WO2017106292A1 - Compositions et méthodes de traitement de maladies rénales - Google Patents

Compositions et méthodes de traitement de maladies rénales Download PDF

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Publication number
WO2017106292A1
WO2017106292A1 PCT/US2016/066576 US2016066576W WO2017106292A1 WO 2017106292 A1 WO2017106292 A1 WO 2017106292A1 US 2016066576 W US2016066576 W US 2016066576W WO 2017106292 A1 WO2017106292 A1 WO 2017106292A1
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WIPO (PCT)
Prior art keywords
mrna
nucleobases
ric pre
seq
retained intron
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PCT/US2016/066576
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English (en)
Inventor
Isabel AZNAREZ
Huw M. Nash
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Cold Spring Harbor Laboratory
Stoke Therapeutics, Inc.
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Publication date
Application filed by Cold Spring Harbor Laboratory, Stoke Therapeutics, Inc. filed Critical Cold Spring Harbor Laboratory
Priority to CA3005249A priority Critical patent/CA3005249A1/fr
Priority to JP2018529237A priority patent/JP7049247B2/ja
Priority to EP16876548.5A priority patent/EP3390666A4/fr
Publication of WO2017106292A1 publication Critical patent/WO2017106292A1/fr
Priority to US16/007,435 priority patent/US11096956B2/en
Priority to US17/379,793 priority patent/US20220118000A1/en
Priority to JP2022013876A priority patent/JP2022062143A/ja

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/33—Alteration of splicing

Definitions

  • Kidney disease is a debilitating and potentially fatal group of conditions associated with the damaging of the kidneys.
  • the kidney is vital for many functions in the body including, but not limited to cleansing of the blood by removing waste and excess fluid, maintaining the balance of salt and minerals in the blood, and regulation of blood pressure. Incidence of kidney disease often results in the buildup of fluid and waste products, vomiting, weakness, poor sleep and shortness of breath, and ultimately could lead to death. While kidney transplantation can often prevent mortality, the odds of receiving a donor kidney is typically low.
  • kidney diseases While there are a large number of diseases and conditions associated with the kidney, a subset of kidney diseases have been shown to proceed via a deficiency in the expression of a gene, and in turn, a deficiency in the gene product.
  • CTNS CTNS
  • PAX2 CYP24A1
  • PPARD PPARD
  • ASO antisense oligomer
  • the kidney disease is infantile nephropathic cystinosis, late-onset cystinosis, focal segmental glomerulosclerosis 7, Papillorenal syndrome, or infantile hypercalcemia.
  • a method of increasing expression of a target protein by cells having a retained-intron-containing pre-mRNA comprising a retained intron, an exon flanking the 5' splice site of the retained intron, an exon flanking the 3' splice site of the retained intron, and wherein the RIC pre-mRNA encodes the target protein
  • the method comprising contacting the cells with an antisense oligomer (ASO) complementary to a targeted portion of the RIC pre-mRNA encoding the target protein, whereby the retained intron is constitutively spliced from the RIC pre-mRNA encoding the target protein, thereby increasing the level of mRNA encoding the target protein, and increasing the expression of the target protein in the cells, wherein the target protein is cystinosin, protein paired box gene 2 protein, protein cytochrome P450 family 24, subfamily A, polypeptide 1, or peroxisome pro
  • ASO antisense oligomer
  • the target protein is cystinosin, protein paired box gene 2 protein, protein cytochrome P450 family 24, subfamily A, polypeptide 1, or peroxisome proliferator activated receptor delta.
  • the target protein or the functional RNA is a compensating protein or a compensating functional RNA that functionally augments or replaces a target protein or functional RNA that is deficient in amount or activity in the subject.
  • the cells are in or from a subject having a condition caused by a deficient amount or activity of the target protein.
  • the deficient amount of the target protein is caused by haploinsufficiency of the target protein, wherein the subject has a first allele encoding a functional target protein, and a second allele from which the target protein is not produced, or a second allele encoding a nonfunctional target protein, and wherein the antisense oligomer binds to a targeted portion of a RIC pre-mRNA transcribed from the first allele.
  • the subject has a condition caused by a disorder resulting from a deficiency in the amount or function of the target protein, wherein the subject has a first mutant allele from which the target protein is produced at a reduced level compared to production from a wild-type allele, the target protein is produced in a form having reduced function compared to an equivalent wild- type protein, or the target protein is not produced, and a second mutant allele from which the target protein is produced at a reduced level compared to production from a wild-type allele, the target protein is produced in a form having reduced function compared to an equivalent wild-type protein, or the target protein is not produced, and wherein when the subject has a first mutant allele a(iii), the second mutant allele is b(i) or b(ii), and wherein when the subject has a second mutant allele b(iii), the first mutant allele is a(i) or a(ii), and wherein the RIC pre-mRNA is
  • the target protein is produced in a form having reduced function compared to the equivalent wild-type protein.
  • the target protein is produced in a form that is fully-functional compared to the equivalent wild-type protein.
  • the targeted portion of the RIC pre-mRNA is in the retained intron within the region +6 relative to the 5' splice site of the retained intron to -16 relative to the 3' splice site of the retained intron.
  • the targeted portion of the RIC pre-mRNA is in the retained intron within the region +69 relative to the 5' splice site of the retained intron to -79 relative to the 3' splice site of the retained intron.
  • the target protein is cystinosin.
  • the targeted portion of the RIC pre-mRNA is in the retained intron within the region +500 relative to the 5' splice site of the retained intron to -500 relative to the 3' splice site of the retained intron.
  • the targeted portion of the RIC pre-mRNA comprises a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complimentary to any one of SEQ ID NOs 8624-10068.
  • the targeted portion of the RIC pre-mRNA comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a region comprising at least 8 contiguous nucleic acids of SEQ ID NO 10748 or SEQ ID NO 10734.
  • the ASO comprises a sequence with at least about 80%, 85%, 90%, 95%,
  • the RIC pre-mRNA comprises a sequence with at least about 80%, 85%,
  • the RIC pre-mRNA is encoded by a genetic sequence with at least about
  • the target protein is paired box gene 2 protein.
  • the targeted portion of the RIC pre-mRNA is in the retained intron within the region +500 relative to the 5' splice site of the retained intron to -500 relative to the 3' splice site of the retained intron.
  • the targeted portion of the RIC pre-mRNA comprises a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complimentary to any one of SEQ ID NOs. 6953-8623.
  • the targeted portion of the RIC pre-mRNA comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a region comprising at least 8 contiguous nucleic acids of SEQ ID NO 10738 or SEQ ID NO 10740.
  • the ASO comprises a sequence with at least about 80%, 85%, 90%, 95%,
  • the RIC pre-mRNA comprises a sequence with at least about 80%, 85%,
  • the RIC pre-mRNA is encoded by a genetic sequence with at least about
  • the target protein is protein cytochrome P450 family 24, subfamily A, polypeptide 1.
  • the targeted portion of the RIC pre-mRNA is in the retained intron within the region +500 relative to the 5' splice site of the retained intron to -500 relative to the 3' splice site of the retained intron.
  • the targeted portion of the RIC pre-mRNA comprises a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complimentary to any one of SEQ ID NOs. 9520-10733.
  • the targeted portion of the RIC pre-mRNA comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a region comprising at least 8 contiguous nucleic acids of SEQ ID NO 10748, SEQ ID NO 10734, SEQ ID NO 10746, SEQ ID NO 10745 or SEQ ID NO 10741.
  • the ASO comprises a sequence with at least about 80%, 85%, 90%, 95%,
  • the RIC pre-mRNA comprises a sequence with at least about 80%, 85%,
  • the RIC pre-mRNA is encoded by a genetic sequence with at least about
  • the target protein is peroxisome proliferator activated receptor delta.
  • the targeted portion of the RIC pre-mRNA is in the retained intron within the region +500 relative to the 5' splice site of the retained intron to -500 relative to the 3' splice site of the retained intron.
  • the targeted portion of the RIC pre-mRNA comprises a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complimentary to any one of SEQ ID NOs 20-6952.
  • the targeted portion of the RIC pre-mRNA comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a region comprising at least 8 contiguous nucleic acids of SEQ ID NO 10736, SEQ ID NO 10747, SEQ ID NO 10739, SEQ ID
  • the ASO comprises a sequence with at least about 80%, 85%, 90%, 95%,
  • the RIC pre-mRNA comprises a sequence with at least about 80%, 85%,
  • the RIC pre-mRNA is encoded by a genetic sequence with at least about
  • the targeted portion of the RIC pre-mRNA is in the retained intron within: the region +6 to +100 relative to the 5' splice site of the retained intron; or the region -16 to -100 relative to the 3 ' splice site of the retained intron.
  • the antisense oligomer targets a portion of the RIC pre-mRNA that is within the region about 100 nucleotides downstream of the 5' splice site of the at least one retained intron, to about 100 nucleotides upstream of the 3' splice site of the at least one retained intron
  • the targeted portion of the RIC pre-mRNA is within: the region +2e to -4e in the exon flanking the 5 ' splice site of the retained intron; or the region +2e to -4e in the exon flanking the 3' splice site of the retained intron.
  • the antisense oligomer does not increase the amount of the target protein or the functional RNA by modulating alternative splicing of pre-mRNA transcribed from a gene encoding the functional RNA or target protein.
  • the antisense oligomer does not increase the amount of the target protein or the functional RNA by modulating aberrant splicing resulting from mutation of the gene encoding the target protein or the functional RNA.
  • the RIC pre-mRNA was produced by partial splicing of a full-length pre- mRNA or partial splicing of a wild-type pre-mRNA.
  • the mRNA encoding the target protein or functional RNA is a full-length mature mRNA, or a wild-type mature mRNA.
  • the target protein produced is full-length protein, or wild-type protein.
  • the total amount of the mRNA encoding the target protein or functional RNA produced in the cell contacted with the antisense oligomer is increased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 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
  • the total amount of target protein produced by the cell contacted with the antisense oligomer is increased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5- fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1 -fold, at least about 1.5- fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 1.1 to about 10-fold,
  • the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.
  • the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-0-methyl, a 2'-Fluoro, or a 2'-0-methoxyethyl moiety.
  • the antisense oligomer comprises at least one modified sugar moiety.
  • each sugar moiety is a modified sugar moiety.
  • the antisense oligomer consists of from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases,
  • the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%), at least 98%, at least 99%, or 100%, complementary to the targeted portion of the RIC pre-mRNA encoding the protein.
  • the cell comprises a population of RIC pre-mRNAs transcribed from the gene encoding the target protein or functional RNA, wherein the population of RIC pre-mRNAs comprises two or more retained introns, and wherein the antisense oligomer binds to the most abundant retained intron in the population of RIC pre-mRNAs.
  • the binding of the antisense oligomer to the most abundant retained intron induces splicing out of the two or more retained introns from the population of RIC pre-mRNAs to produce mRNA encoding the target protein or functional RNA.
  • the cell comprises a population of RIC pre-mRNAs transcribed from the gene encoding the target protein or functional RNA, wherein the population of RIC pre-mRNAs comprises two or more retained introns, and wherein the antisense oligomer binds to the second most abundant retained intron in the population of RIC pre-mRNAs.
  • the binding of the antisense oligomer to the second most abundant retained intron induces splicing out of the two or more retained introns from the population of RIC pre- mRNAs to produce mRNA encoding the target protein or functional RNA.
  • the condition is a disease or disorder.
  • the disease or disorder is a kidney disease.
  • the kidney disease is infantile nephropathic cystinosis, late-onset cystinosis, focal segmental glomerulosclerosis 7, Papillorenal syndrome, or infantile hypercalcemia.
  • the target protein and the RIC pre-mRNA are encoded by a gene, wherein the gene is CTNS, PAX2, CYP24A1 or PPARD.
  • the method further comprises assessing protein expression.
  • the subject is a human.
  • the subject is a non-human animal.
  • the subject is a fetus, an embryo, or a child.
  • the cells are ex vivo.
  • the antisense oligomer is administered by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection of the subject.
  • the 9 nucleotides at -3e to -le of the exon flanking the 5' splice site are identical to each other.
  • +1 to +6 of the retained intron are identical to the corresponding wild-type sequence.
  • the 16 nucleotides at -15 to -1 of the retained intron and +le of the exon flanking the 3' splice site are identical to the corresponding wild-type sequence.
  • an antisense oligomer as used in a method described herein.
  • an antisense oligomer comprising a sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs 20-10733.
  • a pharmaceutical composition comprising an antisense oligomer described herein and an excipient.
  • provided herein is a method of treating a subject in need thereof by administering a pharmaceutical composition deribed herein by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.
  • composition comprising an antisense oligomer for use in a method of increasing expression of a target protein or a functional RNA by cells to treat a kidney disease in a subject in need thereof associated with a deficient protein or deficient functional RNA, wherein the deficient protein or deficient functional RNA is deficient in amount or activity in the subject, wherein the antisense oligomer enhances constitutive splicing of a retained intron-containing pre-mRNA (RIC pre- mRNA) encoding the target protein or the functional RNA, wherein the target protein is: the deficient protein; or a compensating protein which functionally augments or replaces the deficient protein or in the subject; and wherein the functional RNA is: the deficient RNA; or a compensating functional RNA which functionally augments or replaces the deficient functional RNA in the subject; wherein the RIC pre-mRNA comprises a retained intron, an exon flanking the 5' splice site and an ex
  • the kidney disease is infantile nephropathic cystinosis, late-onset cystinosis, focal segmental glomerulosclerosis 7, Papillorenal syndrome, or infantile hypercalcemia.
