CA3005247A1 - Antisense oligomers for treatment of polycystic kidney disease - Google Patents

Abstract

Provided herein are methods and compositions for increasing the expression of PC-2, and for treating a subject in need thereof, e.g., a subject with deficient PC-2 protein expression or a subject having Polycystic Kidney Disease (PKD).

Description

ANTISENSE OLIGOMERS FOR TREATMENT OF POLYCYSTIC KIDNEY DISEASE
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional Application No.
62/267,252, filed December 14, 2015, which application is incorporated herein by reference.
REFERENCE TO A SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on December 9, 2016, is named 47991 707 601 SL.txt and is 253,332 bytes in size.
BACKGROUND OF THE INVENTION

[0003] Autosomal dominant polycystic kidney disease (ADPKD), is one of the most common inherited renal cystic diseases, conditions characterized by the development of renal cysts and a variety of extrarenal manifestations (Tones and Harris, 2009, Kidney International (2009) 76, 149-168). Patients suffering from ADPKD generally develop end-stage renal disease (ESRD) by age 70, which ultimately requires interventions such as renal dialysis. The prevalence of ADPKD at birth is estimated to be between 1:400 and 1:1,000, affecting about 600,000 people in the US.

[0004] Mutations in either the 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 (Tones and Harris, 2009).
SUMMARY OF THE INVENTION

[0005] Disclosed herein, in some embodiments, are methods of treating Polycystic Kidney Disease in a subject in need thereof, by increasing the expression of a target protein or functional RNA by cells of the subject, wherein the cells have a retained-intron-containing pre-mRNA (RIC 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 RNA in the cells of the subject.

[0006] Also disclosed herein, in some embodiments, are methods of increasing expression of a target protein, wherein the target protein is PC-2, by cells having a retained-intron-containing pre-mRNA (RIC
pre-mRNA), the RIC pre-mRNA comprising a retained intron, an exon flanking the 5' splice site of the retained intron, an exon flanking the 3' splice site of the retained intron, and wherein the RIC pre-mRNA
encodes 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.

[0007] In some embodiments of any of the aforementioned methods, the target protein is PC-2. In some embodiments, 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. In some embodiments, the cells are in or from a subject having a condition caused by a deficient amount or activity of PC-2 protein.

[0008] In some embodiments of any of the aforementioned methods, the deficient amount of the target protein is caused by haploinsufficiency of the target protein, wherein the subject has a first allele encoding a functional target protein, and a second allele from which the target protein is not produced, or a second allele encoding a nonfunctional target protein, and wherein the antisense oligomer binds to a targeted portion of a RIC pre-mRNA transcribed from the first allele. In some embodiments, 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 first mutant allele is (a)(i) or (a)(ii), and wherein the RIC pre-mRNA is transcribed from either the first mutant allele that is (a)(i) or (a)(ii), and/or the second allele that is (b)(i) or (b)(ii). In some embodiments, the target protein is produced in a form having reduced function compared to the equivalent wild-type protein. In some embodiments, the target protein is produced in a form that is fully-functional compared to the equivalent wild-type protein.

[0009] In some embodiments of any of the aforementioned methods, 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. In some embodiments, 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. In some embodiments, the targeted portion of the RIC pre-mRNA is in the retained intron within: (a) the region -4e to -1,054e 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. In some embodiments, the target protein is PC-2.

[0010] In some embodiments of any of the aforementioned methods, the RIC pre-mRNA comprises a sequence with at least about 80%, 85%, 90%, 950, 97%, or 10000 sequence identity to SEQ ID NO: 2. In some embodiments, the RIC pre-mRNA is encoded by a genetic sequence with at least about 80%, 85%, 90%, 950, 97%, or 1000o sequence identity to SEQ ID NO: 1. In some embodiments, the targeted portion of the RIC pre-mRNA comprises a sequence with at least 80%, 85%, 90%, 950, 97%, or 1000o sequence identity to a region comprising at least 8 contiguous nucleic acids of SEQ ID NO: 281. In some embodiments, the ASO comprises a sequence that is at least about 80%, 85%, 90%, 950, 97%, or 1000o complimentary to any one of SEQ ID NOs: 3-280. In some embodiments, 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. In some embodiments, the ASO comprises a sequence that is at least about 80%, 85%, 90%, 950, 97%, or 1000o complimentary to any one of SEQ ID NOs: 3-280. In some embodiments, 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.
In some embodiments, the ASO comprises a sequence that is at least about 80%, 85%, 90%, 950, 97%, or 1000o complimentary to any one of SEQ ID NOs: 3-43. In some embodiments, 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. In some embodiments, the ASO comprises a sequence that is at least about 80%, 85%, 90%, 950, 97%, or 1000o complimentary to any one of SEQ ID NOs:44-140. In some embodiments, 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. In some embodiments, the ASO comprises a sequence that is at least about 80%, 85%, 90%, 950, 97%, or 1000o complimentary to any one of SEQ ID
NOs: 141-237. In some embodiments, 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. In some embodiments, the ASO comprises a sequence that is at least about 80%, 85%, 90%, 950, 97%, or 1000o complimentary to any one of SEQ
ID NOs: 238-280.
100111 In some embodiments of any of the aforementioned methods, 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. In some embodiments, the mRNA
encoding the target protein or functional RNA is a full-length mature mRNA, or a wild-type mature mRNA. In some embodiments, the target protein produced is full-length protein, or wild-type protein. In some embodiments, 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

