WO2017106211A1 - Antisense oligomers for treatment of polycystic kidney disease - Google Patents

Antisense oligomers for treatment of polycystic kidney disease Download PDF

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Publication number
WO2017106211A1
WO2017106211A1 PCT/US2016/066417 US2016066417W WO2017106211A1 WO 2017106211 A1 WO2017106211 A1 WO 2017106211A1 US 2016066417 W US2016066417 W US 2016066417W WO 2017106211 A1 WO2017106211 A1 WO 2017106211A1
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WIPO (PCT)
Prior art keywords
nucleobases
mrna
fold
retained intron
protein
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PCT/US2016/066417
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English (en)
French (fr)
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 CA3005247A priority Critical patent/CA3005247A1/en
Priority to EP16876500.6A priority patent/EP3389782A4/en
Priority to JP2018529219A priority patent/JP2018538287A/ja
Publication of WO2017106211A1 publication Critical patent/WO2017106211A1/en
Priority to US16/007,435 priority patent/US11096956B2/en
Priority to US17/379,793 priority patent/US20220118000A1/en
Priority to JP2022000102A priority patent/JP2022046723A/ja

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    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/33—Alteration of splicing
    • C—CHEMISTRY; METALLURGY
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • ADPKD Autosomal dominant polycystic kidney disease
  • ESRD end-stage renal disease
  • PKD1 or PKD2 gene have been shown to manifest as ADPKD, with mutations in PKD2 being responsible for the late onset form of ADPKD.
  • the PKD1 and PKD2 genes encode the PC-1 and PC-2 proteins, respectively. These proteins are believed to be essential to maintain the differentiated phenotype of the tubular epithelium (Torres and Harris, 2009).
  • RIC pre-mRNA retained-intron-containing pre-mRNA
  • the RIC pre-mRNA comprising a retained intron, an exon flanking the 5' splice site, an exon flanking the 3' splice site, and wherein the RIC pre-mRNA encodes the target protein or functional RNA
  • the method comprising contacting the cells of the subject with an antisense oligomer (ASO) complementary to a targeted portion of the RIC pre-mRNA encoding the target protein or functional RNA, whereby the retained intron is constitutively spliced from the RIC pre-mRNA encoding the target protein or functional RNA, thereby increasing the level of mRNA encoding the target protein or functional RNA, and increasing the expression of the target protein or functional
  • ASO antisense oligomer
  • a target protein wherein the target protein is PC-2
  • the target protein is PC-2
  • cells having a retained-intron-containing pre-mRNA RIC pre-mRNA
  • the RIC pre-mRNA comprising a retained intron, an exon flanking the 5' splice site of the retained intron, an exon flanking the 3' splice site of the retained intron, and wherein the RIC pre-mRNA encodes PC-2 protein
  • the method comprising contacting the cells with an antisense oligomer (ASO) complementary to a targeted portion of the RIC pre-mRNA encoding PC-2 protein, whereby the retained intron is constitutively spliced from the RIC pre-mRNA encoding PC-2 protein, thereby increasing the level of mRNA encoding PC-2 protein, and increasing the expression of PC-2 protein in the cells.
  • ASO antisense oligomer
  • the target protein is PC-2.
  • 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 PC-2 protein.
  • the deficient amount of the target protein is caused by haplo insufficiency of the target protein, wherein the subject has a first allele encoding a functional target protein, and a second allele from which the target protein is not produced, or a second allele encoding a nonfunctional target protein, and wherein the antisense oligomer binds to a targeted portion of a RIC pre-mRNA transcribed from the first allele.
  • the subject has a condition caused by a disorder resulting from a deficiency in the amount or function of the target protein, wherein the subject has (a) a first mutant allele from which (i) the target protein is produced at a reduced level compared to production from a wild-type allele, (ii) the target protein is produced in a form having reduced function compared to an equivalent wild-type protein, or (iii) the target protein is not produced, and (b) a second mutant allele from which (i) the target protein is produced at a reduced level compared to production from a wild-type allele, (ii) the target protein is produced in a form having reduced function compared to an equivalent wild-type protein, or (iii) the target protein is not produced, and wherein when the subject has a first mutant allele (a)(iii), the second mutant allele is (b)(i) or (b)(ii), and wherein when the subject has a second mutant allele (b)(iii),
  • the targeted portion of the RIC pre- mRNA is in the retained intron within the region +6 relative to the 5 ' splice site of the retained intron to - 16 relative to the 3 ' splice site of the retained intron. In some embodiments, the targeted portion of the RIC pre-mRNA is in the retained intron within: (a) the region +6 to +497 relative to the 5' splice site of the retained intron; or (b) the region -16 to -496 relative to the 3' splice site of the retained intron.
  • the targeted portion of the RIC pre-mRNA is within: (a) the region +2e to -4e in the exon flanking the 5' splice site of the retained intron; or (b) the region +2e to -4e in the exon flanking the 3' splice site of the retained intron.
  • the targeted portion of the RIC pre-mRNA is in the retained intron within: (a) the region -4e to -l,054e relative to the 5 ' splice site of the retained intron; (b) the region +6 to +499 relative to the 5' splice site of the retained intron; (c) the region -16 to - 496 relative to the 3 ' splice site of the retained intron; or (d) the region +2e to +1,912e relative to the 3' splice site of the retained intron.
  • the target protein is PC-2.
  • the RIC pre-mRNA comprises a sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 2.
  • the RIC pre -mRNA is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 1.
  • the targeted portion of the RIC pre-mRNA comprises a sequence with at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 contiguous nucleic acids of SEQ ID NO: 281.
  • the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complimentary to any one of SEQ ID NOs: 3-280.
  • the targeted portion of the RIC pre-mRNA is within the region -204e to +497 relative to the 5 ' splice site of the retained intron 5 or within the region -496 to +212e relative to the 3' splice site of the retained intron 5.
  • the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complimentary to any one of SEQ ID NOs: 3-280.
  • the targeted portion of the RIC pre-mRNA is in exon 5 within the region -204e to -4e relative to the 5 ' splice site of the retained intron 5.
  • the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complimentary to any one of SEQ ID NOs: 3-43.
  • the targeted portion of the RIC pre-mRNA is in retained intron 5 within the region +6 to +497 relative to the 5' splice site of the retained intron.
  • the ASO comprises a sequence that is at least about 80%, 85%, 90%), 95%), 97%), or 100%) complimentary to any one of SEQ ID NOs:44-140.
  • the targeted portion of the RIC pre-mRNA is in retained intron 5 within the region -16 to -496 relative to the 3' splice site of the retained intron.
  • the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complimentary to any one of SEQ ID NOs: 141-237.
  • the targeted portion of the RIC pre-mRNA is in exon 6 within the region +2e to +212e relative to the 3' splice site of the retained intron 5.
  • the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complimentary to any one of SEQ ID NOs: 238-280.
  • the antisense oligomer does not increase the amount of the target protein or the functional RNA by modulating alternative splicing of pre- mRNA transcribed from a gene encoding the functional RNA or target protein. In some embodiments, the antisense oligomer does not increase the amount of the target protein or the functional RNA by modulating aberrant splicing resulting from mutation of the gene encoding the target protein or the functional RNA. In some embodiments, the RIC pre-mRNA was produced by partial splicing of a full- length pre-mRNA or partial splicing of a wild-type pre-mRNA.