  • composition comprising an antisense oligomer for use in a method of treating a condition associated with a target protein in a subject in need thereof, the method comprising the step of increasing expression of the target protein by cells of the subject, wherein the cells have a retained-intron-containing pre-mRNA (RIC pre-mRNA) comprising a retained intron, an exon flanking the 5' splice site of the retained intron, an exon flanking the 3' splice site of the retained intron, and wherein the RIC pre-mRNA encodes the target protein, the method comprising contacting the cells with the antisense oligomer, whereby the retained intron is constitutively spliced from the RIC pre- mRNA transcripts encoding the target protein, thereby increasing the level of mRNA encoding the target protein or functional RNA, and increasing the expression of the target protein, in the cells of the subject.
  • RIC pre-mRNA retained-intron-containing pre-mRNA
  • the target protein is cystinosin, protein paired box gene 2 protein, protein cytochrome P450 family 24, subfamily A, polypeptide 1, or peroxisome proliferator activated receptor delta.
  • the condition is a disease or disorder.
  • the disease or disorder is a kidney disease.
  • the kidney disease is infantile nephropathic cystinosis, late-onset cystinosis, focal segmental glomerulosclerosis 7, Papillorenal syndrome, or infantile hypercalcemia.
  • the target protein and RIC pre-mRNA are encoded by a gene, wherein the gene is CTNS, PAX2, CYP24A1 or PPARD.
  • the antisense oligomer targets a portion of the RIC pre-mRNA that is in the retained intron within the region +6 relative to the 5' splice site of the retained intron to -16 relative to the 3' splice site of the retained intron.
  • the targeted portion of the RIC pre-mRNA is in the retained intron within the region +69 relative to the 5' splice site of the retained intron to -79 relative to the 3' splice site of the retained intron.
  • the target protein is cystinosin.
  • the targeted portion of the RIC pre-mRNA is in the retained intron within the region +500 relative to the 5' splice site of the retained intron to -500 relative to the 3 Of the retained intron.
  • the targeted portion of the RIC pre-mRNA comprises a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complimentary to any one of SEQ ID NOs 8624-10068.
  • the targeted portion of the RIC pre-mRNA comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a region comprising at least 8 contiguous nucleic acids of SEQ ID NO 10748 or SEQ ID NO 10734.
  • the ASO comprises a sequence with at least about 80%, 85%, 90%, 95%,
  • the RIC pre-mRNA comprises a sequence with at least about 80%, 85%,
  • the RIC pre-mRNA is encoded by a genetic sequence with at least about
  • the target protein is paired box gene 2 protein.
  • the targeted portion of the RIC pre-mRNA is in the retained intron within the region +500 relative to the 5' splice site of the retained intron to -500 relative to the 3' splice site of the retained intron.
  • the targeted portion of the RIC pre-mRNA comprises a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complimentary to any one of SEQ ID NOs. 6953-8623.
  • the targeted portion of the RIC pre-mRNA comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a region comprising at least 8 contiguous nucleic acids of SEQ ID NO 10738 or SEQ ID NO 10740.
  • the ASO comprises a sequence with at least about 80%, 85%, 90%, 95%,
  • the RIC pre-mRNA comprises a sequence with at least about 80%, 85%,
  • the RIC pre-mRNA is encoded by a genetic sequence with at least about
  • the target protein is protein cytochrome P450 family 24, subfamily A, polypeptide 1.
  • the targeted portion of the RIC pre-mRNA is in the retained intron within the region +500 relative to the 5' splice site of the retained intron to -500 relative to the 3' splice site of the retained intron.
  • the targeted portion of the RIC pre-mRNA comprises a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complimentary to any one of SEQ ID NOs. 9520-10733.
  • the targeted portion of the RIC pre-mRNA comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a region comprising at least 8 contiguous nucleic acids of SEQ ID NO 10748, SEQ ID NO 10734, SEQ ID NO 10746, SEQ ID NO 10745 or SEQ ID NO 10741.
  • the ASO comprises a sequence with at least about 80%, 85%, 90%, 95%,
  • the RIC pre-mRNA comprises a sequence with at least about 80%, 85%,
  • the RIC pre-mRNA is encoded by a genetic sequence with at least about
  • the target protein is peroxisome proliferator activated receptor delta.
  • the targeted portion of the RIC pre-mRNA is in the retained intron within the region +500 relative to the 5' splice site of the retained intron to -500 relative to the 3' splice site of the retained intron.
  • the targeted portion of the RIC pre-mRNA comprises a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complimentary to any one of SEQ ID NOs 20-6952.
  • the targeted portion of the RIC pre-mRNA comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a region comprising at least 8 contiguous nucleic acids of SEQ ID NO 10736, SEQ ID NO 10747, SEQ ID NO 10739, SEQ ID
  • the ASO comprises a sequence with at least about 80%, 85%, 90%, 95%,
  • the RIC pre-mRNA comprises a sequence with at least about 80%, 85%,
  • the RIC pre-mRNA is encoded by a genetic sequence with at least about
  • the antisense oligomer targets a portion of the RIC pre-mRNA that is in the retained intron within: the region +6 to +100 relative to the 5' splice site of the retained intron; or the region -16 to -100 relative to the 3' splice site of the retained intron.
  • the antisense oligomer targets a portion of the RIC pre-mRNA that is within the region about 100 nucleotides downstream of the 5' splice site of the at least one retained intron, to about 100 nucleotides upstream of the 3' splice site of the at least one retained intron.
  • the targeted portion of the RIC pre-mRNA is within: the region +2e to - 4e in the exon flanking the 5' splice site of the retained intron; or the region +2e to -4e in the exon flanking the 3 ' splice site of the retained intron.
  • the antisense oligomer does not increase the amount of target protein or functional RNA by modulating alternative splicing of the pre-mRNA transcribed from a gene encoding the target protein or functional RNA.
  • the antisense oligomer does not increase the amount of the functional RNA or functional protein by modulating aberrant splicing resulting from mutation of the gene encoding the target protein or functional RNA.
  • the RIC pre-mRNA was produced by partial splicing from a full-length pre-mRNA or a wild-type pre-mRNA.
  • the mRNA encoding the target protein or functional RNA is a full-length mature mRNA, or a wild-type mature mRNA.
  • the target protein produced is full-length protein, or wild-type protein.
  • the retained intron is a rate-limiting intron.
  • said retained intron is the most abundant retained intron in said RIC pre- mRNA.
  • the retained intron is the second most abundant retained intron in said RIC pre-mRNA.
  • the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.
  • said antisense oligomer is an antisense oligonucleotide.
  • the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-0-methyl, a 2'-Fluoro, or a 2'-0-methoxyethyl moiety.
  • the antisense oligomer comprises at least one modified sugar moiety.
  • each sugar moiety is a modified sugar moiety.
  • the antisense oligomer consists of from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases,
  • the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or is 100% complementary to the targeted portion of the RIC pre-mRNA encoding the protein.
  • a pharmaceutical composition comprising an antisense oligomer described herein, and an excipient.
  • a pharmaceutical composition comprising: an antisense oligomer that hybridizes to a target sequence of a deficient CTNS, PAX2, CYP24A1 or PPARD mRNA transcript, wherein the deficient CTNS, PAX2, CYP24A1 or PPARD mRNA transcript comprises a retained intron, wherein the antisense oligomer induces splicing out of the retained intron from the deficient CTNS, PAX2, CYP24A1 or PPARD mRNA transcript; and a pharmaceutical acceptable excipient.
  • the deficient CTNS, PAX2, CYP24A1 or PPARD mRNA transcript is a CTNS, PAX2, CYP24A1 or PPARD RIC pre-mRNA transcript.
  • the targeted portion of the CTNS, PAX2, CYP24A1 or PPARD RIC pre- mRNA transcript is in the retained intron within the region +500 relative to the 5' splice site of the retained intron to -500 relative to the 3' spliced site of the retained intron.
  • the CTNS, PAX2, CYP24A1 or PPARD RIC pre-mRNA transcript is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 1-4.
  • the CTNS, PAX2, CYP24A1 or PPARD RIC pre-mRNA transcript compnses a sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 5-19.
  • the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.
  • the antisense oligomer is an antisense oligonucleotide.
  • the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-0-methyl, a 2'-Fluoro, or a 2'-0-methoxyethyl moiety.
  • the antisense oligomer comprises at least one modified sugar moiety.
  • the antisense oligomer comprises from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to
  • 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 CTNS, PAX2, CYP24A1 or PPARD RIC pre-mRNA transcript.
  • the targeted portion of the CTNS, PAX2, CYP24A1 or PPARD RIC pre- mRNA transcript is within a sequence selected from SEQ ID NOs: 10034-10748.
  • the antisense oligomer comprises a nucleotide sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 20-10733.
  • the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NOs: 20-10733.
  • the pharmaceutical composition is formulated for intrathecal injection, intracerebro ventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.
  • a method of inducing processing of a deficient CTNS, PAX2, CYP24A1 or PPARD mRNA transcript to facilitate removal of a retained intron to produce a fully processed CTNS, PAX2, CYP24A1 or PPARD mRNA transcript that encodes a functional form of a CTNS, PAX2, CYP24A1 or PPARD protein the method comprising: contacting an antisense oligomer to a target cell of a subject; hybridizing the antisense oligomer to the deficient CTNS, PAX2, CYP24A1 or PPARD mRNA transcript, wherein the deficient CTNS, PAX2, CYP24A1 or PPARD mRNA transcript is capable of encoding the functional form of a
  • the retained intron is an entire retained intron.
  • the deficient CTNS, PAX2, CYP24A1 or PPARD mRNA transcript is a CTNS, PAX2, CYP24A1 or PPARD RIC pre-mRNA transcript.
  • a method of treating a subject having a condition caused by a deficient amount or activity of CTNS, PAX2, CYP24A1 or PPARD protein comprising administering to the subject an antisense oligomer comprising a nucleotide sequence with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 20- 10733.
  • FIG. 1 depicts an schematic representation of an exemplary retained-intron-containing (RIC) pre-mRNA transcript.
  • the 5' splice site consensus sequence is indicated with underlined letters (letters are nucleotides; upper case: exonic portion and lower case: intronic portion) from -3e to -le and +1 to +6 (numbers labeled "e” are exonic and unlabeled numbers are intronic).
  • the 3' splice site consensus sequence is indicated with underlined letters (letters are nucleotides; upper case: exonic portion and lower case: intronic portion) from -15 to -1 and +le (numbers labeled "e” are exonic and unlabeled numbers are intronic).
  • Intronic target regions for ASO screening comprise nucleotides +6 relative to the 5' splice site of the retained intron (arrow at left) to -16 relative to the 3' splice site of the retained intron (arrow at right).
  • intronic target regions for ASO screening comprise nucleotides +6 to +100 relative to the 5' splice site of the retained intron and -16 to -100 relative to the 3' splice site of the retained intron.
  • Exonic target regions comprise nucleotides +2e to -4e in the exon flanking the 5' splice site of the retained intron and +2e to -4e in the exon flanking the 3 ' splice site of the retained intron.
  • n or N denote any nucleotide
  • y denotes pyrimidine.
  • the sequences shown represent consensus sequences for mammalian splice sites and individual introns and exons need not match the consensus sequences at every position.
  • FIG. 2A depicts an exemplary schematic representation of the Targeted Augmentation of Nuclear Gene Output (TANGO) approach.
  • FIG. 2A shows a cell divided into nuclear and cytoplasmic compartments.
  • a pre-mRNA transcript of a target gene consisting of exons (rectangles) and introns (connecting lines) undergoes splicing to generate an mRNA, and this mRNA is exported to the cytoplasm and translated into target protein.
  • the splicing of intron 1 is inefficient and a retained intron-containing (RIC) pre-mRNA accumulates primarily in the nucleus, and if exported to the cytoplasm, is degraded, leading to no target protein production.
  • RIC retained intron-containing
  • FIG. 2B depicts an exemplary schematic representation of the Targeted Augmentation of Nuclear Gene Output (TANGO) approach.
  • FIG. 2B shows an example of the same cell as in FIG. 2A divided into nuclear and cytoplasmic compartments.
  • Treatment with an antisense oligomer (ASO) promotes the splicing of intron 1 and results in an increase in mRNA, which is in turn translated into higher levels of target protein.
  • ASO antisense oligomer
  • FIG. 3A depicts a schematic of the ReSeq Genes for CTNS corresponding to NM 004937 and NM_001031681.
  • the Percent Intron Retention (PIR) of the circled intron is shown.
  • FIG. 3B depicts a schematic of the ReSeq Genes for CTNS corresponding to NM 004937 and NM 001031681. The PIR of the circled intron is shown.
  • FIG. 4A depicts a schematic of the ReSeq Genes for CYP24A1 corresponding to NM 000782 and NM 001128915. The PIR of the circled intron is shown.
  • FIG. 4B depicts a schematic of the ReSeq Genes for CYP24A1 corresponding to NM 000782 and NM 001128915. The PIR of the circled intron is shown.