11 PCT/US2016/066417 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-fold, or at least about 10-fold, compared to the total amount of the mRNA encoding the target protein or functional RNA
produced in a control cell. In some embodiments, 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-fold, or at least about 10-fold, compared to the total amount of target protein produced by a control cell.
100121 In some embodiments of any of the aforementioned methods, the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage. In some embodiments, the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-0-methyl, a 2'-Fluoro, or a 2'-0-methoxyethyl moiety. In some embodiments, the antisense oligomer comprises at least one modified sugar moiety. In some embodiments, each sugar moiety is a modified sugar moiety. In some embodiments, the antisense oligomer consists of from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases. In some embodiments, the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, complementary to the targeted portion of the RIC pre-mRNA encoding the protein.
[0013] In some embodiments of any of the aforementioned methods, 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.
In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the method further comprises assessing PC-2 protein expression.
[0014] In some embodiments of any of the aforementioned methods, the antisense oligomer binds to a targeted portion of a PKD2 RIC pre-mRNA, wherein the targeted portion is in a sequence selected from SEQ ID NOs: 3-280. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. In some embodiments, the subject is a fetus, an embryo, or a child. In some embodiments, the cells are ex vivo. In some embodiments, the antisense oligomer is administered by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection of the subject. In some embodiments, the 9 nucleotides at -3e to -le of the exon flanking the 5' splice site and +1 to +6 of the retained intron, are identical to the corresponding wild-type sequence. In some embodiments, 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. Disclosed herein, in some embodiments, are antisense oligomers as described in any of the aforementioned methods.
[0015] Disclosed herein, in some embodiments, are 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.
[0016] Also disclosed herein, in some embodiments, are pharmaceutical compositions comprising any of the aforementioned antisense oligomers and an excipient.
[0017] Disclosed herein, in some embodiments, are methods of treating a subject in need thereof, by administering any of the aforementioned pharmaceutical compositions by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.
[0018] Disclosed herein, in some embodiments, are 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 Polycystic Kidney Disease in a subject in need thereof, associated with a deficient protein or deficient functional RNA, wherein the deficient protein or deficient functional RNA is deficient in amount or activity in the subject, wherein the antisense oligomer enhances constitutive splicing of a retained intron-containing pre-mRNA (RIC pre-mRNA) encoding the target protein or the functional RNA, wherein the target protein is: (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 encoding the target protein or the functional RNA, thereby increasing production or activity of the target protein or the functional RNA in the subject.
100191 Disclosed herein, in some embodiments, are 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 constitutively 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. In some embodiments, the condition is a disease or disorder. In some embodiments, the disease or disorder is Polycystic Kidney Disease. In some embodiments, the target protein and RIC pre-mRNA are encoded by the PKD2 gene.
[0020] In some embodiments of any of the aforementioned compositions, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the targeted portion of the RIC pre-mRNA is within: (a) the region -4e to -1,054e 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. In some embodiments, the target protein is PC-2.
In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.
[0021] In some embodiments of any of the aforementioned compositions, 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. In some embodiments, the mRNA encoding the target protein or functional RNA is a full-length mature mRNA, or a wild-type mature mRNA. In some embodiments, the target protein produced is full-length protein, or wild-type protein. In some embodiments, the retained intron is a rate-limiting intron. In some embodiments, the retained intron is the most abundant retained intron in said RIC pre-mRNA. In some embodiments, the retained intron is the second most abundant retained intron in said RIC pre-mRNA.
[0022] In some embodiments of any of the aforementioned compositions, the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage. In some embodiments, the antisense oligomer is an antisense oligonucleotide. In some embodiments, the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-0-methyl, a 2'-Fluoro, or a 2'-0-methoxyethyl moiety. In some embodiments, the antisense oligomer comprises at least one modified sugar moiety. In some embodiments, each sugar moiety is a modified sugar moiety. In some embodiments, the antisense oligomer consists of from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.
100231 Disclosed herein, in some embodiments, are pharmaceutical compositions comprising any of the aforementioned antisense oligomers and an excipient. Disclosed herein, in some embodiments, are methods of treating a subject in need thereof, by administering the aforementioned pharmaceutical compositions by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.
100241 Disclosed herein, in some embodiments, are pharmaceutical 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. In some embodiments, the deficient PKD2 mRNA transcript is a PKD2 RIC pre-mRNA transcript. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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, to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases. In some embodiments, the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or is 100%
complementary to a targeted portion of the PKD2 RIC pre-mRNA transcript. In some embodiments, the targeted portion of the PKD2 RIC pre-mRNA transcript is within SEQ ID NO: 281.
In some embodiments, 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. In some embodiments, the antisense oligomer comprises a nucleotide sequence selected from SEQ
ID NOs: 3-280. In some embodiments, the pharmaceutical composition is formulated for intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.
[0025] Disclosed herein, in some embodiments, are methods of inducing processing of a deficient PKD2 mRNA transcript to facilitate removal of a retained intron to produce a fully processed PKD2 mRNA
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. In some embodiments, the retained intron is an entire retained intron. In some embodiments, the deficient PKD2 mRNA
transcript is a PKD2 pre-mRNA
transcript.
[0026] Disclosed herein, in some embodiments, are methods of treating a subject having a condition caused by a deficient amount or activity of PC-2 protein comprising administering to the subject an antisense oligomer comprising a nucleotide sequence with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID
NOs: 3-280.
INCORPORATION BY REFERENCE
[0027] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] The novel features of the invention are set forth with particularity in the appended claims. A
better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings.
[0029] FIG. 1 depicts an exemplary schematic representation of a retained-intron-containing (RIC) pre-mRNA transcript. In FIG.1, 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). In embodiments, 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.
[0030] 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 (connecting lines) undergoes splicing to generate an mRNA, and this mRNA is exported to the cytoplasm and translated into target protein. For this target gene, the splicing of intron 1 is inefficient and a retained intron-containing (RIC) pre-mRNA accumulates primarily in the nucleus, and if exported to the cytoplasm, is degraded, leading to no target protein production. FIG. 2B shows an example of the same cell divided into nuclear and cytoplasmic compartments. Treatment with an antisense oligomer (ASO) promotes the splicing of intron 1 and results in an increase in mRNA, which is in turn translated into higher levels of target protein.
[0031] 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). At the bottom of this panel, 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. Higher read density is detected for intron 5 (pointed by the arrow) 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 are retained in the nucleus and are not exported out to the cytoplasm.
The read density for intron 5 in renal epithelial cells is shown in detail in the lower panel.
[0032] FIG. 4 depicts an exemplary PKD2 gene intron 5 (IVS 5) ASO walk. A
graphic representation of the ASO walk performed for PKD2 IVS 5 targeting sequences immediately downstream of the 5' splice site or upstream of the 3' splice site using 2'-0-Me AS0s, PS backbone, is shown. ASOs were designed to cover these regions by shifting 5 nucleotides at a time. The PKD2 exon-intron structure is drawn to scale.
[0033] 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.
DETAILED DESCRIPTION OF THE INVENTION
[0034] Individual introns in primary transcripts of protein-coding genes having more than one intron are spliced from the primary transcript with different efficiencies. In most cases only the fully spliced mRNA

is exported through nuclear pores for subsequent translation in the cytoplasm.
Unspliced and partially spliced transcripts are detectable in the nucleus. It is generally thought that nuclear accumulation of transcripts that are not fully spliced is a mechanism to prevent the accumulation of potentially deleterious mRNAs in the cytoplasm that may be translated to protein. For some genes, splicing of the least efficient intron is a rate-limiting post-transcriptional step in gene expression, prior to translation in the cytoplasm.
[0035] Substantial levels of partially-spliced transcripts of the 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. These 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. These compositions and methods utilize antisense oligomers (AS0s) that promote constitutive splicing at an intron splice site of a retained-intron-containing PKD2 pre-mRNA that accumulates in the nucleus. Thus, in embodiments, PC-2 protein is increased using the methods of the invention to treat a disease caused by PC-2 deficiency.
[0036] In other embodiments, the methods of the invention are used to increase PC-2 production to treat a condition in a subject in need thereof. In embodiments, 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. In embodiments, the condition is a caused by a PC-2 haploinsufficiency.
Polycystic Kidney Disease [0037] Polycystic Kidney Disease (PKD) is an autosomal dominant multisystem disorder characterized by the evolution of renal cysts and a variety of extrarenal manifestations (Tones and Harris, 2009). The main clinical and pathological findings are renal disease due to the development and enlargement of renal cysts, which results in renal manifestations such an increase in kidney volume that correlates directly with the increase in the cyst volume. Other 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. While 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 (Tones and Harris, 2009).
[0038] Mutations in either the PKD1 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 (Tones 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:
Q13563-1, 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.
The PKD2 mRNA
sequence is set forth at NCBI Reference Sequence: NM 000297.3.2, described by Yang Y, etal., 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.
[0039] 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 (Tones and Harris, 2009). Mutations such as nonsense and insertions/deletions are associated with the classic ADPKD2 phenotype display functional haploinsufficiency. A PKD missense mutation that results in expression of the PC-2-D5 11V protein was predicted to be indistinguishable from wild-type PC-2 in terms of stability (Reynolds, etal., 1999, J. Am. Soc. Nephrol. 10: 2342-2351). The PC-2-D5 11V
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.
Retained Intron Containing Pre-mRNA (RIC Pre-mRNA) [0040] In embodiments, 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).
PKD2 Nuclear Transcripts [0041] As described herein in the Examples, the PKD2 gene was analyzed for intron-retention events and retention of intron 5 was observed. RNA sequencing (RNAseq), 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).

