Compositions and methods for modulating CFTR

Abstract

In some aspects, the present disclosure provides compositions, methods, and kits related to agents that regulate the expression of CFTR protein.The agents provided herein can modify CFTR gene or regulate the process of CFTR pre-mRNA.In some embodiments, the compositions, methods, and kits provided herein can be applied to the treatment of cystic fibrosis.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 412,771, filed October 3, 2022, which is incorporated herein by reference. [Background technology]

[0002] Cystic fibrosis (CF) is a life-shortening autosomal recessive disease that is most common in populations of Northern European descent, with a frequency of 1 in 2,000–3,000 live births. Despite advances in CF treatment, there is no cure. Cystic fibrosis can be caused by pathogenic mutations in the CFTR gene, a 250-kilogram genomic sequence that encodes a mature epithelial cell protein composed of 1,480 amino acids.

[0003] Premature termination codon (PTC) mutations in the CFTR gene represent the largest class of cystic fibrosis (CF)-causing mutations for which no cure exists. PTC mutations can result in a hypofunctional or nonfunctional protein product and can induce a dramatic reduction in the CFTR mRNA template via nonsense-mediated mRNA decay (NMD). Certain C-terminal truncated CFTR proteins can retain some chloride channel function in cells. Therefore, without wishing to be bound by theory, inhibiting NMD to increase truncated protein expression is an attractive approach for treating diseases or conditions caused by PTC mutations near the 3' end of CFTR. However, given the important role of NMD as a global quality control mechanism for cells, a CFTR-specific mechanism that circumvents NMD is desirable. Summary of the Invention

[0004] In some aspects, the present disclosure provides a method for regulating the expression of a CFTR gene in a cell, the method comprising contacting a cell with an agent or a vector encoding the agent, wherein the cell contains a pre-mRNA, the pre-mRNA is transcribed from the CFTR gene and contains a first intron that contains an alternative polyadenylation site, and the agent modifies the CFTR gene or regulates pre-mRNA processing, thereby increasing the level of processed mRNA that is processed from the pre-mRNA and lacks the nucleic acid sequence of the pre-mRNA downstream of the first intron.In some embodiments, the cell is a human cell, and the CFTR gene is a human gene.In some embodiments, the first intron is intron 22. In some embodiments, the first intron is located in the region from GRCh38.p14 / hg38:chr7:117,627,771 to GRCh38.p14 / hg38:chr7:117,642,437. In some embodiments, the agent removes a nucleic acid sequence of the CFTR gene downstream of the first intron from the genome of the cell. In some embodiments, the nucleic acid sequence removed from the genome is located from GRCh38.p14 / hg38:chr7:117,642,438 to GRCh38.p14 / hg38:chr7:117,668,665. In some embodiments, the agent comprises a gene editing agent based on CRISPR / Cas9, TALEN, zinc finger, or any combination thereof. In some embodiments, the agent comprises a pair of guide RNAs, wherein the pair of guide RNAs comprises the sequences of SEQ ID NOs: 94 and 95, respectively. In some embodiments, the agent removes from the pre-mRNA a nucleic acid sequence of the pre-mRNA downstream of the first intron. In some embodiments, the nucleic acid sequence removed from the pre-mRNA is located at GRCh38.p14 / hg38:chr7:117,642,438 to GRCh38.p14 / hg38:chr7:117,668,665.In some embodiments, the agent inhibits splicing out of the first intron from the pre-mRNA.

[0005] In some aspects, the present disclosure provides a method for modulating expression of a CFTR gene in a cell, comprising contacting a cell with an agent or a vector encoding the agent, wherein the cell comprises a pre-mRNA transcribed from the CFTR gene and comprises a first intron comprising an alternative polyadenylation site, and wherein the agent inhibits splicing out of the first intron from the pre-mRNA during splicing of the pre-mRNA in the cell. In some embodiments, the cell is a human cell and the CFTR gene is a human gene. In some embodiments, the first intron is intron 22. In some embodiments, the first intron is located in the region from GRCh38.p14 / hg38:chr7:117,627,771 to GRCh38.p14 / hg38:chr7:117,642,437. In some embodiments, the agent increases the level of processed mRNA that is processed from the pre-mRNA and lacks the nucleic acid sequence of the pre-mRNA downstream of the first intron. In some embodiments, the nucleic acid sequence of the pre-mRNA downstream of the first intron is located in the region from GRCh38.p14 / hg38:chr7:117,642,438 to GRCh38.p14 / hg38:chr7:117,668,665.

[0006] In some embodiments, the level of processed mRNA is increased in the cells by at least about 1.5, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, or at least about 20-fold compared to corresponding cells not contacted with the agent or agent-encoding vector. In some embodiments, the level of processed mRNA is increased in the cells by at least about 10-fold compared to corresponding cells not contacted with the agent or agent-encoding vector.

[0007] In some embodiments, the processed mRNA comprises, in 5' to 3' order, 22 exons, an intron sequence encoding 9 amino acids, a stop codon, and an alternative 3' untranslated region. In some embodiments, the intron sequence encoding 9 amino acids is transcribed from a genomic sequence located between GRCh38.p14 / hg38:chr7:117,627,771 and GRCh38.p14 / hg38:chr7:117,627,797. In some embodiments, the processed mRNA is polyadenylated at an alternative polyadenylation site in a nucleic acid sequence transcribed from a genomic sequence located between GRCh38.p14 / hg38:chr7:117,627,771 and GRCh38.p14 / hg38:chr7:117,642,437.

[0008] In some embodiments, the agent increases the level of a truncated CFTR protein in a cell lacking an amino acid sequence expressed from an exon sequence of the CFTR gene downstream of the first intron. In some embodiments, the level of the truncated CFTR protein in the cell is increased by at least about 1.5, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, or at least about 20-fold compared to a corresponding cell not contacted with the agent or a vector encoding the agent. In some embodiments, the truncated CFTR protein in the cell has a chloride channel conductivity that is less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% of the chloride channel conductivity of a wild-type CFTR protein.

[0009] In some embodiments, the methods provided herein further include contacting the cell with a second agent. In some embodiments, the second agent comprises a modulator of CFTR protein that enhances chloride conductance of CFTR in the cell. In some embodiments, the second agent comprises ivacaftor, lumacaftor, tezacaftor, elexacaftor, a combination of lumacaftor and ivacaftor, a combination of tezacaftor and ivacaftor, or a combination of elexacaftor, ivacaftor, and tezacaftor. In some embodiments, the second agent increases the chloride channel conductance of the truncated CFTR protein by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 950%, at least 1000% improvement.In some embodiments, the second agent restores the chloride channel conductance of the truncated CFTR protein to approximately at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more of the chloride channel conductance of the wild-type CFTR protein.

[0010] In some embodiments, the agent (a) binds to the 5' splice site of the first intron, (b) binds to the 3' splice site of the first intron, (c) binds to the branch point of the 3' splice site of the first intron, or (d) interferes with a splicing factor involved in splicing out of the first intron.

[0011] In some embodiments, the agent comprises a polynucleotide sequence that is at least 80% complementary to at least 8 contiguous nucleic acids of a sequence selected from the group consisting of SEQ ID NOs: 6-36. In some embodiments, the agent comprises a polynucleotide sequence that is at least 80% complementary to at least 10, 12, 14, or 16 contiguous nucleic acids of a sequence selected from the group consisting of SEQ ID NOs: 6-36. In some embodiments, the agent comprises a polynucleotide sequence that is at least 80% complementary to at least 18 contiguous nucleic acids of a sequence selected from the group consisting of SEQ ID NOs: 6-36. In some embodiments, the agent comprises a polynucleotide sequence that is 100% complementary to a sequence selected from the group consisting of SEQ ID NOs: 6-36. In some embodiments, the agent comprises a polynucleotide sequence that has at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 37-64. In some embodiments, the agent comprises a polynucleotide sequence that comprises at least 8 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 37-64.

[0012] In some aspects, the disclosure provides methods comprising contacting a cell with an agent or a vector encoding the agent, wherein the agent comprises a polynucleotide sequence at least 80% complementary to a sequence selected from the group consisting of SEQ ID NOs: 6-36.

[0013] In some aspects, the disclosure provides methods comprising contacting a cell with an agent or a vector encoding the agent, wherein the agent comprises a polynucleotide sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 37-64.

[0014] In some aspects, the disclosure provides methods comprising contacting a cell with an agent or a vector encoding the agent, wherein the agent comprises at least 8 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 37-64.

[0015] In some embodiments, the agent polynucleotide sequence has at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 37-64. In some embodiments, the agent polynucleotide sequence has 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 37-64. In some embodiments, the agent polynucleotide sequence comprises at least 10, 12, 14, or 16 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 37-64. In some embodiments, the agent polynucleotide sequence comprises at least 18 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 37-64. In some embodiments, the agent polynucleotide sequence comprises at least 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 37-64.

[0016] In some embodiments, the agent is an antisense oligomer. In some embodiments, the antisense oligomer comprises a backbone modification, a modified sugar moiety, or a combination thereof. In some embodiments, the antisense oligomer comprises a phosphorothioate linkage or a phosphorodiamidate linkage. In some embodiments, each internucleotide linkage of the antisense oligomer is a phosphorothioate linkage. In some embodiments, the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl moiety, a 2'-fluoro moiety, a 2'-O-methoxyethyl moiety, or a 2'-NMA moiety. In some embodiments, the antisense oligomer comprises at least one modified sugar moiety. In some embodiments, each nucleotide of the antisense oligomer comprises a modified sugar moiety. In some embodiments, each nucleotide of the antisense oligomer comprises a 2'-O-methoxyethyl moiety. In some embodiments, the antisense oligomer comprises at least one modified nucleobase. In some embodiments, the antisense oligomer comprises hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, or 5-hydroxymethoylcytosine.

[0017] In some embodiments, the antisense oligomer comprises from 8 to 50 nucleobases, from 8 to 40 nucleobases, from 8 to 35 nucleobases, from 8 to 30 nucleobases, from 8 to 25 nucleobases, from 8 to 20 nucleobases, from 8 to 15 nucleobases, from 10 to 50 nucleobases, from 10 to 40 nucleobases, from 10 to 35 nucleobases, from 10 to 30 nucleobases, from 10 to 25 nucleobases, from 10 to 20 nucleobases, from 10 to 15 nucleobases, from 12 to 50 nucleobases, from 12 to 40 nucleobases, from 12 to 35 nucleobases, from 12 to 30 The length of the antisense oligomer is 12-25 nucleobases, 12-20 nucleobases, 12-15 nucleobases, 15-50 nucleobases, 15-40 nucleobases, 15-35 nucleobases, 15-30 nucleobases, 15-25 nucleobases, 15-20 nucleobases, 15-19 nucleobases, 15-18 nucleobases, 15-16 nucleobases, 16-20 nucleobases, 16-19 nucleobases, 16-18 nucleobases, 17-20 nucleobases, 17-19 nucleobases, or 18-20 nucleobases. In some embodiments, the antisense oligomer comprises a sequence set forth in any one of SEQ ID NOs: 66-93.

[0018] In some embodiments, the method includes contacting the cell with a vector, wherein the vector comprises a viral vector encoding the agent. In some embodiments, the viral vector comprises an adenoviral vector, an adeno-associated viral (AAV) vector, a lentiviral vector, a herpes simplex viral (HSV) viral vector, or a retroviral vector.

[0019] In some embodiments, the CFTR gene comprises a mutation downstream of the first intron. In some embodiments, the mutation downstream of the first intron is a nonsense mutation. In some embodiments, the CFTR gene comprises a mutation that results in the presence of an in-frame premature stop codon downstream of the first intron. In some embodiments, at least one allele of the CFTR gene in the cell is selected from the group consisting of c.3717G>A, c.3717+4A>G, c.3717+5G>A, c.3717+40A>G, c.3718-2477C>T, c.3718-1G>A, c.3718-3T>G, c.3719T>G, c.3731G>A, c.3737C>T, c.3744delA, c.3745G>A, c.3747delG, c.3752G>A, c.3761T>G, c.376 3T>C, c.3764C>A, c.3773dupT, c.3806T>A, c.3808delG, c.3808G>A, c.3822G>A, c.3846G>A, c.[3846G>A, 3848G>T], c.3848G>T , c.3872A>G, c.3873G>C, c.3873+1G>A, c.(3873+1_3874-1)_(3963+1_3964-1)del, c.3873+2T>C, 3876delA, c.3883_3886delAT TT, c.3883delA, c.3889dupT, c.3891dupT, c.3908delA, c.3909C>G, c.3929G>A, c.3937C>T, c.(3963+1_3964-1)_(*1_?)del, c .3964-78_4242+577del, c.3971T>C, c.3988C>T, c.4004T>C, c.4036_4042del, c.4046G>A, c.4077_4080delTGTTinsAA, c.4086d upT, c.4097T>A, c.4111G>T, c.4124A>C, c.4127_4131delTGGAT, c.4144C>T, c.4147dupA, c.4197_4198delCT, c.4231C>T, c.4234C>T, c.4242+1G>T, c.4242+1G>A, c.4251delA, c.4300_4301dup, c.4364C>G, c.4426C>T, and c.4439T>C.

[0020] In some aspects, the disclosure provides compositions comprising an agent or a vector encoding an agent, wherein when present in a human cell containing a pre-mRNA transcribed from the CFTR gene and comprising a first intron comprising an alternative polyadenylation site, the agent modifies the CFTR gene or regulates processing of the pre-mRNA, thereby increasing the level of processed mRNA that is processed from the pre-mRNA and lacks the nucleic acid sequence of the pre-mRNA downstream of the first intron.

[0021] In some embodiments, the cell is a human cell and the CFTR gene is a human gene. In some embodiments, the first intron is intron 22. In some embodiments, the first intron is located in the region of GRCh38.p14 / hg38:chr7:117,627,771 to GRCh38.p14 / hg38:chr7:117,642,437. In some embodiments, the agent removes a nucleic acid sequence of the CFTR gene downstream of the first intron from the genome of the cell. In some embodiments, the removed nucleic acid sequence is located in the region of GRCh38.p14 / hg38:chr7:117,642,438 to GRCh38.p14 / hg38:chr7:117,668,665. In some embodiments, the agent comprises a gene editing agent based on CRISPR / Cas9, TALEN, zinc finger, or any combination thereof. In some embodiments, the agent comprises a pair of guide RNAs, the pair of guide RNAs comprising the sequences of SEQ ID NOs: 94 and 95, respectively. In some embodiments, the agent removes a nucleic acid sequence of the pre-mRNA downstream of the first intron from the pre-mRNA. In some embodiments, the removed nucleic acid sequence is located at GRCh38.p14 / hg38:chr7:117,642,438 to GRCh38.p14 / hg38:chr7:117,668,665. In some embodiments, the agent suppresses splicing out from the first intron.

[0022] In some aspects, the disclosure provides a composition comprising an agent or a vector encoding the agent, wherein when present in a cell containing a pre-mRNA transcribed from the CFTR gene and including a first intron containing an alternative polyadenylation site, the agent inhibits splicing out from the first intron. In some embodiments, the cell is a human cell and the CFTR gene is a human gene. In some embodiments, the first intron is intron 22. In some embodiments, the first intron is located in the region from GRCh38.p14 / hg38:chr7:117,627,771 to GRCh38.p14 / hg38:chr7:117,642,437. In some embodiments, the agent increases the level of processed mRNA that is processed from the pre-mRNA and lacks the nucleic acid sequence of the pre-mRNA downstream of the first intron. In some embodiments, the nucleic acid sequence of the pre-mRNA downstream of the first intron is located in the region from GRCh38.p14 / hg38:chr7:117,642,438 to GRCh38.p14 / hg38:chr7:117,668,665.

[0023] In some embodiments, the level of processed mRNA is increased in the cells by at least about 1.5, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, or at least about 20-fold compared to corresponding cells not contacted with the agent or agent-encoding vector. In some embodiments, the level of processed mRNA is increased in the cells by at least about 10-fold compared to corresponding cells not contacted with the agent or agent-encoding vector. In some embodiments, the processed mRNA comprises, in 5' to 3' order, 22 exons, an intron sequence encoding 9 amino acids, a stop codon, and an alternative 3' untranslated region. In some embodiments, the intron sequence encoding the nine amino acids is transcribed from genomic sequences located between chr7:117,627,771 and GRCh38.p14 / hg38:chr7:117,627,797. In some embodiments, the processed mRNA is polyadenylated at an alternative polyadenylation site in the nucleic acid sequence transcribed from genomic sequences located between GRCh38.p14 / hg38:chr7:117,627,771 and GRCh38.p14 / hg38:chr7:117,642,437. In some embodiments, the agent increases the level of a truncated CFTR protein in a cell lacking amino acid sequences expressed from exon sequences of the CFTR gene downstream of the first intron. In some embodiments, the level of truncated CFTR protein in the cells is increased by at least about 1.5, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, or at least about 20-fold compared to corresponding cells not contacted with the agent or a vector encoding the agent.In some embodiments, the truncated CFTR protein in the cell has a chloride channel conductance that is less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% of the chloride channel conductance of the wild-type CFTR protein.

[0024] In some embodiments, the compositions provided herein further comprise a second agent. In some embodiments, the second agent comprises a CFTR protein modulator that enhances chloride conductance of CFTR in cells. In some embodiments, the second agent comprises ivacaftor, lumacaftor, tezacaftor, elexacaftor, a combination of lumacaftor and ivacaftor, a combination of tezacaftor and ivacaftor, or a combination of elexacaftor, ivacaftor, and tezacaftor. In some embodiments, the second agent increases the chloride channel conductance of the truncated CFTR protein by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 950%, at least 1000% improvement. In some embodiments, the second agent restores the chloride channel conductance of the truncated CFTR protein to approximately at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more of the chloride channel conductance of the wild-type CFTR protein.

[0025] In some embodiments, the agent (a) binds to the 5' splice site of the first intron, (b) binds to the 3' splice site of the first intron, (c) binds to the branch point of the 3' splice site of the first intron, or (d) interferes with a splicing factor involved in splicing out of the first intron.

[0026] In some embodiments, the agent comprises a polynucleotide sequence that is at least 80% complementary to at least 8 contiguous nucleic acids of a sequence selected from the group consisting of SEQ ID NOs: 6-36. In some embodiments, the agent comprises a polynucleotide sequence that is at least 80% complementary to at least 10, 12, 14, or 16 contiguous nucleic acids of a sequence selected from the group consisting of SEQ ID NOs: 6-36. In some embodiments, the agent comprises a polynucleotide sequence that is at least 80% complementary to at least 18 contiguous nucleic acids of a sequence selected from the group consisting of SEQ ID NOs: 6-36. In some embodiments, the agent comprises a polynucleotide sequence that is 100% complementary to a sequence selected from the group consisting of SEQ ID NOs: 6-36. In some embodiments, the agent comprises a polynucleotide sequence that has at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 37-64. In some embodiments, the agent comprises a polynucleotide sequence that comprises at least 8 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 37-64.

[0027] In some aspects, the disclosure provides a composition comprising an agent or a vector encoding an agent, wherein the agent comprises a polynucleotide sequence that is at least 80% complementary to a sequence selected from the group consisting of SEQ ID NOs: 6-36.

[0028] In some aspects, the disclosure provides a composition comprising an agent or a vector encoding an agent, wherein the agent comprises a polynucleotide sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 37-64.

[0029] In some aspects, the disclosure provides a composition comprising an agent or a vector encoding an agent, wherein the agent comprises at least 8 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 37-64.

[0030] In some embodiments, the agent polynucleotide sequence has at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 37-64. In some embodiments, the agent polynucleotide sequence has 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 37-64. In some embodiments, the agent polynucleotide sequence comprises at least 10, 12, 14, or 16 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 37-64. In some embodiments, the agent polynucleotide sequence comprises at least 18 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 37-64. In some embodiments, the agent polynucleotide sequence comprises at least 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 37-64. In some embodiments, the agent polynucleotide sequence is at least 80% complementary to at least 8 contiguous nucleic acids of a sequence selected from the group consisting of SEQ ID NOs: 6-36. In some embodiments, the agent polynucleotide sequence is at least 80% complementary to at least 10, 12, 14, or 16 contiguous nucleic acids of a sequence selected from the group consisting of SEQ ID NOs: 6-36. In some embodiments, the agent polynucleotide sequence is at least 80% complementary to at least 18 contiguous nucleic acids of a sequence selected from the group consisting of SEQ ID NOs: 6-36. In some embodiments, the agent polynucleotide sequence is 100% complementary to a sequence selected from the group consisting of SEQ ID NOs: 6-36. In some embodiments, the agent is an antisense oligomer. In some embodiments, the antisense oligomer comprises a backbone modification, a modified sugar moiety, or a combination thereof. In some embodiments, the antisense oligomer comprises a phosphorothioate linkage or a phosphorodiamidate linkage. In some embodiments, each internucleotide linkage of the antisense oligomer is a phosphorothioate linkage. In some embodiments, the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl moiety, a 2'-fluoro moiety, a 2'-O-methoxyethyl moiety, or a 2'-NMA moiety.In some embodiments, the antisense oligomer comprises at least one modified sugar moiety. In some embodiments, each nucleotide of the antisense oligomer comprises a modified sugar moiety. In some embodiments, each nucleotide of the antisense oligomer comprises a 2'-O-methoxyethyl moiety. In some embodiments, the antisense oligomer comprises at least one modified nucleobase. In some embodiments, the antisense oligomer comprises hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, or 5-hydroxymethycytosine.

[0031] In some embodiments, the antisense oligomer comprises from 8 to 50 nucleobases, from 8 to 40 nucleobases, from 8 to 35 nucleobases, from 8 to 30 nucleobases, from 8 to 25 nucleobases, from 8 to 20 nucleobases, from 8 to 15 nucleobases, from 10 to 50 nucleobases, from 10 to 40 nucleobases, from 10 to 35 nucleobases, from 10 to 30 nucleobases, from 10 to 25 nucleobases, from 10 to 20 nucleobases, from 10 to 15 nucleobases, from 12 to 50 nucleobases, from 12 to 40 nucleobases, from 12 to 35 nucleobases, from 12 to 30 The length of the antisense oligomer is 12-25 nucleobases, 12-20 nucleobases, 12-15 nucleobases, 15-50 nucleobases, 15-40 nucleobases, 15-35 nucleobases, 15-30 nucleobases, 15-25 nucleobases, 15-20 nucleobases, 15-19 nucleobases, 15-18 nucleobases, 15-16 nucleobases, 16-20 nucleobases, 16-19 nucleobases, 16-18 nucleobases, 17-20 nucleobases, 17-19 nucleobases, or 18-20 nucleobases. In some embodiments, the antisense oligomer comprises a sequence set forth in any one of SEQ ID NOs: 66-93. In some embodiments, the composition comprises a vector, wherein the vector comprises a viral vector encoding an agent. In some embodiments, the viral vector comprises an adenoviral vector, an adeno-associated viral (AAV) vector, a lentiviral vector, a herpes simplex viral (HSV) viral vector, or a retroviral vector.