  • FIG. 5 depicts a schematic of the ReSeq Genes for PAX2 corresponding to NM 001304569, NM_000278, NM_003990, NM_003988, NM_003987 and NM_003989.
  • FIG. 6A depicts a schematic of the RefSeq Genes for PPARD corresponding to PPARD:
  • NM_006238 NM_177435, NM_001171818, NM_001171819 and NM_001171820.
  • PIR Percent Intron Retention
  • FIG. 6C depicts a schematic of the RefSeq Genes for PPARD corresponding to PPARD:
  • FIG. 6D depicts a schematic of the RefSeq Genes for PPARD corresponding to PPARD:
  • FIG. 6E depicts a schematic of the RefSeq Genes for PPARD corresponding to PPARD:
  • FIG. 6G depicts a schematic of the RefSeq Genes for PPARD corresponding to PPARD:
  • Kidney failure is a debilitating condition that is estimated to affect more than 600,000 in the United States alone. In many cases, renal dialysis is able to prolong life, with an estimated 400,000 patients receiving renal dialysis according to a 2009 study. Still, the only hope for many afflicted with kidney failure is a kidney transplant, though the donor pool is only sufficient to treat a small portion of those in need. Therefore, the odds of receiving a transplant is low, and there are few treatments available to ameliorate the condition for those unable to receive a transplant. Therefore, there exists a need for compositions and methods for treating kidney diseases. [00178] Individual introns in primary transcripts of protein-coding genes having more than one intron are spliced from the primary transcript with different efficiencies.
  • compositions and methods for upregulating splicing of one or more retained introns that are rate-limiting for the nuclear stages of gene expression to increase steady-state production of fully-spliced, mature mRNA, and thus, translated protein levels.
  • compositions and methods utilize antisense oligomers (ASOs) that promote constitutive splicing at an intron splice site of a retained-intron-containing pre-mRNA that accumulates in the nucleus.
  • ASOs antisense oligomers
  • Kidney diseases that can be treated by the invention described herein are diseases where a subject is deficient in a gene product, where deficiency in a gene product causes the kidney disease.
  • CTNS, PAX2, CYP24A1 and PPARD pre-mRNA species comprise at least one retained intron.
  • the present invention provides compositions and methods for upregulating splicing of one or more retained CTNS, PAX2, CYP24A1 or PPARD introns that are rate-limiting for the nuclear stages of gene expression to increase steady-state production of fully-spliced, mature mRNA, and thus, translated CTNS, PAX2, CYP24A1 or PPARD protein levels.
  • compositions and methods can utilize antisense oligomers (ASOs) that promote constitutive splicing at intron splice sites of a retained-intron- containing CTNS, PAX2, CYP24A1 or PPARD pre-mRNA (RIC pre-mRNA) that accumulates in the nucleus.
  • ASOs antisense oligomers
  • CTNS, PAX2, CYP24A1 or PPARD protein can be increased using the methods of the invention to treat a condition caused by CTNS, PAX2, CYP24A1 or PPARD deficiency.
  • the methods of the invention can be used to increase CTNS, PAX2, CYP24A1 or PPARD production to treat a condition in a subject in need thereof.
  • the subject has a condition in which CTNS, PAX2, CYP24A1 or PPARD is not necessarily deficient relative to wild-type, but where an increase in CTNS, PAX2, CYP24A1 or PPARD mitigates the condition nonetheless.
  • the condition can be caused by a CTNS, PAX2, CYP24A1 or PPARD haploinsufficiency.
  • the invention described herein can be used to treat the autosomal recessive kidney disorder infantile nephropathic cystinosis.
  • Infantile nephropathic cystinosis is characterized by the accumulation of cysteine in lysosomes.
  • Clinical manifestations of infantile nephropathic cystinosis include low blood sugar and electrolytes, excessive excretion of proteins in the urine, slow body growth, weak bones, hypothyroidism, blindness, muscle weakness, pulmonary dysfunction and kidney failure.
  • infantile nephropathic cystinosis is diagnosed in infancy.
  • the invention described herein can be used to treat the autosomal recessive kidney disorder late-onset cystinosis.
  • Late onset cystinosis has a similar clinical manifestation as the infantile form, though the late onset form typically manifests in either late adolescence or early adulthood.
  • CTNS protein cystinosin
  • the CTNS gene which is located at 17ql3.2 and spans 12 exons, codes for the CTNS protein. Mutations in the CTNS gene resulting in deficient amounts of CTNS protein have been shown to be responsible for the progression of both infantile nephropathic cystinosis and late-onset cystinosis.
  • a G169D missense mutation was discovered in patients with infantile nephropathic cystinosis. This missense mutation resulted in diminished levels of CTNS, which provided a positive link between deficiency in CTNS and the progression of infantile nephropathic cystinosis.
  • the invention described herein can be used to treat the autosomal dominant kidney disorder focal segmental glomerulosclerosis 7 (FSGS7).
  • FSGS7 is one of the leading causes of kidney failure in adults.
  • FSGS7 is characterized by edema, hypoalbuminemia, hyperlipidemia and hypertension, which ultimately results in kidney failure.
  • the invention described herein can be used to treat the autosomal dominant kidney disorder Papillorenal syndrome (PAPRS).
  • PAPRS is characterized by hypoplastic kidneys, hypodysplasia, multicystic dysplastic kidney, oligomeganephronia, renal insufficiency and vesicoureteral reflux.
  • PAX2 protein paired box gene 2
  • the PAX2 gene which is located at 10q24.31 and spans 12 exons, codes for the PAX2 protein. Mutations in the PAX2 gene resulting in deficient amounts of PAX2 protein have been shown to be responsible for the progression of both FSGS7 and PAPRS.
  • a G76S missense mutation was discovered in 5 generations of a family afflicted with PAPRS. This missense mutation resulted in diminished levels of PAX2 protein, which provided a positive link between deficiency in PAX2 protein and the progression of PAPRS.
  • infantile Hypercalcemia is characterized by elevated calcium levels in the blood, which can result in renal stones, bone pain, abdominal pain, nausea, vomiting, polyuria and psychiatric conditions such as depression, anxiety, cognitive dysfunction, insomnia and coma.
  • CYP24A1 protein cytochrome P450 family 24, subfamily A, polypeptide 1
  • the CYP24A1 gene which is located at 20ql3.2 and spans 2 exons, codes for the CYP24A1 protein. Mutations in the CYP24A1 gene resulting in deficient amounts of CYP24A1 protein have been shown to be responsible for the progression of infantile hypercalcemia. In one study, a number of children diagnosed with infantile hypercalcemia were examined. Children displaying an R396W or an E322K missense mutation were shown to have complete ablation of CYP24A1 activity, which children displaying an L409S mutation retained small, but measurable levels. This finding provides a positive link between diminished
  • CYP24A1 protein and the clinical manifestations of infantile hypercalcemia.
  • the invention described herein can be used to treat the lipid metabolism deficiency, chronic kidney disease (CKD), end-stage renal disease (ESRD) or cardiovascular disease (CVD) caused by deficiency in the protein peroxisome proliferator activated receptor delta (PPARD).
  • CKD chronic kidney disease
  • ESRD end-stage renal disease
  • CVD cardiovascular disease
  • PPARD protein peroxisome proliferator activated receptor delta
  • RIC Pre-mRNA Retained Intron Containing Pre-mRNA
  • the methods of the present invention exploit the presence of retained-intron- containing pre-mRNA (RIC pre-mRNA) transcribed from a gene and encoding a protein that is found to be deficient in a disease described herein, in the cell nucleus.
  • Splicing of the identified RIC pre-mRNA species to produce mature, fully-spliced, mRNA is induced using ASOs that stimulate splicing out of the retained introns.
  • the resulting mature mRNA can be exported to the cytoplasm and translated, thereby increasing the amount of protein in the patient's cells and alleviating symptoms of a disease or condition described herein.
  • the methods of the present invention can exploit the presence of retained-intron-containing pre-mRNA (RIC pre-mRNA) transcribed from the CTNS, PAX2, CYP24A1 or PPARD gene and encoding CTNS, PAX2, CYP24A1 or PPARD protein, in the cell nucleus.
  • Splicing of CTNS, PAX2, CYP24A1 or PPARD RIC pre-mRNA species to produce mature, fully-spliced, CTNS, PAX2, CYP24A1 or PPARD mRNA can be induced using ASOs that stimulate splicing out of the retained introns.
  • the resulting mature CTNS, PAX2, CYP24A1 or PPARD mRNA can be exported to the cytoplasm and translated, thereby increasing the amount of CTNS, PAX2, CYP24A1 or PPARD protein in the patient's cells and alleviating symptoms of the CNS disease or conditions caused by deficiency in CTNS, PAX2, CYP24A1 or PPARD.
  • This method is known as Targeted Augmentation of Nuclear Gene Output (TANGO).
  • a retained intron is an intron that is identified as a retained intron based on a determination of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%, retention.
  • a retained intron is an intron that is identified as a retained intron based on a determination of about 5% to about 100%, about 5% to about 95%, about 5% to about 90%, about 5% to about 85%, about 5% to about 80%, about 5% to about 75%, about 5% to about 70%, about 5% to about 65%, about 5% to about 60%, about 5% to about 65%, about 5% to about 60%, about 5% to about 55%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 10% to about 100%, about 10% to about 95%, about 10% to about 90%, about 10% to about 85%, about 10% to about 80%, about 10% to about 75%, about 10% to about 70%, about 10% to about 65%, about 10% to about 60%, about 10% to about 65%, about 10% to about 60%, about 10% to about 5
  • the CTNS intron numbering corresponds to the mRNA sequence at
  • the targeted portion of the CTNS RIC pre-mRNA is in intron 9 and/or 10. In embodiments, the targeted portion of the CTNS RIC pre-mRNA is in intron 10. In embodiments, the percent retained intron can be 18%. In embodiments, the targeted portion of the CTNS RIC pre-mRNA is in intron 9. In embodiments, the percent retained intron can be 10%. In embodiments, hybridization of an ASO to the targeted portion of the RIC pre-mRNA results in enhanced splicing at the splice site (5' splice site or 3 ' splice site) of retained intron 9 and/or 10 and subsequently increases CTNS protein production.
  • the intron numbering may change in reference to a different CTNS isoform sequence.
  • One of skill in the art can determine the corresponding intron number in any isoform based on an intron sequence provided herein or using the number provided in reference to the mRNA sequence at NM OO 1031681 or NM_001031681.
  • One of skill in the art also can determine the sequences of flanking exons in any CTNS isoform for targeting using the methods of the invention, based on an intron sequence provided herein or using the intron number provided in reference to the mRNA sequence at NM_001031681 or NM_001031681.
  • the PAX2 intron numbering corresponds to the mRNA sequence at
  • the targeted portion of the PAX2 RIC pre-mRNA is in intron 1 or 2.
  • hybridization of an ASO to the targeted portion of the RIC pre-mRNA results in enhanced splicing at the splice site (5' splice site or 3 ' splice site) of retained intron 1 or 2 and subsequently increases PAX2 protein production. It is understood that the intron numbering may change in reference to a different PAX2 isoform sequence.
  • One of skill in the art can determine the corresponding intron number in any isoform based on an intron sequence provided herein or using the number provided in reference to the mRNA sequence at NM_001304569, NM_000278, NM_003990, NM_003988, NM_003987 or NM_003989.
  • One of skill in the art also can determine the sequences of flanking exons in any PAX2 isoform for targeting using the methods of the invention, based on an intron sequence provided herein or using the intron number provided in reference to the mRNA sequence at NM_001304569, NM_000278, NM_003990, NM_003988, NM_003987 or NM_003989.
  • the CYP24A1 intron numbering corresponds to the mRNA sequence at NM_000782 or NM_001128915.
  • the targeted portion of the CYP24A1 RIC pre-mRNA is in intron 10 and/or 9 or 11.
  • the targeted portion of the CYP24A1 RIC pre-mRNA is in intron 9.
  • the targeted portion of the CYP24A1 RIC pre-mRNA is in intron 11.
  • the percent retained intron can be 23%.
  • the targeted portion of the CYP24A1 RIC pre-mRNA is in intron 10.
  • the percent retained intron can be 50%.
  • hybridization of an ASO to the targeted portion of the RIC pre-mRNA results in enhanced splicing at the splice site (5' splice site or 3 ' splice site) of retained intron 10 and/or 9 or 11 and subsequently increases CYP24A1 protein production. It is understood that the intron numbering may change in reference to a different CYP24A1 isoform sequence.
  • One of skill in the art can determine the corresponding intron number in any isoform based on an intron sequence provided herein or using the number provided in reference to the mRNA sequence at NM_000782 or NM_001128915.
  • One of skill in the art also can determine the sequences of flanking exons in any CYP24A1 isoform for targeting using the methods of the invention, based on an intron sequence provided herein or using the intron number provided in reference to the mRNA sequence at NM_000782 or NM_001128915.
  • the PPARD intron numbering corresponds to the mRNA sequence at NM_006238, NM_177435, NM_001171818, NM_001171819 or NM_001171820.
  • the targeted portion of the PPARD RIC pre-mRNA is in intron 3 or 4 or 5 or 6 or 7 and/or 2 or 3 or 4 or 5 or 6 or 7 or 8.