12 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, etal., 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.
[0042] In some embodiments, 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. In some embodiments, 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.
[0043] In some embodiments, 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 of a 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.
[0044] In some embodiments, 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.
[0045] In some embodiments, the ASO targets an intron 5 sequence upstream (or 5') from the 3' splice site of a 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 of a 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.
[0046] In some embodiments, 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

13 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.
[0047] In embodiments, 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.
In embodiments, hybridization of an ASO to the targeted portion of the RIC pre-mRNA results in enhanced splicing at the splice site (5' splice site or 3' splice site) of retained intron 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. One of skill in the art can determine the corresponding intron number in any PKD2 isoform based on an intron sequence provided herein or using the intron number provided in reference to the mRNA sequence at NM 000297.3. One of skill in the art also can determine the sequences of flanking exons in any PKD2 isoform for targeting using the methods of the invention, based on an intron sequence provided herein or using the intron number provided in reference to the mRNA
sequence at NM 000297.3. In embodiments, the compositions and methods of the present invention are used to increase the expression of any known PKD2 isoform, e.g., as described in the 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.
PC-2 Protein Expression [0048] As described above, PC-2 mutations in ADPKD are spread across the entire protein, with 95 PC-2 truncating mutations having been reported in the ADPKD Mutation Database (PKD
Foundation).
[0049] In embodiments, the methods described herein are used to increase the production of a functional PC-2 protein. As used herein, 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. In embodiments, the methods are used to increase the production of a partially functional PC-2 protein. As used herein, 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. In some embodiments, 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.
[0050] In embodiments, 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. In such an embodiment, 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. In another such embodiment, the subject has a first allele encoding a functional PC-2 protein, and a second allele encoding a nonfunctional PC-2 protein. In another such embodiment, the subject has a first allele encoding a functional PC-2 protein, and a second allele encoding a partially

14 functional PC-2 protein. In any of these embodiments, 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-2 protein in the cells of the subject.
[0051] In embodiments, 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.
[0052] In related embodiments, the method is a method of using an ASO to increase the expression of a protein or functional RNA. In embodiments, 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.
[0053] In embodiments, the RIC pre-mRNA transcript that encodes the protein that is causative of the disease or condition is targeted by the ASOs described herein. In some embodiments, a RIC pre-mRNA
transcript that encodes a protein that is not causative of the disease is targeted by the ASOs. For example, 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. In some embodiments, the function of the second protein is able to compensate for the mutation or deficiency of the first protein.
[0054] In embodiments, the subject has:
(a) a first mutant allele from which (i) the PC-2 protein is produced at a reduced level compared to production from a wild-type allele, (ii) the PC-2 protein is produced in a form having reduced function compared to an equivalent wild-type protein, or (iii) the PC-2 protein or functional RNA is not produced; and (b) a second mutant allele from which (i) the PC-2 protein is produced at a reduced level compared to production from a wild-type allele, (ii) the PC-2 protein is produced in a form having reduced function compared to an equivalent wild-type protein, or (iii) the PC-2 protein is not produced, and wherein the RIC pre-mRNA is transcribed from the first allele and/or the second allele. In these embodiments, 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. In these embodiments, 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).
[0055] In embodiments, 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.
[0056] In embodiments, 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. In embodiments, 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. In embodiments, 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.
[0057] In embodiments, 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. In embodiments, 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. In embodiments, a subject treated using the methods of the invention has a PKD2 whole gene deletion, in one allele.
[0058] In embodiments, the subject has a PC-2 missense mutation selected from M1K, P24L, R28P, A35D, R6ON, S8OL, 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,1448K, I452V, F482C, Y487H, D5 11V, V516L, L517R, V519M, A552P, I556V, N578D, N580K, M583I, A615T, F629S, C632R, R638C, L656W, L715I, I758V, R798C, M800L, S804N, R807Q, R848Q, D886G, R893G, V909I, D919N, T931M, R945H
or S949F. In embodiments, the subject has a PC-2 deletion mutation selected from EX l_EX13del, IVS2 3'(ABCG2)del8Okb*, IVS2 3'(ABCG2)de198kb, IVS4+1452 IVS5-965de15722, S378de1, F605de1, IVS9_31de128kb, 2182_2183delAG, L736_N737de12 or R878de1. In embodiments, the subject has PC-2 duplication mutation Ex3dup*. In embodiments, a subject having any PC-2 mutation known in the art and described in the literature, e.g., by Chang, etal., 2005, Ren Fail 27: 95-100, is treated using the methods and compositions of the present invention.

Use of TANGO for Increasing PC-2 Protein Expression [0059] As described above, in embodiments, Targeted Augmentation of Nuclear Gene Output (TANGO) is used in the methods of the invention to increase expression of a PC-2 protein. In these embodiments, 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 (AS0s) that are complementary to a targeted portion of the RIC pre-mRNA. 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.
[0060] The terms "pre-mRNA," and "pre-mRNA transcript" may be used interchangeably and refer to any pre-mRNA species that contains at least one intron. In embodiments, pre-mRNA or pre-mRNA
transcripts comprise a 5'-7-methylguanosine cap and/or a poly-A tail. In embodiments, pre-mRNA or pre-mRNA transcripts comprise both a 5'-7-methylguanosine cap and a poly-A
tail. In some embodiments, the pre-mRNA transcript does not comprise a 5'-7-methylguanosine cap and/or a poly-A
tail. A pre-mRNA transcript is a non-productive messenger RNA (mRNA) molecule if it is not translated into a protein (or transported into the cytoplasm from the nucleus).
[0061] As used herein, 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. In some embodiments, the retained intron is the most abundant intron in RIC pre-mRNA encoding the target protein. In embodiments, 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. In embodiments, 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. In embodiments, a mature mRNA encoding the target protein is thereby produced.
The terms "mature mRNA," and "fully-spliced mRNA," are used interchangeably herein to describe a fully processed mRNA
encoding a target protein (e.g., mRNA that is exported from the nucleus into the cytoplasm and translated into target protein) or a fully processed functional RNA. The term "productive mRNA," also can be used to describe a fully processed mRNA encoding a target protein. In embodiments, the targeted region is in a retained intron that is the most abundant intron in a RIC pre-mRNA encoding the PC-2 protein. In embodiments, the most retained intron in a RIC pre-mRNA encoding the PC-2 protein is intron 5.
[0062] In embodiments, a retained intron is an intron that is identified as a retained intron based on a determination of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%, retention. In embodiments, a retained intron is an intron that is identified as a retained intron based on a determination of about 5% to about 10000, 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 450, about 5% to about 40%, about 5% to about 350, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 10% to about 1000o, about 10% to about 95%, about 10% to about 90%, about 10% to about 85%, about 10% to about 80%, about 10% to about 75%, about 10% to about 70%, about 10% to about 65%, about 10% to about 60%, about 10% to about 55%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about