[0032] In some aspects, the present disclosure provides methods of treating a subject in need thereof, the method comprising contacting cells of the subject with a composition provided herein. In some embodiments, the cells are ex vivo. In some embodiments, the cells are in vivo. In some embodiments, the method comprises administering the composition to the subject by intranasal administration, intratracheal administration, intravenous injection, intraperitoneal injection, intramuscular injection, intrathecal injection, subcutaneous injection, oral administration, synovial injection, intracerebroventricular injection, intravitreal administration, subretinal injection, topical application, or implantation. In some embodiments, the method comprises administering the composition to the subject via the respiratory route. In some embodiments, the method further comprises administering a second agent to the subject. In some embodiments, the second agent comprises a modulator of CFTR protein that enhances chloride conductance of CFTR in the cell. In some embodiments, the second medicament comprises ivacaftor, lumacaftor, tezacaftor, elexacaftor, a combination of lumacaftor and ivacaftor, a combination of tezacaftor and ivacaftor, or a combination of elexacaftor, ivacaftor, and tezacaftor. In some embodiments, the second medicament comprises a mucolytic agent for airway clearance, optionally selected from the group consisting of acetylcysteine, ambroxol, bromhexine, carbocysteine, erdosteine, mecysteine, dornase alfa, hypertonic saline, and mannitol. In some embodiments, the second medicament comprises a bronchodilator, optionally the bronchodilator is albuterol. In some embodiments, the second medicament comprises an immunosuppressant. In some embodiments, the immunosuppressant is a corticosteroid selected from the group consisting of beclomethasone, budesonide, formoterol, ciclesonide, fluticasone, salmeterol, umeclidinium, vilanterol, mometasone, or mometasone, prednisone, and methylprednisolone, and pharmaceutically acceptable salts thereof.In some embodiments, the immunosuppressant is a nonsteroidal immunosuppressant selected from the group consisting of polyclonal antilymphocyte antibodies, monoclonal antilymphocyte antibodies, interleukin-2 (IL-2) receptor antagonists, calcineurin inhibitors, cell cycle inhibitors, mammalian target of rapamycin (mTOR) inhibitors, methotrexate, cyclophosphamide, anthracyclines, and taxanes.

[0033] In some embodiments, the method treats a disease or disorder caused by a mutation in the CFTR gene in a subject. In some embodiments, the CFTR gene contains a mutation downstream of the first intron. In some embodiments, the mutation downstream of the first intron is a nonsense mutation. In some embodiments, the CFTR gene contains a mutation that results in the presence of an in-frame premature stop codon downstream of the first intron.In some embodiments, at least one allele of the CFTR gene in the subject's cells is selected from the group consisting of c.3717G>A, c.3717+4A>G, c.3717+5G>A, c.3717+40A>G, c.3718-2477C>T, c.3718-1G>A, c.3718-3T>G, c.3719T>G, c.3731G>A, c.3737C>T, c.3744delA, c.3745G>A, c.3747delG, c.3752G>A, c.3761T>G, c.3763T>C, c.3764C>A, c.3773dupT, c.3806T>A, c.3808delG, c.3808G>A, c.3822G>A, c.3846G>A, c.[3846G>A, 3848G>T], c.38 48G>T, c.3872A>G, c.3873G>C, c.3873+1G>A, c.(3873+1_3874-1)_(3963+1_3964-1)del, c.3873+2T>C, c.3883_3886delATTT, c.3883delA, c.3889dupT, c.3891dupT, c.3908delA, c.3909C>G, c.3929G>A, c.3937C>T, c.(3963+1_3964-1)_(*1_?)del, c.39 64-78_4242+577del, c.3971T>C, c.3988C>T, c.4004T>C, c.4036_4042del, c.4046G>A, c.4077_4080delTGTTinsAA, c.4086dup and c.4439T>C. In some embodiments, the method ameliorates or prevents one or more symptoms associated with cystic fibrosis.

[0034] In some aspects, the present disclosure provides a pharmaceutical composition comprising (a) a pharmaceutically acceptable excipient or carrier and (b) a composition provided herein. In some embodiments, the pharmaceutical composition is formulated for intranasal administration, intratracheal administration, intravenous injection, intraperitoneal injection, intramuscular injection, intrathecal injection, subcutaneous injection, oral administration, synovial injection, intracerebroventricular injection, intravitreal administration, subretinal injection, topical application, or implantation. In some embodiments, the pharmaceutical composition is formulated for administration via the respiratory route. In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent comprises a modulator of CFTR protein that enhances chloride conductance of CFTR in cells. In some embodiments, the second agent comprises ivacaftor, lumacaftor, tezacaftor, elexacaftor, a combination of lumacaftor and ivacaftor, a combination of tezacaftor and ivacaftor, or a combination of elexacaftor, ivacaftor, and tezacaftor. In some embodiments, the second medicament comprises a mucolytic agent for airway clearance, optionally selected from the group consisting of acetylcysteine, ambroxol, bromhexine, carbocysteine, erdosteine, mecysteine, dornase alfa, hypertonic saline, and mannitol. In some embodiments, the second medicament comprises a bronchodilator, optionally the bronchodilator is albuterol. In some embodiments, the second medicament comprises an immunosuppressant. In some embodiments, the immunosuppressant is a corticosteroid selected from the group consisting of beclomethasone, budesonide, formoterol, ciclesonide, fluticasone, salmeterol, umeclidinium, vilanterol, mometasone, or mometasone, prednisone, and methylprednisolone, and pharmaceutically acceptable salts thereof.In some embodiments, the immunosuppressant is a nonsteroidal immunosuppressant selected from the group consisting of polyclonal antilymphocyte antibodies, monoclonal antilymphocyte antibodies, interleukin-2 (IL-2) receptor antagonists, calcineurin inhibitors, cell cycle inhibitors, mammalian target of rapamycin (mTOR) inhibitors, methotrexate, cyclophosphamide, anthracyclines, and taxanes.

[0035] In some aspects, the present disclosure provides a kit comprising (a) a composition provided herein or a pharmaceutical composition provided herein, and (b) instructions for use of the composition or pharmaceutical composition.

[0036] In some aspects, the present disclosure provides a kit comprising (a) a composition or pharmaceutical composition provided herein and (b) a second therapeutic agent. In some embodiments, the second therapeutic agent comprises a modulator of CFTR protein that enhances chloride conductance of CFTR in cells. In some embodiments, the second therapeutic agent comprises ivacaftor, lumacaftor, tezacaftor, elexacaftor, a combination of lumacaftor and ivacaftor, a combination of tezacaftor and ivacaftor, or a combination of elexacaftor, ivacaftor, and tezacaftor. In some embodiments, the second therapeutic agent comprises a mucolytic agent for airway clearance, optionally selected from the group consisting of acetylcysteine, ambroxol, bromhexine, carbocysteine, erdosteine, mecysteine, dornase alfa, hypertonic saline, and mannitol. In some embodiments, the second medicament comprises a bronchodilator, optionally the bronchodilator is albuterol. In some embodiments, the second medicament comprises an immunosuppressant. In some embodiments, the immunosuppressant is a corticosteroid selected from the group consisting of beclomethasone, budesonide, formoterol, ciclesonide, fluticasone, salmeterol, umeclidinium, vilanterol, mometasone, or mometasone, prednisone, and methylprednisolone, and pharmaceutically acceptable salts thereof. In some embodiments, the immunosuppressant is a nonsteroidal immunosuppressant selected from the group consisting of a polyclonal antilymphocyte antibody, a monoclonal antilymphocyte antibody, an interleukin-2 (IL-2) receptor antagonist, a calcineurin inhibitor, a cell cycle inhibitor, a mammalian target of rapamycin (mTOR) inhibitor, methotrexate, cyclophosphamide, an anthracycline, and a taxane. In some embodiments, the kit further comprises instructions for use of the composition or pharmaceutical composition and instructions for use of a second therapeutic agent.

[0037] Incorporation by Reference 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 explanation of the drawings]

