13 The method of any one of embodiments 3 to 5 or 12, wherein the targeted portion is about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream of genomic site selected from the group consisting of: GRCh38/hg38: chr1 243564285; GRCh38/hg38: chr19 13236449; GRCh38/hg38: chr21 43059730; GRCh38/hg38: chr1 207775745; GRCh38/hg38: chr1 196675529; GRCh38/hg38: chr15 9299
  • Embodiment 14 The method of any one of embodiments 3 to 13, wherein the targeted portion is located in an intronic region between two canonical exonic regions of the mRNA encoding the target protein, and wherein the intronic region contains the NMD exon.
  • Embodiment 15 The method of any one of embodiments 3 to 14, wherein the targeted portion at least partially overlaps with the NMD exon.
  • Embodiment 17 The method of any one of embodiments 3 to 16, wherein the targeted portion comprises 5′ NMD exon-intron junction or 3′ NMD exon-intron junction.
  • Embodiment 18 The method of any one of embodiments 3 to 16, wherein the targeted portion is within the NMD exon.
  • Embodiment 19 The method of any one of embodiments 1 to 18, wherein the targeted portion comprises about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more consecutive nucleotides of the NMD exon.
  • Embodiment 20 The method of any one of embodiments 1 to 19, wherein the mRNA encoding the target protein comprises a sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 135-191.
  • Embodiment 21 The method of any one of embodiments 1 to 20, wherein the mRNA encoding the target protein is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1-5, 12, 19-21, 25, 26, 28, 30, 33, 35, 38, 40, 41, 44, 45, 51, 53, 55-57, and 192-211.
  • Embodiment 23 The method of any one of embodiments 1 to 22, wherein the agent is an antisense oligomer (ASO) and wherein the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complementary to at least 8 contiguous nucleic acids of a sequence selected from the group consisting of SEQ ID NOs: 135-191.
  • ASO antisense oligomer
  • Embodiment 25 The method of any one of embodiments 3 to 23, wherein the targeted portion of the mRNA is upstream or downstream of the non-sense mediated RNA decay-inducing exon selected from the group consisting of: GRCh38/hg38: chr1 243564285 243564388; GRCh38/hg38: chr19 13236449 13236618; GRCh38/hg38: chr21 43059730 43060012; GRCh38/hg38: chr1 207775610 207775745; GRCh38/hg38: chr1 196675450 196675529; GRCh38/hg38: chr15 92998149 92998261; GRCh38/hg38: chr16 28479644 28479765; GRCh38/hg38: chr6 33183634 33183698; GRCh38/hg38: chr2 227296487 227
  • Embodiment 26 The method of any one of embodiments 3 to 23, wherein the targeted portion of the mRNA comprises an exon-intron junction of exon selected from the group consisting of: GRCh38/hg38: chr1 243564285 243564388; GRCh38/hg38: chr19 13236449 13236618; GRCh38/hg38: chr21 43059730 43060012; GRCh38/hg38: chr1 207775610 207775745; GRCh38/hg38: chr1 196675450 196675529; GRCh38/hg38: chr15 92998149 92998261; GRCh38/hg38: chr16 28479644 28479765; GRCh38/hg38: chr6 33183634 33183698; GRCh38/hg38: chr2 227296487 227296526; GRCh38/hg
  • Embodiment 28 The method of any one of embodiments 1 to 27, wherein the therapeutic agent promotes exclusion of the NMD exon from the processed mRNA encoding the target protein.
  • Embodiment 29 The method of embodiment 28, wherein exclusion of the NMD exon from the processed mRNA encoding the target protein in the cell contacted with the therapeutic agent is increased by about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 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
  • Embodiment 30 The method of embodiment 28 or 29, wherein the therapeutic agent increases the level of the processed mRNA encoding the target protein in the cell.
  • Embodiment 31 The method of any one of embodiments 28 to 30, wherein the level of the processed mRNA encoding the target protein produced in the cell contacted with the therapeutic agent is increased by about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 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
  • Embodiment 32 The method of any one of embodiments 28 to 31, wherein the therapeutic agent increases the expression of the target protein in the cell.
  • Embodiment 33 The method of any one of embodiments 28 to 32, wherein a level of the target protein produced in the cell contacted with the therapeutic agent is increased by about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 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
  • Embodiment 35 The method of embodiment 34, wherein the disease or condition is associated with haploinsufficiency of a gene encoding the target protein, and 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 produced at a reduced level, or a second allele encoding a nonfunctional target protein or a partially functional target protein.