15% to about 1000o, about 15% to about 95%, about 15% to about 90%, about 15% to about 85%, about 15% to about 80%, about 1500 to about 75%, about 150o to about 70%, about 150o to about 65%, about 150o to about 60%, about 15% to about 55%, about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, about 20% to about 1000o, about 20% to about 95%, about 20% to about 90%, about 20% to about 85%, about 20% to about 80%, about 20% to about 75%, about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 550, about 20% to about 500o, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 25% to about 1000o, about 25% to about 95%, about 25% to about 90%, about 25% to about 85%, about 25% to about 80%, about 25% to about 75%, about 25% to about 70%, about 25% to about 65%, about 25% to about 60%, about 25% to about 550 , about 25% to about 500o, about 25% to about 45%, about 25% to about 40%, or about 25% to about 35%, retention.
[0063] As used herein, 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.
Thus, as used herein, 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).
[0064] In embodiments of any of the compositions and methods provided herein, "comprising" may be replaced with "consisting essentially of" or "consisting of." The phrase "consisting essentially of' is used herein to require the specified feature(s) (e.g., nucleobase sequence) as well as those which do not materially affect the character or function of the claimed invention. As used herein, 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).
[0065] In embodiments, an ASO is complementary to a targeted region that is within a non-retained intron in a RIC pre-mRNA. In embodiments, 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. In embodiments, 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. As used to identify the location of a region or sequence, "within" is understood to include the residues at the positions recited. For example, a region +6 to +100 includes the residues at positions +6 and +100. In embodiments, fully-spliced (mature) RNA encoding the target protein is thereby produced.
[0066] In embodiments, the retained intron of the RIC pre-mRNA is an inefficiently spliced intron. As used herein, "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. The term "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.
[0067] In embodiments, 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. In embodiments, 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. As used herein, the "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. As used herein, the "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).
[0068] 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. As used herein, "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. In some embodiments, 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.
[0069] As used herein, 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.
[0070] In embodiments, 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 ELISA.
100711 In embodiments, 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. In embodiments, 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 5-fold, or at least about 10-fold, compared to the amount of target protein produced by a control compound.
A control compound can be, for example, an oligonucleotide that is not complementary to the targeted portion of the RIC pre-mRNA.
[0072] In some embodiments, 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. In some embodiments, 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. In embodiments, 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, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold compared to the amount of mature RNA produced in an untreated cell, e.g., an untreated cell or a cell treated with a control compound. A control compound can be, for example, an oligonucleotide that is not complementary to the targeted portion of the PKD2 RIC pre-mRNA.

Constitutive Splicing of a Retained Intron from a RIC pre-mRNA
[0073] 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. In particular, the methods and compositions as described herein induce the constitutive splicing of a retained intron from a PKD2 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.
[0074] 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, as used herein, 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. For example, constitutive splicing of a retained intron, as induced using the methods and antisense oligonucleotides provided herein, 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.
[0075] In embodiments, 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.
[0076] In some embodiments, 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. In these embodiments, 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.
[0077] In other embodiments, 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. In these embodiments, 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.