[0038] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings. [Figure 1A] Figure 1 shows a schematic of W1282X CFTR mRNA processing and a therapeutic strategy to promote exon 22-truncated mRNA (E22-truncated mRNA) levels to regulate the expression of eligible CFTR variants. As shown in Figure 1A, nascent W1282X CFTR mRNA can be processed into two types of mature transcripts. One transcript, the exon 22-truncated mRNA, results from the use of an alternative polyadenylation (ApA) site in intron 22, is able to escape NMD, and lacks a premature termination codon (PTC)-containing exon 23. Another transcript, the FL W1282X CFTR mRNA transcript, contains a PTC in exon 23 and a normal poly(A) tail after the 3'UTR in exon 27, making it susceptible to NMD. Without wishing to be bound by any particular theory, as shown in Figure 1B, methods according to some embodiments of the present disclosure can be used to suppress splicing out of intron 22, thereby increasing the level of exon 22-truncated mRNA transcripts and thus increasing exon 22 CFTR protein expression. Figure 1C lists 13 CFTR variants present in exons 23-27 that result in PTC, including variants that cause CF and variants that exhibit various clinical outcomes. These CFTR variants are not approved for treatment with Trikafta at the time of this application. [Figure 1B]Figure 1 shows a schematic of W1282X CFTR mRNA processing and a therapeutic strategy to promote exon 22-truncated mRNA (E22-truncated mRNA) levels to regulate the expression of eligible CFTR variants. As shown in Figure 1A, nascent W1282X CFTR mRNA can be processed into two types of mature transcripts. One transcript, the exon 22-truncated mRNA, results from the use of an alternative polyadenylation (ApA) site in intron 22, is able to escape NMD, and lacks a premature termination codon (PTC)-containing exon 23. Another transcript, the FL W1282X CFTR mRNA transcript, contains a PTC in exon 23 and a normal poly(A) tail after the 3'UTR in exon 27, making it susceptible to NMD. Without wishing to be bound by any particular theory, as shown in Figure 1B, methods according to some embodiments of the present disclosure can be used to suppress splicing out of intron 22, thereby increasing the level of exon 22-truncated mRNA transcripts and thus increasing exon 22 CFTR protein expression. Figure 1C lists 13 CFTR variants present in exons 23-27 that result in PTC, including variants that cause CF and variants that exhibit various clinical outcomes. These CFTR variants are not approved for treatment with Trikafta at the time of this application. [Figure 1C]Figure 1 shows a schematic of W1282X CFTR mRNA processing and a therapeutic strategy to promote exon 22-truncated mRNA (E22-truncated mRNA) levels to regulate the expression of eligible CFTR variants. As shown in Figure 1A, nascent W1282X CFTR mRNA can be processed into two types of mature transcripts. One transcript, the exon 22-truncated mRNA, results from the use of an alternative polyadenylation (ApA) site in intron 22, is able to escape NMD, and lacks a premature termination codon (PTC)-containing exon 23. Another transcript, the FL W1282X CFTR mRNA transcript, contains a PTC in exon 23 and a normal poly(A) tail after the 3'UTR in exon 27, making it susceptible to NMD. Without wishing to be bound by any particular theory, as shown in Figure 1B, methods according to some embodiments of the present disclosure can be used to suppress splicing out of intron 22, thereby increasing the level of exon 22-truncated mRNA transcripts and thus increasing exon 22 CFTR protein expression. Figure 1C lists 13 CFTR variants present in exons 23-27 that result in PTC, including variants that cause CF and variants that exhibit various clinical outcomes. These CFTR variants are not approved for treatment with Trikafta at the time of this application. [Figure 2A]Figure 2A shows the discovery of 3' truncated transcripts in 16HBEge-W1282X and 16HBEge-R1162X cells, indicated by low sequence coverage of the 3'-terminal exons (e.g., exons 23-27) of the CFTR transcript. All sequence counts were normalized to wild-type 16HBE cells, and sequence coverage data for the CFTR exon 7 to exon 27 region are shown. Sequencing results from the 16HBEge-Y122X cell line showed a coverage profile similar to that of wild-type cells, as no splicing liability was expected. Both R553X and G542X showed reduced coverage in exon 12, consistent with previous experiments. Figure 2B shows an exemplary sequence of a truncated 16HBEge-W1282X CFTR transcript terminating within intron 22 due to an approximately 140-bp extension into the 3' intron. The 16HBEge-W1282X transcript ended with a series of unaligned adenosine nucleotides. Consistent with this finding, a consensus ApA (alternative polyadenylation) motif was identified within intron 22 near the polyadenylation site described above, indicating ApA usage. Figure 2C shows an IGV screen capture of CFTR 3' RACE reads from 16HBE14o cells aligned to the Hg38 reference genome. A close-up of the IGV screen capture shows the location of the putative ApA hexanucleotide and CA cleavage site in the black box. Figure 2D shows the CFTR genomic locus with intron 22 ApA. The intron is not to scale. A representation of the E22-truncated mRNA shows the alternative 3' UTR and polyA tail. The E22-truncated protein representation shows an additional nine alternative amino acids and the deleted NBD2. [Figure 2B]Figure 2A shows the discovery of 3' truncated transcripts in 16HBEge-W1282X and 16HBEge-R1162X cells, indicated by low sequence coverage of the 3'-terminal exons (e.g., exons 23-27) of the CFTR transcript. All sequence counts were normalized to wild-type 16HBE cells, and sequence coverage data for the CFTR exon 7 to exon 27 region are shown. Sequencing results from the 16HBEge-Y122X cell line showed a coverage profile similar to that of wild-type cells, as no splicing liability was expected. Both R553X and G542X showed reduced coverage in exon 12, consistent with previous experiments. Figure 2B shows an exemplary sequence of a truncated 16HBEge-W1282X CFTR transcript terminating within intron 22 due to an approximately 140-bp extension into the 3' intron. The 16HBEge-W1282X transcript ended with a series of unaligned adenosine nucleotides. Consistent with this finding, a consensus ApA (alternative polyadenylation) motif was identified within intron 22 near the polyadenylation site described above, indicating ApA usage. Figure 2C shows an IGV screen capture of CFTR 3' RACE reads from 16HBE14o cells aligned to the Hg38 reference genome. A close-up of the IGV screen capture shows the location of the putative ApA hexanucleotide and CA cleavage site in the black box. Figure 2D shows the CFTR genomic locus with intron 22 ApA. The intron is not to scale. A representation of the E22-truncated mRNA shows the alternative 3' UTR and polyA tail. The E22-truncated protein representation shows an additional nine alternative amino acids and the deleted NBD2. [Figure 2C]Figure 2A shows the discovery of 3' truncated transcripts in 16HBEge-W1282X and 16HBEge-R1162X cells, indicated by low sequence coverage of the 3'-terminal exons (e.g., exons 23-27) of the CFTR transcript. All sequence counts were normalized to wild-type 16HBE cells, and sequence coverage data for the CFTR exon 7 to exon 27 region are shown. Sequencing results from the 16HBEge-Y122X cell line showed a coverage profile similar to that of wild-type cells, as no splicing liability was expected. Both R553X and G542X showed reduced coverage in exon 12, consistent with previous experiments. Figure 2B shows an exemplary sequence of a truncated 16HBEge-W1282X CFTR transcript terminating within intron 22 due to an approximately 140-bp extension into the 3' intron. The 16HBEge-W1282X transcript ended with a series of unaligned adenosine nucleotides. Consistent with this finding, a consensus ApA (alternative polyadenylation) motif was identified within intron 22 near the polyadenylation site described above, indicating ApA usage. Figure 2C shows an IGV screen capture of CFTR 3' RACE reads from 16HBE14o cells aligned to the Hg38 reference genome. A close-up of the IGV screen capture shows the location of the putative ApA hexanucleotide and CA cleavage site in the black box. Figure 2D shows the CFTR genomic locus with intron 22 ApA. The intron is not to scale. A representation of the E22-truncated mRNA shows the alternative 3' UTR and polyA tail. The E22-truncated protein representation shows an additional nine alternative amino acids and the deleted NBD2. [Figure 2D]Figure 2A shows the discovery of 3' truncated transcripts in 16HBEge-W1282X and 16HBEge-R1162X cells, indicated by low sequence coverage of the 3'-terminal exons (e.g., exons 23-27) of the CFTR transcript. All sequence counts were normalized to wild-type 16HBE cells, and sequence coverage data for the CFTR exon 7 to exon 27 region are shown. Sequencing results from the 16HBEge-Y122X cell line showed a coverage profile similar to that of wild-type cells, as no splicing liability was expected. Both R553X and G542X showed reduced coverage in exon 12, consistent with previous experiments. Figure 2B shows an exemplary sequence of a truncated 16HBEge-W1282X CFTR transcript terminating within intron 22 due to an approximately 140-bp extension into the 3' intron. The 16HBEge-W1282X transcript ended with a series of unaligned adenosine nucleotides. Consistent with this finding, a consensus ApA (alternative polyadenylation) motif was identified within intron 22 near the polyadenylation site described above, indicating ApA usage. Figure 2C shows an IGV screen capture of CFTR 3' RACE reads from 16HBE14o cells aligned to the Hg38 reference genome. A close-up of the IGV screen capture shows the location of the putative ApA hexanucleotide and CA cleavage site in the black box. Figure 2D shows the CFTR genomic locus with intron 22 ApA. The intron is not to scale. A representation of the E22-truncated mRNA shows the alternative 3' UTR and polyA tail. The E22-truncated protein representation shows an additional nine alternative amino acids and the deleted NBD2. [Figure 3] 1 is a table showing expression levels of E22 truncated mRNA and full-length CFTR mRNA in wild-type and W1282X airway and intestinal cells. [Figure 4]Figure 1 shows the predicted effect of exon 22 truncation on the CFTR protein. The truncated mRNA sequence is approximately 3,857 base pairs (bp) long, containing the first 22 exons and approximately 140 bp of intron 22 sequence. The first 27 bp of intron 22 sequence encodes 9 amino acids in frame, followed by a stop codon and an alternative 3' UTR (untranslated region). Thus, the truncated CFTR protein resulting from the 3' truncated exon 22 mRNA-ApA may contain the first 1,239 amino acids of WT CFTR plus several additional amino acids (approximately 9) encoded by intronic sequences within intron 22. [Figure 5] A graph plotting the percent of exon 22 truncated mRNA transcripts normalized to full-length CFTR transcripts ("FL CFTR"). A higher fraction of exon 22 truncated mRNA over FL CFTR was observed in 16HBEge-W1282X and R1162X cells compared to the fraction in wild-type cells (WT), and addition of SMG-1 inhibitor significantly reduced this fraction in the cells, indicating NMD escape in transcript processing. [Figure 6A]A schematic diagram of the RNA stability test is shown. Briefly, the percentage of remaining mRNA was monitored using droplet digital PCR (ddPCR) after actinomycin D treatment in the mutant cell line W1282X-I22-SAd, which was generated by disrupting the splice acceptor site in intron 22. W1282X-I22-SAd cells produced three CFTR transcripts: exon 22-truncated mRNA, full-length CFTR mRNA containing the PTC ("FL-W1282X transcript"), and exon 23-skipped CFTR mRNA. The top panel of Figure 6B shows a graph plotting the mRNA levels of these three transcripts in W1282X-I22-SAd cells at steady state. The exon 22-truncated mRNA transcript was found to be present at levels more than four-fold higher than those in the W1282X parent strain. The bottom panel of Figure 6B shows exemplary results from the RNA stability test. The amount of each CFTR mRNA species assessed at the first time point (t = 0) was set to 1 (100%), and the remaining mRNA fraction was measured by ddPCR. mRNA half-life was calculated from exponential decay. The t of the exon 22-truncated mRNA was estimated to be approximately 4.24 h, significantly longer than the t of the FL-W1282X transcript (<<<2 h). The exon 23-skipped mRNA transcript showed a long half-life (t = 10.61 h), comparable to that of the wild-type CFTR transcript. [Figure 6B]A schematic diagram of the RNA stability test is shown. Briefly, the percentage of remaining mRNA was monitored using droplet digital PCR (ddPCR) after actinomycin D treatment in the mutant cell line W1282X-I22-SAd, which was generated by disrupting the splice acceptor site in intron 22. W1282X-I22-SAd cells produced three CFTR transcripts: exon 22-truncated mRNA, full-length CFTR mRNA containing the PTC ("FL-W1282X transcript"), and exon 23-skipped CFTR mRNA. The top panel of Figure 6B shows a graph plotting the mRNA levels of these three transcripts in W1282X-I22-SAd cells at steady state. The exon 22-truncated mRNA transcript was found to be present at levels more than four-fold higher than those in the W1282X parent strain. The bottom panel of Figure 6B shows exemplary results from the RNA stability test. The amount of each CFTR mRNA species assessed at the first time point (t = 0) was set to 1 (100%), and the remaining mRNA fraction was measured by ddPCR. mRNA half-life was calculated from exponential decay. The t of the exon 22-truncated mRNA was estimated to be approximately 4.24 h, significantly longer than the t of the FL-W1282X transcript (<<<2 h). The exon 23-skipped mRNA transcript showed a long half-life (t = 10.61 h), comparable to that of the wild-type CFTR transcript. [Figure 7A]Figure 7A shows exemplary results from a transepithelial chloride conductance assay (TECC-24 assay) of transiently expressed CFTR variants under Trikafta treatment. Figure 7A shows results from a TECC-24 assay for exon 22-truncated CFTR variants and F508delCFTR variants under Trikafta treatment. Compared to the untreated group, F508del CFTR function was enhanced in vitro by the addition of Trikafta, as indicated by an increase in the area under the curve (AUC) of CFTR-mediated chloride current. Similarly, exon 22-truncated CFTR function was also enhanced by Trikafta. Trikafta was observed to restore F508del CFTR function to approximately 35% of wild-type function and enhance exon 22-truncated CFTR function to approximately 15% of wild-type function. Figure 7B shows results from a TECC-24 assay for FRT cells overexpressing the exon 22-truncated cDNA. [Figure 7B] Figure 7A shows exemplary results from a transepithelial chloride conductance assay (TECC-24 assay) of transiently expressed CFTR variants under Trikafta treatment. Figure 7A shows results from a TECC-24 assay for exon 22-truncated CFTR variants and F508delCFTR variants under Trikafta treatment. Compared to the untreated group, F508del CFTR function was enhanced in vitro by the addition of Trikafta, as indicated by an increase in the area under the curve (AUC) of CFTR-mediated chloride current. Similarly, exon 22-truncated CFTR function was also enhanced by Trikafta. Trikafta was observed to restore F508del CFTR function to approximately 35% of wild-type function and enhance exon 22-truncated CFTR function to approximately 15% of wild-type function. Figure 7B shows results from a TECC-24 assay for FRT cells overexpressing the exon 22-truncated cDNA. [Figure 8]A schematic diagram of the Δ23-27 (or Del23-27) gene editing model, 16HBE14o cells, is shown, which has a genomic deletion of the region spanning from the 5' portion of intron 22 to after the 3' UTR. The resulting cell genome contains exons 1-22, followed by approximately 13.5 kb of intron 22, and then the intergenic region upstream of CTTNBP2. [Figure 9] Sequencing results are shown for three exemplary Delta23-27 gene-editing model cell lines: 2-H07, 3-B09, and 3-D01. Exon 22-truncated CFTR mRNA transcripts (black bars) from the three Del23-27 clonal lines and 16HBE14o cells were assayed using ddPCR. Full-length CFTR was measured from 16HBE14o cells (gray bars). All three Del23-27 cell lines contained significantly higher levels of exon 22-truncated CFTR mRNA compared to the parental 16HBE14o line, approximately 12-fold higher than in the parental cells. The levels of exon 22-truncated CFTR mRNA in all three Del23-27 cell lines were higher than the levels of full-length WT CFTR transcripts in the parental 16HBE14o cells. [Figure 10]Figure 1 shows exemplary Western blot results of truncated forms of CFTR protein detected from Del23-27 clonal lines (2-H07, 3-B09, 3-D01) using α-CFTR UNC596 antibody (raised against the epitope of Exon 22: 1204-1211 aa). Reactivity against exon 22 was detected in two bands (bands C and B) in all Del23-27 cells. Seven leftmost lanes: 2-H07, 3-B09, 3-D01, and Z23-27. Western blot analysis of gene edited (+ / -) VX-445 / VX-661 and 16HBE14o- parental lines. Six rightmost lanes: PNGaseF-treated (deglycosylated) W1282X, exon 22 truncated form, cDNA overexpression control from HEK293 cells, and Δ23-27. The 2-H07 and 16HBE14o parental strains were diluted to produce comparable band intensities. ACTB and Na / K-ATPase loading controls are grayed out for PNGaseF-treated samples. Na / K-ATPase was blotted as a loading control. VX-445 / VX-661 (3 / 3 μM) treatment did not significantly affect the levels of truncated CFTR protein. [Figure 11A]Figure 11 shows exemplary results from a TECC-24 assay performed in various cell types treated with DMSO (vehicle) or pretreated with VX-445 / VX-661 (3 / 3 μM) for 48 hours. Figure 11A shows the results for WT16HBE14o cells. In the presence of VX-445 / VX-661, Del23-27 CFTR protein function was restored by VX-770, as shown by chloride conductance induced by VX-770 and inhibited by CFTR(inh)-172. All samples were treated in the assay with VX-770 (1 μM). As shown in Figure 11B, CFTR function in the three Del23-27 cell lines, 2-H07, 3-B09, and 3-D01, was restored to approximately 25%, 17%, and 18% of WT function, respectively, much higher than that of F508Del, which was enhanced by VX-809 / VX-770 (approximately 5% of WT). Figure 11C shows the TECC-24 Ieq assay results for 16HBE14- and CFF-16HBEge-W1282X, as well as dose-escalation of CFF-16HBEge-W1282X-Δ23-27-2H07(+ / -)VX-445 / VX-661 3 / 3 μM. [Figure 11B]Figure 11 shows exemplary results from a TECC-24 assay performed in various cell types treated with DMSO (vehicle) or pretreated with VX-445 / VX-661 (3 / 3 μM) for 48 hours. Figure 11A shows the results for WT16HBE14o cells. In the presence of VX-445 / VX-661, Del23-27 CFTR protein function was restored by VX-770, as shown by chloride conductance induced by VX-770 and inhibited by CFTR(inh)-172. All samples were treated in the assay with VX-770 (1 μM). As shown in Figure 11B, CFTR function in the three Del23-27 cell lines, 2-H07, 3-B09, and 3-D01, was restored to approximately 25%, 17%, and 18% of WT function, respectively, much higher than that of F508Del, which was enhanced by VX-809 / VX-770 (approximately 5% of WT). Figure 11C shows the TECC-24 Ieq assay results for 16HBE14- and CFF-16HBEge-W1282X, as well as dose-escalation of CFF-16HBEge-W1282X-Δ23-27-2H07(+ / -)VX-445 / VX-661 3 / 3 μM. [Figure 11C]Figure 11 shows exemplary results from a TECC-24 assay performed in various cell types treated with DMSO (vehicle) or pretreated with VX-445 / VX-661 (3 / 3 μM) for 48 hours. Figure 11A shows the results for WT16HBE14o cells. In the presence of VX-445 / VX-661, Del23-27 CFTR protein function was restored by VX-770, as shown by chloride conductance induced by VX-770 and inhibited by CFTR(inh)-172. All samples were treated in the assay with VX-770 (1 μM). As shown in Figure 11B, CFTR function in the three Del23-27 cell lines, 2-H07, 3-B09, and 3-D01, was restored to approximately 25%, 17%, and 18% of WT function, respectively, much higher than that of F508Del, which was enhanced by VX-809 / VX-770 (approximately 5% of WT). Figure 11C shows the TECC-24 Ieq assay results for 16HBE14- and CFF-16HBEge-W1282X, as well as dose-escalation of CFF-16HBEge-W1282X-Δ23-27-2H07(+ / -)VX-445 / VX-661 3 / 3 μM. [Figure 12A] A schematic diagram of ASO sequence design is shown. Steric-blocking ASOs were designed to target the intron 22 donor site (black bars) or acceptor site (gray bars) via a 10-step, 1-nucleotide "walk" tiled scheme. Figure 12B shows two graphs demonstrating increased expression of exon 22-truncated CFTR mRNA in 16HBE14o-WT cells after administration of exemplary ASOs targeting the intron 22 donor (ASO SD, middle) or acceptor (ASO SA, bottom). SD10 (black bars, top) and SA8 (black bars, bottom) increased the amount of FL CFTR mRNA by approximately 37% and the amount of exon 22-truncated CFTR mRNA by approximately 25%, respectively, in WT cells. Scrambled ASOs, off-target ASOs (ASOs targeting CEP290 mRNA), and untreated cells were included in the experiment as controls. [Figure 12B]A schematic diagram of ASO sequence design is shown. Steric-blocking ASOs were designed to target the intron 22 donor site (black bars) or acceptor site (gray bars) via a 10-step, 1-nucleotide "walk" tiled scheme. Figure 12B shows two graphs demonstrating increased expression of exon 22-truncated CFTR mRNA in 16HBE14o-WT cells after administration of exemplary ASOs targeting the intron 22 donor (ASO SD, middle) or acceptor (ASO SA, bottom). SD10 (black bars, top) and SA8 (black bars, bottom) increased the amount of FL CFTR mRNA by approximately 37% and the amount of exon 22-truncated CFTR mRNA by approximately 25%, respectively, in WT cells. Scrambled ASOs, off-target ASOs (ASOs targeting CEP290 mRNA), and untreated cells were included in the experiment as controls. [Figure 13A] 13A shows bar graphs depicting the changes in exon 22-truncated CFTR mRNA after treating 16HBEge-W1282X cells with various doses of exemplary ASOs. Figure 13A shows that both SA08 and SD10 ASOs individually modulated CFTR mRNA processing and increased the amount of exon 22-truncated CFTR mRNA. Treatment with the two ASOs in combination further increased the amount of exon 22-truncated CFTR mRNA. Figure 13B shows the effects of exemplary ASOs at various doses. SD-10 and SA-08 ASOs were administered at up to 100 μM and 10 μM, respectively. [Figure 13B]13A shows bar graphs depicting the changes in exon 22-truncated CFTR mRNA after treating 16HBEge-W1282X cells with various doses of exemplary ASOs. Figure 13A shows that both SA08 and SD10 ASOs individually modulated CFTR mRNA processing and increased the amount of exon 22-truncated CFTR mRNA. Treatment with the two ASOs in combination further increased the amount of exon 22-truncated CFTR mRNA. Figure 13B shows the effects of exemplary ASOs at various doses. SD-10 and SA-08 ASOs were administered at up to 100 μM and 10 μM, respectively. [Figure 14] 1 shows Western blot analysis of deglycosylated exon 22-truncated CFTR protein expression in 16HBEge-W1282X cells treated with exemplary ASOs alone or in combination (e.g., SD10; SD10 and SA08) in the presence of drug vehicle or corrector VX-445 / 661. Expression of exon 22-truncated CFTR protein was increased by both treatment with SD10 alone and treatment with the SD10 / SA08 combination, whereas treatment with correctors VX-445 / VX-661 did not significantly affect expression of exon 22-truncated CFTR protein. [Figure 15A]Figure 15A shows the effect of ASOs on 16HBEge-W1282X cells. Figure 15A shows exemplary chloride conductance traces from a TECC-24 assay performed with ASOs and trikafta administration in 16HBEge-W1282X cells. Treatment with exemplary ASOs, SD10 and SA8, induced much greater chloride channel conductance in the presence of trikafta (VX-445 / VX-661 / VX-770). Figure 15B shows the enhancement of chloride conductance AUC (percentage of WT CFTR AUC) normalized to WT CFTR in 16HBEge-W1282X cells after treatment with exemplary ASOs, with or without trikafta treatment. All ASO treatments increased the percent of WT CFTR AUC compared to vehicle alone. In the presence of trikafta, ASOs further enhanced the percent of WT CFTR AUC in a dose-dependent manner. The combination of the two ASOs significantly increased the percentage of WT AUC compared to either ASO alone. The greatest effect was observed in the 2 μM SA8 / 10 μM SD10 group with Trikafta treatment, which enhanced chloride conductance AUC of 16HBEge-W1282X cells to approximately 13.4% of WT. Figure 15C shows the TECC-24 assay results for various ASO doses. CFF-16HBEge W1282X cells were treated with DMSO (vehicle), SD-10, or SA-08 alone or in combination for 48 hours, with or without 48-hour pretreatment with VX-445 / VX-661 (3 / 3 μM). All samples were treated with VX-770 (3 μM) in the assay. The combination of the two ASOs significantly increased chloride conductance compared to either ASO alone. The greatest effect was observed in the 10 μM SA8 / 100 uM SD10 group with Trikafta treatment. [Figure 15B]Figure 15A shows the effect of ASOs on 16HBEge-W1282X cells. Figure 15A shows exemplary chloride conductance traces from a TECC-24 assay performed with ASOs and trikafta administration in 16HBEge-W1282X cells. Treatment with exemplary ASOs, SD10 and SA8, induced much greater chloride channel conductance in the presence of trikafta (VX-445 / VX-661 / VX-770). Figure 15B shows the enhancement of chloride conductance AUC (percentage of WT CFTR AUC) normalized to WT CFTR in 16HBEge-W1282X cells after treatment with exemplary ASOs, with or without trikafta treatment. All ASO treatments increased the percent of WT CFTR AUC compared to vehicle alone. In the presence of trikafta, ASOs further enhanced the percent of WT CFTR AUC in a dose-dependent manner. The combination of the two ASOs significantly increased the percentage of WT AUC compared to either ASO alone. The greatest effect was observed in the 2 μM SA8 / 10 μM SD10 group with Trikafta treatment, which enhanced chloride conductance AUC of 16HBEge-W1282X cells to approximately 13.4% of WT. Figure 15C shows the TECC-24 assay results for various ASO doses. CFF-16HBEge W1282X cells were treated with DMSO (vehicle), SD-10, or SA-08 alone or in combination for 48 hours, with or without 48-hour pretreatment with VX-445 / VX-661 (3 / 3 μM). All samples were treated with VX-770 (3 μM) in the assay. The combination of the two ASOs significantly increased chloride conductance compared to either ASO alone. The greatest effect was observed in the 10 μM SA8 / 100 uM SD10 group with Trikafta treatment. [Figure 15C]Figure 15A shows the effect of ASOs on 16HBEge-W1282X cells. Figure 15A shows exemplary chloride conductance traces from a TECC-24 assay performed with ASOs and trikafta administration in 16HBEge-W1282X cells. Treatment with exemplary ASOs, SD10 and SA8, induced much greater chloride channel conductance in the presence of trikafta (VX-445 / VX-661 / VX-770). Figure 15B shows the enhancement of chloride conductance AUC (percentage of WT CFTR AUC) normalized to WT CFTR in 16HBEge-W1282X cells after treatment with exemplary ASOs, with or without trikafta treatment. All ASO treatments increased the percent of WT CFTR AUC compared to vehicle alone. In the presence of trikafta, ASOs further enhanced the percent of WT CFTR AUC in a dose-dependent manner. The combination of the two ASOs significantly increased the percentage of WT AUC compared to either ASO alone. The greatest effect was observed in the 2 μM SA8 / 10 μM SD10 group with Trikafta treatment, which enhanced chloride conductance AUC of 16HBEge-W1282X cells to approximately 13.4% of WT. Figure 15C shows the TECC-24 assay results for various ASO doses. CFF-16HBEge W1282X cells were treated with DMSO (vehicle), SD-10, or SA-08 alone or in combination for 48 hours, with or without 48-hour pretreatment with VX-445 / VX-661 (3 / 3 μM). All samples were treated with VX-770 (3 μM) in the assay. The combination of the two ASOs significantly increased chloride conductance compared to either ASO alone. The greatest effect was observed in the 10 μM SA8 / 100 uM SD10 group with Trikafta treatment. [Figure 16] Bar graph showing exon 22-truncated CFTR mRNA levels in primary W1282X+ / + HBEs after 2 or 3 weeks of treatment with exemplary ASOs. In both long-term treatment groups, exon 22-truncated CFTR mRNA expression increased dramatically compared to no ASO treatment. [Figure 17]Representative Ieq traces of chloride conductance from a TECC-24 assay are shown. Transepithelial electrical resistance (TEER) was consistent across all experimental groups, and no toxicity was observed with repeated treatment. Stronger chloride conductance (Ieq) traces were observed after 2 and 3 weeks of treatment with the exemplary ASO. [Figure 18] 1 is a bar graph showing that 2- and 3-week long-term ASO treatment increased the normalized chloride conductance AUC measured in primary W1282X+ / + HBE cells and improved the function of Exon22-truncated CFTR protein. [Figure 19] Bar graph showing changes in exon 22 truncated CFTR mRNA after treatment of fully differentiated primary W1282X+ / + HBEs. Cells were treated with DMSO (vehicle) or SD-10 or SA-08, alone or in combination, for 48 hours in the TECC-24 assay, with or without 48-hour VX-445 / VX-661 (3 / 3 μM) pretreatment. All samples were treated with VX-770 (1 μM) in the assay. Treatment with SD-10 (100 μM) and SA-08 (10 μM) resulted in the highest amount of E22 truncated mRNA. [Figure 20A] Figure 20A shows CFTR protein expression in primary W1282X+ / + HBE cells after treatment with ASOs and drugs. Figure 20A shows a Western blot analysis from fully differentiated HBE W1282X+ / + cells at air-liquid interphase (ALI) after the TECC-24 assay described above. CFTR UNC596 and beta-actin (ACTB) were used to detect CFTR, respectively, and served as loading controls. Figure 20B shows bar graphs of CFTR B and C normalized to (-) ASO control (-) VX-445 / VX-661 (3 / 3 μM). Administered ASOs increased the expression of E22-truncated CFTR protein. [Figure 20B]Figure 20A shows CFTR protein expression in primary W1282X+ / + HBE cells after treatment with ASOs and drugs. Figure 20A shows a Western blot analysis from fully differentiated HBE W1282X+ / + cells at air-liquid interphase (ALI) after the TECC-24 assay described above. CFTR UNC596 and beta-actin (ACTB) were used to detect CFTR, respectively, and served as loading controls. Figure 20B shows bar graphs of CFTR B and C normalized to (-) ASO control (-) VX-445 / VX-661 (3 / 3 μM). Administered ASOs increased the expression of E22-truncated CFTR protein. [Figure 21] Results of the TECC-24 assay from fully differentiated HBE W1282X+ / + at the ALI are shown, following 48-hour treatment with DMSO (vehicle) or SD-10 or SA-08, alone or in combination, with or without pretreatment with VX-445 / VX-661 (3 / 3 μM) for 48 hours. All samples were treated in the assay with VX-770 (1 μM). Data are expressed as the ratio of %WT HBE14 (AUC / min) W1282X(FSK+VX-770) / WT(FSK). ASO treatment significantly restored chloride conductance of mutant CFTR. Detailed Description of the Invention

[0039] In some aspects, the present disclosure relates to compositions, methods, and kits that include agents that modulate the expression of the CFTR gene. The agents provided herein can modify the CFTR gene or modulate the processing of pre-mRNA transcribed from the CFTR gene (CFTR pre-mRNA).

[0040] In some cases, the agent increases the production of exon 22-truncated CFTR mRNA, thus increasing the expression of C-terminally truncated CFTR protein. The C-terminally truncated CFTR protein produced by translation of exon 22-truncated CFTR mRNA may have partial function, for example, compared to wild-type CFTR protein, and have a chloride channel conductance that is less than 100% (e.g., 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or up to 5%) of the chloride channel conductance of the wild-type CFTR protein. In some embodiments, the present disclosure relates to compositions, methods, and kits that include promoting the expression of C-terminally truncated CFTR protein produced by translation of exon 22-truncated CFTR mRNA in cells and restoring CFTR protein function in cells by simultaneous or subsequent treatment of the cells with a CFTR modifying or enhancing agent, for example, an agent that enhances the chloride channel conductance of the CFTR protein.

[0041] In some cases, the compositions, methods, and kits provided herein include an agent (e.g., an antisense oligomer (ASO)) that can modulate splicing events around intron 22 of CFTR pre-mRNA in a cell, thereby increasing the level of exon 22-truncated CFTR mRNA in the cell. In some cases, the compositions, methods, and kits provided herein include an agent (e.g., an antisense oligomer (ASO)) that can promote alternative polyadenylation (ApA) usage within intron 22 of CFTR pre-mRNA in a cell, thereby increasing the level of exon 22-truncated CFTR mRNA in the cell. In various embodiments, the level of C-terminally truncated CFTR protein can be increased using the methods of the present disclosure to treat diseases and disorders associated with one or more genetic mutations in the CFTR gene located downstream (3' direction) of intron 22.

[0042] As shown in Figure 1A, in some cases, nascent pre-mRNA transcripts of some mutant CFTRs (e.g., W1282X CFTR mRNA) can be processed into two types of mature mRNA transcripts. One transcript, the full-length CFTR mRNA transcript, contains a PTC in exon 23 caused by a genetic mutation, such as W1282X in this case. The full-length PTC-containing CFTR mRNA is polyadenylated at the normal polyadenylation site in the 3' untranslated region (3'UTR) of the CFTR pre-mRNA and may be susceptible to NMD due to the presence of the PTC in exon 23. Another transcript, the exon 22-truncated CFTR mRNA transcript, lacks the exon downstream of intron 22 and is polyadenylated at one of the alternative polyadenylation sites within intron 22. The exon 22-truncated CFTR mRNA transcript lacks the PTC-containing exon 23 and can escape NMD in cells. C-terminally truncated CFTR proteins produced from exon 22-truncated CFTR mRNA transcripts can be regulated by CFTR correctors and enhancers, such as trikafta. Without wishing to be bound by theory, as shown in Figure 1B, methods according to some embodiments of the present disclosure can be used to increase the level of exon 22-truncated CFTR mRNA transcripts by blocking splicing of exons 22-23, thereby increasing the expression of C-terminally truncated CFTR proteins, e.g., exon 22-truncated CFTR proteins.

[0043] In some cases, the methods provided herein regulate expression of the CFTR gene in a cell. The method can include contacting a cell with an agent or a vector encoding the agent, wherein the cell contains a pre-mRNA, the pre-mRNA being transcribed from the CFTR gene and containing a first intron that includes an alternative polyadenylation site, and the agent modifies the CFTR gene or regulates processing of the pre-mRNA, thereby increasing the level of processed mRNA that is processed from the pre-mRNA and lacks the nucleic acid sequence of the pre-mRNA downstream of the first intron.

[0044] In some cases, the method includes contacting an agent or a vector encoding the agent with a cell, wherein the cell includes a pre-mRNA transcribed from the CFTR gene and includes a first intron comprising an alternative polyadenylation site, and wherein the agent inhibits splicing out of the first intron from the pre-mRNA during splicing of the pre-mRNA in the cell.

[0045] In some cases, the method includes contacting a cell with an agent or a vector encoding the agent, wherein the agent comprises a polynucleotide sequence that is at least 80% complementary to a sequence selected from the group consisting of SEQ ID NOs: 6-36.

[0046] In some cases, the method includes contacting a cell with an agent or a vector encoding the agent, wherein the agent comprises a polynucleotide sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 37-64.

[0047] In some cases, the method includes contacting a cell with an agent or a vector encoding the agent, wherein the agent comprises at least 8 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 37-64.

[0048] In some cases, the method includes contacting a cell with an agent or a vector encoding the agent, wherein the agent comprises a polynucleotide comprising a sequence set forth in any one of SEQ ID NOs: 66-93. In some cases, the method includes contacting a cell with an agent or a vector encoding the agent, wherein the agent comprises a polynucleotide consisting of a sequence set forth in any one of SEQ ID NOs: 66-93.

[0049] In some cases, the methods provided herein include an agent or a vector encoding the agent, wherein when the agent is present in a human cell containing a pre-mRNA transcribed from the CFTR gene and including a first intron containing an alternative polyadenylation site, the agent modifies the CFTR gene or regulates processing of the pre-mRNA, thereby increasing the level of processed mRNA that is processed from the pre-mRNA and lacks the nucleic acid sequence of the pre-mRNA downstream of the first intron.

[0050] In some cases, the composition comprises an agent or a vector encoding the agent, wherein when the agent is present in a cell containing a pre-mRNA transcribed from the CFTR gene and containing a first intron that contains an alternative polyadenylation site, the agent inhibits splicing out of the first intron.

[0051] In some cases, the composition comprises an agent or a vector encoding the agent, wherein the agent comprises a polynucleotide sequence that is at least 80% complementary to a sequence selected from the group consisting of SEQ ID NOs: 6-36.

[0052] In some cases, the composition comprises an agent or a vector encoding the agent, wherein the agent comprises a polynucleotide sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 37-64.

[0053] In some cases, the composition comprises an agent or a vector encoding the agent, wherein the agent comprises at least 8 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 37-64.

[0054] In some cases, the composition comprises an agent or a vector encoding the agent, wherein the agent comprises a polynucleotide comprising a sequence set forth in any one of SEQ ID NOs: 66-93. In some cases, the composition comprises an agent or a vector encoding the agent, wherein the agent comprises a polynucleotide consisting of a sequence set forth in any one of SEQ ID NOs: 66-93.

[0055] In some cases, the methods provided herein are for treating a subject in need thereof. The treatment methods provided herein can include contacting the subject's cells with the compositions provided herein. In some cases, the methods include administering to the subject a second therapeutic agent simultaneously or sequentially.

[0056] In some cases, the pharmaceutical compositions provided herein comprise a pharmaceutically acceptable excipient or carrier and a composition provided herein.

[0057] In some cases, the kits provided herein include a composition or pharmaceutical composition disclosed herein and instructions for use of the composition or pharmaceutical composition. In some cases, the kits provided herein include a composition or pharmaceutical composition disclosed herein and a second therapeutic agent.