  • 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
  • the total amount of target protein produced by the cell contacted with the antisense oligomer is increased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5 -fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7- fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1 -fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5
  • the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.
  • 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, 1 1 to 50 nucleobases, 1 1 to 40 nucleobases, 1 1 to 40
  • nucleobases 1 1 to 35 nucleobases, 1 1 to 30 nucleobases, 1 1 to 25 nucleobases, 1 1 to 20 nucleobases, 1 1 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30
  • 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-mR A 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 at least one retained intron, 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 at least one retained intron 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 method further comprises assessing PC-2 protein expression.
  • the antisense oligomer binds to a targeted portion ⁇ ⁇ ⁇ 2 RIC pre-mRNA, wherein the targeted portion is in a sequence selected from SEQ ID NOs: 3-280.
  • 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 and +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.
  • antisense oligomers as described in any of the aforementioned methods.
  • antisense oligomers comprising a sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 3-280.
  • compositions comprising any of the aforementioned antisense oligomers and an excipient.
  • compositions comprising an antisense oligomer for use in a method of increasing expression of a target protein or a functional RNA by cells to treat
  • a deficient protein or deficient functional RNA comprising a deficient protein or deficient functional RNA, wherein the deficient protein or deficient functional RNA is deficient in amount or activity in the subject, wherein the antisense oligomer enhances constitutive splicing of a retained intron- containing pre-mRNA (RIC pre-mRNA) encoding the target protein or the functional RNA, wherein the target protein is: (a) the deficient protein; or (b) a compensating protein which functionally augments or replaces the deficient protein or in the subject; and wherein the functional RNA is: (c) the deficient RNA; or (d) a compensating functional RNA which functionally augments or replaces the deficient functional RNA in the subject; wherein the RIC pre-mRNA comprises a retained intron, an exon flanking the 5' splice site and an exon flanking the 3' splice site, and wherein the retained intron is spliced from the RIC pre-mRNA
  • compositions comprising an antisense oligomer for use in a method of treating a condition associated with PC-2 protein in a subject in need thereof, the method comprising the step of increasing expression of PC-2 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 PC-2 protein, the method comprising contacting the cells with the antisense oligomer, whereby the retained intron is constitutive ly spliced from the RIC pre- mRNA transcripts encoding PC-2 protein, thereby increasing the level of mRNA encoding the PC-2 protein, and increasing the expression of PC-2 protein, in the cells of the subject.
  • the condition is a retained-intron-containing pre-mRNA (RIC pre-mRNA) compris
  • the antisense oligomer targets a portion of the RIC pre-mRNA that is in the retained intron within the region +6 relative to the 5' splice site of the retained intron to -16 relative to the 3' splice site of the retained intron. In some embodiments, the antisense oligomer targets a portion of the RIC pre-mRNA that is in the retained intron within: (a) the region +6 to +497 relative to the 5' splice site of the retained intron; or (b) the region -16 to -496 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: (a) the region +2e to -4e in the exon flanking the 5' splice site of the retained intron; or (b) the region +2e to -4e in the exon flanking the 3' splice site of the retained intron.
  • the targeted portion of the RIC pre-mRNA is within: (a) the region -4e to -l,054e relative to the 5' splice site of the retained intron; (b) the region +6 to +499 relative to the 5' splice site of the retained intron; (c) the region -16 to -496 relative to the 3' splice site of the retained intron; or (d) the region +2e to +l,912e relative to the 3' splice site of the retained intron.
  • the target protein is PC-2.
  • the RIC pre-mRNA comprises a sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 2.
  • the RIC pre-mRNA is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 1.
  • the targeted portion of the RIC pre-mRNA comprises a sequence with at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 contiguous nucleic acids of SEQ ID NO: 281.
  • the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complimentary to any one of SEQ ID NOs: 3-280.
  • the targeted portion of the RIC pre-mRNA is within the region -204e to +497 relative to the 5 ' splice site of the retained intron 5 or within the region -496 to +212e relative to the 3 ' splice site of the retained intron 5.
  • the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complimentary to any one of SEQ ID NOs: 3-280.
  • the targeted portion of the RIC pre-mRNA is in exon 5 within the region -204e to -4e relative to the 5 ' splice site of the retained intron 5.
  • the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complimentary to any one of SEQ ID NOs: 3-43.
  • the targeted portion of the RIC pre-mRNA is in retained intron 5 within the region +6 to +497 relative to the 5' splice site of the retained intron.
  • the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complimentary to any one of SEQ ID NOs:44-140.
  • the targeted portion of the RIC pre-mRNA is in retained intron 5 within the region - 16 to -496 relative to the 3' splice site of the retained intron.
  • the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complimentary to any one of SEQ ID NOs: 141-237.
  • the targeted portion of the RIC pre-mRNA is in exon 6 within the region +2e to +212e relative to the 3' splice site of the retained intron 5.
  • the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complimentary to any one of SEQ ID NOs: 238-280.
  • the antisense oligomer does not increase the amount of target protein or functional RNA by modulating alternative splicing of the pre- mRNA transcribed from a gene encoding the target protein or functional RNA. In some embodiments, the antisense oligomer does not increase the amount of the functional RNA or functional protein by modulating aberrant splicing resulting from mutation of the gene encoding the target protein or functional RNA. In some embodiments, the RIC pre-mRNA was produced by partial splicing from a full-length pre- mRNA or a wild-type pre-mRNA.
  • 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.
  • the 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.
  • 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.
  • 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 nucleobase
  • compositions comprising any of the aforementioned antisense oligomers and an excipient.
  • methods of treating a subject in need thereof, by administering the aforementioned pharmaceutical compositions by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.
  • compositions comprising: an antisense oligomer that hybridizes to a target sequence of a deficient PKD2 mRNA transcript, wherein the deficient PKD2 mRNA transcript comprises a retained intron, wherein the antisense oligomer induces splicing out of the retained intron from the deficient PKD2 mRNA transcript; and a pharmaceutical acceptable excipient.
  • the deficient PKD2 mRNA transcript is a PKD2 RIC pre- mRNA transcript.
  • the targeted portion of the PKD2 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 PKD2 RIC pre-mRNA transcript is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 1.
  • the PKD2 RIC pre-mRNA transcript comprises a sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2.
  • the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage. In some embodiments, the antisense oligomer is an antisense oligonucleotide. In some embodiments,n the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-0-methyl, a 2'-Fluoro, or a 2'-0-methoxyethyl moiety. In some embodiments, the antisense oligomer comprises at least one modified sugar moiety.
  • the antisense oligomer comprises from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11
  • 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 PKD2 RIC pre-mRNA transcript.
  • the targeted portion of the PKD2 RIC pre-mRNA transcript is within SEQ ID NO: 281.
  • the antisense oligomer comprises a nucleotide sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 3-280.
  • the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NOs: 3-280.
  • the pharmaceutical composition is formulated for intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.