[0078] "Correct removal" of the retained intron by constitutive splicing refers to removal of the entire intron, without removal of any part of an exon.
[0079] In embodiments, 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. In embodiments, modulation of alternative or aberrant splicing is determined based on an increase or decrease in the amount of the spliced species of interest of at least 10% or 1.1-fold. In embodiments, modulation is determined based on an increase or decrease at a level that is at least 10% to 100% or 1.1 to 10-fold, as described herein regarding determining an increase in mRNA encoding PC-2 protein in the methods of the invention.
[0080] In embodiments, 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. In embodiments, 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. In embodiments, the PKD2 RIC pre-mRNA was produced by partial splicing of a full-length PKD2 pre-mRNA. In these embodiments, 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.
[0081] In embodiments, the mRNA encoding PC-2 protein is a full-length mature mRNA, or a wild-type mature mRNA. In these embodiments, 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.
Ant/sense Oligomers [0082] One aspect of the present disclosure is a composition comprising antisense oligomers that enhances splicing by binding to a targeted portion of a PKD2 RIC pre-mRNA. As used herein, the terms "ASO" and "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. Typically, if they hybridize to a site other than the intended (targeted) nucleic acid sequence, they hybridize to a limited number of sequences that are not a target nucleic acid (to a few sites other than a target nucleic acid). Design of an ASO can take into consideration the occurrence of the nucleic acid sequence of the targeted portion of the pre-mRNA transcript or a sufficiently similar nucleic acid sequence in other locations in the genome or cellular pre-mRNA or transcriptome, such that the likelihood the ASO will bind other sites and cause "off-target" effects is limited. Any antisense oligomers known in the art, for example in PCT
Application No.
PCT/US2014/054151, published as WO 2015/035091, titled "Reducing Nonsense-Mediated mRNA
Decay," can be used to practice the methods described herein.
[0083] In some embodiments, ASOs "specifically hybridize" to or are "specific"
to a target nucleic acid or a targeted portion of a RIC pre-mRNA. Typically such hybridization occurs with a 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. At a given ionic strength and pH, the Tm is the temperature at which 50% of a target sequence hybridizes to a complementary oligonucleotide.
[0084] 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. The sequence of an antisense oligomer (ASO) need not be 100% complementary to that of its target nucleic acid to hybridize. In certain embodiments, 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. For example, 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. In this example, 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, etal., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).
[0085] 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.
[0086] 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). The ASOs may comprise naturally-occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination of two or three of the preceding. The term "naturally occurring nucleotides" includes deoxyribonucleotides and ribonucleotides. The term "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.
[0087] 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. Examples of modified nucleobases include, without limitation, hypoxanthine, xanthine, 7-me thylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethoylcytosine.
[0088] The ASOs described herein also comprise a backbone structure that connects the components of an oligomer. The term "backbone structure" and "oligomer linkages" may be used interchangeably and refer to the connection between monomers of the ASO. In naturally occurring oligonucleotides, 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, etal., Nucleic Acids Res.
14:9081 (1986); Stec, etal., J. Am. Chem. Soc. 106:6077 (1984), Stein, etal., Nucleic Acids Res.
16:3209 (1988), Zon, etal., Anti Cancer Drug Design 6:539 (1991); Zon, etal., Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stec, etal., U.S. Pat. No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90:543 (1990).
In some embodiments, 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. In some embodiments, the backbone modification is a phosphorothioate linkage. In some embodiments, the backbone modification is a phosphoramidate linkage.
[0089] In embodiments, 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. For example, 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. In embodiments, an ASO used in the methods of the disclosure, including, but not limited to, any of the ASOs set forth herein in Table 1, comprises an ASO having phosphorus internucleotide linkages that are not random. In embodiments, a composition used in the methods of the invention comprises a pure diastereomeric ASO. In embodiments, 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 10000, about 90% to about 1000o, about 91% to about 1000o, about 92% to about 1000o, about 93% to about 1000o, about 94% to about 1000o, about 95% to about 1000o, about 96% to about 1000o, about 97% to about 1000o, about 98%
to about 10000 , or about 99% to about 1000o.
[0090] In embodiments, the ASO has a nonrandom mixture of Rp and Sp configurations at its phosphorus internucleotide linkages. For example, it has been suggested that a mix of Rp and Sp is required in antisense oligonucleotides to achieve a balance between good activity and nuclease stability (Wan, etal., 2014, "Synthesis, biophysical properties and biological activity of second generation antisense oligonucleotides containing chiral phosphorothioate linkages,"
Nucleic Acids Research 42(22):
13456-13468, incorporated herein by reference). In embodiments, an ASO used in the methods of the invention, including, but not limited to, any of the ASOs set forth herein in Table 1, comprises about 5-1000o Rp, at least about 50 Rp, at least about 10% Rp, at least about 1500 Rp, at least about 20% Rp, at least about 25% Rp, at least about 30% Rp, at least about 350 Rp, at least about 40% Rp, at least about 45% Rp, at least about 5000 Rp, at least about 550 Rp, at least about 60% Rp, at least about 65% Rp, at least about 70% Rp, at least about 750 Rp, at least about 80% Rp, at least about 85% Rp, at least about 90% Rp, or at least about 950 Rp, with the remainder Sp, or about 100% Rp. In embodiments, an ASO
used in the methods of the invention, including, but not limited to, any of the ASOs set forth herein in Table 1, 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 450 to about 100% Rp, about 5000 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 750 to about 100% Rp, about 80% to about 100% Rp, about 85% to about 100% Rp, about 90% to about 100% Rp, or about 950 to about 100% Rp, about 20% to about 80%
Rp, about 25% to about 750 Rp, about 30% to about 70% Rp, about 40% to about 60% Rp, or about 450 to about 55% Rp, with the remainder Sp.
[0091] In embodiments, an ASO used in the methods of the invention, including, but not limited to, any of the ASOs set forth herein in Table 1, 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 350 Sp, at least about 40% Sp, at least about 450 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 750 Sp, at least about 80% Sp, at least about 85% Sp, at least about 90% Sp, or at least about 950 Sp, with the remainder Rp, or about 100% Sp. In embodiments, an ASO used in the methods of the invention, including, but not limited to, any of the ASOs set forth herein in Table 1, 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 350 to about 100% Sp, about 40% to about 100% Sp, about 450 to about 10000 Sp, about 50% to about 1000o Sp, about 55% to about 1000o Sp, about 60% to about 1000o Sp, about 65% to about 1000o Sp, about 70% to about 1000o Sp, about 75% to about 1000o Sp, about 80%
to about 1000o Sp, about 85% to about 1000o Sp, about 90% to about 1000o Sp, or about 95% to about 1000o 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.
[0092] 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. In some embodiments, the sugar moiety modification is selected from 2'-0-Me, 2'F, and 2'MOE. In some embodiments, the sugar moiety modification is an extra bridge bond, such as in a locked nucleic acid (LNA). In some embodiments the sugar analog contains a morpholine ring, such as phosphorodiamidate morpholino (PMO). In some embodiments, the sugar moiety comprises a ribofuransyl or 2'deoxyribofuransyl modification. In some embodiments, the sugar moiety comprises 2'4'-constrained 2'0-methyloxyethyl (cM0E) modifications. In some embodiments, the sugar moiety comprises cEt 2', 4' constrained 2'-0 ethyl BNA modifications. In some embodiments, the sugar moiety comprises tricycloDNA (tcDNA) modifications. In some embodiments, the sugar moiety comprises ethylene nucleic acid (ENA) modifications. In some embodiments, the sugar moiety comprises MCE
modifications. Modifications are known in the art and described in the literature, e.g., by Jarver, etal., 2014, "A Chemical View of Oligonucleotides for Exon Skipping and Related Drug Applications," Nucleic Acid Therapeutics 24(1):
37-47, incorporated by reference for this purpose herein.
[0093] In some examples, each monomer of the ASO is modified in the same way, for example each linkage of the backbone of the ASO comprises a phosphorothioate linkage or each ribose sugar moiety comprises a 2'0-methyl modification. Such modifications that are present on each of the monomer components of an ASO are referred to as "uniform modifications." In some examples, a combination of different modifications may be desired, for example, an ASO may comprise a combination of phosphorodiamidate linkages and sugar moieties comprising morpholine rings (morpholinos).
Combinations of different modifications to an ASO are referred to as "mixed modifications" or "mixed chemistries."
[0094] In some embodiments, 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 moipholino (PMO). In some embodiments, the ASO comprises a peptide nucleic acid (PNA). Any of the ASOs or any component of an ASO (e.g., a nucleobase, sugar moiety, backbone) described herein may be modified in order to achieve desired properties or activities of the ASO or reduce undesired properties or activities of the ASO. For example, 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.
[0095] In some embodiments, the ASOs are comprised of 2'-0-(2-methoxyethyl) (MOE) phosphorothioate-modified nucleotides. 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, etal., J Pharmacol Exp Ther. 2001;
296(3):898-904.
[0096] Methods of synthesizing ASOs will be known to one of skill in the art.
Alternatively or in addition, ASOs may be obtained from a commercial source.
[0097] Unless specified otherwise, 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.
Similarly, the right-hand end or direction of a nucleic acid sequence (single or double stranded) is the 3' end or direction. Generally, a region or sequence that is 5' to a reference point in a nucleic acid is referred to as "upstream," and a region or sequence that is 3' to a reference point in a nucleic acid is referred to as "downstream." Generally, the 5' direction or end of an mRNA is where the initiation or start codon is located, while the 3' end or direction is where the termination codon is located. In some aspects, 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. For example, a reference point (e.g., an exon-exon junction in mRNA) may be designated as the "zero" site, and a nucleotide that is directly adjacent and upstream of the reference point is designated "minus one," e.g., "4," while a nucleotide that is directly adjacent and downstream of the reference point is designated "plus one," e.g., ,.+1 [0098] In some embodiments, 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). In some embodiments, 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. In some embodiments, 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). In some embodiments, 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. In some aspects, 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, about +6 to about +210, about +6 to about +200, about +6 to about +190, about +6 to about +180, about +6 to about +170, about +6 to about +160, about +6 to about +150, about +6 to about +140, about +6 to about +130, about +6 to about +120, about +6 to about +110, about +6 to about +100, about +6 to about +90, about +6 to about +80, about +6 to about +70, about +6 to about +60, about +6 to about +50, about +6 to about +40, about +6 to about +30, or about +6 to about +20 relative to 5' splice site of the retained intron.
100991 In some embodiments, 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). In some embodiments, 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. In some embodiments, 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). In some embodiments, 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. In some aspects, 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 -110e, about -4e to about -100e, 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.
1001001ln some embodiments, 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). In some embodiments, 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. In some embodiments, 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. In some aspects, 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 -250, about -16 to about -240, about -16 to about -230, about -16 to about -220, about -16 to about -210, about -16 to about -200, about -16 to about -190, about -16 to about -180, about -16 to about -170, about -16 to about -160, about -16 to about -150, about -16 to about -140, about -16 to about -130, about -16 to about -120, about -16 to about -110, about -16 to about -100, about -16 to about -90, about -16 to about -80, about -16 to about -70, about -16 to about -60, about -16 to about -50, about -16 to about -40, or about -16 to about -30 relative to 3' splice site of the retained intron.
1001011ln some embodiments, the ASOs are complementary to a targeted region of a PKD2 RIC pre-mRNA 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). In some embodiments, 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. In some embodiments, the ASO is not complementary to nucleotides +le relative to the 3' splice site (the first nucleotide located downstream of the 3' splice site). In some embodiments, the ASOs may be complementary to a targeted portion of a PKD2 RIC pre-mRNA that is within the region between nucleotides +2e and +212e relative to the 3' splice site of the retained intron. In some aspects, 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 +110e, 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 of the retained intron.
1001021 In embodiments, 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.
[00103] The ASOs may be of any length suitable for specific binding and effective enhancement of splicing. In some embodiments, the ASOs consist of 8 to 50 nucleobases. For example, the ASO may be 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, or 50 nucleobases in length. In some embodiments, the ASOs consist of more than 50 nucleobases.
In some embodiments, 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, to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, 12 to 15 nucleobases, 13 to 50 nucleobases, 13 to 40 nucleobases, 13 to 35 nucleobases, 13 to 30 nucleobases, 13 to 25 nucleobases, 13 to 20 nucleobases, 14 to 50 nucleobases, 14 to 40 nucleobases, 14 to 35 nucleobases, 14 to 30 nucleobases, 14 to 25 nucleobases, 14 to 20 nucleobases, 15 to 50 nucleobases, 15 to 40 nucleobases, 15 to 35 nucleobases, 15 to 30 nucleobases, 15 to 25 nucleobases, 15 to 20 nucleobases, 20 to 50 nucleobases, 20 to 40 nucleobases, 20 to 35 nucleobases, 20 to 30 nucleobases, 20 to 25 nucleobases, 25 to 50 nucleobases, 25 to 40 nucleobases, 25 to 35 nucleobases, or 25 to 30 nucleobases in length. In some embodiments, the ASOs are 18 nucleotides in length. In some embodiments, the ASOs are 15 nucleotides in length. In some embodiments, the ASOs are 25 nucleotides in length.
[00104] In some embodiments, 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.
[00105] In embodiments, 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.
Oligonucleotides comprising lipophilic moieties and preparation methods have been described in the published literature. In embodiments, 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. 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. In embodiments, the conjugate is attached to the 3' end of the antisense oligonucleotide. Methods of preparing oligonucleotide conjugates are described, e.g., in U.S.
Pat. No. 8,450,467, "Carbohydrate conjugates as delivery agents for oligonucleotides," incorporated by reference herein.
[00106] In some embodiments, the nucleic acid to be targeted by an ASO is a PKD2 RIC pre-mRNA
expressed in a cell, such as a eukaryotic cell. In some embodiments, the term "cell" may refer to a population of cells. In some embodiments, the cell is in a subject. In some embodiments, the cell is isolated from a subject. In some embodiments, the cell is ex vivo. In some embodiments, the cell is a condition or disease-relevant cell or a cell line. In some embodiments, the cell is in vitro (e.g., in cell culture).