[0058] CFTR The CFTR gene encodes the cystic fibrosis transmembrane conductance regulator (CFTR protein), a member of the ATP-binding cassette (ABC) transporter superfamily. ABC proteins can transport various molecules across extracellular and intracellular membranes. Generally, ABC genes are divided into seven distinct subfamilies (ABC1, MDR / TAP, MRP, ALD, OABP, GCN20, and White). CFTR protein is a member of the MRP subfamily, which may be involved in multidrug resistance. CFTR protein functions as a chloride channel and can regulate the regulation of other transport pathways. In some cases, mutations in the CFTR gene are associated with autosomal recessive disorders, such as cystic fibrosis and congenital bilateral aplasia of the vas deferens. Alternatively, spliced ​​CFTR transcript variants have been described, many of which may result from mutations in this gene.

[0059] The human (Homo sapiens) CFTR gene (Gene ID: 1080) is located on chromosome 7 and can be defined by the chromosomal coordinates GRCh38.p14 / hg38:chr7:117,480,025 to GRCh38.p14 / hg38:chr7:117,668,665. CFTR is also designated as Ensembl:ENSG00000001626;MIM:602421;AllianceGenome:HGNC:1884. CFTR is also designated as CF;MRP7;ABC35;ABCC7;CFTR / MRP;TNR-CFTR;DJ76005.1. The CFTR transcript has 27 exons and is designated as NCBI Reference Sequence NM_000492.4; ACCESSION: NM_000492, and Ensembl: ENST00000003084.11. The human CFTR protein is designated as NCBI Reference Sequence: NP000483.3.

[0060] The CFTR protein can form a transmembrane chloride channel, the function of which can be regulated by phosphorylation mediated by cAMP-dependent phosphokinase. In the presence of adenosine triphosphate (ATP), phosphorylation of CFTR expressed on the cell membrane triggers channel opening, allowing approximately 10 chloride ions per minute to exit the cell through the channel formed by the CFTR protein. Certain CFTR gene mutations can result in the production of defective CFTR protein that cannot be processed normally by the endoplasmic reticulum for effective transport to the cell membrane. The few mutant CFTR protein molecules that reach the cell membrane may be dysfunctional and therefore unable to transport chloride ions, resulting in the accumulation of chloride ions and associated water molecules in epithelial cells and a lack of hydration in extracellular mucus and secretions.

[0061] CFTR mutations can be classified according to the abnormalities they may cause, including dysfunctional protein translation, cellular processing, or CFTR channel gating. Missense (single amino acid substitution) mutations account for 38.74% of all known CFTR mutations detected worldwide, frameshift (insertion or deletion) mutations account for 16.25%, splicing (incorrect intron splicing) mutations account for 10.93%, and nonsense (premature stop codon) mutations account for 8.41%. CFTR gene mutations can be classified into six distinct classes that roughly correspond to specific types of CFTR dysfunction. Generally, mutations in classes I-III can cause more severe disease than those in classes IV-VI. The clinical symptoms of CF caused by any combination of mutations may vary, likely due to the effects of gene modifiers. For example, the genotype-phenotype correlation may be weak for CF associated with pulmonary disease but stronger for CF types associated with pancreatic insufficiency. Because several new therapies have recently been developed that target CF disease caused by specific classes of CFTR mutations, mutation characterization may be useful in guiding initial therapy for some patients.

[0062] There are five different types of CFTR mutations. Class I mutations: defective protein production. This type of defect can be caused by nonsense, frameshift, or splice site mutations, resulting in premature termination of the messenger RNA (mRNA) transcript and the absence of full-length CFTR protein. Non-limiting examples include G542X, W1282X, R553X, 621+G>T, and 1717-1G>A. Class II mutations: defective protein processing. This class of mutations can cause abnormal post-translational processing of the CFTR protein, preventing the protein from translocating to the correct cellular location, as exemplified by the F508del mutation, which is present in approximately 50% of CF patients in a homozygous state and in at least 90% of CF patients in a heterozygous state. Class III mutations: defective regulation. These mutations cause reduced channel activity even when ATP levels are adequate. Many mutations can alter the NF-κB ATP-binding regions (designated NBD1 and NBD2), resulting in some variants retaining varying degrees of sensitivity to nucleotide binding. Mutations resulting in the CFTR substitution G551D, which can abolish ATP binding, are the most common Class III mutations in Caucasian populations. Other CFTR mutations within the region encoding the CFTR R domain may also fall into this category. Class IV mutations: Defective conduction. CFTR proteins with these mutations are produced and transported correctly to the cell surface. However, the rate of ion flux and the duration of channel openness may be reduced compared to normal CFTR proteins, even though chloride currents are generated in response to cAMP stimulation. Mutations resulting in an amino acid substitution (R117H) in the CFTR protein are the most common Class IV mutations in Caucasian populations. Class V mutations: Decreased amount of functional CFTR protein.This includes some mutations that can alter mRNA stability and other types of mutations that can alter the stability of the mature CFTR protein (the latter are sometimes classified separately in an additional class, Class VI). Class VI mutations: Reduced CFTR stability. This class includes Phe508del, which can cause substantial plasma membrane instability and is rescued by most correctors (rPhe508del).

[0063] In some embodiments, the function of various CFTR proteins can be evaluated by assays known to those skilled in the art (e.g., electrophysiological assays). Exemplary electrophysiological assays can include transepithelial chloride conductance assays (e.g., TECC-24 assays). In some embodiments, the function of various CFTR proteins can be evaluated by comparing the area under the curve (AUC) of induced chloride conductance obtained in electrophysiological assays. The AUC of various mutant cell lines (e.g., 16HBEge-W1282X cells) can be normalized to the AUC obtained from cells expressing WT CFTR protein with or without treatment and expressed as either a percentage of WT CFTR (%WT CFTR) or a ratio to WT CFTR (variant / WT CFTR). Many cell lines can be used to evaluate the function of CFTR proteins. Variants of human bronchial epithelial (HBE) cell lines, including wild-type, mutant, primary and immortalized cell lines, can be used in electrophysiology assays to assess CFTR channel function.

[0064] In some cases, the function of the C-terminally truncated CFTR protein is less than 100% of the function of the WT CFTR protein. For example, the function of the C-terminally truncated CFTR protein can be at most 95%, at most 90%, at most 85%, at most 80%, at most 70%, at most 60%, at most 50%, at most 40%, at most 30%, at most 20%, at most 10%, at most 5%, at most 2%, at most 1% of that of the WT CFTR protein, or as assessed by an assay known to one of skill in the art (e.g., an electrophysiological assay).

[0065] In the classic form of CF, patients may exhibit clinical disease in one or more organ systems (as described below) and may have elevated sweat chloride (≥ 60 mmol / L). Most of these patients may have evidence of disease in multiple organ systems (pancreas, upper and lower respiratory tract, and male reproductive tract). CFTR-related disorders (CFTR-RD) may refer to clinical disease limited to only one organ system associated with some evidence of CFTR dysfunction that does not meet the full genetic or functional criteria for a CF diagnosis. CFTR-RD includes pulmonary disorders, such as disseminated bronchiectasis. CFTR-RD includes disorders of the gastrointestinal tract, including CF-related pancreatic insufficiency, CF-related pancreatitis, CF-related diabetes, CF-related liver disease, and gallbladder disease. CFTR-RD includes disorders of the reproductive tract, including congenital bilateral absence of the vas deferens (CBAVD). Clinical manifestations may include isolated obstructive rhinosinusitis, chronic pancreatitis, or adult pulmonary disease. In cystic fibrosis, disrupted transport of chloride ions and other CFTR-influenced ions (e.g., sodium and bicarbonate) can result in thick, viscous secretions in the lungs, pancreas, liver, intestines, or reproductive tract, or any combination thereof. Alternatively, or in addition, disrupted transport of chloride ions and other CFTR-influenced ions can result in increased salt content in sweat gland secretions. CF symptoms can vary among individuals of different age groups. Infants and children may exhibit respiratory symptoms, meconium ileus, growth failure, or any combination thereof. Typical respiratory symptoms of CF can include persistent productive cough, hyperinflation of lung fields on chest radiographs, and pulmonary function tests consistent with obstructive airway disease. Other clinical manifestations of lung disease can include microorganism infection, bronchiectasis, airway hyperreactivity, allergic bronchopulmonary aspergillosis, obstructive sleep apnea, and pulmonary hypertension.Patients who present with CF later in life are more likely to have atypical symptoms, including sinus disease, pancreatic disease, infertility, musculoskeletal disorders, recurrent venous thrombosis, anemia, electrolyte abnormalities, nephrolithiasis, and aquagenic wrinkling.

[0066] Processing of CFTR PRE-MRNA In some embodiments, compositions, methods, and kits are provided herein that include agents that regulate the processing of pre-mRNA (CFTR pre-mRNA) encoding the CFTR protein and that include a first intron containing an alternative polyadenylation site. Pre-mRNA processing in cells can include splicing of the pre-mRNA followed by polyadenylation of the resulting mRNA transcript. The terms "mature mRNA," "fully spliced ​​mRNA," and "processed mRNA" are used interchangeably herein to describe fully processed mRNA processed from pre-mRNA. In some cases, agents provided herein suppress splicing out of the first intron (e.g., intron 22) of the CFTR pre-mRNA. Alternatively, or in addition, agents provided herein promote polyadenylation of the CFTR mRNA transcript at an alternative polyadenylation site within the first intron. In some cases, the CFTR pre-mRNA contains a mutation that can result in the presence of a PTC downstream of exon 22 when the pre-mRNA is processed into a mature CFTR mRNA transcript, which can result in NMD of the processed CFTR mRNA transcript.

[0067] Splicing and nonsense-mediated mRNA decay Intervening sequences, or introns, are removed by a highly dynamic RNA-protein complex called the spliceosome, which orchestrates complex interactions among the primary transcript, small nuclear RNAs (snRNAs), and numerous proteins. The spliceosome assembles ad hoc on each intron in an ordered manner, starting with recognition of the 5' splice site (5'ss) by the U1 snRNA or the 3' splice site (3'ss) by the U2 pathway. This is accompanied by binding of the U2 auxiliary factor (U2AF) to the 3'ss region to promote U2 binding to the branch point sequence (BPS). U2AF is a stable heterodimer composed of a U2AF2-encoded 65 kD subunit (U2AF65), which binds to the polypyrimidine tract (PPT), and a U2AF1-encoded 35 kD subunit (U2AF35), which interacts with a highly conserved AG dinucleotide at the 3' end and stabilizes U2AF65 binding. In addition to the BPS / PPT unit and 3'ss / 5'ss, accurate splicing requires auxiliary sequences or structures, known as intron or exon splicing enhancers or silencers, that activate or repress splice site recognition. These elements enable authentic splice sites to be recognized among the vast excess of cryptic or pseudo sites in the genomes of higher eukaryotes, which share the same sequence but are orders of magnitude more numerous than authentic sites. They often have regulatory functions, but the precise mechanisms of their activation and repression are poorly understood.

[0068] The decision to splice or not can typically be modeled as a stochastic rather than a deterministic process; therefore, even the most well-defined splicing signals can occasionally splice incorrectly. However, under normal conditions, pre-mRNA splicing proceeds with surprisingly high fidelity. This is due, in part, to the activity of adjacent cis-acting auxiliary exon and intron splicing regulatory elements (ESRs or ISRs). Typically, these functional elements are classified as either exon or intron splicing enhancers (ESEs or ISEs) or silencers (ESSs or ISSs) based on their ability to stimulate or inhibit splicing, respectively. There is now evidence that some auxiliary cis-acting elements, such as the positioning of the complex between the U1 snRNP and the 5' ss, can act by influencing the dynamics of spliceosome assembly, but it is highly likely that many elements function in concert with trans-acting RNA-binding proteins (RBPs). For example, the serine-rich and arginine-rich families of RBPs (SR proteins) are conserved families of proteins that play important roles in exon definition. SR proteins facilitate exon recognition by recruiting components of the pre-spliceosome to adjacent splice sites or by antagonizing the effects of ESSs in the vicinity. The repressive effects of ESSs can be mediated by members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family, which can alter the recruitment of core splicing factors to adjacent splice sites. In addition to their role in splicing regulation, silencer elements have been suggested to play a role in suppressing pseudo-exons, a set of decoy intronic splice sites that have the typical spacing of exons but lack a functional open reading frame.ESEs and ESSs, in concert with their cognate trans-acting RBPs, are key components of the splicing regulatory chain that determines when, where, and how mRNAs are assembled from their precursors.

[0069] Sequences marking exon-intron boundaries are degenerate signals of varying strength that occur frequently within human genes. In multi-exon genes, different pairs of splice sites can be linked together in many different combinations, creating a diverse array of transcripts from a single gene. This is commonly referred to as alternative pre-mRNA splicing. Although most mRNA isoforms produced by alternative splicing can be exported from the nucleus and translated into functional polypeptides, different mRNA isoforms from a single gene can vary greatly in their translation efficiency. mRNA isoforms with a premature termination codon (PTC) at least 50 bp upstream of the exon junction complex are likely to be targeted for degradation by the nonsense-mediated mRNA decay (NMD) pathway.

[0070] Mutations in traditional (BPS / PPT / 3'ss / 5'ss) and auxiliary splicing motifs can cause aberrant splicing, such as exon skipping, cryptic (or pseudo) exon inclusion, or splice site activation, which significantly contribute to human morbidity and mortality. Both aberrant and alternative splicing patterns can be influenced by natural DNA variants in exons and introns.

[0071] Given that exon-intron boundaries can occur at any of three codon positions, it is clear that only a subset of alternative splicing events can maintain the standard open reading frame. For example, only exons evenly divisible by three can be skipped or included in the mRNA without any change in the reading frame. Splicing events that are out of phase induce frameshifts. Unless reversed by downstream events, frameshifts inevitably result in one or more PTCs, possibly leading to their subsequent degradation by NMD. NMD is a translation-coupled mechanism that eliminates mRNAs containing PTCs. NMD may function as a surveillance pathway present in all eukaryotes. NMD can reduce errors in gene expression by eliminating mRNA transcripts containing premature stop codons. Translation of these aberrant mRNAs can, in some cases, result in gain-of-function or dominant-negative activity of the resulting protein. NMD targets not only transcripts with PTCs but also a wide range of mRNA isoforms expressed from many endogenous genes, suggesting that NMD is a master regulator that drives both fine and coarse regulation of steady-state RNA levels in cells.

[0072] Alternative polyadenylation In some aspects, provided herein are compositions that modify alternative polyadenylation during processing of CFTR pre-mRNA into mature mRNA.

[0073] Alternative polyadenylation (APA) is an RNA processing mechanism that can generate distinct 3' ends on mRNAs and other RNA polymerase II transcripts. It is widespread across all eukaryotic species and is recognized as a major mechanism of gene regulation. APA can exhibit tissue specificity and may be important for cell proliferation and differentiation. The role of APA can cover a range of diverse cellular processes, including mRNA metabolism, protein diversification, and protein localization, and can be generally involved in gene regulation. The molecular mechanisms underlying APA include fluctuations in the concentrations of core processing factors and RNA-binding proteins, as well as transcription-based regulation.

[0074] Regulation of CFTR pre-mRNA processing In some embodiments, methods, compositions, and kits are provided herein for agents that regulate the processing of CFTR pre-mRNA transcripts into mature CFTR mRNA transcripts. CFTR pre-mRNA can contain a first intron that contains an alternative polyadenylation site. In some cases, agents provided herein regulate the processing of CFTR pre-mRNA, thereby increasing the level of processed mRNA that is processed from pre-mRNA and lacks the nucleic acid sequence of pre-mRNA downstream of the first intron that contains the alternative polyadenylation site. In some cases, the first intron is intron 22, such as the intron from GRCh38.p14 / hg38:chr7:117,627,771 to GRCh38.p14 / hg38:chr7:117,642,437.

[0075] In some cases, the agents provided herein remove from the pre-mRNA a nucleic acid sequence of the pre-mRNA downstream of the first intron, e.g., a nucleic acid sequence located downstream of intron 22 of the CFTR pre-mRNA. In some cases, the removed nucleic acid sequence is located at GRCh38.p14 / hg38:chr7:117,642,438 to GRCh38.p14 / hg38:chr7:117,668,665.

[0076] In some cases, the agents provided herein inhibit the splicing out of the first intron from the CFTR pre-mRNA, for example, by inhibiting splicing at the 5' splice site, the 3' splice site, or both of the first intron. In some cases, the agents provided herein inhibit the splicing out of intron 22 from the CFTR pre-mRNA. For example, the agents provided herein inhibit splicing at the 5' splice site, the 3' splice site, or both of intron 22 of the CFTR pre-mRNA.

[0077] Without wishing to be bound by any particular theory, suppression of splicing out from intron 22 from the CFTR pre-mRNA may provide an opportunity for polyadenylation of the mRNA transcript at one of the alternative polyadenylation sites located within intron 22, which may then result in truncation of the mature mRNA transcript, e.g., the generation of an exon 22-truncated CFTR mRNA (a CFTR mRNA without exon sequence downstream of exon 22).

[0078] Alternatively, or additionally, without wishing to be bound by any particular theory, inhibition of splicing out of intron 22 from the CFTR pre-mRNA may inhibit splicing at one or more splice sites downstream of intron 22, which may then result in truncation of the mature mRNA transcript, e.g., the generation of an exon 22-truncated CFTR mRNA transcript.

[0079] In some cases, upon processing, the exon 22-truncated CFTR mRNA transcript, which is the processed mRNA processed from the CFTR pre-mRNA, includes, in 5' to 3' order, 22 exons, an intron sequence encoding nine amino acids, a stop codon, and an alternative 3' untranslated region. The 22 exons can be canonical exons 1 through 22 of the CFTR pre-mRNA. The intron sequence encoding the nine amino acids can be a nucleic acid sequence located within intron 22 of the CFTR pre-mRNA. In some cases, the intron sequence encoding the nine amino acids is transcribed from a genomic sequence located at GRCh38.p14 / hg38:chr7:117,627,771 to GRCh38.p14 / hg38:chr7:117,627,797. In some cases, a processed mRNA that does not have the nucleic acid sequence of a CFTR pre-mRNA downstream of the first intron, e.g., an exon 22-truncated CFTR mRNA, is polyadenylated at an alternative polyadenylation site within the first intron, e.g., an alternative polyadenylation site in a nucleic acid sequence transcribed from a genomic sequence located between GRCh38.p14 / hg38:chr7:117,627,771 and GRCh38.p14 / hg38:chr7:117,642,437.

[0080] In some cases, an agent provided herein binds to the 5' splice site of the first intron (e.g., intron 22) of CFTR pre-mRNA. In some cases, an agent provided herein binds to the 3' splice site of the first intron (e.g., intron 22) of CFTR pre-mRNA. In some cases, an agent provided herein binds to the branch point of the 3' splice site of the first intron (e.g., intron 22) of CFTR pre-mRNA. In some cases, an agent provided herein interferes with a splicing factor involved in splicing from the first intron (e.g., intron 22) of CFTR pre-mRNA. In some cases, the agent interferes with a splicing factor involved in splicing of the first intron (e.g., intron 22) of CFTR pre-mRNA by binding to (a) the 5' splice site of the first intron, (b) the 3' splice site of the first intron, (c) the branch point of the 3' splice site of the first intron, or (d) any combination thereof. In some cases, the agent provided herein binds to a targeting moiety located within the first intron (e.g., intron 22) of CFTR pre-mRNA. In some cases, the agent provided herein binds to a targeting moiety located within the exon immediately preceding the first intron (e.g., exon 22) of CFTR pre-mRNA. In some cases, the agent provided herein binds to a targeting moiety located within the exon immediately following the first intron (e.g., exon 23) of CFTR pre-mRNA. In some cases, the agents provided herein bind to a targeting moiety located at the junction between the first intron (e.g., intron) 22 and the exon immediately preceding the first intron or the exon immediately following the first intron, e.g., the exon 22-intron 22 junction or the intron 22-exon 23 junction.

[0081] As provided herein, when contacted with a cell, the agent of the present disclosure can increase the level of processed mRNA that does not have the nucleic acid sequence of CFTR pre-mRNA downstream of the first intron containing an alternative polyadenylation site (e.g., intron 22) in the cell. For example, the level of processed mRNA that does not have the nucleic acid sequence of CFTR pre-mRNA downstream of the first intron is increased by at least about 1.5, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, or at least about 20 times in the cell compared to a corresponding cell not contacted with the agent. In some cases, the level of processed mRNA is increased by at least about 10 times in the cell compared to a corresponding cell not contacted with the agent.

[0082] As provided herein, an agent of the disclosure, upon contact with a cell, can increase the level of a truncated CFTR protein in the cell that is translated from a processed mRNA that does not have the nucleic acid sequence of the CFTR pre-mRNA downstream of the first intron (e.g., intron 22) that contains an alternative polyadenylation site, and that lacks the amino acid sequence expressed from the exon sequence of the CFTR gene downstream of the first intron. In some cases, the level of truncated CFTR protein in the cell is increased by at least about 1.5, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 120, at least about 150, at least about 180, at least about 200, at least about 250, at least about 300, at least about 400, at least about 500, at least about 750, or at least about 1000 fold compared to a corresponding cell not contacted with the agent. In some cases, the level of the truncated CFTR protein in the cells is increased by at least about 10-fold compared to corresponding cells not contacted with the agent.

[0083] In some cases, the level of truncated CFTR protein in the cells is increased by about 1.5, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 120, about 150, about 180, about 200, about 250, about 300, about 400, about 500, about 750, or about 1000-fold compared to corresponding cells not contacted with the agent. In some cases, the level of truncated CFTR protein in the cells is increased by about 10-fold compared to corresponding cells not contacted with the agent.

[0084] In some cases, the truncated CFTR protein in the cell has a chloride channel conductance that is less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% of the chloride channel conductance of the wild-type CFTR protein.

[0085] In some cases, the chloride channel conductance of the truncated CFTR protein is increased by a modulator of the CFTR protein that enhances the chloride conductance of CFTR in a cell. In some cases, the modulator of the CFTR protein comprises ivacaftor, lumacaftor, tezacaftor, elexacaftor, a combination of lumacaftor and ivacaftor, a combination of tezacaftor and ivacaftor, or a combination of elexacaftor, ivacaftor, and tezacaftor.

[0086] In some cases, the modulator of a CFTR protein increases the chloride channel conductance of a truncated CFTR protein by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, An increase of at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 950%, at least 1000%. In some cases, the modulator of a CFTR protein increases the chloride channel conductance of the truncated CFTR protein by about 1.5, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 120, about 150, about 180, about 200, about 250, about 300, about 400, about 500, about 750, or about 1000 times.

[0087] In some cases, a modulator of a CFTR protein restores chloride channel conductance of a truncated CFTR protein by approximately at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more.

[0088] TARGET NUCLEIC ACIDS AND AGENTS In some embodiments, provided herein are compositions and methods relating to agents (e.g., antisense oligomers) or vectors encoding agents that target a targeting portion of a nucleic acid encoding a CFTR protein, e.g., a CFTR pre-mRNA or a CFTR gene, in a cell.