  • a deficient PKD2 mR A transcript to facilitate removal of a retained intron to produce a fully processed PKD2 mR A transcript that encodes a functional form of a PC-2 protein
  • the method comprising: (a) contacting an antisense oligomer to a target cell of a subject; (b) hybridizing the antisense oligomer to the deficient PKD2 mRNA transcript, wherein the deficient PKD2 mRNA transcript is capable of encoding the functional form of a PC-2 protein and comprises at least one retained intron; (c) removing the at least one retained intron from the deficient PKD2 mRNA transcript to produce the fully processed PKD2 mRNA transcript that encodes the functional form of PC-2 protein; and (d) translating the functional form of PC-2 protein from the fully processed PKD2 mRNA transcript.
  • the retained intron is an entire retained intron.
  • RNA molecules comprising a nucleotide sequence with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 3-280.
  • FIG. 1 depicts an exemplary schematic representation of a 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.
  • FIGS. 2A-B depict a schematic representation of the Targeted Augmentation of Nuclear Gene Output (TANGO) approach.
  • FIG. 2A shows a cell divided into nuclear and cytoplasmic compartments. In the nucleus, a pre-mRNA transcript of a target gene consisting of exons (rectangles) and introns
  • FIG. 2B shows an example of the same cell divided into nuclear and cytoplasmic compartments.
  • Treatment with an antisense oligomer (ASO) promotes the splicing of intron 1 and results in an increase in mRNA, which is in turn translated into higher levels of target protein.
  • ASO antisense oligomer
  • FIG. 3 depicts intron-retention in the PKD2 gene with intron 5 shown in detail.
  • the identification of intron-retention events in the PKD2 gene using RNA sequencing (RNAseq) is shown, visualized in the UCSC genome browser.
  • the upper panel shows the read density corresponding to the PKD2 transcript expressed in renal epithelial cells and localized in either the cytoplasmic (top) or nuclear fraction (bottom).
  • a graphic representation of the PKD2 gene is shown to scale. The read density is shown as peaks. The highest read density corresponds to exons (black boxes), while no reads are observed for the majority of the introns in either cellular fraction.
  • FIG. 4 depicts an exemplary PKD2 gene intron 5 (IVS 5) ASO walk.
  • IVS 5 PKD2 gene intron 5
  • ASOs were designed to cover these regions by shifting 5 nucleotides at a time.
  • the PKD2 exon-intron structure is drawn to scale.
  • FIG. 5 depicts a schematic of the RefSeq Gene for PKD2 corresponding to NM_000297.
  • the Percent Intron Retention (PIR) of intron 5 is detailed.
  • introns in primary transcripts of protein-coding genes having more than one intron are spliced from the primary transcript with different efficiencies. In most cases only the fully spliced mRNA is exported through nuclear pores for subsequent translation in the cytoplasm. Unspliced and partially spliced transcripts are detectable in the nucleus. It is generally thought that nuclear accumulation of transcripts that are not fully spliced is a mechanism to prevent the accumulation of potentially deleterious mR As in the cytoplasm that may be translated to protein. For some genes, splicing of the least efficient intron is a rate-limiting post-transcriptional step in gene expression, prior to translation in the cytoplasm.
  • PKD2 gene which encodes the PC-2 protein that is deficient in the debilitating genetic disease, Polycystic Kidney Disease, have been discovered in the nucleus of human cells.
  • PKD2 pre-mRNA species comprise at least one retained intron.
  • the present invention provides compositions and methods for upregulating splicing of one or more retained PKD2 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 PC-2 protein levels.
  • compositions and methods utilize antisense oligomers (ASOs) that promote constitutive splicing at an intron splice site of a retained-intron-containing PKD2 pre-mRNA that accumulates in the nucleus.
  • ASOs antisense oligomers
  • PC-2 protein is increased using the methods of the invention to treat a disease caused by PC-2 deficiency.
  • the methods of the invention are used to increase PC-2 production to treat a condition in a subject in need thereof.
  • the subject has condition in which PC-2 is not necessarily deficient relative to wild-type, but where an increase in PC-2 mitigates the condition nonetheless.
  • the condition is a caused by a PC-2 haploinsufficiency.
  • PTD Polycystic Kidney Disease
  • PKD manifestations include hypertension; endothelial vasodilation; constrictive nitric oxide synthase activity; polycystic liver disease; vascular manifestations including intracranial aneurysms, thoracic aortic dissections and coronary artery aneurysms; and progressive renal failure that leads to end-stage renal disease (ESRD) by age 70.
  • ESRD end-stage renal disease
  • PKD can be diagnosed in utero or at birth through the use of fetal ultrasonography
  • PKD is classically diagnosed later in life through the detection of renal cysts as determined by renal ultrasound.
  • the worldwide PKD prevalence is estimated to be between 1 :400 and 1 : 1000, with a male/female sex ratio of ⁇ 1.2 (Torres and Harris, 2009).
  • Mutations in either the PKD I or PKD2 gene have been reported to cause PKD. Mutations in PKD1 typically manifest earlier in life than mutations in PKD2 (age at ESRD 54.3 vs. 74.0 years for PKD1 and PKD2, respectively) and typically result in a more severe disease state due to the appearance of cysts at a younger age (Torres and Harris, 2009). Due to this difference in pathophysiology, the late onset form of PKD generally arises from mutations in the PKD2 gene.
  • PKD2 encodes the PC-2 protein, a 968 amino acid protein containing a short N-terminal cytoplasmic region with a ciliary motif, 6 transmembrane domains and a short C-terminal portion.
  • the human genomic sequence of the PKD2 gene is set forth at NCBI Gene ID 5311, and the protein at UniProtKB/Swiss-Prot: Q 13563-1, described by, e.g. , Mochizuki T, et al. , 1996, "PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein," Science 272: 1339-1342, incorporated by reference herein.
  • PKD2 mRNA sequence is set forth at NCBI Reference Sequence: NM_000297.3.2, described by Yang Y, et al , 2015, "Oligomerization of the polycystin-2 C-terminal tail and effects on its Ca2+ binding properties," J. Bio. Chem. 290 (16), 10544-10554, both incorporated by reference herein. Mutations in PKD2 cause late onset autosomal dominant PKD (ADPKD), the most prevalent of the inherited renal cystic diseases.
  • ADPKD late onset autosomal dominant PKD
  • the PKD2 gene consists of 15 exons and is located on chromosome 4p22.1. PKD2 mutations in PKD are spread across the entire protein, with 95 truncating mutations of PKD2 reported in the ADPKD Mutation Database (maintained by the PKD Foundation, 8330 Ward Parkway, Suite 510, Kansas City, MO 64114). Because a homozygous deficiency in PKD2 is predicted to be incompatible with live birth, haploinsufficiency is the most likely mechanism of ADPKD disease manifestation (Torres and Harris, 2009). Mutations such as nonsense and insertions/deletions are associated with the classic ADPKD2 phenotype display functional haploinsufficiency.
  • PC-2-D51 IV protein A PKD missense mutation that results in expression of the PC-2-D51 IV protein was predicted to be indistinguishable from wild-type PC-2 in terms of stability (Reynolds, et al , 1999, J. Am. Soc. Nephrol. 10: 2342-2351).
  • the PC-2-D51 IV variant despite its stability, was shown to be dysfunctional due to a predicted disruption in its ability to act as an ion channel. Thus, even stable variants can cause the phenotype if the nascent activity is disrupted.
  • the disease is described, e.g., by OMIM #613095 (Online Mendelian Inheritance in Man, Johns Hopkins University, 1966-2015), incorporated by reference herein.