  • Embodiment 36 The method of any one of embodiments 2 to 35, wherein the disease or condition is selected from the group consisting of: Sotos syndrome 1; Beckwith-Wiedemann syndrome; Migraine, familial hemiplegic, 1; Episodic ataxia, type 2; Epileptic encephalopathy, childhood-onset; Wagner syndrome 1; Optic atrophy type 1; Alport syndrome; Arrhythmogenic right ventricular dysplasia 9; Neurofibromatosis type 1; Epileptic encephalopathy, early infantile, 11; Seizures, benign familial infantile, 3; Cognitive impairment with or without cerebellar ataxia; Epileptic encephalopathy, early infantile, 13; Seizures, benign familial infantile, 5; Pathway (CNS); 16p11.2 deletion syndrome?; Mental retardation, autosomal dominant 1; Retinitis pigmentosa 18; Retinitis pigmentosa 31; Deafness, autosomal dominant 13; Cone-rod retinal dystrophy-2
  • Embodiment 37 The method of any one of embodiments 2 to 36, wherein the disease or condition is associated with an autosomal recessive mutation of a gene encoding the target protein, wherein the subject has a first allele encoding from which:
  • Embodiment 38 The method of embodiment 37, wherein the disease or condition is selected from the group consisting of: Alport syndrome; Ceroid lipofuscinosis, neuronal, 3; Galactose epimerase deficiency; Homocystinuria, B6-responsive and nonresponsive types; Methyl Malonic Aciduria; Propionic acidemia; Retinitis pigmentosa 59; Tay-Sachs disease; Insensitivity to pain, congenital; and HSAN2D, autosomal recessive.
  • the disease or condition is selected from the group consisting of: Alport syndrome; Ceroid lipofuscinosis, neuronal, 3; Galactose epimerase deficiency; Homocystinuria, B6-responsive and nonresponsive types; Methyl Malonic Aciduria; Propionic acidemia; Retinitis pigmentosa 59; Tay-Sachs disease; Insensitivity to pain, congenital; and HSAN2D
  • Embodiment 39 The method of any one of embodiments 34 to 39, wherein the therapeutic agent promotes exclusion of the NMD exon from the processed mRNA encoding the target protein and increases the expression of the target protein in the cell.
  • Embodiment 40 The method of any one of embodiments 1 to 27, wherein the therapeutic agent inhibits exclusion of the NMD exon from the processed mRNA encoding the target protein.
  • Embodiment 41 The method of embodiment 40, wherein exclusion of the NMD exon from the processed mRNA encoding the target protein in the cell contacted with the therapeutic agent is decreased by about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 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
  • Embodiment 42 The method of embodiment 40 or 41, wherein the therapeutic agent decreases the level of the processed mRNA encoding the target protein in the cell.
  • Embodiment 43 The method of any one of embodiments 40 to 42, wherein the level of the processed mRNA encoding the target protein in the cell contacted with the therapeutic agent is decreased by about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 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
  • Embodiment 44 The method of any one of embodiments 40 to 43, wherein the therapeutic agent decreases the expression of the target protein in the cell.
  • Embodiment 45 The method of any one of embodiments 40 to 44, wherein a level of the target protein produced in the cell contacted with the therapeutic agent is decreased by about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 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
  • Embodiment 46 The method of any one of embodiments 2 to 27 or 40 to 45, wherein the disease or condition is induced by a gain-of-function mutation in the target protein
  • Embodiment 47 The method of embodiment 46, wherein the subject has an allele from which the target protein is produced at an increased level, or an allele encoding a mutant target protein that exhibits increased activity in the cell.
  • Embodiment 48 The method of embodiment 46 or 47, wherein the therapeutic agent inhibits exclusion of the NMD exon from the processed mRNA encoding the target protein and decreases the expression of the target protein in the cell.
  • Embodiment 49 The method of embodiment 40, wherein the target protein comprises SCN8A.
  • Embodiment 50 The method of embodiment 49, wherein the disease or condition comprises a central nervous system disease.
  • Embodiment 51 The method of embodiment 50, wherein the disease or condition comprises epilepsy.
  • Embodiment 53 The method of any one of embodiments 1 to 52, wherein the therapeutic agent is an antisense oligomer (ASO) and wherein the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.
  • ASO antisense oligomer
  • Embodiment 54 The method of any one of embodiments 1 to 53, wherein the therapeutic agent is an antisense oligomer (ASO) and wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2′-O-methyl, a 2′-Fluoro, or a 2′-O-methoxyethyl moiety.