[0089] In some cases, agents provided herein bind to a targeting moiety located within the first intron (e.g., intron 22) of CFTR pre-mRNA. In some cases, agents provided herein bind to a targeting moiety located within the exon immediately preceding the first intron (e.g., exon 22) of CFTR pre-mRNA. In some cases, agents provided herein bind to a targeting moiety located within the exon immediately following the first intron (e.g., exon 23) of CFTR pre-mRNA. In some cases, agents provided herein bind to a targeting moiety located at a junction between the first intron (e.g., intron) 22 and the exon immediately preceding the first intron or the exon immediately following the first intron, for example, at the exon 22-intron 22 junction or the intron 22-exon 23 junction.

[0090] When a polynucleic acid polymer sequence is referenced, one skilled in the art will understand that one or more substitutions, optionally two substitutions, can be tolerated in the sequence so as to maintain the ability to hybridize to a target sequence, or, if substitutions are present in the target sequence, the ability to be recognized as a target sequence. References to sequence identity can be determined by BLAST sequence alignment using standard / default parameters. For example, a sequence can have 99% identity and still function according to the present disclosure. In other embodiments, a sequence can have 98% identity and still function according to the present disclosure. In another embodiment, a sequence can have 95% identity and still function according to the present disclosure. In another embodiment, a sequence can have 90% identity and still function according to the present disclosure.

[0091] Polynucleotide sequences, including oligomers such as oligonucleotides, are "complementary" to one another if 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 the strands of the first polynucleotide and the second polynucleotide. Complementarity (the degree to which one polynucleotide is complementary to another) can be quantified by the proportion (e.g., percentage) of bases on opposing strands that are predicted to form hydrogen bonds with each other according to generally accepted base-pairing rules. The sequence of a polynucleotide sequence, such as an antisense oligomer (ASO), need not be 100% complementary to that of its target nucleic acid in order to hybridize. In certain embodiments, agents (e.g., ASOs) 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 the target region within the target nucleic acid sequence to which they are targeted. For example, an ASO in which 18 of 20 nucleobases of an oligomeric compound are complementary to the target region and therefore specifically hybridize exhibits 90 percent complementarity. In this example, the remaining non-complementary nucleobases can be clustered together or interspersed with complementary nucleobases and do not need to be adjacent to each other or to complementary nucleobases. The percent complementarity of an agent (such as an ASO) with a region of a target nucleic acid can be routinely determined using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul, et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656, the entire contents of which are incorporated herein by reference).

[0092] In some cases, the target nucleic acid is a portion of the sequence of SEQ ID NO: 1 or 2. In some cases, the target nucleic acid has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to SEQ ID NO: 1 or 2. In some cases, the target nucleic acid has about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1 or 2. In some cases, the target nucleic acid is a portion of the sequence of SEQ ID NO: 1 or 2. In some cases, the target nucleic acid has a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least eight contiguous nucleic acids of SEQ ID NO: 1 or 2. In some cases, the target nucleic acid has a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least eight contiguous nucleic acids of SEQ ID NO: 1 or 2. In some cases, the target nucleic acid has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least 10, 12, 14, or 16 contiguous nucleic acids of SEQ ID NO: 1 or 2. In some cases, the target nucleic acid has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least 18 contiguous nucleic acids of SEQ ID NO: 1 or 2.

[0093] In some cases, the target nucleic acid is a portion of exon 22 of CFTR pre-mRNA. In some cases, the target nucleic acid has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to SEQ ID NO: 6. In some cases, the target nucleic acid has about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6. In some cases, the target nucleic acid is a portion of the sequence of SEQ ID NO: 6. In some cases, the target nucleic acid has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least eight contiguous nucleic acids of SEQ ID NO: 6. In some cases, the target nucleic acid has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least eight contiguous nucleic acids of SEQ ID NO: 6. In some cases, the target nucleic acid has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least 10, 12, 14, or 16 contiguous nucleic acids of SEQ ID NO: 6. In some cases, the target nucleic acid has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least 18 contiguous nucleic acids of SEQ ID NO: 6.

[0094] In some cases, the target nucleic acid is a portion of exon 23 of CFTR pre-mRNA. In some cases, the target nucleic acid has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to SEQ ID NO: 7. In some cases, the target nucleic acid has about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7. In some cases, the target nucleic acid is a portion of the sequence of SEQ ID NO: 7. In some cases, the target nucleic acid has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least eight contiguous nucleic acids of SEQ ID NO: 7. In some cases, the target nucleic acid has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least eight contiguous nucleic acids of SEQ ID NO: 7. In some cases, the target nucleic acid has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least 10, 12, 14, or 16 contiguous nucleic acids of SEQ ID NO: 7. In some cases, the target nucleic acid has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least 18 contiguous nucleic acids of SEQ ID NO: 7.

[0095] In some cases, the target nucleic acid is a portion of intron 22 of CFTR pre-mRNA. In some cases, the target nucleic acid has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to SEQ ID NO: 8. In some cases, the target nucleic acid has about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 8. In some cases, the target nucleic acid is a portion of the sequence of SEQ ID NO: 8. In some cases, the target nucleic acid has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least eight contiguous nucleic acids of SEQ ID NO: 8. In some cases, the target nucleic acid has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least eight contiguous nucleic acids of SEQ ID NO: 8. In some cases, the target nucleic acid has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least 10, 12, 14, or 16 contiguous nucleic acids of SEQ ID NO: 8. In some cases, the target nucleic acid has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least 18 contiguous nucleic acids of SEQ ID NO:8.

[0096] In some cases, the target nucleic acid comprises an exon-intron junction of the CFTR pre-mRNA. In some cases, the target nucleic acid comprises a junction between exon 22 and intron 22 of the CFTR pre-mRNA. In some cases, the target nucleic acid comprises a junction between intron 22 and exon 23 of the CFTR pre-mRNA.

[0097] In some cases, the target nucleic acid is a portion of exon 22 of the CFTR gene. In some cases, the target nucleic acid has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to SEQ ID NO: 3. In some cases, the target nucleic acid has about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3. In some cases, the target nucleic acid is a portion of the sequence of SEQ ID NO: 3. In some cases, the target nucleic acid has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least eight contiguous nucleic acids of SEQ ID NO: 3. In some cases, the target nucleic acid has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least eight contiguous nucleic acids of SEQ ID NO: 3. In some cases, the target nucleic acid has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least 10, 12, 14, or 16 contiguous nucleic acids of SEQ ID NO: 3. In some cases, the target nucleic acid has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least 18 contiguous nucleic acids of SEQ ID NO: 3.

[0098] In some cases, the target nucleic acid is a portion of exon 23 of the CFTR gene. In some cases, the target nucleic acid has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to SEQ ID NO: 4. In some cases, the target nucleic acid has about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some cases, the target nucleic acid is a portion of the sequence of SEQ ID NO: 4. In some cases, the target nucleic acid has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least eight contiguous nucleic acids of SEQ ID NO: 4. In some cases, the target nucleic acid has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least eight contiguous nucleic acids of SEQ ID NO: 4. In some cases, the target nucleic acid has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least 10, 12, 14, or 16 contiguous nucleic acids of SEQ ID NO: 4. In some cases, the target nucleic acid has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least 18 contiguous nucleic acids of SEQ ID NO: 4.

[0099] In some cases, the target nucleic acid is a portion of intron 22 of the CFTR gene. In some cases, the target nucleic acid has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to SEQ ID NO: 5. In some cases, the target nucleic acid has about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some cases, the target nucleic acid is a portion of the sequence of SEQ ID NO: 5. In some cases, the target nucleic acid has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least eight contiguous nucleic acids of SEQ ID NO: 5. In some cases, the target nucleic acid has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least eight contiguous nucleic acids of SEQ ID NO: 5. In some cases, the target nucleic acid has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least 10, 12, 14, or 16 contiguous nucleic acids of SEQ ID NO: 5. In some cases, the target nucleic acid has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least 18 contiguous nucleic acids of SEQ ID NO: 5.

[0100] In some cases, the target nucleic acid comprises an exon-intron junction of the CFTR gene. In some cases, the target nucleic acid comprises a junction between exon 22 and intron 22 of the CFTR gene. In some cases, the target nucleic acid comprises a junction between intron 22 and exon 23 of the CFTR gene.

[0101] In some cases, the target nucleic acid has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to any one of SEQ ID NOs: 9-36. In some cases, the target nucleic acid has about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 9-36. In some cases, the target nucleic acid is a portion of the sequence of any one of SEQ ID NOs: 9-36. In some cases, the target nucleic acid has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least 8 contiguous nucleic acids of any one of SEQ ID NOs: 9-36. In some cases, the target nucleic acid has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least 8 contiguous nucleic acids of any one of SEQ ID NOs: 9-36. In some cases, the target nucleic acid has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least 10, 12, 14, or 16 contiguous nucleic acids of any one of SEQ ID NOs: 9-36. In some cases, the target nucleic acid has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least 18 contiguous nucleic acids of any one of SEQ ID NOs: 9-36.

[0102] The agents provided herein can comprise a polynucleotide sequence. The polynucleotide sequence of the agent can be complementary to a target sequence located within the CFTR gene or CFTR pre-mRNA.

[0103] In some cases, the agent comprises a polynucleotide sequence complementary to a portion of exon 22 of the CFTR gene or CFTR pre-mRNA. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to SEQ ID NO: 3 or 6. In some cases, the agent comprises a polynucleotide sequence that is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to SEQ ID NO: 3 or 6. In some cases, the agent comprises a polynucleotide sequence complementary to a portion of the sequence of SEQ ID NO: 3 or 6. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least eight contiguous nucleic acids of SEQ ID NO: 3 or 6. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least eight contiguous nucleic acids of SEQ ID NO: 3 or 6. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least 10, 12, 14, or 16 contiguous nucleic acids of SEQ ID NO: 3 or 6. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least 18 contiguous nucleic acids of SEQ ID NO: 3 or 6.

[0104] In some cases, the agent comprises a polynucleotide sequence complementary to a portion of exon 23 of the CFTR gene or CFTR pre-mRNA. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to SEQ ID NO: 4 or 7. In some cases, the agent comprises a polynucleotide sequence that is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to SEQ ID NO: 4 or 7. In some cases, the agent comprises a polynucleotide sequence complementary to a portion of the sequence of SEQ ID NO: 4 or 7. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least eight contiguous nucleic acids of SEQ ID NO: 4 or 7. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least eight contiguous nucleic acids of SEQ ID NO: 4 or 7. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least 10, 12, 14, or 16 contiguous nucleic acids of SEQ ID NO: 4 or 7. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least 18 contiguous nucleic acids of SEQ ID NO: 4 or 7.

[0105] In some cases, the agent comprises a polynucleotide sequence complementary to intron 22 of the CFTR gene or a portion of the CFTR pre-mRNA. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to SEQ ID NO: 5 or 8. In some cases, the agent comprises a polynucleotide sequence that is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to SEQ ID NO: 5 or 8. In some cases, the agent comprises a polynucleotide sequence complementary to a portion of the sequence of SEQ ID NO: 5 or 8. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least eight contiguous nucleic acids of SEQ ID NO: 5 or 8. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least eight contiguous nucleic acids of SEQ ID NO: 5 or 8. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least 10, 12, 14, or 16 contiguous nucleic acids of SEQ ID NO: 5 or 8. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least 18 contiguous nucleic acids of SEQ ID NO: 5 or 8.

[0106] In some cases, the agent comprises a polynucleotide sequence complementary to an exon-intron junction of the CFTR gene or CFTR pre-mRNA. In some cases, the agent comprises a polynucleotide sequence complementary to a junction between exon 22 and intron 22 of the CFTR gene or CFTR pre-mRNA. In some cases, the agent comprises a polynucleotide sequence complementary to a junction between intron 22 and exon 23 of the CFTR gene or CFTR pre-mRNA.

[0107] Agents provided herein may comprise a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to any one of SEQ ID NOs: 9-36. In some cases, the agent comprises a polynucleotide sequence that is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to any one of SEQ ID NOs: 9-36. In some cases, the agent comprises a polynucleotide sequence complementary to a portion of the sequence of any one of SEQ ID NOs: 9-36. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least 8 contiguous nucleic acids of any one of SEQ ID NOs: 9-36. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least 8 contiguous nucleic acids of any one of SEQ ID NOs: 9-36. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least 10, 12, 14, or 16 contiguous nucleic acids of any one of SEQ ID NOs: 9-36. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least 18 contiguous nucleic acids of any one of SEQ ID NOs: 9-36.

[0108] In some cases, the agent comprises a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to any one of SEQ ID NOs: 37-64. In some cases, the agent comprises a polynucleotide sequence having about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 37-64. In some cases, the agent comprises a polynucleotide sequence having a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least 8 contiguous nucleic acids of any one of SEQ ID NOs: 37-64. In some cases, the agent comprises a polynucleotide sequence having a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least 8 contiguous nucleic acids of any one of SEQ ID NOs: 37-64. In some cases, the agent comprises a polynucleotide sequence having a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least 10, 12, 14, or 16 contiguous nucleic acids of any one of SEQ ID NOs: 37-64. In some cases, the agent comprises a polynucleotide sequence having a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least 18 contiguous nucleic acids of any one of SEQ ID NOs: 37-64.

[0109] In some cases, the agent provided herein targets the nucleic acid that encodes CFTR protein in cells.In some cases, the nucleic acid that encodes CFTR protein in cells (for example, CFTR gene or CFTR pre-mRNA) is variant, and for example, has one or more mutations compared with the wild-type nucleic acid that encodes wild-type CFTR protein (for example, wild-type CFTR gene or wild-type CFTR pre-mRNA).In some cases, the CFTR variant has one or more mutations at the downstream position of intron 22 of CFTR gene or pre-mRNA.The results are CFTR c.3717G>A c.3717+4A>G c.3717+5 G>A、c.3717+40A>G、c.3718-2477C>T、c.3718-1G>A、c.3718-3 T>G、c.3719T>G、c.3731G>A、c.3737C>T、c.3744delA、c.3745G >A、c.3747delG、c.3752G>A、c.3761T>G、c.3763T>C、c.3764C> A、c.3773dupT、c.3806T>A、c.3808delG、c.3808G>A、c.3822G> A、c.3846G>A、c.[3846G>A、3848G>T]、c.3848G>T、c.3872A>G、 c.3873G>C、c.3873+1G>A、c.(3873+1_3874-1)_(3963+1_3964-1)del、c.3873+2T>C、3876delA、c.3883_3886delATTT、c.388 3delA、c.3889dupT、c.3891dupT、c.3908delA、c.3909C>G、c.3929G>A、c.3937C>T、c.(3963+1_3964-1)_(*1_?)del、c.3964- 78_4242+577del、c.3971T>C、c.3988C>T、c.4004T>C、c.4036_4042del、c.4046G>A、c.4077_4080delTGTTinsAA、c.4086dupT c.4097T>A c.4111G>T c.4124A>C c.4127_4131delTGGAT c .4144C>T、c.4147dupA、c.4197_4198delCT、c.4231C>T、c.423 4C>T、c.4242+1G>T、c.4242+1G>A、c.4251delA、c.4300_4301d up, c.4364C>G, c.4426C>T, and 1 of c.4439T>C.

[0110] In some cases, at least one allele of the CFTR gene in a cell into which an agent provided herein is introduced is variant, e.g., has one or more mutations downstream of intron 22 of the CFTR gene or pre-mRNA. In some cases, at least one allele of the CFTR gene in a cell into which an agent provided herein is introduced is variant, e.g., has one or more mutations downstream of intron 22 of the CFTR gene or pre-mRNA. In some cases, at least one allele of the CFTR gene in a cell into which an agent provided herein is introduced is variant, e.g., has one or more mutations downstream of intron 22 of the CFTR gene or pre-mRNA. .3761T>G, c.3763T>C, c.3764C>A, c.3773dupT, c.3806T>A, c.3808delG, c.3808G>A, c.3822G>A, c.3846G>A, c.[3846G>A, 3848G> T], c.3848G>T, c.3872A>G, c.3873G>C, c.3873+1G>A, c.(3873+1_3874-1)_(3963+1_3964-1)del, c.3873+2T>C, 3876delA, c.3883 _3886delATTT, c.3883delA, c.3889dupT, c.3891dupT, c.3908delA, c.3909C>G, c.3929G>A, c.3937C>T, c.(3963+1_3964-1)_(*1 _?)del, c.3964-78_4242+577del, c.3971T>C, c.3988C>T, c.4004T>C, c.4036_4042del, c.4046G>A, c.4077_4080delTGTTinsAA, c have one or more mutations such as c.4086dupT, c.4097T>A, c.4111G>T, c.4124A>C, c.4127_4131delTGGAT, c.4144C>T, c.4147dupA, c.4197_4198delCT, c.4231C>T, c.4234C>T, c.4242+1G>T, c.4242+1G>A, c.4251delA, c.4300_4301dup, c.4364C>G, c.4426C>T, or c.4439T>C.

[0111] therapeutic agent ASO (Antisense Oligomers) Provided herein are compositions comprising antisense oligomers that regulate CFTR pre-mRNA processing, for example, by binding to a targeting portion of CFTR pre-mRNA, thereby inhibiting the splicing of an intron (e.g., intron 22). As used herein, the terms "ASO" and "antisense oligomer" are used interchangeably and refer to oligomers, such as polynucleotides, that contain nucleobases that hybridize to a target nucleic acid (e.g., CFTR pre-mRNA) sequence via Watson-Crick base pairing or wobble base pairing (GU). ASOs can have exact or near-complementary sequences to the target sequence (e.g., sufficient complementarity to bind to the target sequence and enhance splicing at the splice site). ASOs are designed to bind (hybridize) to a target nucleic acid (e.g., a targeting portion of a pre-mRNA transcript) and remain hybridized under physiological conditions. Typically, when they hybridize to sites other than the intended (targeted) nucleic acid sequence, they hybridize to a limited number of sequences that are not target nucleic acids (a small number of sites other than target nucleic acids).ASO design can take into account the occurrence of sufficiently similar nucleic acid sequences in the targeting portion of pre-mRNA transcripts, or in other locations in genome or cell pre-mRNA or transcriptome, so that the possibility of ASO binding to other sites and causing " off-target " effects is limited.Any antisense oligomer known in the art can be used to carry out the method described herein, for example, PCT application PCT / US2014 / 05415, entitled " Reducing Nonsense-Mediated mRNA Decay " and published as WO2015 / 035091, which is incorporated herein by reference.

[0112] In some embodiments, the ASO "specifically hybridizes" or is "specific" for the target nucleic acid or the targeted portion of the CFTR pre-mRNA. Typically, such hybridization occurs at a Tm substantially greater than 37°C, preferably at least 50°C, and typically between 60°C and about 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 the target sequence hybridizes to a complementary oligonucleotide.

[0113] An ASO does not need to hybridize to all nucleobases in a target sequence, and the nucleobases it hybridizes to can be continuous or discontinuous.An ASO can hybridize across one or more segments of a pre-mRNA transcript, and thus intervening or adjacent segments are not involved in the hybridization event (for example, a loop structure or a hairpin structure can be formed).In certain embodiments, an ASO hybridizes to non-contiguous 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 nucleobases that the ASO does not hybridize to.

[0114] The ASOs described herein contain nucleobases complementary to nucleobases present in the targeting portion of CFTR pre-mRNA. The term ASO encompasses oligonucleotides and other oligomeric molecules that contain nucleobases capable of hybridizing to complementary nucleobases on the target mRNA but do not contain sugar moieties, such as peptide nucleic acids (PNAs). ASOs can contain naturally occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination of two or three of the foregoing. The term "naturally occurring nucleotides" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" includes nucleotides with modified or substituted sugar groups and / or nucleotides with modified backbones. In some embodiments, all nucleotides in an ASO are modified nucleotides. Chemical modifications of ASOs or components of ASOs that are compatible with the methods and compositions described herein will be apparent to those skilled in the art and can be found, for example, in U.S. Pat. No. 8,258,109 B2, U.S. Pat. No. 5,656,612, U.S. Patent Application Publication No. 2012 / 0190728, and Dias and Stein, Mol. Cancer Ther. 2002, 347-355, which are incorporated by reference in their entireties.

[0115] One or more nucleobases of the ASO can 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 so that it can hydrogen bond with a nucleobase present on the target pre-mRNA. Examples of modified nucleobases include, but are not limited to, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine.

[0116] The ASOs described herein also contain backbone structures that connect the oligomeric components. The terms "backbone structure" and "oligomer linkages" may be used interchangeably and refer to the bonds between the monomers of the ASO. In naturally occurring oligonucleotides, the backbone contains 3'-5' phosphodiester bonds that link the sugar moieties of the oligomer. The backbone structures or oligomeric linkages of the ASOs described herein include (but are not limited to) phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoroamidate, and the like. See, for example, LaPlanche, et al., Nucleic Acids Res. 14:9081 (1986); Stec, et al., J. Am. Chem. Soc. 106:6077 (1984), Stein, et al., Nucleic Acids Res. 16:3209 (1988), Zon, et al., Anti-Cancer Drug Design 6:539 (1991); Zon, et al., Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stec, et al., US Pat. No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90:543 (1990), the entire contents of which are incorporated herein by reference. In some embodiments, the backbone structure of the ASO does not contain phosphorus, but rather contains peptide bonds, such as those found in peptide nucleic acids (PNAs), or linking groups including carbamates, amides, and linear and cyclic hydrocarbon groups. In some embodiments, the backbone modification is a phosphorothioate bond. In some embodiments, the backbone modification is a phosphoramidate bond.

[0117] In embodiments, the stereochemistry at each of the phosphorus internucleotide linkages in the ASO backbone is random. In embodiments, the stereochemistry at each of the phosphorus internucleotide linkages in the ASO backbone is controlled and not random. For example, US2014 / 0194610, "Methods for the Synthesis of Functionalized Nucleic Acids," incorporated herein by reference, describes methods for independently selecting the chiral handedness at each phosphorus atom in a nucleic acid oligomer. In embodiments, ASOs used in the methods of the invention, including but not limited to any of the ASOs described herein in Tables 3A-3B, comprise ASOs with non-random phosphorus internucleotide linkages. In embodiments, the compositions used in the methods of the invention comprise pure diastereomeric ASOs. In embodiments, the compositions used in the methods of the invention comprise an ASO having a diastereomeric purity of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100%.

[0118] In some cases, the antisense oligomer is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to any one of SEQ ID NOs: 9-36. In some cases, the antisense oligomer is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to any one of SEQ ID NOs: 7-36. In some cases, the antisense oligomer is complementary to a portion of the sequence of any one of SEQ ID NOs: 9-36. In some cases, the antisense oligomer has a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least 8 contiguous nucleic acids of any one of SEQ ID NOs: 9-36. In some cases, the antisense oligomer has a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least 8 contiguous nucleic acids of any one of SEQ ID NOs: 9-36. In some cases, the antisense oligomer has a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least 10, 12, 14, or 16 contiguous nucleic acids of any one of SEQ ID NOs: 9-36. In some cases, the antisense oligomer has a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least 18 contiguous nucleic acids of any one of SEQ ID NOs: 9-36.