  • the targeted portion of the PPARD RIC pre-mRNA is in intron 2.
  • the targeted portion of the PPARD RIC pre-mRNA is in intron 3.
  • the targeted portion of the PPARD RIC pre-mRNA is in intron 4.
  • the targeted portion of the PPARD RIC pre-mRNA is in intron 5. In embodiments, the targeted portion of the PPARD RIC pre- mRNA is in intron 6. In embodiments, the targeted portion of the PPARD RIC pre-mRNA is in intron 7. In embodiments, the targeted portion of the PPARD RIC pre-mRNA is in intron 8. In embodiments, hybridization of an ASO to the targeted portion of the RIC pre-mRNA results in enhanced splicing at the splice site (5' splice site or 3 ' splice site) of a retained intron subsequently increases PPARD protein production. It is understood that the intron numbering may change in reference to a different PPARD isoform sequence.
  • One of skill in the art can determine the corresponding intron number in any isoform based on an intron sequence provided herein or using the number provided in reference to the mRNA sequence at NM_006238, NM_177435, NM_001171818, NM_001171819 or NM_001171820.
  • One of skill in the art also can determine the sequences of flanking exons in any PPARD isoform for targeting using the methods of the invention, based on an intron sequence provided herein or using the intron number provided in reference to the mRNA sequence at NM_006238, NM_177435, NM_001171818, NM_001171819 or NM_001171820.
  • the ASOs disclosed herein target a RIC pre-mRNA transcribed from a CTNS, PAX2, CYP24A1 or PPARD genomic sequence. In some embodiments, the ASOs disclosed herein target a CTNS, PAX2, CYP24A1 or PPARD RIC pre-mRNA sequence.
  • the ASO targets a sequence of a RIC pre-mRNA transcript encoded by a CTNS genomic sequence. In some embodiments, the ASO targets a RIC pre-mRNA transcript encoded by a CTNS genomic sequence comprising retained intron 9 and/or 10. In some embodiments, the ASO targets a RIC pre-mRNA encoded by SEQ ID NO: 3. In some embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ ID NO: 16 or 17. In some embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ ID NO: 16 or 17 comprising a retained intron 9 and/or 10. In some embodiments, the ASOs target SEQ ID NO: 10748 and/or 10734.
  • the ASO comprises a sequence of any one of SEQ ID NOs: 20-6952.
  • the ASO targets a sequence of a RIC pre-mRNA transcript encoded by a PAX2 genomic sequence.
  • the ASO targets a RIC pre-mRNA transcript encoded by a PAX2 genomic sequence comprising retained intron 1 or 2.
  • the ASO targets a RIC pre- mRNA transcript encoded by SEQ ID NO: 2.
  • the ASO targets a RIC pre-mRNA transcript of one or more of SEQ ID NO: 10-15.
  • the ASO targets a RIC pre-mRNA transcript of one or more of SEQ ID NO: 10-15 comprising a retained intron 1 or 2.
  • the ASOs target SEQ ID NO: 10740 and/or 10738.
  • the ASO comprises a sequence of any one of SEQ ID NOs: 6953-8623.
  • the ASO targets a sequence of a RIC pre-mRNA transcript encoded by a CYP24A1 genomic sequence. In some embodiments, the ASO targets a RIC pre-mRNA transcript encoded by a CYP24A1 genomic sequence comprising retained intron 10 and/or 9 or 11. In some embodiments, the ASO targets a RIC pre-mRNA transcript encoded by SEQ ID NO: 4. In some embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ ID NO: 18 or 19. In some embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ ID NO: 18 or 19 comprising a retained intron 10 and/or 9 or 11.
  • the ASOs target SEQ ID NOs: 10748, 10734, 10746, 10745 and/or 10741. In some embodiments, the ASO comprises a sequence of any one of SEQ ID NOs: 9520-10733.
  • the ASO targets a sequence of a RIC pre-mRNA transcript encoded by a PPARD genomic sequence. In some embodiments, the ASO targets a RIC pre-mRNA transcript encoded by a PPARD genomic sequence comprising retained intron 3 or 4 or 5 or 6 or 7 and/or 2 or 3 or 4 or 5 or 6 or 7 or 8. In some embodiments, the ASO targets a RIC pre-mRNA transcript encoded by SEQ ID NO: 1. In some embodiments, the ASO targets a RIC pre-mRNA transcript of one or more of SEQ ID NO: 5-9.
  • the ASO targets a RIC pre-mRNA transcript of one or more of SEQ ID NO: 5-9 comprising a retained intron 3 or 4 or 5 or 6 or 7 and/or 2 or 3 or 4 or 5 or 6 or 7 or 8.
  • the ASOs target SEQ ID NO 10736, 10747, 10739, 10743, 10742, 10737, 10735, and/or 10744.
  • the ASO comprises a sequence of any one of SEQ ID NOs: 20-6952.
  • the ASO targets exon 9 and/or 10 of a CTNS RIC pre-mRNA comprising a retained intron 9 and/or 10. In some embodiments, the ASO targets an exon 9 and/or 10 sequence upstream (or 5') from the 5' splice site of a CTNS RIC pre-mRNA comprising a retained intron 9 and/or 10.
  • the ASO targets intron 9 and/or 10 in a CTNS RIC pre-mRNA comprising a retained intron 9 and/or 10. In some embodiments, the ASO targets an intron 9 and/or 10 sequence downstream (or 3') from the 5' splice site of a CTNS RIC pre-mRNA comprising a retained intron 9 and/or 10.
  • the ASO targets an intron 9 and/or 10 sequence upstream (or 5') from the 3' splice site of a CTNS RIC pre-mRNA comprising a retained intron 9 and/or 10.
  • the ASO targets exon 10 and/or 1 1 in a CTNS RIC pre-mRNA comprising a retained intron 9 and/or 10. In some embodiments, the ASO targets an exon 10 and/or 1 1 sequence downstream (or 3') from the 3' splice site of a CTNS RIC pre-mRNA comprising a retained intron 9 and/or 10.
  • the ASO targets exon 1 or 2 of a PAX2 RIC pre-mRNA comprising a retained intron 1 or 2. In some embodiments, the ASO targets an exon 1 or 2 sequence upstream (or 5') from the 5' splice site of a PAX2 RIC pre-mRNA comprising a retained intron 1 or 2.
  • the ASO targets intron 1 or 2 in a PAX2 RIC pre-mRNA comprising a retained intron 1 or 2. In some embodiments, the ASO targets an intron 1 or 2 sequence downstream (or 3') from the 5' splice site of a PAX2 RIC pre-mRNA comprising a retained intron 1 or 2.
  • the ASO targets an intron 1 or 2 sequence upstream (or 5') from the 3' splice site of a PAX2 RIC pre-mRNA comprising a retained intron 1 or 2.
  • the ASO targets exon 2 or 3 in a PAX2 RIC pre-mRNA comprising a retained intron 1 or 2. In some embodiments, the ASO targets an exon 2 or 3 sequence downstream (or 3') from the 3' splice site of a PAX2 RIC pre-mRNA comprising a retained intron 1 or 2.
  • the ASO targets exon 10 and/or 9 or 1 1 of a CYP24A1 RIC pre-mRNA comprising a retained intron 10 and/or 9 or 1 1. In some embodiments, the ASO targets an exon 10 and/or 9 or 1 1 sequence upstream (or 5') from the 5' splice site of a CYP24A1 RIC pre-mRNA comprising a retained intron 10 and/or 9 or 1 1.
  • the ASO targets intron 10 and/or 9 or 1 1 in a CYP24A1 RIC pre-mRNA comprising a retained intron 10 and/or 9 or 1 1. In some embodiments, the ASO targets an intron 10 and/or
  • the ASO targets an intron 10 and/or 9 or 1 1 sequence upstream (or 5') from the 3' splice site of a CYP24A1 RIC pre-mRNA comprising a retained intron 10 and/or 9 or 1 1.
  • the ASO targets exon 1 1 and/or 10 or 12 in a CYP24A1 RIC pre-mRNA comprising a retained intron 10 and/or 9 or 1 1. In some embodiments, the ASO targets an exon 1 1 and/or
  • the ASO targets exon 3 or 4 or 5 or 6 or 7 and/or 2 or 3 or 4 or 5 or 6 or 7 or 8 of a PPARD RIC pre-mRNA comprising a retained intron 3 or 4 or 5 or 6 or 7 and/or 2 or 3 or 4 or 5 or 6 or 7 or 8.
  • the ASO targets an exon 3 or 4 or 5 or 6 or 7 and/or 2 or 3 or 4 or 5 or 6 or 7 or 8 sequence upstream (or 5') from the 5' splice site of a PPARD RIC pre-mRNA comprising a retained intron 3 or 4 or 5 or 6 or 7 and/or 2 or 3 or 4 or 5 or 6 or 7 or 8.
  • the ASO targets intron 3 or 4 or 5 or 6 or 7 and/or 2 or 3 or 4 or 5 or 6 or 7 or 8 in a PPARD RIC pre-mRNA comprising a retained intron 3 or 4 or 5 or 6 or 7 and/or 2 or 3 or 4 or 5 or 6 or 7 or 8.
  • the ASO targets an intron 3 or 4 or 5 or 6 or 7 and/or 2 or 3 or 4 or 5 or 6 or 7 or 8 sequence downstream (or 3') from the 5' splice site of a PPARD RIC pre-mRNA comprising a retained intron 3 or 4 or 5 or 6 or 7 and/or 2 or 3 or 4 or 5 or 6 or 7 or 8.
  • the ASO targets an intron 3 or 4 or 5 or 6 or 7 and/or 2 or 3 or 4 or 5 or 6 or 7 or 8 sequence upstream (or 5') from the 3' splice site oia PPARD RIC pre-mRNA comprising a retained intron 3 or 4 or 5 or 6 or 7 and/or 2 or 3 or 4 or 5 or 6 or 7 or 8.
  • the ASO targets exon 4 or 5 or 6 or 7 or 8 and/or 3 or 4 or 5 or 6 or 7 or 8 or 9 in a PPARD RIC pre-mRNA comprising a retained intron 3 or 4 or 5 or 6 or 7 and/or 2 or 3 or 4 or 5 or 6 or 7 or 8.
  • the ASO targets an exon 4 or 5 or 6 or 7 or 8 and/or 3 or 4 or 5 or 6 or 7 or 8 or 9 sequence downstream (or 3 ') from the 3 ' splice site of a PPARD RIC pre- mRNA comprising a retained intron 3 or 4 or 5 or 6 or 7 and/or 2 or 3 or 4 or 5 or 6 or 7 or 8.
  • the methods described herein are used to increase the production of a functional protein.
  • the term “functional” refers to the amount of activity or function of a protein that is necessary to eliminate any one or more symptoms of a treated condition.
  • the methods are used to increase the production of a partially functional protein.
  • the term “partially functional” refers to any amount of activity or function of the protein that is less than the amount of activity or function that is necessary to eliminate or prevent any one or more symptoms of a disease or condition.
  • a partially functional protein or RNA will have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, 85%, at least 90%, or at least 95% less activity relative to the fully functional protein or RNA.
  • the method is a method of increasing the expression of the protein by cells of a subject having a RIC pre-mRNA encoding the protein, wherein the subject has a condition described herein caused by a deficient amount of activity of a protein described herein.
  • the deficient amount of the protein is caused by haploinsufficiency of the protein.
  • the subject has a first allele encoding a functional protein, and a second allele from which the protein is not produced.
  • the subject has a first allele encoding a functional protein, and a second allele encoding a nonfunctional protein.
  • the subject has a first allele encoding a functional protein, and a second allele encoding a partially functional protein.
  • the antisense oligomer binds to a targeted portion of the RIC pre-mRNA transcribed from the first allele (encoding functional protein), thereby inducing constitutive splicing of the retained intron from the RIC pre-mRNA, and causing an increase in the level of mature mRNA encoding functional protein, and an increase in the expression of the protein in the cells of the subject.
  • the subject has a first allele encoding a functional protein, and a second allele encoding a partially functional protein, and the antisense oligomer binds to a targeted portion of the RIC pre-mRNA transcribed from the first allele or a targeted portion of the RIC pre-mRNA transcribed from the second allele (encoding partially functional protein), thereby inducing constitutive splicing of the retained intron from the RIC pre-mRNA, and causing an increase in the level of mature mRNA encoding the protein, and an increase in the expression of functional or partially functional protein in the cells of the subject.
  • the method is a method of using an ASO to increase the expression of a protein or functional RNA.
  • an ASO is used to increase the expression of a protein described herein in cells of a subject having a RIC pre-mRNA encoding the protein, wherein the subject has a deficiency in the amount or function of the protein.
  • the RIC pre-mRNA transcript that encodes the protein that is causative of the disease or condition is targeted by the ASOs described herein.
  • a RIC pre-mRNA transcript that encodes a protein that is not causative of the disease is targeted by the ASOs.
  • a disease that is the result of a mutation or deficiency of a first protein in a particular pathway may be ameliorated by targeting a RIC pre-mRNA that encodes a second protein, thereby increasing production of the second protein.
  • the function of the second protein is able to compensate for the mutation or deficiency of the first protein (which is causative of the disease or condition).