  • RIC Pre-mRNA Retained Intron Containing Pre-mRNA
  • the methods disclosed herein exploit the presence of retained-intron-containing pre-mRNA (RIC pre-mRNA) transcribed from the PKD2 gene and encoding PC-2 protein, in the cell nucleus. Splicing of the identified PKD2 RIC pre-mRNA species to produce mature, fully-spliced, PKD2 mRNA, is induced using ASOs that stimulate splicing out of the retained introns. The resulting mature PKD2 mRNA can be exported to the cytoplasm and translated, thereby increasing the amount of PC-2 protein in the patient's cells and alleviating symptoms of Polycystic Kidney Disease. This method, described further below, is known as Targeted Augmentation of Nuclear Gene Output (TANGO).
  • TANGO Targeted Augmentation of Nuclear Gene Output
  • RNA sequencing visualized in the UCSC genome browser, showed PKD2 transcripts expressed in renal epithelial cells and localized in either the cytoplasmic or nuclear fraction. In both fractions, reads were not observed for the majority of the introns. However, higher read density was detected for intron 5 in the nuclear fraction compared to the cytoplasmic fraction indicating that splicing efficiency of intron 5 is low, resulting in intron retention.
  • the retained-intron containing pre-mRNA transcripts accumulate primarily in the nucleus and are not translated into the PC-2 protein.
  • the read density for intron 5 indicated 18% intron retention (FIG. 5).
  • the percent intron retention (PIR) value for intron 5 was obtained by averaging four values (23, 13, 22, and 14), each determined in renal epithelial cells using one of four different algorithms.
  • Analysis of the ENCODE data (described by, e.g. , Tilgner, et al , 2012, "Deep sequencing of subcellular RNA fractions shows splicing to be predominantly co -transcriptional in the human genome but inefficient for lncRNAs," Genome Research 22(9): 1616-25) to identify intron retention events did not identify intron 5 as retained.
  • the ASOs disclosed herein target a RIC pre-mRNA transcribed from a PKD2 genomic sequence. In some embodiments, the ASO targets a RIC pre-mRNA transcript from a PKD2 genomic sequence comprising retained intron 5. In some embodiments, the ASO targets a RIC pre- mRNA transcript of SEQ ID NO: 1. In some embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ ID NO: 1 comprising a retained intron 5. In some embodiments, the ASOs disclosed herein target a PKD2 RIC pre-mRNA sequence. In some embodiments, the ASO targets a PKD2 RIC pre-mRNA sequence comprising a retained intron 5.
  • the ASO targets a PKD2 RIC pre-mRNA sequence according to SEQ ID NO: 2. In some embodiments, the ASO targets a PKD2 RIC pre-mRNA sequence according to SEQ ID NO: 2 comprising a retained intron 5. In some embodiments, the ASOs disclosed herein target SEQ ID NO: 281. In some embodiments, the ASO has a sequence according to any one of SEQ ID NOs: 3-280.
  • the ASO targets exon 5 of a PKD2 RIC pre-mRNA comprising a retained intron 5. In some embodiments, the ASO targets an exon 5 sequence upstream (or 5') from the 5' splice site oia PKD2 RIC pre-mRNA comprising a retained intron 5. In some embodiments, the ASO targets an exon sequence about 4 to about 204 nucleotides upstream (or 5') from the 5' splice site of a PKD2 RIC pre-mRNA comprising a retained intron 5. In some embodiments, the ASO has a sequence according to any one of SEQ ID NOs: 3-43.
  • the ASO targets intron 5 in a PKD2 RIC pre-mRNA comprising a retained intron 5. In some embodiments, the ASO targets an intron 5 sequence downstream (or 3') from the 5' splice site of a PKD2 RIC pre-mRNA comprising a retained intron 5. In some embodiments, the ASO targets an intron 5 sequence about 6 to about 497 nucleotides downstream (or 3') from the 5' splice site of a PKD2 RIC pre-mRNA comprising a retained intron 5. In some embodiments, the ASO has a sequence according to any one of SEQ ID NOs: 44-140.
  • the ASO targets an intron 5 sequence upstream (or 5') from the 3' splice site oia PKD2 RIC pre-mRNA comprising a retained intron 5. In some embodiments, the ASO targets an intron 5 sequence about 16 to about 496 nucleotides upstream (or 5') from the 3' splice site oia PKD2 RIC pre-mRNA a comprising retained intron 5. In some embodiments, the ASO has a sequence according to any one of SEQ ID NOs: 141-237.
  • the ASO targets exon 6 in a PKD2 RIC pre-mRNA comprising a retained intron 5. In some embodiments, the ASO targets an exon 6 sequence downstream (or 3') from the 3' splice site of a PKD2 RIC pre-mRNA comprising a retained intron 5. In some embodiments, the ASO targets an exon 6 sequence about 2 to about 212 nucleotides downstream (or 3') from the 3' splice site of a PKD2 RIC pre-mRNA comprising a retained intron 5. In some embodiments, the ASO has a sequence according to any one of SEQ ID NOs: 238-280.
  • the targeted portion of the PKD2 RIC pre-mRNA is in intron 5.
  • the PKD2 intron numbering used herein corresponds to the mRNA sequence at NM_000297.3.
  • 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 5 and subsequently increases PC-2 protein production. It is understood that the intron numbering may change in reference to a different PKD2 mRNA isoform sequence.
  • compositions and methods of the present invention are used to increase the expression of any known PKD2 isoform, e.g.
  • NCBI Gene ID database at Gene ID 5311 (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), incorporated by reference herein.
  • the methods described herein are used to increase the production of a functional PC-2 protein.
  • the term “functional” refers to the amount of activity or function of a PC-2 protein that is necessary to eliminate any one or more symptoms of a treated condition, e.g. , Polycystic Kidney Disease.
  • the methods are used to increase the production of a partially functional PC-2 protein.
  • the term “partially functional” refers to any amount of activity or function of the PC-2 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 PC-2 protein by cells of a subject having a RIC pre-mRNA encoding the PC-2 protein, wherein the subject has Polycystic Kidney Disease caused by a deficient amount of activity of PC-2 protein, and wherein the deficient amount of the PC-2 protein is caused by haploinsufficiency of the PC-2 protein.
  • the subject has a first allele encoding a functional PC-2 protein, and a second allele from which the PC-2 protein is not produced.
  • the subject has a first allele encoding a functional PC-2 protein, and a second allele encoding a nonfunctional PC-2 protein.
  • the subject has a first allele encoding a functional PC-2 protein, and a second allele encoding a partially functional PC-2 protein.
  • the antisense oligomer binds to a targeted portion of the RIC pre-mRNA transcribed from the first allele (encoding functional PC-2 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 PC-2 protein, and an increase in the expression of the PC-
  • the subject has a first allele encoding a functional PC-2 protein, and a second allele encoding a partially functional PC-2 protein, and the antisense oligomer binds to a targeted portion of the RIC pre-mRNA transcribed from the first allele (encoding functional PC-2 protein) or a targeted portion of the RIC pre-mRNA transcribed from the second allele (encoding partially functional PC-2 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 PC-2 protein, and an increase in the expression of functional or partially functional PC-2 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 PC-2 protein in cells of a subject having a RIC pre-mRNA encoding PC-2 protein, wherein the subject has a deficiency e.g. , Polycystic Kidney Disease, in the amount or function of a PC-2 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.