[0119] In some cases, the antisense oligomer has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to any one of SEQ ID NOs: 37-64. In some cases, the antisense oligomer has about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 37-64. In some cases, the antisense oligomer has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least eight contiguous nucleic acids of any one of SEQ ID NOs: 37-64. In some cases, the antisense oligomer has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least eight contiguous nucleic acids of any one of SEQ ID NOs: 37-64. In some cases, the antisense oligomer has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least 10, 12, 14, or 16 contiguous nucleic acids of any one of SEQ ID NOs: 37-64. In some cases, the antisense oligomer has a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% sequence identity to at least 18 contiguous nucleic acids of any one of SEQ ID NOs: 37-64.

[0120] In embodiments, the ASO has a non-random mixture of Rp and Sp configurations in its phosphorus internucleotide linkages. For example, it has been suggested that a mixture of Rp and Sp is required in antisense oligonucleotides or antisense oligomers to achieve a balance between good activity and nuclease stability (Wan, et al., 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, the ASOs used in the methods of the invention, including but not limited to any of the ASOs described herein in SEQ ID NOs: 37-64, comprise about 5-100% Rp, at least about 5% Rp, at least about 10% Rp, at least about 15% Rp, at least about 20% Rp, at least about 25% Rp, at least about 30% Rp, at least about 35% Rp, at least about 40% Rp, at least about 45% Rp, at least about 50% Rp, at least about 55% Rp, at least about 60% Rp, at least about 65% Rp, at least about 70% Rp, at least about 75% Rp, at least about 80% Rp, at least about 85% Rp, at least about 90% Rp, or at least about 95% Rp, with the remainder being Sp, or about 100% Rp.In embodiments, the ASOs used in the methods of the present invention, including, but not limited to, any of the ASOs described herein in SEQ ID NOs: 37-64, have a Rp of about 10% to about 100%, about 15% to about 100%, about 20% to about 100%, about 25% to about 100%, about 30% to about 100%, about 35% to about 100%, about 40% to about 100%, about 45% to about 100%, about 50% to about 100%, about 55% to about 100%, about 60% to about 100%, about 65% to about 100%, about 70% to about 100%, about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, about 95% to about 100%, about 100% to about 100%, about 110% to about 110%, about 120% to about 120%, about 130% to about 130%, about 140% to about 140%, about 150% to about 150%, about 160% to about 160%, about 170% to about 170%, about 180% to about 180%, about 190% to about 190%, about 210% to about 210%, about 220% to about 220%, about 230% to about 230%, about 240% to about 240%, about 250% to about 250%, about 260% to about 260%, about 270% to about 270%, about 280% to about 280%, about 290% to about 29 % to about 100% Rp, about 60% to about 100% Rp, about 65% to about 100% Rp, about 70% to about 100% Rp, about 75% to about 100% Rp, about 80% to about 100% Rp, about 85% to about 100% Rp, about 90% to about 100% Rp, or about 95% to about 100% Rp, about 20% to about 80% Rp, about 25% to about 75% Rp, about 30% to about 70% Rp, about 40% to about 60% Rp, or about 45% to about 55% Rp, with the remainder being Sp.

[0121] In embodiments, the ASOs used in the methods of the invention, including but not limited to any of the ASOs described herein in SEQ ID NOs: 37-64, comprise about 5-100% Sp, at least about 5% Sp, at least about 10% Sp, at least about 15% Sp, at least about 20% Sp, at least about 25% Sp, at least about 30% Sp, at least about 35% Sp, at least about 40% Sp, at least about 45% Sp, at least about 50% Sp, at least about 55% Sp, at least about 60% Sp, at least about 65% Sp, at least about 70% Sp, at least about 75% Sp, at least about 80% Sp, at least about 85% Sp, at least about 90% Sp, or at least about 95% Sp, the remainder being Rp, or about 100% Sp. In embodiments, the ASOs used in the methods of the present invention, including but not limited to any of the ASOs described herein in SEQ ID NOs: 37-64, are from about 10% to about 100% Sp, from about 15% to about 100% Sp, from about 20% to about 100% Sp, from about 25% to about 100% Sp, from about 30% to about 100% Sp, from about 35% to about 100% Sp, from about 40% to about 100% Sp, from about 45 ... Approximately 100% Sp, approximately 40% to approximately 100% Sp, approximately 45% to approximately 100% Sp, approximately 50% to approximately 100% Sp, approximately 55% to approximately 100% Sp, approximately 60% to approximately 100% Sp, approximately 65% ​​to approximately 100% Sp, approximately 70% to approximately 100% Sp, approximately 75% to approximately 100% Sp, approximately 80% to approximately 100% Sp, approximately 85% to approximately 100% Sp. It comprises about 90% to about 100% Sp, or about 95% to about 100% Sp, about 20% to about 80% Sp, about 25% to about 75% Sp, about 30% to about 70% Sp, about 40% to about 60% Sp, or about 45% to about 55% Sp, with the remainder being Rp.

[0122] Any of the ASOs described herein can contain sugar moieties containing ribose or deoxyribose, as found in naturally occurring nucleotides, or modified sugar moieties or sugar analogs containing a morpholine ring. Non-limiting examples of modified sugar moieties include 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'-ON-methyl-acetamido (2'-NMA), 2'-substituted sugars such as 2'F, N3'->P5' phosphoramidate, 2'-dimethylaminooxyethoxy, 2'-dimethylaminoethoxyethoxy, 2'-guanidinium, 2'-O-guanidinium ethyl, carbamate-modified sugars, and bicyclic modified sugars. In some embodiments, the sugar moiety modification is selected from 2'-O-Me, 2'-NMA, 2'F, and 2'MOE. In some embodiments, the sugar moiety modification is an extra crosslink, such as in locked nucleic acids (LNAs). In some embodiments, the sugar analog contains a morpholine ring, such as phosphorodiamidate morpholinos (PMOs). In some embodiments, the sugar moiety comprises a ribofuranyl or 2' deoxyribofuranyl modification. In some embodiments, the sugar moiety comprises a 2',4' constrained 2'O-methyloxyethyl (cMOE) modification. In some embodiments, the sugar moiety comprises a cEt 2',4' constrained 2'-O-ethyl BNA modification. In some embodiments, the sugar moiety comprises a tricycloDNA (tcDNA) modification. In some embodiments, the sugar moiety comprises an ethylene nucleic acid (ENA) modification. In some embodiments, the sugar moiety comprises an MCE modification. Modifications are known in the art and described in the literature, e.g., Arver, et al., 2014, Nucleic Acid Therapeutics 24(1):37-47, incorporated herein by reference for this purpose. "A Chemical View of Oligonucleotides for Exon Skipping and Related Drug Applications," Nucleic Acid Therapeutics 24(1):37-47, which is incorporated herein by reference for this purpose.

[0123] In some embodiments, each monomer of an ASO is modified in the same way, e.g., each linkage in the backbone of the ASO contains a phosphorothioate linkage, or each ribose sugar moiety contains a 2'O-methyl modification. Such modifications present on each of the monomer components of an ASO are referred to as "uniform modifications." In some instances, a combination of different modifications may be desirable; for example, an ASO may contain a combination of phosphorodiamidate linkages and sugar moieties containing morpholine rings (morpholinos). Combinations of different modifications to an ASO are referred to as "mixed modifications" or "mixed chemistries."

[0124] In some embodiments, the ASO comprises one or more backbone modifications. In some embodiments, the ASO comprises one or more sugar moiety modifications. In some embodiments, the ASO comprises one or more backbone modifications and one or more sugar moiety modifications. In some embodiments, the ASO comprises a 2'MOE modification and a phosphorothioate backbone. In some embodiments, the ASO comprises a phosphorodiamidate morpholino (PMO). In some embodiments, the ASO comprises a peptide nucleic acid (PNA). Any of the ASOs described herein, or any component of the ASO (e.g., nucleobase, sugar moiety, backbone), can be modified to achieve a desired property or activity of the ASO or to reduce an undesirable property or activity of the ASO. For example, the ASO, or one or more components of any ASO, can be modified to increase binding affinity for a target sequence in a pre-mRNA transcript, decrease binding to any non-target sequences, decrease degradation by cellular nucleases (i.e., RNase H), improve uptake of the ASO into cells and / or the nucleus of a cell, alter the pharmacokinetics or pharmacodynamics of the ASO, and / or modulate the half-life of the ASO.

[0125] In some embodiments, ASO is composed of 2'-O-(2-methoxyethyl) (MOE) phosphorothioate modified nucleotides.ASO composed of such nucleotides is particularly well suited for the methods disclosed herein, and oligomers with such modifications have been shown to have significantly enhanced resistance to nuclease degradation and increased bioavailability, making them suitable for oral delivery in some embodiments described herein.For example, see Geary, et al., J Pharmacol Exp Ther.2001;296(3):890-7; Geary, et al., J Pharmacol Exp Ther.2001;296(3):898-904, the entire contents of which are incorporated herein by reference.

[0126] In some cases, the antisense oligomer comprises a sequence set forth in any one of SEQ ID NOs: 66-93. In some cases, the antisense oligomer consists of a sequence set forth in any one of SEQ ID NOs: 66-93.

[0127] Methods for synthesizing ASOs are known to those of skill in the art. Alternatively, or in addition, ASOs can be obtained from commercial sources.

[0128] Unless otherwise specified, the left-hand end of a single-stranded nucleic acid sequence (e.g., a pre-mRNA transcript, an oligonucleotide, an ASO, etc.) is the 5' end, and the left-hand direction of a single-stranded or double-stranded nucleic acid sequence is referred to as the 5' direction. Similarly, the right-hand end or direction of a nucleic acid sequence (single-stranded or double-stranded) is referred to as the 3' end or direction. Generally, a region or sequence 5' of a reference point in a nucleic acid is referred to as "upstream," and a region or sequence 3' of 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 codon or start codon is located, and the 3' end or direction is where the termination codon is located. In some embodiments, nucleotides upstream of a reference point in a nucleic acid can be designated by negative numbers, and nucleotides downstream of the reference point can be designated by positive numbers. For example, a reference point (e.g., an exon-exon junction in an mRNA) may be designated as the "zero" site, the nucleotides immediately adjacent to and upstream of the reference point are designated "minus one," while the nucleotides immediately adjacent to and downstream of the reference point are designated "plus one," e.g., "+1."

[0129] In some embodiments, the ASO is complementary to (and binds to) a targeted portion of the CFTR pre-mRNA downstream (3' direction) of the 5' splice site of intron 22 (e.g., the intron located from GRCh38.p14 / hg38:chr7:117,627,771 to GRCh38.p14 / hg38:chr7:117,642,437) in the CFTR pre-mRNA (e.g., in the direction designated by positive numbers relative to the 5' splice site). In some embodiments, the ASO is complementary to a targeting portion of the CFTR pre-mRNA within the region of about +1 to about +500 relative to the 5' splice site of intron 22 (e.g., the intron located from GRCh38.p14 / hg38:chr7:117,627,771 to GRCh38.p14 / hg38:chr7:117,642,437). In some embodiments, the ASO can be complementary to a targeting portion of the CFTR pre-mRNA within the region of nucleotides +6 to +496 relative to the 5' splice site of intron 22 (e.g., the intron located from GRCh38.p14 / hg38:chr7:117,627,771 to GRCh38.p14 / hg38:chr7:117,642,437).In some embodiments, the ASO is located at about +1 to about +500, about +1 to about +490, about +1 to about +480, about +1 to about +470, about +1 to about +510, about +1 to about +520, about +1 to about +560, about +1 to about +580, about +1 to about +590, about +1 to about +600, about +1 to about +610, about +1 to about +620, about +1 to about +630, about +1 to about +640, about +1 to about +650, about +1 to about +660, about +1 to about +670, about +1 to about +680, about +1 to about +690, about +1 to about +700, about +1 to about +710, about +1 to about +720, about +1 to about +730, about +1 to about +740, about +1 to about +750, about +1 to about +760, about +1 to about +770, about +1 to about +780, about +1 to about +79 ... 460, about +1 to about +450, about +1 to about +450, about +1 to about +440, about +1 to about +430, about +1 to about +420, about +1 to about +410, about +1 to about +400, about +1 to about +390, about +1 to about +380, about +1 to about +370, about +1 to about +360, about +1 to about +350, about +1 to about +340, about +1 to about +330, about +1 to about +320, about + 1 to about +310, about +1 to about +300, about +1 to about +290, about +1 to about +280, about +1 to about +270, about +1 to about +260, about +1 to about +250, about +1 to about +240, about +1 to about +230, about +1 to about +220, about +1 to about +210, about +1 to about +200, about +1 to about +190, about +1 to about +180, about +1 to about +170, about +1 to about + 160, about +1 to about +150, about +1 to about +140, about +1 to about +130, about +1 to about +120, about +1 to about +110, about +1 to about +100, about +1 to about +90, about +1 to about +80, about +1 to about +70, about +1 to about +60, about +1 to about +50, about +1 to about +40, about +1 to about +30, or about +1 to about +20. In some embodiments, the ASO is complementary to a targeting portion within a region of about +1 to about +100, about +100 to about +200, about +200 to about +300, about +300 to about +400, or about +400 to about +500 relative to the 5' splice site of intron 22 (e.g., the intron located from GRCh38.p14 / hg38:chr7:117,627,771 to GRCh38.p14 / hg38:chr7:117,642,437).

[0130] In some embodiments, the ASO is complementary to (and binds to) a targeting portion of the CFTR pre-mRNA that is upstream (5' direction) of the 5' splice site of intron 22 (e.g., the intron located from GRCh38.p14 / hg38:chr7:117,627,771 to GRCh38.p14 / hg38:chr7:117,642,437). In some embodiments, the ASO is complementary to a targeting portion of the CFTR pre-mRNA that is within the region from about -4 to about -270 relative to the 5' splice site of intron 22 (e.g., the intron located from GRCh38.p14 / hg38:chr7:117,627,771 to GRCh38.p14 / hg38:chr7:117,642,437). In some embodiments, the ASO may be complementary to a targeted portion of the CFTR pre-mRNA within the region between nucleotides -1 and -264 relative to the 5' splice site of intron 22 (e.g., the intron located from GRCh38.p14 / hg38:chr7:117,627,771 to GRCh38.p14 / hg38:chr7:117,642,437). In some embodiments, the ASO is at a position within the 5' splice site of intron 22 (e.g., the intron located at GRCh38.p14 / hg38:chr7:117,627,771 to GRCh38.p14 / hg38:chr7:117,642,437) that is about -1 to about -270, about -1 to about -260, about -1 to about -250, about -1 to about -240, about -1 to about -230, about -1 to about -220, about -1 to about -210, about -1 to about -20 0, about -1 to about -190, about -1 to about -180, about -1 to about -170, about -1 to about -160, about -1 to about -150, about -1 to about -140, about -1 to about -130, about -1 to about -120, about -1 to about -110, about -1 to about -100, about -1 to about -90, about -1 to about -80, about -1 to about -70, about -1 to about -60, about -1 to about -50, about -1 to about -40, about -1 to about -30, or about -1 to about -20.In some embodiments, the ASO is complementary to a targeting portion within a region of about -1 to about -50, about -50 to about -100, about -100 to about -150, about -150 to about -200, or about -200 to about -250 relative to the 5' splice site of intron 22 (e.g., the intron located from GRCh38.p14 / hg38:chr7:117,627,771 to GRCh38.p14 / hg38:chr7:117,642,437).

[0131] In some embodiments, the ASO is complementary to a targeted region of the CFTR pre-mRNA that is upstream (5' direction) of the 3' splice site of intron 22 (e.g., the intron located from GRCh38.p14 / hg38:chr7:117,627,771 to GRCh38.p14 / hg38:chr7:117,642,437). In some embodiments, the ASO is complementary to a targeted portion of the CFTR pre-mRNA that is within a region from about -1 to about -500 relative to the 3' splice site of intron 22 (e.g., the intron located from GRCh38.p14 / hg38:chr7:117,627,771 to GRCh38.p14 / hg38:chr7:117,642,437). In some embodiments, the ASO is complementary to a targeted portion of the CFTR pre-mRNA within region -1 to -496 relative to the 3' splice site of intron 22 (e.g., the intron located from GRCh38.p14 / hg38:chr7:117,627,771 to GRCh38.p14 / hg38:chr7:117,642,437).In some embodiments, the ASO is at a position between about -1 and about -500, about -1 and about -490, about -1 and about -480, about -1 and about -470, about -1 and about -50 ... about −460, about −1 to about −450, about −1 to about −440, about −1 to about −430, about −1 to about −420, about −1 to about −410, about −1 to about −400, about −1 to about −390, about −1 to about −380, about −1 to about −370, about −1 to about −360, about −1 to about −350, about −1 to about −340, about −1 to about −330, about −1 to about −320, about −1 to about -310, about -1 to about -300, about -1 to about -290, about -1 to about -280, about -1 to about -270, about -1 to about -260, about -1 to about -250, about -1 to about -240, about -1 to about -230, about -1 to about -220, about -1 to about -210, about -1 to about -200, about -1 to about -190, about -1 to about -180, about -1 to about -170, about -1 to about -280 to about -160, about -1 to about -150, about -1 to about -140, about -1 to about -130, about -1 to about -120, about -1 to about -110, about -1 to about -100, about -1 to about -90, about -1 to about -80, about -1 to about -70, about -1 to about -60, about -1 to about -50, about -1 to about -40, or about -1 to about -30. The targeting portion is complementary to a region within about -100 to about -200, about -200 to about -300, about -300 to about -400, or about -400 to about -500 relative to the 3' splice site of intron 22 (e.g., the intron located from GRCh38.p14 / hg38:chr7:117,627,771 to GRCh38.p14 / hg38:chr7:117,642,437).

[0132] In some embodiments, the ASO is complementary to a targeted region of the CFTR pre-mRNA downstream (3' direction) of the 3' splice site of intron 22 (e.g., the intron located from GRCh38.p14 / hg38:chr7:117,627,771 to GRCh38.p14 / hg38:chr7:117,642,437). In some embodiments, the ASO is complementary to a targeted portion of the CFTR pre-mRNA within the region of about +1 to about +100 relative to the 3' splice site of intron 22 (e.g., the intron located from GRCh38.p14 / hg38:chr7:117,627,771 to GRCh38.p14 / hg38:chr7:117,642,437). In some embodiments, the ASO is complementary to a targeting portion within a region from about +1 to about +90, about +1 to about +80, about +1 to about +70, about +1 to about +60, about +1 to about +50, about +1 to about +40, about +1 to about +30, about +1 to about +20, or about +1 to about +10 relative to the 3' splice site of intron 22 (e.g., the intron located from GRCh38.p14 / hg38:chr7:117,627,771 to GRCh38.p14 / hg38:chr7:117,642,437).

[0133] In some embodiments, the targeting portion of the CFTR pre-mRNA is within the region from 100 relative to the 5' splice site of intron 22 (e.g., the intron located from GRCh38.p14 / hg38:chr7:117,627,771 to GRCh38.p14 / hg38:chr7:117,642,437) to -100 relative to the 3' splice site of intron 22 (e.g., the intron located from GRCh38.p14 / hg38:chr7:117,627,771 to GRCh38.p14 / hg38:chr7:117,642,437). In some embodiments, the targeting portion of the CFTR pre-mRNA is within intron 22 (e.g., the intron located from GRCh38.p14 / hg38:chr7:117,627,771 to GRCh38.p14 / hg38:chr7:117,642,437). In some embodiments, the targeting portion of the CFTR pre-mRNA includes exon and intron boundaries.

[0134] ASOs can be of any length suitable for specific binding and effective enhancement of splicing. In some embodiments, the ASO consists of 8 to 50 nucleobases. For example, the ASO can 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 ASO consists of more than 50 nucleobases. In some embodiments, the ASO comprises 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 ... 0 nucleobases, 12-25 nucleobases, 12-20 nucleobases, 12-15 nucleobases, 13-50 nucleobases, 13-40 nucleobases, 13-35 nucleobases, 13-30 nucleobases, 13-25 nucleobases, 13-20 nucleobases, 14-50 nucleobases, 14-40 nucleobases, 14-35 nucleobases, 14-30 nucleobases, 14-25 nucleobases, 14-20 nucleobases, length The ASO may be 15-50 nucleobases, 15-40 nucleobases, 15-35 nucleobases, 15-30 nucleobases, 15-25 nucleobases, 15-20 nucleobases, 20-50 nucleobases, 20-40 nucleobases, 20-35 nucleobases, 20-30 nucleobases, 20-25 nucleobases, 25-50 nucleobases, 25-40 nucleobases, 25-35 nucleobases, or 25-30 nucleobases in length. In some embodiments, the ASO is 18 nucleotides in length. In some embodiments, the ASO is 15 nucleotides in length. In some embodiments, the ASO is 25 nucleotides in length.

[0135] In some embodiments, two or more ASOs are used that have different chemistries but are complementary to the same targeting portion of the CFTR pre-mRNA, hi some embodiments, two or more ASOs are used that are complementary to different targeting portions of the CFTR pre-mRNA.

[0136] In embodiments, the antisense oligonucleotides or antisense oligomers of the present disclosure are chemically linked to one or more moieties or conjugates, such as targeting moieties or other conjugates that enhance the activity or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties, cholesteryl moieties, aliphatic chains, such as dodecanediol or undecyl residues, polyamines, polyethylene glycol chains, or adamantane acetic acid. Oligonucleotides containing lipophilic moieties and preparation methods are described in the published literature. In some embodiments, the antisense oligonucleotides or antisense oligomers are conjugated to moieties, including, but not limited to, abasic nucleotides, aptamers, polyethers, polyamines, polyamides, peptides, polypeptides (e.g., antibodies), carbohydrates, such as N-acetylgalactosamine (GalNAc), N-Ac-Glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate), lipids, or polyhydrocarbon compounds. Conjugates can be linked to one or more of any nucleotide, including antisense oligonucleotides or antisense oligomers, at any of several positions on the sugar, base, or phosphate group, using, for example, a linker, as understood in the art and described in the literature. The linker can include a bivalent or trivalent branched linker. In embodiments, the conjugate is attached to the 3' end of the antisense oligonucleotide or antisense oligomer. Methods for preparing oligonucleotide conjugates are described, for example, in U.S. Patent No. 8,450,467 ("Carbohydrate conjugates as delivery agents for oligonucleotides"), which is incorporated herein by reference. In some embodiments, the antisense oligonucleotides or antisense oligomers of the present disclosure are non-covalently conjugated to moieties, including nanoparticles for nucleic acid drug delivery, known to those skilled in the art.Examples of strategies that can be applied to enhance delivery of the antisense oligonucleotides or antisense oligomers of the present disclosure are described, for example, in Roberts et al., Advances in oligonucleotide drug delivery. Nat Rev Drug Discov 19, 673-694 (2020), which is incorporated herein by reference in its entirety.

[0137] In some embodiments, the nucleic acid targeted by the ASO is a CFTR pre-mRNA expressed in a cell, such as a eukaryotic cell. In some embodiments, the term "cell" can refer to a population of cells. In some embodiments, the cell is within 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 cell or cell line associated with a disease or disorder. In some embodiments, the cell is in vitro (e.g., cell culture).