  • the subject has:
  • the protein is produced at a reduced level compared to production from a wild- type allele
  • the protein is produced in a form having reduced function compared to an
  • the protein is produced at a reduced level compared to production from a wild- type allele
  • the protein is produced in a form having reduced function compared to an
  • the protein is not produced, and wherein the RIC pre-mRNA is transcribed from the first allele and/or the second allele.
  • the ASO binds to a targeted portion of the RIC pre-mRNA transcribed from the first allele or the second allele, thereby inducing constitutive splicing of the retained intron from the RIC pre- mRNA, and causing an increase in the level of mRNA encoding a protein and an increase in the expression of the target protein or functional RNA in the cells of the subject.
  • the target protein or functional RNA having an increase in expression level resulting from the constitutive splicing of the retained intron from the RIC pre-mRNA is either in a form having reduced function compared to the equivalent wild-type protein (partially-functional), or having full function compared to the equivalent wild-type protein (fully-functional).
  • the level of mRNA encoding a protein described herein is increased 1.1 to 10- fold, when compared to the amount of mRNA encoding the protein that is produced in a control cell, e.g., one that is not treated with the antisense oligomer or one that is treated with an antisense oligomer that does not bind to the targeted portion of the RIC pre-mRNA.
  • the condition caused by a deficient amount or activity of a protein is not a condition caused by alternative or aberrant splicing of the retained intron to which the ASO is targeted.
  • the condition caused by a deficient amount or activity of the protein is not a condition caused by alternative or aberrant splicing of any retained intron in a RIC pre-mRNA encoding the protein.
  • alternative or aberrant splicing may occur in a pre-mRNA transcribed from the gene, however the compositions and methods of the invention do not prevent or correct this alternative or aberrant splicing.
  • a subject treated using the methods of the invention expresses a partially functional protein from one allele, wherein the partially functional protein is caused by a frameshift mutation, a nonsense mutation, a missense mutation, or a partial gene deletion.
  • a subject treated using the methods of the invention expresses a nonfunctional protein from one allele, wherein the nonfunctional protein is caused by a frameshift mutation, a nonsense mutation, a missense mutation, a partial gene deletion, in one allele.
  • a subject treated using the methods of the invention has a whole gene deletion, in one allele.
  • Targeted Augmentation of Nuclear Gene Output is used in the methods of the invention to increase expression of a protein.
  • a retained-intron-containing pre-mRNA encoding a protein is present in the nucleus of a cell.
  • Cells having a RIC pre-mRNA that comprises a retained intron, an exon flanking the 5' splice site, and an exon flanking the 3' splice site, encoding the protein are contacted with antisense oligomers (ASOs) that are complementary to a targeted portion of the RIC pre-mRNA.
  • ASOs antisense oligomers
  • Hybridization of the ASOs to the targeted portion of the RIC pre-mRNA results in enhanced splicing at the splice site (5' splice site or 3 ' splice site) of the retained intron and subsequently increases target protein production.
  • pre-mRNA and “pre-mRNA transcript” may be used interchangeably and refer to any pre-mRNA species that contains at least one intron.
  • pre-mRNA or pre-mRNA transcripts comprise a 5'-7-methylguanosine cap and/or a poly-A tail.
  • pre-mRNA or pre-mRNA transcripts comprise both a 5'-7-methylguanosine cap and a poly- A tail.
  • the pre-mRNA transcript does not comprise a 5'-7-methylguanosine cap and/or a poly- A tail.
  • a pre-mRNA transcript is a non-productive messenger RNA (mRNA) molecule if it is not translated into a protein (or transported into the cytoplasm from the nucleus).
  • a "retained-intron-containing pre-mRNA” (“RIC pre-mRNA”) is a pre-mRNA transcript that contains at least one retained intron.
  • the RIC pre-mRNA contains a retained intron, an exon flanking the 5' splice site of the retained intron, an exon flanking the 3 ' splice site of the retained intron, and encodes the target protein.
  • An "RIC pre-mRNA encoding a target protein” is understood to encode the target protein when fully spliced.
  • a “retained intron” is any intron that is present in a pre- mRNA transcript when one or more other introns, such as an adjacent intron, encoded by the same gene have been spliced out of the same pre-mRNA transcript.
  • the retained intron is the most abundant intron in RIC pre-mRNA encoding the target protein.
  • the retained intron is the most abundant intron in a population of RIC pre-mRNAs transcribed from the gene encoding the target protein in a cell, wherein the population of RIC pre-mRNAs comprises two or more retained introns.
  • an antisense oligomer targeted to the most abundant intron in the population of RIC pre-mRNAs encoding the target protein induces splicing out of two or more retained introns in the population, including the retained intron to which the antisense oligomer is targeted or binds.
  • a mature mRNA encoding the target protein is thereby produced.
  • the terms "mature mRNA,” and “fully-spliced mRNA,” are used interchangeably herein to describe a fully processed mRNA encoding a target protein (e.g., mRNA that is exported from the nucleus into the cytoplasm and translated into target protein) or a fully processed functional RNA.
  • the term "productive mRNA,” also can be used to describe a fully processed mRNA encoding a target protein.
  • the targeted region is in a retained intron that is the most abundant intron in a RIC pre-mRNA encoding the protein.
  • the term “comprise” or variations thereof such as “comprises” or “comprising” are to be read to indicate the inclusion of any recited feature (e.g. in the case of an antisense oligomer, a defined nucleobase sequence) but not the exclusion of any other features.
  • the term “comprising” is inclusive and does not exclude additional, unrecited features (e.g. in the case of an antisense oligomer, the presence of additional, unrecited nucleobases).
  • compositions and methods comprising
  • consisting may be replaced with “consisting essentially of " or “consisting of.”
  • the phrase “consisting essentially of is used herein to require the specified feature(s) (e.g. nucleobase sequence) as well as those which do not materially affect the character or function of the claimed invention.
  • the term “consisting” is used to indicate the presence of the recited feature (e.g. nucleobase sequence) alone (so that in the case of an antisense oligomer consisting of a specified nucleobase sequence, the presence of additional, unrecited nucleobases is excluded).
  • the targeted region is in a retained intron that is the second most abundant intron in a RIC pre-mRNA encoding a protein described herein.
  • the second most abundant retained intron may be targeted rather than the most abundant retained intron due to the uniqueness of the nucleotide sequence of the second most abundant retained intron, ease of ASO design to target a particular nucleotide sequence, and/or amount of increase in protein production resulting from targeting the intron with an ASO.
  • the retained intron is the second most abundant intron in a population of RIC pre-mRNAs transcribed from the gene encoding the target protein in a cell, wherein the population of RIC pre-mRNAs comprises two or more retained introns.
  • an antisense oligomer targeted to the second most abundant intron in the population of RIC pre-mRNAs encoding the target protein induces splicing out of two or more retained introns in the population, including the retained intron to which the antisense oligomer is targeted or binds.
  • fully-spliced (mature) RNA encoding the target protein is thereby produced.
  • an ASO is complementary to a targeted region that is within a non-retained intron in a RIC pre-mRNA.
  • the targeted portion of the RIC pre-mRNA is within: the region +6 to +100 relative to the 5' splice site of the non-retained intron; or the region -16 to -100 relative to the 3 ' splice site of the non-retained intron.
  • the targeted portion of the RIC pre- mRNA is within the region +100 relative to the 5' splice site of the non-retained intron to -100 relative to the 3 ' splice site of the non-retained intron.
  • RNA encoding the target protein is thereby produced.
  • the retained intron of the RIC pre-mRNA is an inefficiently spliced intron.
  • "inefficiently spliced” may refer to a relatively low frequency of splicing at a splice site adjacent to the retained intron (5' splice site or 3 ' splice site) as compared to the frequency of splicing at another splice site in the RIC pre-mRNA.
  • inefficiently spliced may also refer to the relative rate or kinetics of splicing at a splice site, in which an "inefficiently spliced" intron may be spliced or removed at a slower rate as compared to another intron in a RIC pre-mRNA.
  • the 9-nucleotide sequence at -3e to -le of the exon flanking the 5' splice site and +1 to +6 of the retained intron is identical to the corresponding wild-type sequence.
  • the 16 nucleotide sequence at -15 to -1 of the retained intron and +le of the exon flanking the 3 ' splice site is identical to the corresponding wild-type sequence.
  • wild-type sequence refers to the nucleotide sequence for a gene in the published reference genome deposited in the NCBI repository of biological and scientific information (operated by National Center for Biotechnology Information, National Library of Medicine, 8600 Rockville Pike, Bethesda, MD USA 20894).
  • nucleotide position denoted with an "e” indicates the nucleotide is present in the sequence of an exon (e.g., the exon flanking the 5' splice site or the exon flanking the 3 ' splice site).
  • the methods involve contacting cells with an ASO that is complementary to a portion of a pre- mRNA encoding a protein described herein, resulting in increased expression of the protein.
  • contacting or administering to cells refers to any method of providing an ASO in immediate proximity with the cells such that the ASO and the cells interact.
  • a cell that is contacted with an ASO will take up or transport the ASO into the cell.
  • the method involves contacting a condition or disease- associated or condition or disease-relevant cell with an ASO.
  • the ASO may be further modified or attached (e.g., covalently attached) to another molecule to target the ASO to a cell type, enhance contact between the ASO and the condition or disease-associated or condition or disease- relevant cell, or enhance uptake of the ASO.
  • the term “increasing protein production” or “increasing expression of a target protein” means enhancing the amount of protein that is translated from an mRNA in a cell.
  • a “target protein” may be any protein for which increased expression/production is desired.
  • contacting a cell that expresses a RIC pre-mRNA with an ASO that is complementary to a targeted portion of the RIC pre-mRNA transcript results in a measurable increase in the amount of a protein (e.g., a target protein) encoded by the pre-mRNA.
  • a protein e.g., a target protein
  • Methods of measuring or detecting production of a protein will be evident to one of skill in the art and include any known method, for example, Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA.
  • contacting cells with an ASO that is complementary to a targeted portion of an RIC pre-mRNA transcript results in an increase in the amount of a protein produced by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000%, compared to the amount of the protein produced by a cell in the absence of the ASO/absence of treatment.
  • the total amount of a protein produced by the cell to which the antisense oligomer was contacted is increased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1 -fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-
  • contacting cells with an ASO that is complementary to a targeted portion of an RIC pre-mRNA transcript results in an increase in the amount of mRNA encoding a protein, including the mature mRNA encoding the target protein.
  • the amount of mRNA encoding a protein, or the mature mRNA encoding the protein is increased by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000%, compared to the amount of the protein produced by a cell in the absence of the ASO/absence of treatment.
  • the total amount of the mRNA encoding a protein, or the mature mRNA encoding a protein produced in the cell to which the antisense oligomer was contacted is increased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1 -fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-
  • the methods and antisense oligonucleotide compositions provided herein are useful for increasing the expression of a protein in cells, for example, in a subject having a condition described herein caused by a deficiency in the amount or activity of a protein described herein, by increasing the level of mRNA encoding the protein, or the mature mRNA encoding the protein.
  • the methods and compositions as described herein induce the constitutive splicing of a retained intron from an RIC pre-mRNA transcript encoding the protein, thereby increasing the level of mRNA encoding the protein, or the mature mRNA encoding the protein and increasing the expression of the protein.
  • Constitutive splicing of a retained intron from a RIC pre-mRNA correctly removes the retained intron from the RIC pre-mRNA, wherein the retained intron has wild-type splice sequences.
  • Constitutive splicing does not encompass splicing of a retained intron from a RIC pre-mRNA transcribed from a gene or allele having a mutation that causes alternative splicing or aberrant splicing of a pre-mRNA transcribed from the gene or allele.
  • constitutive splicing of a retained intron does not correct aberrant splicing in or influence alternative splicing of a pre-mRNA to result in an increased expression of a target protein or functional RNA.
  • constitutive splicing correctly removes a retained intron from an RIC pre- mRNA, wherein the RIC pre-mRNA is transcribed from a wild-type gene or allele, or a polymorphic gene or allele, that encodes a fully-functional target protein or functional RNA, and wherein the gene or allele does not have a mutation that causes alternative splicing or aberrant splicing of the retained intron.
  • constitutive splicing of a retained intron from an RIC pre-mRNA encoding a protein correctly removes a retained intron from an RIC pre-mRNA encoding the protein, wherein the RIC pre-mRNA is transcribed from a gene or allele from which the target gene or functional RNA is produced at a reduced level compared to production from a wild-type allele, and wherein the gene or allele does not have a mutation that causes alternative splicing or aberrant splicing of the retained intron.
  • the correct removal of the constitutively spliced retained intron results in production of target protein or functional RNA that is functional when compared to an equivalent wild- type protein or functional RNA.
  • constitutive splicing correctly removes a retained intron from an RIC pre- mRNA, wherein the RIC pre-mRNA is transcribed from a gene or allele that encodes a target protein or functional RNA produced in a form having reduced function compared to an equivalent wild-type protein or functional RNA, and wherein the gene or allele does not have a mutation that causes alternative splicing or aberrant splicing of the retained intron.
  • the correct removal of the constitutively spliced retained intron results in production of partially functional target protein, or functional RNA that is partially functional when compared to an equivalent wild-type protein or functional RNA.
  • an antisense oligomer as described herein or used in any method described herein does not increase the amount of mRNA encoding a protein or the amount of a protein by modulating alternative splicing or aberrant splicing of a pre-mRNA transcribed from the gene.