  • the subject has:
  • the PC-2 protein is produced in a form having reduced function compared to an equivalent wild-type protein
  • the PC-2 protein is produced in a form having reduced function compared to an equivalent wild-type protein
  • 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 PC-2 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 PC-2 protein is increased 1.1 to 10-fold, when compared to the amount of mRNA encoding PC-2 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 PKD2 RIC pre-mRNA.
  • the condition caused by a deficient amount or activity of PC-2 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 PC-2 protein is not a condition caused by alternative or aberrant splicing of any retained intron in a RIC pre-mRNA encoding the PC-2 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 in the pre-mRNA.
  • a subject treated using the methods of the invention expresses a partially functional PC-2 protein from one allele, wherein the partially functional PC-2 protein is caused by a frameshift mutation, a nonsense mutation, a missense mutation, or a partial gene deletion.
  • the partially functional PC-2 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 PC-2 protein from one allele, wherein the nonfunctional PC-2 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 PKD2 whole gene deletion, in one allele.
  • the subject has a PC-2 missense mutation selected from M1K, P24L, R28P, A35D, R60N, S80L, Q107D, R119H, G121A, G135V, A147V, A190T, V262M, W292C, R306Q, L314V, R322W, R322Q, R325P, R325Q, C331S, S332A, Y324C, S349P, A356P, A384P, G390S, W414G, G418V, T419A, R420G, A421S, R440S, T448K, I452V, F482C, Y487H, D511V, V516L, L517R, V519M, A552P, I556V, N578D, N580K, M583I, A615T, F629S, C632R, R
  • the subject has a PC-2 deletion mutation selected from EXl_EX13del, IVS2_3'(ABCG2)del80kb*, IVS2_3'(ABCG2)del98kb, IVS4+1452_IVS5-965del5722, S378del, F605del, IVS9_3'del28kb, 2182_2183delAG, L736_N737del2 or R878del.
  • the subject has PC-2 duplication mutation Ex3dup*.
  • a subject having any PC-2 mutation known in the art and described in the literature, e.g. , by Chang, et al , 2005, Ren Fail 27: 95-100 is treated using the methods and compositions of the present invention.
  • Targeted Augmentation of Nuclear Gene Output is used in the methods of the invention to increase expression of a PC-2 protein.
  • a retained-intron-containing pre-mRNA (RIC pre-mRNA) encoding PC-2 protein is present in the nucleus of a cell.
  • Cells having a PKD2 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 PC-2 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 targeted region is in a retained intron that is the most abundant intron in a RIC pre-mRNA encoding the PC-2 protein.
  • the most retained intron in a RIC pre-mRNA encoding the PC-2 protein is intron 5.
  • 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
  • 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 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°/ Uo about 90°/ 7 o, about 10°/ Uo about 85°/ about 10°/ Uo about 80°/ 7 o, about 10°/ Uo about 75%, about 10°/ Uo about 70°/ about 10°/ Uo about 65°/ about 10°/ Uo about
  • 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).
  • an ASO is complementary to a targeted region that is within a non-retained intron in a RIC pre-mRNA.
  • the targeted portion of the RIC pre-mRNA is within: the region +6 to +100 relative to the 5 ' splice site of the non-retained intron; or the region -16 to -100 relative to the 3' splice site of the non -retained intron.
  • the targeted portion of the RIC pre- mRNA is within the region +100 relative to the 5' splice site of the non -retained intron to -100 relative to the 3' splice site of the non-retained intron.
  • RNA encoding the target protein is thereby produced.
  • the retained intron of the RIC pre-mRNA is an inefficiently spliced intron.
  • "inefficiently spliced” may refer to a relatively low frequency of splicing at a splice site adjacent to the retained intron (5' splice site or 3' splice site) as compared to the frequency of splicing at another splice site in the RIC pre-mRNA.
  • inefficiently spliced may also refer to the relative rate or kinetics of splicing at a splice site, in which an "inefficiently spliced" intron may be spliced or removed at a slower rate as compared to another intron in a RIC pre-mRNA.
  • the 9-nucleotide sequence at -3e to -le of the exon flanking the 5' splice site and +1 to +6 of the retained intron is identical to the corresponding wild -type sequence.
  • the 16 nucleotide sequence at -15 to -1 of the retained intron and +le of the exon flanking the 3' splice site is identical to the corresponding wild -type sequence.
  • wild-type sequence refers to the nucleotide sequence for the PKD2 gene in the published reference genome deposited in the NCBI repository of biological and scientific information.
  • wild-type sequence refers to the canonical sequence for the PKD2 gene found at NCBI Gene ID 5311. Also used herein, a nucleotide position denoted with an “e” indicates the nucleotide is present in the sequence of an exon (e.g., the exon flanking the 5 ' splice site or the exon flanking the 3' splice site).
  • the methods involve contacting cells with an ASO that is complementary to a portion of a pre- mRNA encoding PC-2 protein, resulting in increased expression of PC-2.
  • contacting or administering to cells refers to any method of providing an ASO in immediate proximity with the cells such that the ASO and the cells interact.
  • a cell that is contacted with an ASO will take up or transport the ASO into the cell.
  • the method involves contacting a condition or disease-associated or condition or disease-relevant cell with any of the ASOs described herein.
  • the ASO may be further modified or attached (e.g., covalently attached) to another molecule to target the ASO to a cell type, enhance contact between the ASO and the condition or disease -associated or condition or disease- relevant cell, or enhance uptake of the ASO.
  • the term “increasing protein production” or “increasing expression of a target protein” means enhancing the amount of protein that is translated from an mRNA in a cell.
  • a “target protein” may be any protein for which increased expression/production is desired.
  • contacting a cell that expresses a PKD2 RIC pre-mRNA with an ASO that is complementary to a targeted portion of the PKD2 RIC pre-mRNA transcript results in a measurable increase in the amount of the PC-2 protein (e.g., a target protein) encoded by the pre-mRNA.
  • Methods of measuring or detecting production of a protein will be evident to one of skill in the art and include any known method, for example, Western blotting, flow cytometry, immunofluorescence microscopy, and
  • contacting cells with an ASO that is complementary to a targeted portion of a PKD2 RIC pre-mRNA transcript results in an increase in the amount of PC-2 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 PC-2 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
  • contacting cells with an ASO that is complementary to a targeted portion of a PKD2 RIC pre-mRNA transcript results in an increase in the amount of mRNA encoding PC-2, including the mature mRNA encoding the target protein.
  • the amount of mRNA encoding PC-2 protein, or the mature mRNA encoding the PC-2 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 PC-2 protein, or the mature mRNA encoding PC-2 protein produced in the cell to which the antisense oligomer was contacted is increased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8- fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold
  • the methods and antisense oligonucleotide compositions provided herein are useful for increasing the expression of PC-2 protein in cells, for example, in a subject having Polycystic Kidney Disease caused by a deficiency in the amount or activity of PC-2 protein, by increasing the level of mRNA encoding PC-2 protein, or the mature mRNA encoding PC-2 protein.
  • the methods and compositions as described herein induce the constitutive splicing of a retained intron from aPKD2 RIC pre-mRNA transcript encoding PC-2 protein, thereby increasing the level of mRNA encoding PC-2 protein, or the mature mRNA encoding PC-2 protein and increasing the expression of PC-2 protein.