Pharmaceutical Compositions [00107] Pharmaceutical 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. In embodiments, 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.
[00108] Pharmaceutically acceptable 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. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like.
Representative 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.
[00109] In embodiments, 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.
In embodiments, 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. In embodiments, 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).

[00110] 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. In embodiments, liposomes also include sterically stabilized liposomes, e.g., liposomes comprising one or more specialized lipids.
These specialized lipids result in liposomes with enhanced circulation lifetimes. In embodiments, a sterically stabilized liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. In embodiments, 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. In embodiments, 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.
In embodiments, the penetration enhancer is a surfactant, fatty acid, bile salt, chelating agent, or non-chelating nonsurfactant.
[00111] In embodiments, the pharmaceutical formulation comprises multiple antisense oligonucleotides.
In embodiments, the antisense oligonucleotide is administered in combination with another drug or therapeutic agent. In embodiments, the antisense oligonucleotide is administered with one or more agents capable of promoting penetration of the subject antisense oligonucleotide across the blood-brain barrier by any method known in the art. For example, delivery of agents by administration of an adenovirus vector to motor neurons in muscle tissue is described in U.S. Pat. No. 6,632,427, "Adenoviral-vector-mediated gene transfer into medullary motor neurons," incorporated herein by reference.
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.
[00112] In embodiments, the antisense oligonucleotides are linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties. In embodiments, 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. In embodiments, 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. In embodiments, osmotic blood brain barrier disruption is assisted by infusion of sugars, e.g., meso erythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose, dulcitol, myo-inositol, L(-) fructose, D(-) mannitol, D(+) glucose, D(+) arabinose, D(-) arabinose, cellobiose, D(+) maltose, D(+) raffinose, L(+) rhamnose, D(+) melibiose, D(-) ribose, adonitol, D(+) arabitol, L(-) arabitol, D(+) fucose, L(-) fucose, D(-) lyxose, L(+) lyxose, and L(-) lyxose, or amino acids, e.g., glutamine, lysine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, leucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine, and taurine. Methods and materials for enhancing blood brain barrier penetration are described, e.g., in U.S. Pat. No. 4,866,042, "Method for the delivery of genetic material across the blood brain barrier,"
U.S. Pat. No. 6,294,520, "Material for passage through the blood-brain barrier," and U.S. Pat. No.
6,936,589, "Parenteral delivery systems," each incorporated herein by reference.

[00113] In embodiments, 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.
Oligonucleotides comprising lipophilic moieties, and preparation methods have been described in the published literature. In embodiments, 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. 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. In embodiments, the conjugate is attached to the 3' end of the antisense oligonucleotide. Methods of preparing oligonucleotide conjugates are described, e.g., in U.S.
Pat. No. 8,450,467, "Carbohydrate conjugates as delivery agents for oligonucleotides," incorporated by reference herein.
Treatment of Subjects [00114] Any of the 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. In embodiments, the individual is a human. In embodiments, the individual is a fetus, an embryo, or a child. In other embodiments, the individual may be another eukaryotic organism, such as a plant. In some embodiments, the compositions provided herein are administered to a cell ex vivo.
[00115] In some embodiments, the compositions provided herein are administered to an individual as a method of treating a disease or disorder. In some embodiments, the individual has a genetic disease, such as any of the diseases described herein. In some embodiments, the individual is at risk of having the disease, such as any of the diseases described herein. In some embodiments, the individual is at increased risk of having a disease or disorder caused by insufficient amount of a protein or insufficient activity of a protein. If an individual is "at an increased risk" of having a disease or disorder caused insufficient amount of a protein or insufficient activity of a protein, the method involves preventative or prophylactic treatment. For example, an individual may be at an increased risk of having such a disease or disorder because of family history of the disease. Typically, 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).
[00116] 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.
[00117] Subjects are evaluated for response to treatment using any appropriate markers. In embodiments, 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.
Methods of Identifying Additional ASOs that Enhance Splicing [00118] Also within the scope of the present invention are methods for identifying (determining) additional ASOs that enhance splicing of a PKD2 RIC pre-mRNA, specifically at the target intron. 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.
In some embodiments, the ASO may block or interfere with the binding site(s) of a splicing repressor(s)/silencer. Any method known in the art may be used to identify (determine) an ASO that when hybridized to the target region of the intron results in the desired effect (e.g., enhanced splicing, protein or functional RNA production). These methods also can be used for identifying ASOs that enhance splicing of the retained intron by binding to a targeted region in an exon flanking the retained intron, or in a non-retained intron. An example of a method that may be used is provided below.
[00119] A round of screening, referred to as an ASO "walk" may be performed using ASOs that have been designed to hybridize to a target region of a pre-mRNA. For example, 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). For example, 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. In embodiments, the ASOs can be tiled more closely, e.g., every 1, 2, 3, or 4 nucleotides. Further, the ASOs can be tiled from 100 nucleotides downstream of the 5' splice site, to 100 nucleotides upstream of the 3' splice site. In some embodiments, 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. In some embodiments, 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.
[00120] 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) 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. In some embodiments, 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.
[00121] 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.
[00122] 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.
[00123] As described for the ASO walk above, 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. In some embodiments, 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.
[00124] 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.
Following administration, 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.
EXAMPLES
[00125] The following examples provide illustrative embodiments of the invention. One of ordinary skill in the art will recognize the numerous modifications and variations that may be performed without altering the spirit or scope of the disclosure. Such modifications and variations are encompassed within the scope of the disclosure. The Examples do not in any way limit the disclosure described herein.
Example 1: Identification of intron retention events in PKD2 transcripts by RNAseq using next generation sequencing [00126] Whole transcriptome shotgun sequencing was carried out using next generation sequencing to reveal a snapshot of transcripts produced by the PKD2 gene to identify intron-retention events. For this purpose, polyA+ RNA from nuclear and cytoplasmic fractions of renal epithelial cells was isolated and cDNA libraries constructed using Illumina's TruSeq Stranded mRNA library Prep Kit. The libraries were pair-end sequenced resulting in 100-nucleotide reads that were mapped to the human genome (Feb. 2009, GRCh37/hg19 assembly). The sequencing results for PKD2 are shown in FIG. 3.
Briefly, FIG. 3 shows the mapped reads visualized using the UCSC genome browser (operated by the UCSC Genome Informatics Group (Center for Biomolecular Science & Engineering, University of California, Santa Cruz, 1156 High Street, Santa Cruz, CA 95064) and described by, e.g., Rosenbloom, etal., 2015, "The UCSC
Genome Browser database: 2015 update," Nucleic Acids Research 43, Database Issue, doi:
10.1093/nar/gku1177) and the coverage and number of reads can be inferred by the peak signals. The height of the peaks indicates the level of expression given by the density of the reads in a particular region. A schematic representation of PKD2 (drawn to scale) is provided by the UCSC genome browser (below the read signals) so that peaks can be matched to PKD2 exonic and intronic regions. Based on this display, we identified one intron (intron 5, as indicated) that has high read density in the nuclear fraction of HCN, but very low to no reads in the cytoplasmic fraction of these cells (as shown for intron 5 in the bottom diagram of FIG. 3). This indicates that intron 5 is retained and that the intron-5 containing transcripts remain in the nucleus, suggesting that this retained PKD2 RIC pre-mRNAs is non-productive, as it is not exported out to the cytoplasm.
Example 2: Design of ASO-walk targeting intron 5 of PKD2 [00127] 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. From this design, 2'-0-Me RNA, PS backbone, 18-mer ASOs shifted by 5-nucleotide intervals can be produced and utilized to target PKD2 RIC pre-mRNAs to increase PC-2 protein production.