  • Modulation of alternative splicing or aberrant splicing can be measured using any known method for analyzing the sequence and length of RNA species, e.g., by RT-PCR and using methods described elsewhere herein and in the literature.
  • modulation of alternative or aberrant splicing is determined based on an increase or decrease in the amount of the spliced species of interest of at least 10% or 1.1 -fold.
  • modulation is determined based on an increase or decrease at a level that is at least 10% to 100% or 1.1 to 10-fold, as described herein regarding determining an increase in mRNA encoding the protein in the methods of the invention.
  • the method is a method wherein the RIC pre-mRNA was produced by partial splicing of a wild-type pre-mRNA. In embodiments, the method is a method wherein the RIC pre-mRNA was produced by partial splicing of a full-length wild-type pre-mRNA. In embodiments, the RIC pre- mRNA was produced by partial splicing of a full-length pre-mRNA. In these embodiments, a full-length pre-mRNA may have a polymorphism in a splice site of the retained intron that does not impair correct splicing of the retained intron as compared to splicing of the retained intron having the wild-type splice site sequence.
  • the mRNA encoding a protein is a full-length mature mRNA, or a wild-type mature mRNA.
  • a full-length mature mRNA may have a polymorphism that does not affect the activity of the target protein or the functional RNA encoded by the mature mRNA, as compared to the activity of the protein encoded by the wild-type mature mRNA.
  • composition comprising antisense oligomers that enhances splicing by binding to a targeted portion of an RIC pre-mRNA.
  • ASO antisense oligomer
  • antisense oligomer are used interchangeably and refer to an oligomer such as a
  • polynucleotide comprising nucleobases, that hybridizes to a target nucleic acid ⁇ e.g., an RIC pre-mRNA) sequence by Watson-Crick base pairing or wobble base pairing (G-U).
  • the ASO may have exact sequence complementary to the target sequence or near complementarity ⁇ e.g., sufficient
  • ASOs are designed so that they bind (hybridize) to a target nucleic acid ⁇ e.g., a targeted portion of a pre-mRNA transcript) and remain hybridized under physiological conditions. Typically, if they hybridize to a site other than the intended (targeted) nucleic acid sequence, they hybridize to a limited number of sequences that are not a target nucleic acid (to a few sites other than a target nucleic acid).
  • Design of an ASO can take into consideration the occurrence of the nucleic acid sequence of the targeted portion of the pre-mRNA transcript or a sufficiently similar nucleic acid sequence in other locations in the genome or cellular pre- mRNA or transcriptome, such that the likelihood the ASO will bind other sites and cause "off-target” effects is limited.
  • PCT/US2014/054151 published as WO 2015/035091, titled “Reducing Nonsense-Mediated mRNA Decay,” can be used to practice the methods described herein.
  • ASOs "specifically hybridize” to or are “specific” to a target nucleic acid or a targeted portion of a RIC pre-mRNA.
  • hybridization occurs with a Tm substantially greater than 37°C, preferably at least 50°C, and typically between 60°C to approximately 90°C.
  • Tm substantially greater than 37°C, preferably at least 50°C, and typically between 60°C to approximately 90°C.
  • Such hybridization preferably corresponds to stringent hybridization conditions.
  • the Tm is the temperature at which 50% of a target sequence hybridizes to a complementary oligonucleotide.
  • Oligomers such as oligonucleotides, are "complementary" to one another when hybridization occurs in an antiparallel configuration between two single-stranded 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 noncomplementary nucleobases may be clustered together or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases.
  • Percent complementarity of an ASO with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol, 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).
  • 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
  • an ASO hybridizes to noncontiguous nucleobases in a target pre-mRNA transcript.
  • 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 RIC pre-mRNA.
  • the term ASO embodies oligonucleotides and any other oligomeric molecule that comprises nucleobases capable of hybridizing to a complementary nucleobase on a target mRNA but does not comprise a sugar moiety, such as a peptide nucleic acid (PNA).
  • 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.
  • nucleotides includes deoxyribonucleotides and ribonucleotides.
  • modified nucleotides includes nucleotides with modified or substituted sugar groups and/or having a modified backbone. In some embodiments, all of the nucleotides of the ASO are modified nucleotides.
  • Chemical modifications of ASOs or components of ASOs that are compatible with the methods and compositions described herein will be evident to one of skill in the art and can be found, for example, in U.S. Patent No. 8,258,109 B2, U.S. Patent No. 5,656,612, U.S. Patent Publication No. 2012/0190728, and Dias and Stein, Mol. Cancer Ther. 2002, 1, 347-355, herein incorporated by reference in their entirety.
  • the nucleobase of an ASO may be any naturally occurring, unmodified nucleobase such as adenine, guanine, cytosine, thymine and uracil, or any synthetic or modified nucleobase that is sufficiently similar to an unmodified nucleobase such that it is capable of hydrogen bonding with a nucleobase present on a target pre-mRNA.
  • modified nucleobases include, without limitation, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-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,
  • the backbone structure of the ASO does not contain phosphorous but rather contains peptide bonds, for example in a peptide nucleic acid (PNA), or linking groups including carbamate, amides, and linear and cyclic hydrocarbon groups.
  • PNA peptide nucleic acid
  • the backbone modification is a phosphorothioate linkage. In some embodiments, the backbone modification is a phosphoramidate linkage.
  • the stereochemistry at each of the phosphorus intemucleotide linkages of the ASO backbone is random. In embodiments, the stereochemistry at each of the phosphorus intemucleotide linkages of the ASO backbone is controlled and is not random.
  • U.S. Pat. App. Pub. No. 2014/0194610 "Methods for the Synthesis of Functionalized Nucleic Acids," incorporated herein by reference, describes methods for independently selecting the handedness of chirality at each phosphorous atom in a nucleic acid oligomer.
  • an ASO used in the methods of the invention comprises an ASO having phosphorus intemucleotide linkages that are not random.
  • a composition used in the methods of the invention comprises a pure diastereomeric ASO.
  • a composition used in the methods of the invention comprises an ASO that has diastereomeric purity of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100%.
  • the ASO has a nonrandom mixture of Rp and Sp configurations at its phosphorus intemucleotide linkages.
  • Rp and Sp are required in antisense oligonucleotides to achieve a balance between good activity and nuclease stability.
  • an ASO used in the methods of the invention comprises about 5-100% Rp, at least about 5% Rp, at least about 10% Rp, at least about 15% Rp, at least about 20% Rp, at least about 25% Rp, at least about 30% Rp, at least about 35% Rp, at least about 40% Rp, at least about 45% Rp, at least about 50% Rp, at least about 55% Rp, at least about 60% Rp, at least about 65% Rp, at least about 70% Rp, at least about 75% Rp, at least about 80% Rp, at least about 85% Rp, at least about 90% Rp, or at least about 95% Rp, with the remainder Sp, or about 100% Rp.
  • an ASO used in the methods of the invention comprises about 10% to about 100% Rp, about 15% to about 100% Rp, about 20% to about 100% Rp, about 25% to about 100% Rp, about 30% to about 100% Rp, about 35% to about 100% Rp, about 40% to about 100% Rp, about 45% to about 100% Rp, about 50% to about 100% Rp, about 55% to about 100% Rp, about 60% to about 100% Rp, about 65% to about 100% Rp, about 70% to about 100% Rp, about 75% to about 100% Rp, about 80% to about 100% Rp, about 85% to about 100% Rp, about 90% to about 100% Rp, or about 95% to about 100% Rp, about 20% to about 80% Rp, about 25% to about 75% Rp, about 30% to about 70% Rp, about 40% to about 60% Rp, or about 45% to about 55% Rp, with the remainder Sp.
  • an ASO used in the methods of the invention comprises about 5-100% Sp, at least about 5% Sp, at least about 10% Sp, at least about 15% Sp, at least about 20% Sp, at least about 25% Sp, at least about 30% Sp, at least about 35% Sp, at least about 40% Sp, at least about 45% Sp, at least about 50% Sp, at least about 55% Sp, at least about 60% Sp, at least about 65% Sp, at least about 70% Sp, at least about 75% Sp, at least about 80% Sp, at least about 85% Sp, at least about 90% Sp, or at least about 95% Sp, with the remainder Rp, or about 100% Sp.
  • an ASO used in the methods of the invention comprises about 10% to about 100% Sp, about 15% to about 100% Sp, about 20% to about 100% Sp, about 25% to about 100% Sp, about 30% to about 100% Sp, about 35% to about 100% Sp, about 40% to about 100% Sp, about 45% to about 100% Sp, about 50% to about 100% Sp, about 55% to about 100% Sp, about 60% to about 100% Sp, about 65% to about 100% Sp, about 70% to about 100% Sp, about 75% to about 100% Sp, about 80% to about 100% Sp, about 85% to about 100% Sp, about 90% to about 100% Sp, or about 95% to about 100% Sp, about 20% to about 80% Sp, about 25% to about 75% Sp, about 30% to about 70% Sp, about 40% to about 60% Sp, or about 45% to about 55% Sp, with the remainder Rp.
  • Any of the ASOs described herein may contain a sugar moiety that comprises ribose or deoxyribose, as present in naturally occurring nucleotides, or a modified sugar moiety or sugar analog, including a morpholine ring.
  • modified sugar moieties include 2' substitutions such as 2'-0-methyl (2'-0-Me), 2'-0-methoxyethyl (2'MOE), 2'-0-aminoethyl, 2'F; N3'->P5' phosphoramidate, 2' dimethyl aminooxyethoxy, 2'dimethylaminoethoxyethoxy, 2'-guanidinidium, 2'-0- guanidinium ethyl, carbamate modified sugars, and bicyclic modified sugars.
  • the sugar moiety modification is selected from 2'-0-Me, 2'F, and 2'MOE.
  • the sugar moiety modification is an extra bridge bond, such as in a locked nucleic acid (LNA).
  • the sugar analog contains a morpholine ring, such as phosphorodiamidate morpholino (PMO).
  • the sugar moiety comprises a ribofuransyl or 2'deoxyribofuransyl modification.
  • the sugar moiety comprises 2'4' -constrained 2'O-methyloxyethyl (cMOE) modifications.
  • the sugar moiety comprises cEt 2', 4' constrained 2'-0 ethyl BNA modifications.
  • the sugar moiety comprises tricycloDNA (tcDNA) modifications.
  • the sugar moiety comprises ethylene nucleic acid (ENA) modifications.
  • the sugar moiety comprises MCE modifications. Modifications are known in the art and described in the literature, e.g., by Jarver, et al., 2014, "A Chemical View of Oligonucleotides for Exon Skipping and Related Drug Applications,” Nucleic Acid Therapeutics 24(1): 37-47, incorporated by reference for this purpose herein.
  • each monomer of the ASO is modified in the same way, for example each linkage of the backbone of the ASO comprises a phosphorothioate linkage or each ribose sugar moiety comprises a 2'0-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).
  • the ASO comprises one or more backbone modification. In some embodiments, the ASO comprises one or more sugar moiety modification. In some embodiments, the ASO comprises one or more backbone modification and one or more sugar moiety modification. In some embodiments, the ASO comprises 2'MOE modifications and a phosphorothioate backbone. In some embodiments, the ASO comprises a phosphorodiamidate morpholino (PMO). In some embodiments, the ASO comprises a peptide nucleic acid (PNA).
  • PMO phosphorodiamidate morpholino
  • PNA peptide nucleic acid
  • any of the ASOs or any component of an ASO ⁇ e.g., a nucleobase, sugar moiety, backbone) described herein may be modified in order to achieve desired properties or activities of the ASO or reduce undesired properties or activities of the ASO.
  • an ASO or one or more component of any ASO may be modified to enhance binding affinity to a target sequence on a pre-mRNA transcript; reduce binding to any non-target sequence; reduce degradation by cellular nucleases (i.e., RNase H); improve uptake of the ASO into a cell and/or into the nucleus of a cell; alter the pharmacokinetics or pharmacodynamics of the ASO; and modulate the half-life of the ASO.
  • the ASOs are comprised of 2'-0-(2-methoxyethyl) (MOE)
  • ASOs comprised of such nucleotides are especially well-suited to the methods disclosed herein; oligomers having such modifications have been shown to have significantly enhanced resistance to nuclease degradation and increased bioavailability, making them suitable, for example, for oral delivery in some embodiments described herein. See e.g., Geary et al, J Pharmacol Exp Ther. 2001; 296(3):890-7; Geary et al, J Pharmacol Exp Ther. 2001; 296(3):898-904.
  • ASOs may be obtained from a commercial source.
  • the left-hand end of single-stranded nucleic acid ⁇ e.g., pre-mRNA transcript, oligonucleotide, ASO, etc.
  • 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.
  • a region or sequence that is 5' to a reference point in a nucleic acid is referred to as "upstream”
  • a region or sequence that is 3' to a reference point in a nucleic acid is referred to as "downstream.”
  • 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., while a nucleotide that is directly adjacent and downstream of the reference point is designated “plus one,” e.g.,
  • the ASOs are complementary to (and bind to) a targeted portion of a CTNS, PAX2, CYP24A1 or PPARD RIC pre-mRNA that is downstream (in the 3' direction) of the 5' splice site of the retained intron in a CTNS, PAX2, CYP24A1 or PPARD RIC pre-mRNA ⁇ e.g., the direction designated by positive numbers relative to the 5' splice site) (FIG. 1).