  • Constitutive splicing of a retained intron from a RIC pre-mRNA correctly removes the retained intron from the RIC pre-mRNA, wherein the retained intron has wild-type splice sequences.
  • Constitutive splicing does not encompass splicing of a retained intron from a RIC pre-mRNA transcribed from a gene or allele having a mutation that causes alternative splicing or aberrant splicing of a pre-mRNA transcribed from the gene or allele.
  • constitutive splicing of a retained intron does not correct aberrant splicing in or influence alternative splicing of a pre-mRNA to result in an increased expression of a target protein or functional RNA.
  • constitutive splicing correctly removes a retained intron from a PKD2 RIC pre-mRNA, wherein the PKD2 RIC pre-mRNA is transcribed from a wild-type gene or allele, or a polymorphic gene or allele, that encodes a fully-functional target protein or functional RNA, and wherein the gene or allele does not have a mutation that causes alternative splicing or aberrant splicing of the retained intron.
  • constitutive splicing of a retained intron from a PKD2 RIC pre-mRNA encoding PC-2 protein correctly removes a retained intron from a PKD2 RIC pre-mRNA encoding PC-2 protein, wherein the PKD2 RIC pre-mRNA is transcribed from a gene or allele from which the target gene or functional RNA is produced at a reduced level compared to production from a wild-type allele, and wherein the gene or allele does not have a mutation that causes alternative splicing or aberrant splicing of the retained intron.
  • the correct removal of the constitutively spliced retained intron results in production of target protein or functional RNA that is functional when compared to an equivalent wild-type protein or functional RNA.
  • constitutive splicing correctly removes a retained intron from a PKD2 RIC pre-mRNA, wherein the PKD2 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.
  • "Correct removal" of the retained intron by constitutive splicing refers to removal of the entire intron, without removal of any part of an exon.
  • an antisense oligomer as described herein or used in any method described herein does not increase the amount of mRNA encoding PC-2 protein or the amount of PC-2 protein by modulating alternative splicing or aberrant splicing of a pre -mRNA transcribed from the PKD2 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 PC-2 protein in the methods of the invention.
  • the methods described herein is a method wherein the PKD2 RIC pre-mRNA was produced by partial splicing of a wild-type PKD2 pre-mRNA.
  • the method is a method wherein the PKD2 RIC pre-mRNA was produced by partial splicing of a full-length wild-type PKD2 pre-mRNA.
  • the PKD2 RIC pre-mRNA was produced by partial splicing of a full- length PKD2 pre-mRNA.
  • a full-length PKD2 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 PC-2 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 PC-2 protein encoded by the wild-type mature mRNA.
  • composition comprising antisense oligomers that enhances splicing by binding to a targeted portion of a PKD2 RIC pre-mRNA.
  • ASO antisense oligomer
  • antisense oligomer are used interchangeably and refer to an oligomer such as a
  • polynucleotide comprising nucleobases that hybridize to a target nucleic acid (e.g., a PKD2 RIC pre- mRNA) sequence by Watson -Crick base pairing or wobble base pairing (G-U).
  • the ASO may have exact sequence complementary to the target sequence or near complementarity (e.g., sufficient complementarity to bind the target sequence and enhancing splicing at a splice site).
  • ASOs are designed so that they bind (hybridize) to a target nucleic acid (e.g., a targeted portion of a pre-mRNA transcript) and remain hybridized under physiological conditions.
  • 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 hybridization event (e.g., a loop structure or hairpin structure may be formed). In certain embodiments, an ASO hybridizes to noncontiguous nucleobases in a target pre-mRNA transcript. For example, an ASO can hybridize to nucleobases in a pre-mRNA transcript that are separated by one or more nucleobase(s) to which the ASO does not hybridize.
  • the ASOs described herein comprise nucleobases that are complementary to nucleobases present in a target portion of a 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, 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, phosphoroanilothioate, phosphoraniladate, phosphoramidate, and the like. See e.g., LaPlanche, et al , Nucleic Acids Res.
  • the backbone structure of the ASO does not contain phosphorous but rather contains peptide bonds, for example in a peptide nucleic acid (PNA), or linking groups including carbamate, amides, and linear and cyclic hydrocarbon groups.
  • PNA peptide nucleic acid
  • the backbone modification is a phosphorothioate linkage. In some embodiments, the backbone modification is a phosphoramidate linkage.
  • the stereochemistry at each of the phosphorus internucleotide linkages of the ASO backbone is random. In embodiments, the stereochemistry at each of the phosphorus internucleotide 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 disclosure comprises an ASO having phosphorus internucleotide linkages that are not random.
  • a composition used in the methods of the invention comprises a pure diastereomeric ASO.
  • a composition used in the methods of the invention comprises an ASO that has diastereomeric purity of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100% , or about 99% to about 100%.
  • the ASO has a nonrandom mixture of Rp and Sp configurations at its phosphorus internucleotide linkages.
  • Rp and Sp are required in antisense oligonucleotides to achieve a balance between good activity and nuclease stability.
  • an ASO used in the methods of the invention comprises about 5- 100% Rp, at least about 5% Rp, at least about 10% Rp, at least about 15% Rp, at least about 20% Rp, at least about 25% Rp, at least about 30% Rp, at least about 35% Rp, at least about 40% Rp, at least about 45% Rp, at least about 50% Rp, at least about 55% Rp, at least about 60% Rp, at least about 65% Rp, at least about 70% Rp, at least about 75% Rp, at least about 80% Rp, at least about 85% Rp, at least about 90% Rp, or at least about 95% Rp, with the remainder Sp, or about 100% Rp.
  • an ASO used in the methods of the invention comprises about 10% to about 100% Rp, about 15% to about 100% Rp, about 20% to about 100% Rp, about 25% to about 100% Rp, about 30% to about 100% Rp, about 35% to about 100% Rp, about 40% to about 100% Rp, about 45% to about 100% Rp, about 50% to about 100% Rp, about 55% to about 100% Rp, about 60% to about 100% Rp, about 65% to about 100% Rp, about 70% to about 100% Rp, about 75% to about 100% Rp, about 80% to about 100% Rp, about 85% to about 100% Rp, about 90% to about 100% Rp, or about 95% to about 100% Rp, about 20% to about 80% Rp, about 25% to about 75% Rp, about 30% to about 70% Rp, about 40% to about 60% Rp, or about 45% to about 55% Rp, with the remainder Sp.
  • an ASO used in the methods of the invention comprises about 5-100% Sp, at least about 5% Sp, at least about 10% Sp, at least about 15% Sp, at least about 20% Sp, at least about 25% Sp, at least about 30% Sp, at least about 35% Sp, at least about 40% Sp, at least about 45% Sp, at least about 50% Sp, at least about 55% Sp, at least about 60% Sp, at least about 65% Sp, at least about 70% Sp, at least about 75% Sp, at least about 80% Sp, at least about 85% Sp, at least about 90% Sp, or at least about 95% Sp, with the remainder Rp, or about 100% Sp.