[0138] In some embodiments, the ASO is a salt of a nucleotide. In some embodiments, the ASO is a salt of a nucleotide fully phosphorothioate-linked oligonucleotide. In some embodiments, the ASO is a salt of a nucleotide in which a salt is linked to a phosphate bond. In some embodiments, the ASO is a salt of a nucleotide fully phosphorothioate-linked oligonucleotide, the salt being linked to a phosphate bond. In some embodiments, the ASO is a sodium salt of a nucleotide. In some embodiments, the ASO is a sodium salt of a nucleotide fully phosphorothioate-linked oligonucleotide. In some embodiments, the ASO is a sodium salt of a nucleotide in which a sodium salt is linked to a phosphate bond. In some embodiments, the ASO is a potassium salt of a nucleotide. In some embodiments, the ASO is a potassium salt of a nucleotide fully phosphorothioate-linked oligonucleotide. In some embodiments, the ASO is a potassium salt of a nucleotide fully phosphorothioate-linked oligonucleotide. In some embodiments, the ASO is a potassium salt of a nucleotide fully phosphorothioate-linked oligonucleotide in which a potassium salt is linked to a phosphate bond. In some embodiments, the ASO is a potassium salt of a nucleotide fully phosphorothioate-linked oligonucleotide in which a potassium salt is linked to a phosphate bond.

[0139] In some embodiments, the ASO is an undecasodium salt of a 12-nucleotide (12-mer). In some embodiments, the ASO is a dodecasodium salt of a 13-nucleotide (13-mer). In some embodiments, the ASO is a tridecasodium salt of a 14-nucleotide (14-mer). In some embodiments, the ASO is a tetradecasodium salt of a 15-nucleotide (15-mer). In some embodiments, the ASO is a pentadeca sodium salt of a 16-nucleotide (16-mer). In some embodiments, the ASO is a hexadeca sodium salt of a 17-nucleotide (17-mer). In some embodiments, the ASO is a heptadeca sodium salt of an 18-nucleotide (18-mer). In some embodiments, the ASO is an octadeca sodium salt of a 19-nucleotide (19-mer). In some embodiments, the ASO is a non-decasodium salt of a 20-nucleotide (20-mer). In some embodiments, the ASO is an icosodium salt of a 21-nucleotide (21-mer). In some embodiments, the ASO is a henicosa sodium salt of a 22-nucleotide (22-mer). In some embodiments, the ASO is a docosa sodium salt of a 23-nucleotide (23-mer). In some embodiments, the ASO is a tricosa sodium salt of a 24-nucleotide (24-mer). In some embodiments, the ASO is a tetracosa sodium salt of a 25-nucleotide (25-mer). In some embodiments, the ASO is a pentacosa sodium salt of a 26-nucleotide (26-mer). In some embodiments, the ASO is a hexacosa sodium salt of a 27-nucleotide (27-mer). In some embodiments, the ASO is a heptacosa sodium salt of a 28-nucleotide (28-mer). In some embodiments, the ASO is an octacosa sodium salt of a 29-nucleotide (29-mer). In some embodiments, the ASO is a non-cosa sodium salt of a 30-nucleotide (30-mer). In some embodiments, the ASO is a triacontriacont sodium salt of a 31-nucleotide (31-mer).In some embodiments, the ASO is a hentriaconta sodium salt of a 32-nucleotide (32-mer). In some embodiments, the ASO is a dotriaconta sodium salt of a 33-nucleotide (33-mer). In some embodiments, the ASO is a tritriaconta sodium salt of a 34-nucleotide (34-mer). In some embodiments, the ASO is a tetratriaconta sodium salt of a 35-nucleotide (35-mer). In some embodiments, the ASO is a pentatriaconta sodium salt of a 36-nucleotide (36-mer). In some embodiments, the ASO is a hexatriaconta sodium salt of a 37-nucleotide (37-mer). In some embodiments, the ASO is a heptatriaconta sodium salt of a 38-nucleotide (38-mer). In some embodiments, the ASO is an octatriaconta sodium salt of a 39-nucleotide (39-mer). In some embodiments, the ASO is a non-triaconta sodium salt of a 40-nucleotide (40-mer). In some embodiments, the ASO is a 41-nucleotide (41-mer) tetracone sodium salt. In some embodiments, the ASO is a 42-nucleotide (42-mer) henatetracone sodium salt. In some embodiments, the ASO is a 43-nucleotide (43-mer) donatetracone sodium salt. In some embodiments, the ASO is a 44-nucleotide (44-mer) trinatetracone sodium salt. In some embodiments, the ASO is a 45-nucleotide (45-mer) tetracone sodium salt. In some embodiments, the ASO is a 46-nucleotide (46-mer) pentatetracone sodium salt. In some embodiments, the ASO is a 47-nucleotide (47-mer) hexatetracone sodium salt. In some embodiments, the ASO is a 48-nucleotide (48-mer) heptatetracone sodium salt. In some embodiments, the ASO is a 49-nucleotide (49-mer) octatetracone sodium salt. In some embodiments, the ASO is a non-tetrasodium salt of a 50-nucleotide (50-mer).In some embodiments, the ASO is a 51-nucleotide (51-mer) pentacone sodium salt.

[0140] In some embodiments, the ASO is a non-decapotassium salt of a 12-nucleotide (12-mer). In some embodiments, the ASO is a dodecapotassium salt of a 13-nucleotide (13-mer). In some embodiments, the ASO is a tridecapotassium salt of a 14-nucleotide (14-mer). In some embodiments, the ASO is a tetradecapotassium salt of a 15-nucleotide (15-mer). In some embodiments, the ASO is a pentadecapotassium salt of a 16-nucleotide (16-mer). In some embodiments, the ASO is a hexadecapotassium salt of a 17-nucleotide (17-mer). In some embodiments, the ASO is a heptadecapotassium salt of an 18-nucleotide (18-mer). In some embodiments, the ASO is an octadecapotassium salt of a 19-nucleotide (19-mer). In some embodiments, the ASO is a non-adecapotassium salt of a 20-nucleotide (20-mer). In some embodiments, the ASO is an icosapomo potassium salt of a 21-nucleotide (21-mer). In some embodiments, the ASO is a henicosapotassium salt of a 22-nucleotide (22-mer). In some embodiments, the ASO is a docosapotassium salt of a 23-nucleotide (23-mer). In some embodiments, the ASO is a tricosapotassium salt of a 24-nucleotide (24-mer). In some embodiments, the ASO is a tetracosapotassium salt of a 25-nucleotide (25-mer). In some embodiments, the ASO is a pentacosapotassium salt of a 26-nucleotide (26-mer). In some embodiments, the ASO is a hexacosapotassium salt of a 27-nucleotide (27-mer). In some embodiments, the ASO is a heptacosapotassium salt of a 28-nucleotide (28-mer). In some embodiments, the ASO is an octacosapotassium salt of a 29-nucleotide (29-mer). In some embodiments, the ASO is a non-cosaponin potassium salt of a 30-nucleotide (30-mer). In some embodiments, the ASO is a triacontamin potassium salt of a 31-nucleotide (31-mer).In some embodiments, the ASO is a hentriakonta potassium salt of a 32-nucleotide (32-mer). In some embodiments, the ASO is a dotriakonta potassium salt of a 33-nucleotide (33-mer). In some embodiments, the ASO is a tritriakonta potassium salt of a 34-nucleotide (34-mer). In some embodiments, the ASO is a tetratriakonta potassium salt of a 35-nucleotide (35-mer). In some embodiments, the ASO is a pentatriakonta potassium salt of a 36-nucleotide (36-mer). In some embodiments, the ASO is a hexatriakonta potassium salt of a 37-nucleotide (37-mer). In some embodiments, the ASO is a heptatriakonta potassium salt of a 38-nucleotide (38-mer). In some embodiments, the ASO is an octatriakonta potassium salt of a 39-nucleotide (39-mer). In some embodiments, the ASO is a nonatriakonta potassium salt of a 40-nucleotide (40-mer). In some embodiments, the ASO is a tetracontapotassium salt of a 41-nucleotide (41-mer). In some embodiments, the ASO is a heptatetracontapotassium salt of a 42-nucleotide (42-mer). In some embodiments, the ASO is a dotetracontapotassium salt of a 43-nucleotide (43-mer). In some embodiments, the ASO is a tritetracontapotassium salt of a 44-nucleotide (44-mer). In some embodiments, the ASO is a tetratetracontapotassium salt of a 45-nucleotide (45-mer). In some embodiments, the ASO is a pentatetracontapotassium salt of a 46-nucleotide (46-mer). In some embodiments, the ASO is a hexatetracontapotassium salt of a 47-nucleotide (47-mer). In some embodiments, the ASO is a heptatetracontapotassium salt of a 48-nucleotide (48-mer). In some embodiments, the ASO is an octatetracontapotassium salt of a 49-nucleotide (49-mer). In some embodiments, the ASO is a non-tetracontaminant potassium salt of a 50-nucleotide (50-mer).In some embodiments, the ASO is a pentacontamin potassium salt of a 51-nucleotide (51-mer).

[0141] vector In some embodiments, vectors encoding the agents provided herein are provided herein. The vectors can express agents comprising the polynucleotide sequences disclosed herein. For example, the vectors can be viral vectors that express a polynucleotide sequence that binds to a targeting portion of a gene or pre-mRNA (e.g., a CFTR gene or a CFTR pre-mRNA encoding a CFTR protein) encoding a target peptide sequence. The methods provided herein can be adapted to contact a cell with a vector encoding an agent, such as an oligonucleotide, so that the agent binds to a gene or pre-mRNA in the cell and thus can regulate the processing of the gene or pre-mRNA. In some cases, the vector encoding the agent includes plasmid DNA, a viral vector, and a bacterial vector. In some cases, the viral vector includes an adenoviral vector, an adeno-associated viral (AAV) vector, a lentiviral vector, a herpes simplex virus (HSV) viral vector, a retroviral vector, a parvovirus-based vector, or any applicable viral vector.

[0142] Gene editing In some aspects, provided herein are agents that can be used for gene editing.

[0143] In some cases, the agent comprises or forms part of a gene editing tool configured to modify the CFTR gene. For example, the agent can remove nucleic acid sequences in the CFTR gene downstream of the first intron (e.g., intron 22) that contain an alternative adenylation site. In some cases, the nucleic acid sequences removed by the agent from the CFTR gene are located at GRCh38.p14 / hg38:chr7:117,642,438 to GRCh38.p14 / hg38:chr7:117,668,665. Thus, the level of processed mRNA that encodes the CFTR protein and lacks the nucleic acid sequences downstream of the first intron is increased.

[0144] In some cases, the gene editing tool comprises a vector, e.g., a viral vector, for gene editing based on CRISPR-Cas9, TALEN, zinc finger, or other applicable technology.

[0145] In some cases, a gene editing agent provided herein comprises a polynucleotide sequence complementary to a target nucleic acid located in the CFTR gene. For example, the agent may comprise a guide RNA complementary to a target nucleic acid located in the CFTR gene. As shown in FIG. 8, in some embodiments, the guide RNA targets a genomic region 5' to exon 23 of the CFTR gene. In some embodiments, the guide RNA targets a genomic region within intron 22 of the CFTR gene. In some embodiments, the guide RNA targets a genomic region of the CFTR gene 3' downstream of exon 27. In some embodiments, the guide RNA targets a genomic region downstream of the 3'UTR of the CFTR gene. In some cases, the gene editing agent comprises multiple guide RNAs. In some cases, the gene editing agent comprises at least two guide RNAs. In some cases, the guide RNA comprises the sequence of SEQ ID NO: 94. In some cases, the guide RNA comprises the sequence of SEQ ID NO: 95. In some cases, the agent for gene editing comprises a pair of guide RNAs, each of which comprises the sequence of SEQ ID NOs: 94 and 95.

[0146] In some cases, the agent for gene editing comprises a polynucleotide sequence complementary to a portion of exon 23 of the CFTR gene. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to SEQ ID NO: 4. In some cases, the agent comprises a polynucleotide sequence that is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to SEQ ID NO: 4. In some cases, the agent comprises a polynucleotide sequence complementary to a portion of the sequence of SEQ ID NO: 4. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least eight contiguous nucleic acids of SEQ ID NO: 4. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least eight contiguous nucleic acids of SEQ ID NO: 4. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least 10, 12, 14, or 16 contiguous nucleic acids of SEQ ID NO: 4. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least 18 contiguous nucleic acids of SEQ ID NO:4.

[0147] In some cases, the agent for gene editing comprises a polynucleotide sequence complementary to a portion of exon 22 of the CFTR gene. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to SEQ ID NO: 3. In some cases, the agent comprises a polynucleotide sequence that is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to SEQ ID NO: 3. In some cases, the agent comprises a polynucleotide sequence complementary to a portion of the sequence of SEQ ID NO: 3. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least eight contiguous nucleic acids of SEQ ID NO: 3. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least eight contiguous nucleic acids of SEQ ID NO: 3. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least 10, 12, 14, or 16 contiguous nucleic acids of SEQ ID NO: 3. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least 18 contiguous nucleic acids of SEQ ID NO:3.

[0148] In some cases, the agent for gene editing comprises a polynucleotide sequence complementary to a portion of intron 22 of the CFTR gene. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to SEQ ID NO: 5. In some cases, the agent comprises a polynucleotide sequence that is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to SEQ ID NO: 5. In some cases, the agent comprises a polynucleotide sequence complementary to a portion of the sequence of SEQ ID NO: 5. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least eight contiguous nucleic acids of SEQ ID NO: 5. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least eight contiguous nucleic acids of SEQ ID NO: 5. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least 10, 12, 14, or 16 contiguous nucleic acids of SEQ ID NO: 5. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least 18 contiguous nucleic acids of SEQ ID NO:5.

[0149] In some cases, the agent for gene editing further comprises a polynucleotide sequence complementary to a portion of the 3'UTR of the CFTR gene. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to SEQ ID NO: 65. In some cases, the agent comprises a polynucleotide sequence that is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to SEQ ID NO: 65. In some cases, the agent comprises a polynucleotide sequence complementary to a portion of the sequence of SEQ ID NO: 65. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least eight contiguous nucleic acids of SEQ ID NO: 65. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least eight contiguous nucleic acids of SEQ ID NO: 65. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least 10, 12, 14, or 16 contiguous nucleic acids of SEQ ID NO: 65. In some cases, the agent comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% complementary to at least 18 contiguous nucleic acids of SEQ ID NO: 65.

[0150] Target treatment Any of the compositions provided herein can be administered to an individual. "Individual" can be used interchangeably with "subject" or "patient." An individual can be a mammal, e.g., a human, or an animal, e.g., a non-human primate, rodent, rabbit, rat, mouse, horse, donkey, goat, cat, dog, cow, pig, or sheep. In embodiments, the individual is a human. In embodiments, the individual is a fetus, embryo, or child. In other embodiments, the individual can be another eukaryotic organism, such as a plant. In some embodiments, the compositions provided herein are administered to cells ex vivo.

[0151] 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 a 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 an insufficient amount of a protein or an insufficient activity of a protein. In cases where an individual is "at an increased risk" of having a disease or disorder that causes an insufficient amount of a protein or an insufficient activity of a protein, the method includes preventative or prophylactic treatment. For example, an individual may be at increased risk of having such a disease or disorder due to a family history of the disease. Typically, individuals at increased risk of having such a disease or disorder benefit from preventative treatment (e.g., by preventing or delaying the onset or progression of the disease or disorder). In embodiments, a fetus is treated in utero, for example, by administering an ASO composition to the fetus directly or indirectly (e.g., via the mother).

[0152] In some cases, the therapeutic agent is a drug provided herein. For example, the therapeutic agent can include an antisense oligomer. In some cases, the therapeutic agent includes a vector, such as a viral vector, that expresses an oligonucleotide that binds to a target region of a pre-mRNA (e.g., a CFTR pre-mRNA encoding a CFTR protein) that encodes a target peptide sequence. The method provided herein can be adapted to contact a drug, such as a vector encoding an oligonucleotide, with a cell, so that the drug binds to the pre-mRNA in the cell and regulates the processing of the pre-mRNA. In some cases, the viral vector includes an adenovirus vector, an adeno-associated virus (AAV) vector, a lentivirus vector, a herpes simplex virus (HSV) virus vector, a retrovirus vector, a parvovirus-based vector, or any applicable viral vector. In some cases, the therapeutic agent includes a gene editing tool configured to modify the gene encoding the target peptide sequence so that the gene region encoding the inefficient translation region is deleted. In some cases, the gene editing tool comprises a vector, e.g., a viral vector, for gene editing based on CRISPR-Cas9, TALEN, zinc finger, or other applicable technology.

[0153] The suitable administration route of the agent of the present disclosure can vary depending on the cell type that the agent is desired to be delivered to.Multiple tissues and organs are affected by cystic fibrosis, and lungs are the most prominently affected tissue.The agent of the present disclosure can be administered to patients parenterally, for example, by intranasal administration, intratracheal administration, intravenous injection, intraperitoneal injection, intramuscular injection, intrathecal injection, subcutaneous injection, oral administration, intrasynovial injection, intraventricular injection, intravitreal administration, subretinal injection, topical application or implantation.

[0154] In some embodiments, subjects treated with the present methods and compositions are assessed for improvement in their condition using any method known and described in the art.

[0155] Any of the compositions provided herein can be administered to an individual (e.g., a human subject) to treat one or more symptoms associated with cystic fibrosis or related complications. In some cases, any of the compositions provided herein can be administered to an individual to reduce the risk of developing cystic fibrosis or one or more symptoms associated with cystic fibrosis or related complications. In some cases, one or more symptoms improved or prevented by the compositions or methods of the presently disclosed subject matter include isolated obstructive azoospermia, chronic sinusitis, chronic pancreatitis, pulmonary dysfunction, thick secretions in the lungs, pancreas, liver, intestines, and reproductive tract, elevated sweat chloride, and increased salt content in sweat gland secretions. In some cases, the one or more symptoms ameliorated or prevented by the compositions or methods of the presently disclosed subject matter include respiratory and / or pulmonary symptoms such as persistent productive cough, hyperinflation of the lung fields on chest radiograph, microbial infection, bronchiectasis, airway hyperreactivity, allergic bronchopulmonary aspergillosis, obstructive sleep apnea, and pulmonary hypertension. In some cases, the compositions or methods of the presently disclosed subject matter reduce the risk of developing atypical symptoms that can occur in later life in cystic fibrosis patients, including sinus disease, pancreatic disease, infertility, musculoskeletal disorders, recurrent venous thrombosis, anemia, electrolyte abnormalities, nephrolithiasis, and hydropic wrinkles.

[0156] Any of the compositions provided herein can be administered to an individual to treat a CFTR-related disorder (CFTR-RD). CFTR-RD can include one or more clinical entities related to CFTR dysfunction that may not meet the diagnostic criteria for CF. CFTR-RD can be caused by impaired CFTR channel function in multiple affected organs (e.g., lung, pancreas, liver, reproductive organs) and can be associated with impaired pulmonary function, poor nutritional status, and chest infections. CFTR-RD can include disorders of the gastrointestinal (GI) tract, including CF-related pancreatic insufficiency, CF-related pancreatitis, CF-related diabetes, CF-related liver disease, and gallbladder disease. CFTR-RD includes disorders of the reproductive tract, including congenital bilateral absence of the vas deferens (CBAVD), and disorders of the lung, including disseminated bronchiectasis.

[0157] In some cases, the compositions provided herein can improve CFTR-RD by restoring CFTR-associated ion transport in the lungs and improving lung function. In some cases, the compositions provided herein can directly address the underlying cause of CFTR-RD by enhancing CFTR-associated ion transport in affected organs (e.g., lungs, pancreas, liver, reproductive organs). Any of the compositions provided herein can be administered to an individual (e.g., a male human subject) to treat one or more symptoms associated with congenital bilateral absence of the vas deferens (CBAVD). In some cases, congenital bilateral absence of the vas deferens (CBAVD) in a subject is associated with one or more mutations in the CFTR gene. A subject suffering from congenital bilateral absence of the vas deferens may have delayed or underdeveloped development of the vas deferens (the ducts that carry sperm from the testes). In some cases, the compositions provided herein promote the development of the vas deferens in a subject or treat one or more dysfunctions of the vas deferens in a subject.

[0158] When referring to increasing partially functional, functional CFTR protein levels, the increase can be clinically significant. The increase can be relative to the level of partially functional, functional CFTR protein in a subject without treatment, or relative to the amount of CFTR protein in a population of similar subjects. It is generally accepted that 5% of wild-type CFTR function can be therapeutically relevant; for example, if a therapeutic intervention restores CFTR function in a patient to a level that is at least 5% of wild-type CFTR function, the intervention is therapeutically effective, e.g., ameliorating, eliminating, or preventing one or more symptoms associated with a lack of CFTR activity and / or function from which the patient suffers. The increase can be at least 10% or more CFTR protein function compared to the average subject or pre-treatment subject. The increase can be at least 20% or more CFTR protein compared to the average subject or pre-treatment subject. The increase can be at least 40% or more CFTR protein compared to the average subject or pre-treatment subject. The increase can be at least 50% or more CFTR protein compared to the average subject or pre-treatment subject. The increase can be at least 80% or more CFTR protein relative to the average subject or the subject before treatment. The increase can be at least 100% or more CFTR protein relative to the average subject or the subject before treatment.

[0159] Therapies that promote exon 22 excision, as described herein, can be used to treat many CFTR mutations 3' downstream of the first exon 22ApA site. For example, Figure 1C lists 13 mutations in exons 23-27 that can result in PTC, all of which are potential candidates for exon 22 excision therapy. Non-limiting exemplary CFTR mutations to which the subject compositions, methods, and kits are applicable include c.3717G>A, c.3717+4A>G, c.3717+5G>A, c.3717+40A>G, c.3718-2477C>T, c.3718-1G>A, c.3718-3T>G, c.3719T>G, c.3731G>A, c.3737C>T, c.3744delA, c.3745G>A, c.3747delG, c.3752G>A, c.3761T>G , c.3763T>C, c.3764C>A, c.3773dupT, c.3806T>A, c.3808delG, c.3808G>A, c.3822G>A, c.3846G>A, c.[3846G>A, 3848G>T], c. 3848G>T, c.3872A>G, c.3873G>C, c.3873+1G>A, c.(3873+1_3874-1)_(3963+1_3964-1)del, c.3873+2T>C, 3876delA, c.3883_3 886delATTT, c.3883delA, c.3889dupT, c.3891dupT, c.3908delA, c.3909C>G, c.3929G>A, c.3937C>T, c.(3963+1_3964-1)_(* 1_?)del, c.3964-78_4242+577del, c.3971T>C, c.3988C>T, c.4004T>C, c.4036_4042del, c.4046G>A, c.4077_4080delTGTTin sAA, c.4086dupT, c.4097T>A, c.4111G>T, c.4124A>C, c.4127_4131delTGGAT, c.4144C>T, c.4147dupA, c.4197_4198delCT, c. Includes 4231C>T, c.4234C>T, c.4242+1G>T, c.4242+1G>A, c.4251delA, c.4300_4301dup, c.4364C>G, c.4426C>T, or c.4439T>C.