  • the ASOs are complementary to a targeted portion of the CTNS, PAX2, CYP24A1 or PPARD RIC pre- mRNA that is within the region 1 to +4000, 1 to +3500, 1 to +3000, 1 to +2500, 1 to +2000, 1 to +1500, 1 to +1000, 1 to +500, +2 to +4000, +2 to +3500, +2 to +3000, +2 to +2500, +2 to +2000, +2 to +1500, +2 to +1000, +2 to +500, +2 to +400, +2 to +300, +2 to +200, +6 to +4000, +6 to +3500, +6 to +3000, +6 to +2500, +6 to +2000, +6 to +1500, +6 to +1000, +6 to +500, +6 to +400, +6 to +300, +6 to +200, or +6 to +100 relative to the 5' splice site of the retained intron.
  • the ASO is not complementary to nucleotides +1 to +5 relative to the 5' splice site (the first five nucleotides located downstream of the 5' splice site).
  • the ASOs may be complementary to a targeted portion of a CTNS, PAX2, CYP24A1 or PPARD RIC pre-mRNA that is within the region between nucleotides +6 and +50 relative to the 5' splice site of the retained intron.
  • the ASOs are complementary to a targeted portion of the CTNS, PAX2, CYP24A1 or PPARD RIC pre-mRNA that is within the region +6 to +90, +6 to +80, +6 to +70, +6 to +60, +6 to +50, +6 to +40, +6 to +30, or +6 to +20 relative to 5' splice site of the retained intron.
  • the ASOs are complementary to a targeted region of a CTNS, PAX2, CYP24A1 or PPARD RIC pre-mRNA that is upstream (5' relative) of the 3 ' splice site of the retained intron in an RIC pre-mRNA (e.g., in the direction designated by negative numbers) (FIG. 1).
  • the ASOs are complementary to a targeted portion of the CTNS, PAX2, CYP24A1 or PPARD RIC pre-mRNA that is within the region -16 to -4000, -16 to -3000, -16 to -2000, -16 to -1000, -16 to -500, -16 to -400, -16 to -300, -6 to -200, or -16 to -100 relative to the 3 ' splice site of the retained intron.
  • the ASO is not complementary to nucleotides -1 to -15 relative to the 3 ' splice site (the first 15 nucleotides located upstream of the 3 ' splice site).
  • the ASOs are complementary to a targeted portion of the CTNS, PAX2, CYP24A1 or PPARD RIC pre- mRNA that is within the region -16 to -50 relative to the 3 ' splice site of the retained intron. In some aspects, the ASOs are complementary to a targeted portion of the CTNS, PAX2, CYP24A1 or PPARD RIC pre-mRNA that is within the region -16 to -90, -16 to -80, -16 to -70, -16 to -60, -16 to -50, -16 to - 40, or -16 to -30 relative to 3 ' splice site of the retained intron.
  • the targeted portion of the RIC pre-mRNA is within the region +100 relative to the 5' splice site of the retained intron to -100 relative to the 3 ' splice site of the retained intron.
  • the ASOs are complementary to a targeted portion of a CTNS, PAX2, CYP24A1 or PPARD RIC pre-mRNA that is within the exon flanking the 5' splice site (upstream) of the retained intron (FIG. 1).
  • the ASOs are complementary to a targeted portion of the RIC pre-mRNA that is within the region +2e to -4e in the exon flanking the 5' splice site of the retained intron.
  • the ASOs are not complementary to nucleotides -le to -3e relative to the 5' splice site of the retained intron.
  • the ASOs are complementary to a targeted portion of the CTNS, PAX2, CYP24A1 or PPARD RIC pre-mRNA that is within the region -4e to-lOOe, -4e to -90e, -4e to -80e, -4e to -70e, -4e to -60e, -4e to -50e, -4 to -40e, -4e to -30e, or -4e to -20e relative to the 5' splice site of the retained intron.
  • the ASOs are complementary to a targeted portion of a CTNS, PAX2, CYP24A1 or PPARD RIC pre-mRNA that is within the exon flanking the 3 ' splice site (downstream) of the retained intron (FIG. 1). In some embodiments, the ASOs are complementary to a targeted portion to the CTNS, PAX2, CYP24A1 or PPARD RIC pre-mRNA that is within the region +2e to -4e in the exon flanking the 3 ' splice site of the retained intron. In some embodiments, the ASOs are not complementary to nucleotide +le relative to the 3' splice site of the retained intron.
  • the ASOs are complementary to a targeted portion of the CTNS, PAX2, CYP24A1 or PPARD RIC pre-mRNA that is within the region+2e to +100e, +2e to +90e, +2e to +80e, +2e to +70e, +2e to +60e, +2e to +50e, +2e to +40e, +2e to +30e, or +2 to +20e relative to the 3' splice site of the retained intron.
  • the ASOs may be of any length suitable for specific binding and effective enhancement of splicing. In some embodiments, the ASOs consist of 8 to 50 nucleobases.
  • the ASO may be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, or 50 nucleobases in length. In some embodiments, the ASOs consist of more than 50 nucleobases.
  • 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
  • the ASOs are 30 nucleotides in length. In some embodiments, the ASOs are 29 nucleotides in length. In some embodiments, the ASOs are 28 nucleotides in length. In some embodiments, the ASOs are 27 nucleotides in length. In some embodiments, the ASOs are 26 nucleotides in length. In some embodiments, the ASOs are 25 nucleotides in length. In some embodiments, the ASOs are 24 nucleotides in length. In some embodiments, the ASOs are 23 nucleotides in length. In some embodiments, the ASOs are 22 nucleotides in length. In some embodiments, the ASOs are 21 nucleotides in length.
  • two or more ASOs with different chemistries but complementary to the same targeted portion of the RIC pre-mRNA are used. In some embodiments, two or more ASOs that are complementary to different targeted portions of the RIC pre-mRNA are used.
  • the antisense oligonucleotides of the invention are chemically linked to one or more moieties or conjugates, e.g., a targeting moiety or other conjugate that enhances the activity or cellular uptake of the oligonucleotide.
  • moieties include, but are not limited to, a lipid moiety, e.g., as a cholesterol moiety, a cholesteryl moiety, an aliphatic chain, e.g., dodecandiol or undecyl residues, a polyamine or a polyethylene glycol chain, or adamantane acetic acid.
  • the antisense oligonucleotide is conjugated with a moiety including, but not limited to, an abasic nucleotide, a polyether, a polyamine, a polyamide, a peptides, a carbohydrate, e.g., N- acetylgalactosamine (GalNAc), N-Ac-Glucosamine (GluNAc), or mannose ⁇ e.g., mannose-6-phosphate), a lipid, or a 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
  • Conjugates can be linked to one or more of any nucleotides comprising the antisense oligonucleotide at any of several positions on the sugar, base or phosphate group, as understood in the art and described in the literature, e.g., using a linker.
  • Linkers can include a bivalent or trivalent branched linker.
  • the conjugate is attached to the 3' end of the antisense oligonucleotide.
  • the nucleic acid to be targeted by an ASO is an RIC pre-mRNA expressed in a cell, such as a eukaryotic cell.
  • 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).
  • compositions or formulations comprising the antisense oligonucleotide of the described compositions and for use in any of the described methods can be prepared according to conventional techniques well known in the pharmaceutical industry and described in the published literature.
  • a pharmaceutical composition or formulation for treating a subject comprises an effective amount of any antisense oligomer as described above, or a pharmaceutically acceptable salt, solvate, hydrate or ester thereof, and a pharmaceutically acceptable diluent.
  • the antisense oligomer of a pharmaceutical formulation may further comprise a pharmaceutically acceptable excipient, diluent or carrier.
  • salts are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., and are commensurate with a reasonable benefit/risk ratio. (See, e.g., S. M. Berge, et al., J. Pharmaceutical Sciences, 66: 1-19 (1977), incorporated herein by reference for this purpose.
  • the salts can be prepared in situ during the final isolation and purification of the compounds, or separately by reacting the free base function with a suitable organic acid.
  • Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other documented methodologies such as ion exchange.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid
  • organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other documented methodologies such as ion exchange.
  • salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, 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,
  • 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 invention includes, but is not limited to, a solution, emulsion, microemulsion, foam or liposome- containing formulation (e.g., cationic or noncationic liposomes).
  • the pharmaceutical composition or formulation of the present invention may comprise one or more penetration enhancer, carrier, excipients or other active or inactive ingredients as appropriate and well known to those of skill in the art or described in the published literature.
  • liposomes also include sterically stabilized liposomes, e.g., liposomes comprising one or more specialized lipids. These specialized lipids result in liposomes with enhanced circulation lifetimes.
  • a sterically stabilized liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety.
  • a surfactant is included in the pharmaceutical formulation or compositions.
  • the present invention employs a penetration enhancer to effect the efficient delivery of the antisense oligonucleotide, e.g., to aid diffusion across cell membranes and /or enhance the permeability of a lipophilic drug.
  • the penetration enhancers is 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 antisense oligonucleotide is administered with one or more agents capable of promoting penetration of the subject antisense oligonucleotide across the blood-brain barrier by any method known in the art. For example, delivery of agents by administration of an adenovirus vector to motor neurons in muscle tissue is described in U.S. Pat. No. 6,632,427, "Adenoviral -vector- mediated gene transfer into medullary motor neurons,” incorporated herein by reference.
  • vectors directly to the brain e.g., the striatum, the thalamus, the hippocampus, or the substantia nigra
  • Delivery of vectors directly to the brain e.g., the striatum, the thalamus, the hippocampus, or the substantia nigra, is described, e.g., in U.S. Pat. No. 6,756,523, "Adenovirus vectors for the transfer of foreign genes into cells of the central nervous system particularly in brain," incorporated herein by reference.
  • the antisense oligonucleotides are linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties.
  • the antisense oligonucleotide is coupled to a substance, known in the art to promote penetration or transport across the blood-brain barrier, e.g., an antibody to the transferrin receptor.
  • the antisense oligonucleotide is linked with a viral vector, e.g., to render the antisense compound more effective or increase transport across the blood-brain barrier.
  • the antisense oligonucleotides of the invention are chemically linked to one or more moieties or conjugates, e.g., a targeting moiety or other conjugate that enhances the activity or cellular uptake of the oligonucleotide.
  • moieties include, but are not limited to, a lipid moiety, e.g., as a cholesterol moiety, a cholesteryl moiety, an aliphatic chain, e.g., dodecandiol or undecyl residues, a polyamine or a polyethylene glycol chain, or adamantane acetic acid.
  • the antisense oligonucleotide is conjugated with a moiety including, but not limited to, an abasic nucleotide, a polyether, a polyamine, a polyamide, a peptides, a carbohydrate, e.g., N- acetylgalactosamine (GalNAc), N-Ac-Glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate), a lipid, or a 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.
  • compositions provided herein may be administered to an individual.
  • “Individual” may be used interchangeably with “subject” or "patient.”
  • An individual may be a mammal, for example a human or animal such as a non-human primate, a rodent, a rabbit, a rat, a mouse, a horse, a donkey, a goat, a cat, a dog, a cow, a pig, or a sheep.
  • the individual is a human.
  • the individual is a fetus, an embryo, or a child.
  • the individual may be another eukaryotic organism, such as a plant.
  • the compositions provided herein are administered to a cell ex vivo.
  • the compositions provided herein are administered to an individual as a method of treating a disease or disorder.
  • the individual has a genetic disease, such as any of the diseases described herein.
  • the individual is at risk of having the disease, such as any of the diseases described herein.
  • the individual is at increased risk of having a disease or disorder caused by insufficient amount of a protein or insufficient activity of a protein. If an individual is "at an increased risk" of having a disease or disorder caused insufficient amount of a protein or insufficient activity of a protein, the method involves preventative or prophylactic treatment.
  • an individual may be at an increased risk of having such a disease or disorder because of family history of the disease.
  • individuals at an increased risk of having such a disease or disorder benefit from prophylactic treatment ⁇ e.g., by preventing or delaying the onset or progression of the disease or disorder).
  • Suitable routes for administration of ASOs of the present invention may vary depending on cell type to which delivery of the ASOs is desired. Multiple tissues and organs can be affected in a condition described herein, with the kidney being the most significantly affected tissue.
  • the ASOs of the present invention may be administered to patients parenterally, for example, by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. In embodiments, delivery is to the kidney.
  • a fetus is treated in utero, e.g., by administering the ASO composition to the fetus directly or indirectly (e.g., via the mother).
  • ASOs that specifically hybridize to different nucleotides within the target region of the pre-mRNA may be screened to identify (determine) ASOs that improve the rate and/or extent of splicing of the target intron.
  • the ASO may block or interfere with the binding site(s) of a splicing repressor(s)/silencer.
  • Any method known in the art may be used to identify (determine) an ASO that when hybridized to the target region of the intron results in the desired effect ⁇ e.g., enhanced splicing, protein or functional RNA production). These methods also can be used for identifying ASOs that enhance splicing of the retained intron by binding to a targeted region in an exon flanking the retained intron, or in a non-retained intron. An example of a method that may be used is provided below.