Table 1. List of ASOs targeting the PKD2 gene Gene Pre-mRNA ASOs Retained Target Sequence SEQ ID NO: SEQ ID NO: Intron SEQ ID NO:
PKD2 PKD2:NM 000297 SEQ ID NOs: 3-280 Intron 5 SEQ ID NO: 281 SEQ ID NO: 1 SEQ ID NO: 2 Example 3: Improved splicing efficiency via ASO-targeting of PKD2 intron 5 increases transcript levels [00128] 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)) are mock-transfected, or transfected with the targeting ASOs described in Table 1.
Cells are transfected using Lipofectamine RNAiMax transfection reagent (Thermo Fisher) according to manufacturer's specifications. Briefly, 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. To quantify the amount of splicing at the intron of interest, quantitative PCR is carried out using 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^-(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.
[00129] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only.
Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims (118)

What is claimed is:

1. A method of treating Polycystic Kidney Disease in a subject in need thereof, by increasing the expression of a target protein or functional RNA by cells of the subject, wherein the cells have a retained-intron-containing pre-mRNA (RIC 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 RNA in the cells of the subject.

2. A method of increasing expression of a target protein, wherein the target protein is PC-2, by cells having a retained-intron-containing pre-mRNA (RIC pre-mRNA), the RIC pre-mRNA comprising a retained intron, an exon flanking the 5' splice site of the retained intron, an exon flanking the 3' splice site of the retained intron, and wherein the RIC pre-mRNA encodes 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.

3. The method of claim 1, wherein the target protein is PC-2.

4. The method of claim 1, wherein 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.

5. The method of claim 2, wherein the cells are in or from a subject having a condition caused by a deficient amount or activity of PC-2 protein.

6. The method of any one of claims 1 to 5, wherein the deficient amount of the target protein is caused by haploinsufficiency of the target protein, wherein the subject has a first allele encoding a functional target protein, and a second allele from which the target protein is not produced, or a second allele encoding a nonfunctional target protein, and wherein the antisense oligomer binds to a targeted portion of a RIC pre-mRNA transcribed from the first allele.

7. The method of any one of claims 1 to 5, wherein the subject has a condition caused by a disorder resulting from a deficiency in the amount or function of the target protein, wherein the subject has (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 first mutant allele is (a)(i) or (a)(ii), and wherein the RIC pre-mRNA is transcribed from either the first mutant allele that is (a)(i) or (a)(ii), and/or the second allele that is (b)(i) or (b)(ii).

8. The method of claim 7, wherein the target protein is produced in a form having reduced function compared to the equivalent wild-type protein.

9. The method of claim 7, wherein the target protein is produced in a form that is fully-functional compared to the equivalent wild-type protein.

10. The method of any one of claims 1 to 9, wherein 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.

11. The method of any one of claims 1 to 9, wherein 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.

12. The method of any one of claims 1 to 9, wherein 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.

13. The method of any of claims 1 to 9, wherein the targeted portion of the RIC pre-mRNA is in the retained intron within:

(a) the region -4e to -1,054e 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.

14. The method of any one of claims 1 to 13, wherein the target protein is PC-2.

15. The method of claim 14, wherein 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.

16. The method of claim 14, wherein 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.

17. The method of claim 14, wherein 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.

18. The method of claim 14, wherein 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.

19. The method of claim 14, wherein 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.

20. The method of claim 19, wherein 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.

21. The method of claim 14, wherein 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.

22. The method of claim 21, wherein 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.

23. The method of claim 14, wherein the targeted portion of the RIC pre-mRNA is in retained intron within the region +6 to +497 relative to the 5' splice site of the retained intron.

24. The method of claim 23, wherein 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.

25. The method of claim 14, wherein 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.

26. The method of claim 25, wherein 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.

27. The method of claim 14, wherein 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.

28. The method of claim 27, wherein 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.

29. The method of any one of claims 1 to 28, wherein 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.

30. The method of any one of claims 1 to 29, wherein 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.

31. The method of any one of claims 1 to 30, wherein the RIC pre-mRNA was produced by partial splicing of a full-length pre-mRNA or partial splicing of a wild-type pre-mRNA.

32. The method of any one of claims 1 to 31, wherein the mRNA encoding the target protein or functional RNA is a full-length mature mRNA, or a wild-type mature mRNA.

33. The method of any one of claims 1 to 32, wherein the target protein produced is full-length protein, or wild-type protein.

34. The method of any one of claims 1 to 33, wherein the total amount of the mRNA encoding the target protein or functional RNA produced in the cell contacted with the antisense oligomer is increased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold, compared to the total amount of the mRNA encoding the target protein or functional RNA
produced in a control cell.

35. The method of any one of claims 1 to 34, wherein the total amount of target protein produced by the cell contacted with the antisense oligomer is increased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold, compared to the total amount of target protein produced by a control cell.

36. The method of any one of claims 1 to 35, wherein the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.

37. The method of any one of claims 1 to 36, wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, a 2'-Fluoro, or a 2'-O-methoxyethyl moiety.

38. The method of any one of claims 1 to 37, wherein the antisense oligomer comprises at least one modified sugar moiety.

39. The method of claim 38, wherein each sugar moiety is a modified sugar moiety.

40. The method of any one of claims 1 to 39, wherein the antisense oligomer consists of from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.

41. The method of any one of claims 1 to 40 wherein the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, complementary to the targeted portion of the RIC pre-mRNA encoding the protein.

42. The method of any one of claims 1 to 41, wherein 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.

43. The method of claim 42, whereby 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.

44. The method of any one of claims 1 to 43, wherein 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.

45. The method of claim 44, whereby 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.

46. The method of any one of claims 1 to 45 wherein the method further comprises assessing PC-2 protein expression.

47. The method of any one of claims 1 to 46, wherein the antisense oligomer binds to a targeted portion of a PKD2 RIC pre-mRNA, wherein the targeted portion is in a sequence selected from SEQ ID
NOs: 3-280.