  • ASO antisense oligomer
  • Embodiment 59 The method of any one of embodiments 1 to 58, wherein the method further comprises assessing mRNA level or expression level of the target protein.
  • Embodiment 60 The method of any one of embodiments 1 to 59, wherein the subject is a human.
  • Embodiment 62 The method of any one of embodiments 2 to 60, wherein the subject is a fetus, an embryo, or a child.
  • Embodiment 63 The method of any one of embodiments 1 to 62, wherein the cells are ex vivo.
  • Embodiment 64 The method of any one of embodiments 2 to 62, wherein the therapeutic agent is administered by intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravitreal, or intravenous injection of the subject.
  • Embodiment 66 The method of any one of embodiments 1 to 65, wherein the second therapeutic agent is a small molecule.
  • Embodiment 68 The method of any one of embodiments 1 to 67, wherein the second therapeutic agent corrects intron retention.
  • Embodiment 69 The method of any one of embodiments 2 to 68, wherein the disease or condition is selected from the group consisting of: 16p11.2 deletion syndrome; Alport syndrome; Arrhythmogenic right ventricular dysplasia 9; Ceroid lipofuscinosis, neuronal, 3; Cognitive impairment with or without cerebellar ataxia; Epileptic encephalopathy, early infantile, 13; Seizures, benign familial infantile, 5; Cone-rod retinal dystrophy-2; Cornelia de Lange; Deafness, autosomal dominant 13; Deafness, autosomal dominant 4A; Peripheral neuropathy, myopathy, hoarseness, and hearing loss; Epilepsy, generalized, with febrile seizures plus, type 7; Febrile seizures, familial, 3B; Insensitivity to pain, congenital; HSAN2D, autosomal recessive; Epileptic encephalopathy, childhood-onset; Epileptic encephalopathy, early infantile, 11; Seizures, benign familial infant
  • FIGS. 2-58 depict identification of different exemplary nonsense-mediated mRNA decay (NMD)-inducing exons in various genes.
  • RT-PCR analysis using cytoplasmic RNA from DMSO-treated or puromycin or cycloheximide-treated human cells and primers in exons can confirm the presence of a band corresponding to an NMD-inducing exon. The identity of the product is confirmed by sequencing. Densitometry analysis of the bands is performed to calculate percent NMD exon inclusion of total transcript. Treatment of cells with cycloheximide or puromycin to inhibit NMD can lead to an increase of the product corresponding to the NMD-inducing exon in the cytoplasmic fraction.
  • FIGS. 59, 62, 65, 69, 72, and 75 depict confirmation of exemplary NIE exons in various gene transcripts using cycloheximide or puromycin treatment, respectively.
  • An ASO walk is performed for NMD exon region targeting sequences immediately upstream of the 3′ splice site, across the 3′ splice site, the NMD exon, across the 5′ splice site, and downstream of the 5′ splice site using 2′-MOE ASOs, PS backbone.
  • ASOs are designed to cover these regions by shifting 5 nucleotides at a time.
  • FIGS. 60, 63, 66, 70, 73, and 76 depict ASO walk for various exemplary NIE exon regions, respectively.
  • ASO walk sequences can be evaluated by for example RT-PCR.
  • PAGE can be used to show SYBR-safe-stained RT-PCR products of mock-treated (Sham), SMN-control ASO treated (SMN), or treated with a 2′-MOE ASO targeting the NMD exon regions as described herein at 20 ⁇ M concentration in human cells by gymnotic uptake.
  • Products corresponding to NMD exon inclusion and full-length are quantified and percent NMD exon inclusion is plotted
  • Full-length products can be normalized to RPL32 internal control and fold-change relative to Sham can be plotted.
  • FIGS. 71 and 78 depict evaluation via RT-PCR of various exemplary ASO walk along exemplary NIE exon regions, respectively.
  • PAGE can be used to show SYBR-safe-stained RT-PCR products of mock-treated (Sham, RNAiMAX alone), or treated with 2′-MOE ASOs targeting NMD exons at 30 nM, 80 nM, and 200 nM concentrations in mouse or human cells by RNAiMAX transfection.
  • Products corresponding to NMD exon inclusion and full-length are quantified and percent NMD exon inclusion can be plotted.
  • the full-length products can also be normalized to HPRT internal control and fold-change relative to Sham can be plotted.

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