  • 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 5' splice site of the retained intron ⁇ e.g., a portion of sequence of the exon located upstream of the target/retained intron) to approximately 100 nucleotides downstream of the 5' splice site of the target/retained intron and/or from approximately 100 nucleotides upstream of the 3' splice site of the retained intron to approximately 100 nucleotides downstream of the 3' splice site of the target/retained intron ⁇ e.g., a portion of sequence of the exon located downstream of the target/retained intron).
  • a first ASO of 15 nucleotides in length may be designed to specifically hybridize to nucleotides +6 to +20 relative to the 5' splice site of the target/retained intron.
  • a second ASO is designed to specifically hybridize to nucleotides +11 to +25 relative to the 5' splice site of the target/retained intron.
  • ASOs are designed as such spanning the target region of the pre-mRNA.
  • the ASOs can be tiled more closely, e.g., every 1, 2, 3, or 4 nucleotides. Further, the ASOs can be tiled from 100 nucleotides downstream of the 5' splice site, to 100 nucleotides upstream of the 3' splice site.
  • One or more ASOs, or a control ASO are delivered, for example by transfection, into a disease- relevant cell line that expresses the target pre-mRNA (e.g., the RIC pre-mRNA described elsewhere herein).
  • the splicing-inducing effects of each of the ASOs may be assessed by any method known in the art, for example by reverse transcriptase (RT)-PCR using primers that span the splice junction, as described herein (see “Identification of intron-retention events").
  • a reduction or absence of the RT-PCR product produced using the primers spanning the splice junction in ASO-treated cells as compared to in control ASO-treated cells indicates that splicing of the target intron has been enhanced.
  • the splicing efficiency, the ratio of spliced to unspliced pre-mRNA, the rate of splicing, or the extent of splicing may be improved using the ASOs described herein.
  • the amount of protein or functional RNA that is encoded by the target pre-mRNA can also be assessed to determine whether each ASO achieved the desired effect (e.g. , enhanced protein production). Any method known in the art for assessing and/or quantifying protein production, such as Western blotting, flow cytometry,
  • a second round of screening referred to as an ASO "micro-walk” may be performed using ASOs that have been designed to hybridize to a target region of a pre-mRNA.
  • the ASOs used in the ASO micro-walk are tiled every 1 nucleotide to further refine the nucleotide acid sequence of the pre- mRNA that when hybridized with an ASO results in enhanced splicing.
  • Regions defined by ASOs that promote splicing of the target intron are explored in greater detail by means of an ASO "micro-walk", involving ASOs spaced in 1-nt steps, as well as longer ASOs, typically 18-25 nt.
  • the ASO micro-walk is performed by delivering one or more ASOs, or a control ASO (an ASO with a scrambled sequence, sequence that is not expected to hybridize to the target region), for example by transfection, into a disease-relevant cell line that expresses the target pre-mRNA.
  • the splicing-inducing effects of each of the ASOs may be assessed by any method known in the art, for example by reverse transcriptase (RT)-PCR using primers that span the splice junction, as described herein (see “Identification of intron-retention events").
  • a reduction or absence of the RT-PCR product produced using the primers spanning the splice junction in ASO-treated cells as compared to in control ASO-treated cells indicates that splicing of the target intron has been enhanced.
  • the splicing efficiency, the ratio of spliced to unspliced pre-mRNA, the rate of splicing, or the extent of splicing may be improved using the ASOs described herein.
  • the amount of protein or functional RNA that is encoded by the target pre-mRNA can also be assessed to determine whether each ASO achieved the desired effect (e.g. , enhanced protein production). Any method known in the art for assessing and/or quantifying protein production, such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA, can be used.
  • ASOs that when hybridized to a region of a pre-mRNA result in enhanced splicing and increased protein production may be tested in vivo using animal models, for example transgenic mouse models in which the full-length human gene has been knocked-in or in humanized mouse models of disease.
  • Suitable routes for administration of ASOs may vary depending on the disease and/or the cell types to which delivery of the ASOs is desired.
  • ASOs may be administered, for example, by 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 (efficiency, rate, extent) and protein production by methods known in the art and described herein.
  • the animal models may also be any phenotypic or behavioral indication of the disease or disease severity.
  • Example 1 Identification of intron retention events in transcripts by RNAseq using next generation sequencing
  • Example 2 Identification of intron retention events in gene transcripts by RNAseq using next generation sequencing for retained introns not yet identified
  • a schematic representation of the gene was provided by the UCSC genome browser (below the read signals) so that peaks could be matched to the gene exonic and intronic regions.
  • retained introns were inferred as those that have high read density in the nuclear fraction of renal epithelial cells, but have very low to no reads in the cytoplasmic fraction of these cells. This indicated that the introns are retained and that the retained intron-containing transcripts remained in the nucleus, and suggests that these retained RIC pre-mRNAs are non-productive, as they are not exported out to the cytoplasm.
  • Example 3 Design of ASO-walk targeting a retained intron
  • An ASO walk was designed to target a retained intron using the method described herein.
  • a region immediately downstream of the intron 5' splice site e.g., spanning nucleotides +6 to +69 and a region immediately upstream of intron 3' splice site, e.g., spanning nucleotides -16 to -79 of the intron was targeted with 2'-0-Me RNA, PS backbone, 18-mer ASOs shifted by 5-nucleotide intervals.
  • Table 1 lists exemplary ASOs that were designed and their target sequences.
  • Example 4 Improved splicing efficiency via ASO-targeting of a retained intron increases CTNS transcript levels
  • Ct values from CTNS targeting - AS O-transfected cells are normalized to RPL32 and plotted relative to the normalized qPCR product from mock-treated cells. Results of this analysis indicated that several CTNS targeting ASOs increase gene transcript levels. These results show that inducing splicing of a retained intron in the gene using ASOs leads to an increase in gene expression. Altogether, these results show that improving the splicing efficiency of a rate limiting intron in the CTNS gene using ASOs led to an increase in CTNS gene expression
  • Example 5 Improved splicing efficiency via ASO-targeting of a retained intron increases CYP24A1 transcript levels
  • CYP24A1 To determine whether an increase in expression of CYP24A1 could be achieved by improving splicing efficiency of a retained intron using ASOs, the methods described herein were used.
  • ARPE-19 cells were mock-transfected, or transfected with CYP24A1 targeting ASOs, or a non-targeting ASO control, independently, using RNAiMAX (Invitrogen) delivery reagents.
  • Example 6 Improved splicing efficiency via ASO-targeting of a retained intron increases PPARD transcript levels
  • ARPE- 19 cells were mock-transfected, or transfected with PPARD targeting ASOs, or a non-targeting ASO control, independently, using RNAiMAX (Invitrogen) delivery reagents. Experiments were performed using 80 nM ASOs for 24 hrs (FIG. 6A and B, FIG. 6E and F). Taqman qPCR results showed that several targeting ASOs increase PPARD gene transcript level compared to the mock- transfected.
  • Ct values from PPARD targeting-ASO-transfected cells are normalized to RPL32 and plotted relative to the normalized qPCR product from mock-treated cells. Results of this analysis indicated that several PPARD targeting ASOs increase gene transcript levels. These results show that inducing splicing of a retained intron in the gene using ASOs leads to an increase in gene expression. Altogether, these results show that improving the splicing efficiency of a rate limiting intron in the PPARD gene using ASOs led to an increase in PPARD gene expression.
  • Example 7 Improved splicing efficiency via ASO-targeting of a retained intron increases transcript levels
  • ARPE-19 cells a human retinal epithelium cell line (American Type Culture Collection (ATCC), USA), or Huh-7, a human hepatoma cell line (NIBIOHN, Japan), or SK-N-AS, a human neuroblastoma cell line (ATCC) were mock-transfected, or transfected with the targeting ASOs described in FIGs. 3-6 and Table 1.
  • Cells were transfected using Lipofectamine RNAiMax transfection reagent (Thermo Fisher) according to vendor's specifications.
  • ASOs were plated in 96-well tissue culture plates and combined with RNAiMax diluted in Opti-MEM. Cells were detached using trypsin and resuspended in full medium, and approximately 25,000 cells were added the ASO-transfection mixture. Transfection experiments were carried out in triplicate plate replicates. Final ASO concentration was 80 nM. Media was changed 6h post-transfection, and cells harvested at 24h, using the Cells-to-Ct lysis reagent, supplemented with DNAse (Thermo Fisher), according to vendor's specifications. cDNA was generated with Cells-to-Ct RT reagents (Thermo Fisher) according to vendor's specifications.
  • Taqman assays were carried out according to vendor's specifications, on a QuantStudio 7 Flex Real-Time PCR system (Thermo Fisher).
  • Target gene assay values were normalized to RPL32 (deltaCt) and plate-matched mock transfected samples (delta-delta Ct), generating fold-change over mock quantitation (2 A -(delta-deltaCt). Average fold- change over mock of the three plate replicates was plotted (FIG. 3C, FIG. 4C, FIG.

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Abstract

L'invention concerne des procédés et des compositions pour augmenter l'expression d'une protéine, et pour traiter un sujet en ayant besoin, par exemple un sujet souffrant d'une déficience d'expression de protéine ou un sujet atteint d'une maladie rénale.
PCT/US2016/066576 2015-12-14 2016-12-14 Compositions et méthodes de traitement de maladies rénales WO2017106292A1 (fr)

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CA3005249A CA3005249A1 (fr) 2015-12-14 2016-12-14 Compositions et methodes de traitement de maladies renales
JP2018529237A JP7049247B2 (ja) 2015-12-14 2016-12-14 腎臓病の処置のための組成物と方法
EP16876548.5A EP3390666A4 (fr) 2015-12-14 2016-12-14 Compositions et méthodes de traitement de maladies rénales
US16/007,435 US11096956B2 (en) 2015-12-14 2018-06-13 Antisense oligomers and uses thereof
US17/379,793 US20220118000A1 (en) 2015-12-14 2021-07-19 Antisense oligomers and uses thereof
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US11827886B2 (en) 2014-06-03 2023-11-28 University Of Massachusetts Compositions and methods for modulating dysferlin expression
US10538764B2 (en) 2014-06-16 2020-01-21 University Of Southampton Reducing intron retention
US11891605B2 (en) 2014-06-16 2024-02-06 University Of Southampton Reducing intron retention
US10696969B2 (en) 2014-10-03 2020-06-30 Cold Spring Harbor Laboratory Targeted augmentation of nuclear gene output
US11702660B2 (en) 2015-10-09 2023-07-18 University Of Southampton Modulation of gene expression and screening for deregulated protein expression
US10941405B2 (en) 2015-10-09 2021-03-09 University Of Southampton Modulation of gene expression and screening for deregulated protein expression
US10196639B2 (en) 2015-10-09 2019-02-05 University Of Southampton Modulation of gene expression and screening for deregulated protein expression
US11083745B2 (en) 2015-12-14 2021-08-10 Cold Spring Harbor Laboratory Antisense oligomers for treatment of autosomal dominant mental retardation-5 and Dravet Syndrome
US11096956B2 (en) 2015-12-14 2021-08-24 Stoke Therapeutics, Inc. Antisense oligomers and uses thereof
US11845963B2 (en) 2017-01-23 2023-12-19 Regeneron Pharmaceuticals, Inc. HSD17B13 variants and uses thereof
US11753628B2 (en) 2017-01-23 2023-09-12 Regeneron Pharmaceuticals, Inc. HSD17B13 variants and uses thereof
US11485958B2 (en) 2017-01-23 2022-11-01 Regeneron Pharmaceuticals, Inc. HSD17B13 variants and uses thereof
US11479802B2 (en) 2017-04-11 2022-10-25 Regeneron Pharmaceuticals, Inc. Assays for screening activity of modulators of members of the hydroxy steroid (17-beta) dehydrogenase (HSD17B) family
US10913947B2 (en) 2017-08-25 2021-02-09 Stoke Therapeutics, Inc. Antisense oligomers for treatment of conditions and diseases
US11873490B2 (en) 2017-08-25 2024-01-16 Stoke Therapeutics, Inc. Antisense oligomers for treatment of conditions and diseases
US10683503B2 (en) 2017-08-25 2020-06-16 Stoke Therapeutics, Inc. Antisense oligomers for treatment of conditions and diseases
US11702700B2 (en) 2017-10-11 2023-07-18 Regeneron Pharmaceuticals, Inc. Inhibition of HSD17B13 in the treatment of liver disease in patients expressing the PNPLA3 I148M variation
EP3802829A4 (fr) * 2018-06-08 2022-10-19 University of Massachusetts Oligonucléotides antisens pour restaurer l'expression de la protéine dysferline dans des cellules de patients souffrant d'une dysferlinopathie
EP3969469A4 (fr) * 2019-01-23 2022-11-23 The Florey Institute of Neuroscience and Mental Health Oligonucléotides antisens ciblant des introns de scn2a conservés
WO2021034985A1 (fr) * 2019-08-19 2021-02-25 Stoke Therapeutics, Inc. Compositions et méthodes pour moduler l'épissage et l'expression de protéines
US11940448B2 (en) 2020-03-31 2024-03-26 Seattle Children's Hospital Proteomic screening for lysosomal storage diseases
US11814622B2 (en) 2020-05-11 2023-11-14 Stoke Therapeutics, Inc. OPA1 antisense oligomers for treatment of conditions and diseases

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