48. The method of any one of claims 1 to 47, wherein the subject is a human.

49. The method of any one of claims 1 to 47, wherein the subject is a non-human animal.

50. The method of any one of claims 1 to 48, wherein the subject is a fetus, an embryo, or a child.

51. The method of any one of claims 1 to 47, wherein the cells are ex vivo.

52. The method of any one of claims 1 to 51 wherein the antisense oligomer is administered by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection of the subject.

53. The method of any one of claims 1 to 52, wherein the 9 nucleotides at -3e to -1e of the exon flanking the 5' splice site and +1 to +6 of the retained intron, are identical to the corresponding wild-type sequence.

54. The method of any one of claims 1 to 53, wherein the 16 nucleotides at -15 to -1 of the retained intron and +1e of the exon flanking the 3' splice site are identical to the corresponding wild-type sequence.

55. An antisense oligomer as used in a method of any one of claims 1 to 54.

56. An antisense oligomer comprising a sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NOs: 3-280.

57. A pharmaceutical composition comprising the antisense oligomer of claim 55 or 56 and an and a pharmaceutically acceptable excipient, diluent or carrier.

58. A method of treating a subject in need thereof, by administering the pharmaceutical composition of claim 57 by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.

59. A composition comprising an antisense oligomer for use in a method of increasing expression of a target protein or a functional RNA by cells to treat Polycystic Kidney Disease in a subject in need thereof, associated with a deficient protein or deficient functional RNA, wherein the deficient protein or deficient functional RNA is deficient in amount or activity in the subject, wherein the antisense oligomer enhances constitutive splicing of a retained intron-containing pre-mRNA (RIC
pre-mRNA) encoding the target protein or the functional RNA, wherein the target protein is:
(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 encoding the target protein or the functional RNA, thereby increasing production or activity of the target protein or the functional RNA in the subject.

60. A composition 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 constitutively 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.

61. The composition of claim 60, wherein the condition is a disease or disorder.

62. The composition of claim 61, wherein the disease or disorder is Polycystic Kidney Disease.

63. The composition of claim 62, wherein the target protein and RIC pre-mRNA are encoded by the PKD2 gene.

64. The composition of any one of claims 59 to 63, wherein 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.

65. The composition of any one of claims 59 to 64, wherein 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.

66. The composition of any one of claims 59 to 63, wherein 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.

67. The composition of any one of claims 59 to 63, wherein 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.

68. The composition of any of claims 59 to 63, wherein the targeted portion of the RIC
pre-mRNA is within:
(a) the region -4e to -1,054e 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.

69. The composition of any one of claims 59 to 68, wherein the target protein is PC-2.

70. The composition of claim 69, wherein 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.

71. The composition of claim 69, wherein 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.

72. The composition of claim 69, wherein 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.

73. The composition of claim 69, wherein 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.

74. The composition of claim 69, wherein 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.

75. The composition of claim 74, wherein 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.

76. The composition of claim 69, wherein 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.

77. The composition of claim 76, wherein 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.

78. The composition of claim 69, wherein 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.

79. The composition of claim 78, wherein 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.

80. The composition of claim 69, wherein 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.

81. The composition of claim 80, wherein 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.

82. The composition of claim 69, wherein 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.

83. The composition of claim 82, wherein 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.

84. The composition of any one of claims 59 to 83, wherein 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.

85. The composition of any one of claims 59 to 84, wherein 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.

86. The composition of any one of claims 59 to 85, wherein the RIC pre-mRNA
was produced by partial splicing from a full-length pre-mRNA or a wild-type pre-mRNA.

87. The composition of any one of claims 59 to 86, wherein the mRNA
encoding the target protein or functional RNA is a full-length mature mRNA, or a wild-type mature mRNA.

88. The composition of any one of claims 59 to 87, wherein the target protein produced is full-length protein, or wild-type protein.

89. The composition of any one of claims 59 to 88, wherein the retained intron is a rate-limiting intron.

90. The composition of any one of claims 59 to 89 wherein said retained intron is the most abundant retained intron in said RIC pre-mRNA.

91. The composition of any one of claims 59 to 89, wherein the retained intron is the second most abundant retained intron in said RIC pre-mRNA.

92. The composition of any one of claims 59 to 91, wherein the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.

93. The composition of any one of claims 59 to 92 wherein said antisense oligomer is an antisense oligonucleotide.

94. The composition of any one of claims 59 to 93, wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, a 2'-Fluoro, or a 2'-O-methoxyethyl moiety.

95. The composition of any one of claims 59 to 94, wherein the antisense oligomer comprises at least one modified sugar moiety.

96. The composition of claim 95, wherein each sugar moiety is a modified sugar moiety.

97. The composition of any one of claims 59 to 96, wherein the antisense oligomer consists of from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.

98. A pharmaceutical composition comprising the antisense oligomer of any one of the compositions of claims 59 to 97, and a pharmaceutically acceptable excipient, diluent, or carrier.

99. A method of treating a subject in need thereof, by administering the pharmaceutical composition of claim 98 by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.

100. A pharmaceutical composition 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 pharmaceutically acceptable excipient, diluent, or carrier.

101. The pharmaceutical composition of claim 100, wherein the deficient PKD2 mRNA transcript is a PKD2 RIC pre-mRNA transcript.

102. The pharmaceutical composition of claim 100 or 101, wherein the targeted portion of the PKD2 RIC pre-mRNA transcript is in the retained intron within the region of about +500 relative to the 5' splice site of the retained intron to about -500 relative to the 3' spliced site of the retained intron.

103. The pharmaceutical composition of claim 100 or 101, wherein 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.

104. The pharmaceutical composition of claim 100 or 101, wherein 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.

105. The pharmaceutical composition of claim 100, wherein the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.

106. The pharmaceutical composition of claim 100, wherein the antisense oligomer is an antisense oligonucleotide.

107. The pharmaceutical composition of claim 100, wherein 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'-O-methoxyethyl moiety.

108. The pharmaceutical composition of claim 100, wherein the antisense oligomer comprises at least one modified sugar moiety.

109. The pharmaceutical composition of claim 100, wherein 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, to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.

110. The pharmaceutical composition of claim 100 or 101, wherein the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or is 100% complementary to a targeted portion of the PKD2 RIC pre-mRNA transcript.

111. The pharmaceutical composition of claim 100 or 101, wherein the targeted portion of the PKD2 RIC pre-mRNA transcript is within SEQ ID NO: 281.

112. The pharmaceutical composition of claim 100, wherein the antisense oligomer comprises a nucleotide sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 3-280.

113. The pharmaceutical composition of claim 100, wherein the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NOs: 3-280.

114. The pharmaceutical composition of any one of the claims 100-113, wherein the pharmaceutical composition is formulated for intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.

115. A method of inducing processing of a deficient PKD2 mRNA transcript to facilitate removal of a retained intron to produce a fully processed PKD2 mRNA 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.

116. The method of claim 115, wherein the retained intron is an entire retained intron.

117. The method of claim 115, wherein the deficient PKD2 mRNA transcript is a PKD2 pre-mRNA

transcript.

118.
A method of treating a subject having a condition caused by a deficient amount or activity of PC-2 protein comprising administering to the subject an antisense oligomer comprising a nucleotide sequence with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 3-280.