  • an ASO used in the methods of the invention comprises about 10% to about 100% Sp, about 15% to about 100% Sp, about 20% to about 100% Sp, about 25% to about 100% Sp, about 30% to about 100% Sp, about 35% to about 100% Sp, about 40% to about 100% Sp, about 45% to about 100% Sp, about 50% to about 100% Sp, about 55% to about 100% Sp, about 60% to about 100%
  • Sp about 65% to about 100% Sp, about 70% to about 100% Sp, about 75% to about 100% Sp, about 80% to about 100% Sp, about 85% to about 100% Sp, about 90% to about 100% Sp, or about 95% to about 100% Sp, about 20% to about 80% Sp, about 25% to about 75% Sp, about 30% to about 70% Sp, about 40% to about 60% Sp, or about 45% to about 55% Sp, with the remainder Rp.
  • Any of the ASOs described herein may contain a sugar moiety that comprises ribose or deoxyribose, as present in naturally occurring nucleotides, or a modified sugar moiety or sugar analog, including a morpholine ring.
  • Non-limiting examples of modified sugar moieties include 2' substitutions such as 2'-0-methyl (2'-0-Me), 2'-0-methoxyethyl (2'MOE), 2'-0-aminoethyl, 2'F; N3'->P5 ' phosphoramidate, 2'dimethylaminooxyethoxy, 2'dimethylaminoethoxyethoxy, 2'-guanidinidium, 2'-0- guanidinium ethyl, carbamate modified sugars, and bicyclic modified sugars.
  • 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).
  • LNA locked nucleic acid
  • the sugar analog contains a morpholine ring, such as phosphorodiamidate morpholino (PMO).
  • the sugar moiety comprises a ribofuransyl or 2'deoxyribofuransyl modification.
  • the sugar moiety comprises 2 '4 '-constrained 2'0-methyloxyethyl (cMOE) modifications.
  • the sugar moiety comprises cEt 2', 4' constrained 2'-0 ethyl BNA modifications.
  • the sugar moiety comprises tricycloDNA (tcDNA) modifications.
  • the sugar moiety comprises ethylene nucleic acid (ENA) modifications.
  • the sugar moiety comprises MCE modifications.
  • each monomer of the ASO is modified in the same way, for example each linkage of the backbone of the ASO comprises a phosphorothioate linkage or each ribose sugar moiety comprises a 2'O-methyl modification.
  • Such modifications that are present on each of the monomer components of an ASO are referred to as "uniform modifications.”
  • a combination of different modifications may be desired, for example, an ASO may comprise a combination of phosphorodiamidate linkages and sugar moieties comprising morpholine rings (morpholinos).
  • 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 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.
  • sequences is the 5' end and the left-hand direction of single or double-stranded nucleic acid sequences is referred to as the 5' direction.
  • the right-hand end or direction of a nucleic acid sequence is the 3' end or direction.
  • nucleotides that are upstream of a reference point in a nucleic acid may be designated by a negative number, while nucleotides that are downstream of a reference point may be designated by a positive number.
  • a reference point e.g., an exon-exon junction in mRNA
  • a nucleotide that is directly adjacent and upstream of the reference point is designated “minus one,” e.g., 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 PKD2 RIC pre-mRNA that is downstream (in the 3 ' direction) of the 5 ' splice site of the retained intron in a PKD2 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 PKD2 RIC pre- mRNA that is within the region of about +6 to about +500 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 PKD2 RIC pre-mRNA that is within the region between nucleotides +6 and +497 relative to the 5' splice site of the retained intron.
  • the ASOs are complementary to a targeted portion that is within the region about +6 to about +500, about +6 to about +490, about +6 to about +480, about +6 to about +470, about +6 to about +460, about +6 to about +450, about +6 to about +440, about +6 to about +430, about +6 to about +420, about +6 to about +410, about +6 to about +400, about +6 to about +390, about +6 to about +380, about +6 to about +370, about +6 to about +360, about +6 to about +350, about +6 to about +340, about +6 to about +330, about +6 to about +320, about +6 to about +310, about +6 to about +300, about +6 to about +290, about +6 to about +280, about +6 to about +270, about +6 to about +260, about +6 to about +250, about +6 to about +240, about +6 to about +230, about +6 to about +220,
  • the ASOs are complementary to (and bind to) a targeted portion of a PKD2 RIC pre-mRNA that is upstream (in the 5' direction) of the 5' splice site of the retained intron in a PKD2 RIC pre-mRNA (e.g., the direction designated by negative numbers relative to the 5' splice site) (FIG. 1).
  • the ASOs are complementary to a targeted portion of the PKD2 RIC pre- mRNA that is within the region of about -4e to about -210e relative to the 5' splice site of the retained intron.
  • the ASO is not complementary to nucleotides -le to -3e relative to the 5 ' splice site (the first three nucleotides located upstream of the 5 ' splice site).
  • the ASOs may be complementary to a targeted portion of a PKD2 RIC pre-mRNA that is within the region between nucleotides -4e and about -204e relative to the 5 ' splice site of the retained intron.
  • the ASOs are complementary to a targeted portion that is within the region about -4e to about - 210e, about -4e to about -200e, about -4e to about -190e, about -4e to about -180e, about -4e to about - 170e, about -4e to about -160e, about -4e to about -150e, about -4e to about -140e, about -4e to about - 130e, about -4e to about -120e, about -4e to about -1 lOe, about -4e to about -lOOe, about -4e to about - 90e, about -4e to about -80e, about -4e to about -70e, about -4e to about -60e, about -4e to about -50e, about -4e to about -40e, about -4e to about -30e, or about -4e to about -20e relative to 5' splice site of the retained intron.
  • the ASOs are complementary to a targeted region of a PKD2 RIC pre- mRNA that is upstream (in the 5' direction) of the 3' splice site of the retained intron in a PKD2 RIC pre- mRNA (e.g., in the direction designated by negative numbers) (FIG. 1).
  • the ASOs are complementary to a targeted portion of the PKD2 RIC pre-mRNA that is within the region of about - 16 to about -500 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). In some embodiments, the ASOs are complementary to a targeted portion of the PKD2 RIC pre-mRNA that is within the region -16 to -496 relative to the 3' splice site of the retained intron.
  • the ASOs are complementary to a targeted portion that is within the region about -16 to about -500, about -16 to about -490, about -16 to about -480, about -16 to about -470, about -16 to about -460, about -16 to about -450, about -16 to about -440, about -16 to about -430, about - 16 to about -420, about -16 to about -410, about -16 to about -400, about -16 to about -390, about -16 to about -380, about -16 to about -370, about -16 to about -360, about -16 to about -350, about -16 to about - 340, about -16 to about -330, about -16 to about -320, about -16 to about -310, about -16 to about -300, about -16 to about -290, about -16 to about -280, about -16 to about -270, about -16 to about -260, about - 16 to about -
  • the ASOs are complementary to a targeted region of a PKD2 RIC pre- mR A that is downstream (in the 3 ' direction) of the 3 ' splice site of the retained intron in a PKD2 RIC pre-mRNA (e.g., in the direction designated by positive numbers) (FIG. 1).
  • the ASOs are complementary to a targeted portion of the PKD2 RIC pre-mRNA that is within the region of about +2e to about +220e relative to the 3 ' splice site of the retained intron.
  • the ASO is not complementary to nucleotides +l e relative to the 3 ' splice site (the first nucleotide located downstream of the 3 ' splice site).
  • the ASOs may be complementary to a targeted portion ⁇ ⁇ ⁇ 2 RIC pre-mRNA that is within the region between nucleotides +2e and +212e relative to the 3 ' splice site of the retained intron.
  • the ASOs are complementary to a targeted portion that is within the region about +2e to about +220e, about +2e to about +210e, about +2e to about +200e, about +2e to about +190e, about +2e to about +180e, about +2e to about +170e, about +2e to about +160e, about +2e to about +150e, about +2e to about +140e, about +2e to about +130e, about +2e to about +120e, about +2e to about +1 lOe, about +2e to about +100e, about +2e to about +90e, about +2e to about +80e, about +2e to about +70e, about +2e to about +60e, about +2e to about +50e, about +2e to about +40e, about +2e to about +30e, about +2e to about +20e, or about +2e to about +10e relative to 3 ' splice site
  • the targeted portion of the PKD2 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 may be of any length suitable for specific binding and effective enhancement of splicing.
  • the ASOs consist of 8 to 50 nucleobases.
  • the ASO may be 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, or 50 nucleobases in length.
  • the ASOs consist of more than 50 nucleobases.
  • the ASO is from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 1 1 to 50 nucleobases, 1 1 to 40 nucleobases, 1 1 to 35 nucleobases, 1 1 to 30 nucleobases, 1 1 to 25 nucleobases, 1 1 to 20 nucleobases,
  • 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 a PKD2 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, methanesulfonate, 2 -naphthalene sulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, peroxine sodium
  • 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.
  • a sterically stabilized liposome comprises one or more glyco lipids 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 use of surfactants in drug products, formulations and emulsions is well known in the art.
  • 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 enhancer 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.
  • agents capable of promoting penetration of the subject antisense oligonucleotide across the blood-brain barrier by any method known in the art.
  • delivery of agents by administration of an adenovirus vector to motor neurons in muscle tissue is described in U.S. Pat. No. 6,632,427, "Adenoviral-vector-mediated gene transfer into medullary motor neurons," incorporated herein by reference.
  • 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.
  • Such moieties include, but are not limited to, a lipid moiety, e.g., as a cholesterol moiety, a cholesteryl moiety, an aliphatic chain, e.g., dodecandiol or undecyl residues, a polyamine or a polyethylene glycol chain, or adamantane acetic acid.
  • 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 e.g., 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.
  • compositions provided herein may be administered to an individual.
  • “Individual” may be used interchangeably with “subject” or "patient.”
  • An individual may be a mammal, for example a human or animal such as a non-human primate, a rodent, a rabbit, a rat, a mouse, a horse, a donkey, a goat, a cat, a dog, a cow, a pig, or a sheep.
  • the individual is a human.
  • the individual is a fetus, an embryo, or a child.
  • the individual may be another eukaryotic organism, such as a plant.
  • the compositions provided herein are administered to a cell ex vivo.
  • the compositions provided herein are administered to an individual as a method of treating a disease or disorder.
  • the individual has a genetic disease, such as any of the diseases described herein.
  • the individual is at risk of having the disease, such as any of the diseases described herein.
  • the individual is at increased risk of having a disease or disorder caused by insufficient amount of a protein or insufficient activity of a protein. If an individual is "at an increased risk" of having a disease or disorder caused insufficient amount of a protein or insufficient activity of a protein, the method involves preventative or prophylactic treatment.
  • an individual may be at an increased risk of having such a disease or disorder because of family history of the disease.
  • individuals at an increased risk of having such a disease or disorder benefit from prophylactic treatment (e.g. , by preventing or delaying the onset or progression of the disease or disorder).
  • Suitable routes for administration of ASOs of the present invention may vary depending on cell type to which delivery of the ASOs is desired. Multiple tissues and organs are affected by Polycystic Kidney Disease, 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.
  • Subjects are evaluated for response to treatment using any appropriate markers.
  • subjects with kidney disease are evaluated for response to treatment by measuring specific markers for kidney disease, including creatinine, creatinine clearance, blood pressure, 24-hour urine volume, 24-hour urine protein, vWAg and platelet aggregation by arachidonic acid.
  • specific markers for kidney disease including creatinine, creatinine clearance, blood pressure, 24-hour urine volume, 24-hour urine protein, vWAg and platelet aggregation by arachidonic acid.
  • 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
  • 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.
  • the ASOs can be tiled from 100 nucleotides downstream of the 5' splice site, to 100 nucleotides upstream of the 3' splice site.
  • the ASOs can be tiled from about 210 nucleotides upstream of the 5' splice site, to about 500 nucleotides downstream of the 5' splice site.
  • the ASOs can be tiled from about 500 nucleotides upstream of the 3 ' splice site, to about 220 nucleotides downstream 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 PKD2 transcripts by RNAseq using next generation sequencing
  • FIG. 3 shows the mapped reads visualized using the UCSC genome browser (operated by the UCSC Genome
  • An ASO walk was designed to target intron 5 using the method described herein (FIG. 4; Table 1, SEQ ID NOS: 3 to 280).
  • a region immediately upstream and downstream of the 5' splice site of intron 5, spanning nucleotides +497 to -204e, and a region immediately upstream and downstream of the 3 ' splice site of intron 5, spanning nucleotides -496to +212e were utilized to design ASOs to target retained intron 5 PKD2 RIC pre-mRNAs.
  • Table 1 lists exemplary ASOs that were designed and their target sequences.
  • Example 3 Improved splicing efficiency via ASO-targeting of PKD2 intron 5 increases transcript levels
  • PKD2 intron 5 To determine whether an increase in PKD2 expression could be achieved by improving splicing efficiency of PKD2 intron 5 using ASOs, the method described herein can be used.
  • Cell lines of interest e.g., 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)
  • ATCC American Type Culture Collection
  • Huh-7 Huh-7
  • a human hepatoma cell line NIBIOHN, Japan
  • SK-N-AS a human neuroblastoma cell line
  • ATCC human neuroblastoma cell line
  • ASOs are plated in 96-well tissue culture plates and combined with RNAiMax diluted in Opti-MEM. Cells are detached using trypsin, resuspended in full medium, and approximately 25,000 cells are added to the ASO-transfection mixture. Transfection experiments are carried out in triplicate plate replicates. Final ASO concentration is 80 nM. Media is changed 6h post- transfection, and cells are harvested at 24h, using the Cells-to-Ct lysis reagent, supplemented with DNAse (Thermo Fisher), according to manufacturer's specifications. cDNA is generated with Cells-to-Ct RT reagents (Thermo Fisher) according to manufacturer's specifications.
  • Taqman assays with probes spanning the corresponding exon-exon junction (Thermo Fisher), listed in Table 1.
  • Taqman assays are carried out according to manufacturer's specifications, on a QuantStudio 7 Flex Real-Time PCR system (Thermo Fisher).
  • Target gene assay values are normalized to RPL32 (deltaCt) and plate-matched mock transfected samples (delta-delta Ct), generating fold -change over mock quantitation (2 A -(delta-deltaCt). Average fold-change over mock of the three plate replicates is plotted.
  • ASOs identified as increasing the target gene expression by a threshold amount imply an increase in splicing at that target intron. Together with whole transcriptome data confirming retention of the target intron (FIG. 3), these results confirm that ASOs can improve the splicing efficiency of a rate limiting intron.

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