[0160] Pharmaceutical Composition Pharmaceutical compositions or preparations that comprise the drug of the described compositions, such as antisense oligonucleotides, and that are used in any of the described methods can be prepared according to conventional techniques that are well known in the pharmaceutical industry and are described in published literature.In embodiments, the pharmaceutical compositions or preparations for treating subjects comprise any antisense oligomers described herein, or their pharmaceutically acceptable salts, solvates, hydrates or esters in an effective amount.The pharmaceutical preparations that comprise antisense oligomers can further comprise pharmaceutically acceptable excipients, diluents or carriers.

[0161] Pharmaceutically acceptable salts are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, 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. Salts may be used in conjunction with other salts during the final isolation and purification of the compounds. They can be prepared in situ or separately by reacting the free base form with a suitable organic acid. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or 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, and glucoheptonate. , glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, ethyl methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, phenylpropionate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc.Further pharmaceutically acceptable salts include non-toxic ammonium salts, quaternary ammonium salts, amine cation salts formed, where appropriate, with counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, aryl sulfonates, and the like.

[0162] In some embodiments, the compositions are formulated into any of many possible dosage forms, including, 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. Suspensions may also contain stabilizers. In embodiments, pharmaceutical formulations or compositions of the present disclosure include, but are not limited to, solutions, emulsions, microemulsions, foams, or liposome-containing formulations (e.g., cationic or non-cationic liposomes).

[0163] The pharmaceutical compositions or formulations described herein may include one or more penetration enhancers, carriers, excipients, or other active or inactive ingredients as appropriate and known to those skilled in the art or as described in the published literature. In some embodiments, the liposomes also include sterically stabilized liposomes, such as liposomes containing one or more specialized lipids. These specialized lipids result in liposomes with enhanced circulation life. In some embodiments, the sterically stabilized liposomes contain one or more glycolipids or are derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. In some embodiments, a surfactant is included in the pharmaceutical formulation or composition. The use of surfactants in drug products, formulations, and emulsions is well known in the art. In embodiments, the present disclosure uses penetration enhancers to facilitate efficient delivery of antisense oligonucleotides, for example, to aid diffusion across cell membranes and / or to enhance the permeability of lipophilic drugs. In some embodiments, the penetration enhancer is a surfactant, fatty acid, bile salt, chelating agent, or non-chelating nonsurfactant. In some embodiments, the pharmaceutical composition or formulation can comprise lipid nanoparticles. In some embodiments, the pharmaceutical formulation comprises multiple antisense oligonucleotides. In embodiments, the antisense oligonucleotide is administered in combination with another drug or therapeutic agent.

[0164] In some cases, the pharmaceutical composition is formulated for any suitable administration method for a cystic fibrosis patient, for example, parenteral administration, including intranasal administration, intratracheal administration, intravenous injection, intraperitoneal injection, intramuscular injection, intrathecal injection, subcutaneous injection, oral administration, synovial injection, intracerebroventricular injection, intravitreal administration, subretinal injection, topical application, or implantation. In some cases, intratracheal administration includes administering a pulmonary dosage form, including a form for administration via a dry powder inhaler, a metered dose inhaler, or a nebulizer, either in conventional or nanoparticle and particulate forms.

[0165] Combination treatment In some cases, the methods provided herein include administering a first therapeutic agent and a second therapeutic agent. In some cases, the first agent includes a therapeutic agent provided herein, and the second agent is another agent that is a known agent for the treatment of cystic fibrosis or another disease or condition. In one embodiment, the additional agent is an antibiotic or anti-infective agent (e.g., azithromycin, amoxicillin and clavulanic acid, cloxacillin and dicloxacillin, ticarcillin and clavulanic acid, cephalexin, cefdinir, cefprozil, cefaclor; sulfamethoxazole and trimethoprim, erythromycin / sulfisoxazole, erythromycin, clarithromycin, tetracycline, doxycycline, minocycline, tigecycline, vancomycin, imipenem, meropenem).

[0166] In one embodiment, the additional agent is one or more CFTR modulators. In some cases, the CFTR modulator can improve the processing and transport of one or more variants of CFTR protein, such as lumacaftor, tezacaftor, elexacaftor, or tezacaftor. In some cases, the CFTR modulator can enhance the chloride conductance of one or more variants of CFTR protein on cells, such as ivacaftor. In some embodiments, the one or more CFTR modulators are a combination of an agent that can enhance chloride conductance and an agent that can improve the processing and transport of one or more variants of CFTR protein, such as ivacaftor (KALYDECO®), lumacaftor, lumacaftor / ivacaftor (ORKAMBI®), tezacaftor / ivacaftor (SYMDEKO®), or Trikafta® elexacaftor / ivacaftor / tezacaftor (Trikafta®). In some cases, CFTR modulators are referred to by names used in basic research, such as elexacaftor / VX-661, tezacaftor / VX-445, ivacaftor / VX-770, lumacaftor / VX-809, or trikafta / VX-445 / VX-661 / VX-770, which may be used interchangeably herein.

[0167] In one embodiment, the additional agent is a mucolytic agent for airway clearance, e.g., acetylcysteine, ambroxol, bromhexine, carbocysteine, erdosteine, mecysteine, dornase alfa, hypertonic saline, or mannitol. In one embodiment, the additional agent is a bronchodilator (e.g., albuterol).

[0168] In one embodiment, the additional agent is an immunosuppressant and is a corticosteroid (e.g., an inhaled corticosteroid (e.g., beclomethasone (QVAR®), budesonide (PULMICORT®), budesonide / formoterol (SYMBICORT®), ciclesonide (ALVESCO®), fluticasone (FLOVENT HFA®), fluticasone propionate (FLOVENT DISKUS®), fluticasone furoate (ARNUITY ELLIPTA®), fluticasone propionate / salmeterol (ADVAIR®)), fluticasone furoate / umeclidinium / vilanterol (TRELEGY®), ELLIPTA®), mometasone furoate (ASMANEX®), or mometasone / formoterol (DULERA®), prednisone, or methylprednisolone. In one embodiment, the additional agent is a nonsteroidal immunosuppressant, which may be a biologic, and includes a polyclonal antilymphocyte antibody (e.g., an antilymphocyte globulin (ALG) or anti-thymocyte globulin (ATG) antibody, which may be of horse or rabbit origin, for example), a monoclonal antilymphocyte antibody (e.g., an anti-CD3 antibody (e.g., muromonab or alemtuzumab) or an anti-CD20 antibody (e.g., rituximab)), an interleukin-2 (IL-2) receptor antagonist (e.g., daclizumab or basiliximab).In one embodiment, the additional agent is a nonsteroidal immunosuppressant, which may be a small molecule drug, including a calcineurin inhibitor (e.g., cyclosporin A or tacrolimus), a cell cycle inhibitor (e.g., azathioprine, mycophenolate mofetil (MMF), or mycophenolic acid (MPA)), a mammalian target of rapamycin (mTOR) inhibitor (e.g., sirolimus (rapamycin) or everolimus), methotrexate, cyclophosphamide, anthracyclines (e.g., doxorubicin, idarubicin, aclarubicin, daunorubicin, epirubicin, valrubicin, mitoxantrone, or combinations thereof), a taxane (e.g., TAXOL® (paclitaxel)), and combinations thereof (e.g., a combination of a calcineurin inhibitor, a cell cycle inhibitor, or a corticosteroid).

[0169] In one embodiment, the additional medication is a nutritional supplement such as pancrelipase (pancreatic enzyme replacement) (e.g., Pancrease®, Pancreacarb®, or Ultrase®), Creon®, Liprotamase® (formerly Trizytek®), Aquadeks®, and glutathione inhalation.

[0170] In some embodiments, the ASOs disclosed herein can be used in combination with one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents can include small molecules. For example, the one or more additional therapeutic agents can include small molecules described in US20220241206, the entire contents of which are incorporated herein by reference. In some embodiments, the one or more additional therapeutic agents include ASOs that can be used to inhibit intron splicing-out.

[0171] In some embodiments, the ASOs disclosed herein can be used in combination with one or more additional nucleic acid-based therapeutic agents for treating cystic fibrosis. In some cases, the additional therapeutic agents include agents for replacement therapy (e.g., gene therapy and RNA replacement), agents for gene editing (e.g., CRISPR-Cas9, TALEN, and zinc finger), agents for RNA interference (e.g., siRNA, miRNA), ASOs (e.g., gene silencing ASOs, exon skipping ASOs, or read-through ASOs), and tRNAs (e.g., suppressor tRNAs).

[0172] kit In other aspects, kits are provided herein. The kits disclosed herein may include a composition or pharmaceutical composition disclosed herein and instructions for use of the composition or pharmaceutical composition. The kits may include a composition or pharmaceutical composition disclosed herein and a second therapeutic agent (or additional agent). In some cases, the kits further include one or more additional reagents, such as the immunosuppressant described above, or one or more agents for treating cystic fibrosis described herein. The kits provided herein typically include a label indicating the intended use of the contents of the kit. The term label includes any written or recorded material supplied on or with the kit, or accompanying the kit.

[0173] Methods for identifying additional ASOs Also within the scope of the present disclosure are methods for identifying or determining ASOs that regulate the processing of CFTR pre-mRNA transcripts, for example, the splicing of CFTR pre-mRNA and / or the alternative polyadenylation of CFTR splicing products.For example, the method can include identifying or determining ASOs that process CFTR pre-mRNA transcripts, for example, the splicing of CFTR pre-mRNA and / or the alternative polyadenylation of CFTR splicing products.ASOs that specifically hybridize to different nucleotides within the targeting portion of CFTR pre-mRNA can be screened to identify or determine ASOs that promote the production of exon 22-truncated CFTR mRNA transcripts, for example, ASOs that suppress the splicing of intron 22 and / or promote polyadenylation at alternative polyadenylation sites in intron 22.In some embodiments, the ASOs can interfere with the interaction of one or more splicing factors with CFTR pre-mRNA. Any method known in the art can be used to identify (determine) an ASO that, upon hybridization to a targeted portion of processed mRNA, produces a desired effect (e.g., increasing the level of exon 22-truncated CFTR mRNA, suppressing splicing out from intron 22, or promoting polyadenylation at an alternative polyadenylation site in intron 22). Examples of methods that can be used are provided below.

[0174] A round of screening, referred to as an ASO "walk," can be performed using ASOs designed to hybridize to a target portion of a CFTR pre-mRNA transcript containing a nonsense mutation, e.g., downstream of intron 22. For example, the ASOs used in the ASO walk can be tiled every 5 nucleotides from approximately 100 nucleotides upstream of the region of interest (e.g., a target portion within exon 22, intron 22, and / or exon 23 of the CFTR pre-mRNA) to approximately 100 nucleotides downstream of the region of interest. For example, a first ASO 15 nucleotides in length can be designed to specifically hybridize to the first 18 nucleotides at the 5' end of intron 22 of the CFTR pre-mRNA, e.g., +1 to +15 relative to the 5' end of intron 22 of the CFTR pre-mRNA. A second ASO can be designed to specifically hybridize to nucleotides +6 to +20 relative to the 5' end of intron 22 of the CFTR pre-mRNA. The ASO is designed to span the targeted portion of the CFTR pre-mRNA transcript. In embodiments, the ASO can be tiled more closely, for example, every 1, 2, 3, or 4 nucleotides.

[0175] One or more ASOs, or a control ASO (an ASO with a scrambled sequence, a sequence not expected to hybridize to the target region), are delivered, for example, by transfection, to a disease-related cell line expressing CFTR pre-mRNA. The regulatory effect of each ASO can be evaluated by any method known in the art, for example, by assessing the level of mature CFTR mRNA transcripts in the cells or the expression level of CFTR protein encoded by the processed mRNA. Evaluation of mature CFTR mRNA transcripts can be performed, for example, by reverse transcriptase (RT)-PCR using primers spanning a portion of exon 23. A reduction or absence of longer RT-PCR products produced using primers spanning the region containing the excluded nucleic acid in ASO-treated cells compared to control ASO-treated cells indicates increased levels of processed mRNA lacking the nucleic acid sequence downstream of the first intron containing the alternative adenylation site. In some embodiments, splicing out from intron 22 can be suppressed using the ASOs described herein. The amount of C-terminal truncated CFTR protein encoded by the processed mRNA can also be evaluated to determine whether each ASO achieves the desired effect (e.g., enhancing partial protein expression). Any method known in the art for evaluating and / or quantifying protein production can be used, such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA. In some cases, the function of CFTR protein in cells can also be evaluated to screen for desirable ASOs according to some embodiments of the present disclosure. For example, an increase in chloride channel conductance can indicate an increase in the level of functional or partially functional CFTR protein in cells. In some cases, CFTR correctors / enhancers are used for functional assays.

[0176] A second round of screening, called an ASO "microwalk," can be performed using ASOs designed to hybridize to target regions of the pre-mRNA. The ASOs used in the ASO microwalk are tiled nucleotide-by-nucleotide to further refine the nucleotide sequence of the pre-mRNA that, upon hybridization with the ASO, results in modulation of pre-mRNA processing.

[0177] The region defined by the ASO that promotes splicing of the target intron is explored in more detail by ASO "microwalks," involving ASOs spaced in 1-nt steps, as well as longer ASOs, typically 18-25 nt.

[0178] As described above for ASO walks, ASO microwalks are performed by delivering one or more ASOs, or a control ASO (e.g., an ASO with a scrambled sequence, or a sequence not expected to hybridize to the target region), into a disease-relevant cell line expressing the target pre-mRNA, for example, by transfection. To evaluate the efficacy of candidate ASOs during an ASO microwalk, approaches similar to those used in the ASO walk described above can be used.

[0179] ASOs that hybridize to a region of pre-mRNA, resulting in the regulation of CFTR pre-mRNA processing and the increased production of C-terminal truncated CFTR protein, can be tested in vivo using animal models, such as transgenic mouse models or disease mouse models in which the full-length human CFTR gene (with or without the genetic mutations identified in human cystic fibrosis patients) is knocked in. The appropriate administration route of ASOs can vary depending on the disease and / or the cell type to which ASOs are desired to be delivered. ASOs can be administered, for example, by intranasal administration, intratracheal administration, intravenous injection, intraperitoneal injection, intramuscular injection, intrathecal injection, subcutaneous injection, oral administration, synovial injection, intracerebroventricular injection, intravitreal injection, subretinal injection, topical application, or transplantation. After administration, cells, tissues, and / or organs of model animals can be evaluated to determine the effect of ASO treatment, for example, by evaluating mRNA processing and protein production using methods known in the art and described herein. The animal model can also be any phenotypic or behavioral indicator of disease or disease severity. [Example]

[0180] The following examples are provided to further illustrate some embodiments of the present disclosure, but are not intended to limit the scope of the disclosure, and by their illustrative nature it will be understood that other procedures, methods, or techniques known to those skilled in the art may alternatively be used.

[0181] This study describes a novel, alternative approach based on the use of ASOs that has therapeutic potential to address a subset of CFTR PTC variants present at the 3' end, for which no approved treatment exists. A novel feature of this approach is that it enables CFTR 3' PTC-specific NMD escape without the need for global NMD inhibition. This strategy leverages a mechanistic understanding of the regulatory events that result in two outcomes of intron 22 processing: 1) canonical exon 22 / 23 splicing, which leads to intron 22 excision, or 2) intron 22 ApA site usage (referred to herein as E22 cleavage), which results in a 3'-truncated CFTR mRNA isoform. As shown in Figure 1B, inhibition of canonical exon 22 / 23 splicing promotes intron 22 ApA usage and can promote NMD escape of 3'-end CFTR PTCs (i.e., R1162X and W1282X) and high expression of E22-truncated mRNA via E22-truncated protein. Furthermore, given that the CFTR W1281 truncated protein is known to retain partial function when treated with modulators, the upregulated E22 truncated protein (1259+9aa) may retain some chloride conductance and provide benefit to cystic fibrosis patients with 3'-terminal PTCs.

[0182] method

[0183] Cell culture. Minimum essential medium (MEM), fetal bovine serum (FBS), penicillin / streptomycin, LHC-8 basal medium, 7.5% bovine serum albumin, and TrypLE Express were purchased from Gibco. 16HBE cells were grown in MEM supplemented with 10% FBS and 1% penicillin / streptomycin at 37°C / 5% CO2. Plates / flasks were coated by incubating a thin layer of coating solution (LHC-8 basal medium, 1.34 μl / ml 7.5% bovine serum albumin, 10 μl / ml bovine collagen solution, type 1 (Advanced BioMatrix), 10 μl / ml fibronectin derived from human plasma (Thermo Fisher Scientific)) at 37°C / 5% CO2 for 2–3 h. The coating solution was then completely removed and stored at 4°C. Cells were dissociated with TrypLE Express and centrifuged at 120 × g for 5 min before seeding. Fisher Rat Thyroid (FRT) cells and primary human bronchial epithelial cells were cultured as previously described.

[0184] Generation of gene-edited cell lines. 16HBEge cells were cultured, edited, and cloned as described previously. To generate exon 23-3'UTR deletion clones, the following targeting components of the crRNA guide RNA were used: 5'-TGCTCAGTTATAGTATATAA-3' (SEQ ID NO: 94) and 5'-TTAGTTATCTGTTTAAACTA-3' (SEQ ID NO: 95). PCR amplification using primers spanning the genomic deletion [5'-gactcccctgtccttgttga-3' and 5'-GATCCCACTCCTAGGTCCTTCGA-3'] was used to identify clones with the desired deletion, and primers at the 3' junction [5'-AGCCAGCACAGCCTCTTAGATGC-3' and 5'-GATCCCACTCCTAGGTCCTTCGA-3'] were used to identify clones that were not homozygous for the desired deletion.

[0185] Free uptake ASO treatments in 16HBE. Sterically blocked ASOs were purchased from IDT, with a phosphorothioate (PS) backbone and 2-O-methoxyethyl (2'-MOE) modifications at each nucleotide. Lyophilized sterically blocked ASOs were resuspended to 1 mM in PBS + Mg + Ca and stored in frozen aliquots to minimize the effects of freeze-thaw cycles. 16HBEge and 16HBE14o strains were seeded at 0.150 x 10^6 cells / well in 24-well plates and grown at 37°C / 5% CO2 in MEM supplemented with 10% FBS and 1% penicillin / streptomycin. After 24 h, the medium was refreshed with the addition of ASOs, and RNA was harvested after 48 h of treatment.

[0186] ddPCR isoform quantification. RNA was isolated from treated cells using the Aurum Total RNA Mini Kit. One-step reverse transcriptase and quantitative PCR were performed using the iTaq Universal Probes One Step Kit. All qPCR analyses were performed on a CFX Touch Deep Well real-time PCR detection system and analyzed with Bio-Rad CFX Maestro 1.0 software (version 4.0.2325.0418). ddPCR assays consisted of cDNA or digested genomic DNA, 2x ddPCR Supermix for Probes (without dUTP) (1x final) (Bio-Rad), 40x as-say (1x final), and nuclease-free water in a total reaction volume of 20 μl. cDNA synthesis was performed using the iScript Advanced cDNA Synthesis Kit for RT-qPCR (Bio-Rad). Droplets were generated using a QX200 Droplet Digital PCR system (Bio-Rad), cycled on a CFX Touch Deep Well real-time PCR detection system (Bio-Rad), and analyzed using a QX200 Droplet Digital PCR system Droplet Reader (Bio-Rad). Data were analyzed using Quantasoft software version 1.7.4. ddPCR probes designed against the exon 25 / 26 junction were used as a proxy for full-length WT or W1282X CFTR mRNA, and a probe designed against the exon 22 / intron 22 junction, including the first 140 bp of intron 22, was used as a proxy for exon 22-truncated CFTR mRNA.

[0187] Western blot analysis. Western blot analysis was performed as previously described, except that the loading control, Na+ / K+-ATPase, was detected with an antibody from Santa Cruz Biotechnology (sc-48345) 2.8.1.

[0188] Electrophysiology. Clonal lines were seeded at a density of 4.5 × 10 cells cm−2 onto HTS Transwell 24-well filter inserts pre-coated with human type IV collagen. Cells were grown as submerged cultures in MEM (Gibco, 11095) containing 10% FBS and 1% Pen / Strep and incubated at 37°C and 5% CO2. Cells were treated from both the basolateral and apical sides with fresh medium containing either control (vehicle) or test substance for the treatment times indicated in the figure legends. After a total of 7 days, 16HBE cells typically formed electrically dense epithelia with transepithelial resistances (Rt) of 200–600 Ω·cm−2, and CFTR-mediated Cl equivalent currents (Ieq) were determined as follows:

[0189] Prior to functional (Ieq) studies, the MEM was replaced with fresh HEPES-buffered (pH 7.4) solution (assay buffer). Chloride ion driving force was established by application of a basolateral-to-apical chloride ion gradient (see buffer composition below). Cell plates were mounted on an automated robotic assay platform and equilibrated at approximately 36°C for 90 minutes. After equilibration, transepithelial voltage (Vt) and resistance (Rt) were monitored at approximately 5-minute intervals using a 24-channel transepithelial current clamp amplifier (TECC-24, EP Design, Bertem, Belgium). The electrode potential difference for each pair of Ag / AgCl voltage electrodes was also monitored at 5-minute intervals by taking voltage measurements from a control plate containing matching buffer and 16HBE cells left untreated. After correcting for series resistance and (electrode) voltage offset, which are unrelated to Vt, Ieq was calculated from the Vt and Rt values ​​using Ohm's law. Ieq traces are plotted as mean ± SD (n = 3). The first four data points reflect baseline Ieq currents before sequential stimulation of CFTR with forskolin (10 μM) and VX-770 / ivacaftor (1 μM). The last six data points were recorded in the presence of the CFTR inhibitor CFTRinh-172 (20 μM). Agonists / antagonists were prediluted to 10x concentrations in assay buffer and added to the basolateral (forskolin) or apical (forskolin, VX-770 / ivacaftor, and CFTRinh-172) side of the membrane (assay plate only). The CFTR-mediated change in Ieq (i.e., the area under the curve (AUC) between the addition of delta forskolin, delta VX-770, delta CFTRinh-172, or forskolin and CFTRinh-172) is used as a measure of functional CFTR surface expression or treatment-related functional rescue of mutant CFTR.

[0190] Assay buffer: CFTR-mediated transepithelial currents were recorded using a Cl concentration gradient. The basolateral solution contained (mM): 137 NaCl, 4 KCl, 1.8 CaCl, 1 MgCl, 10 HEPES, and D-glucose adjusted to pH 7.4 with NaOH / HCl ([Cl-]total: 146.6 mM). The apical solution was matched to the basolateral solution except (mM): 137 Na-gluconate replaced 137 NaCl ([Cl-]total: 9.6 mM). Example 1: Use of intron 22 alternative polyadenylation (ApA) results in a naturally occurring, low-expressing CFTR exon 22 truncated CFTR mRNA isoform (e22 truncated mRNA) and truncated protein (e22 truncated protein) that is insensitive to nonsense-mediated decay (NMD).

[0191] To achieve deep and uniform exon coverage across the CFTR mRNA transcript, enriched RNA sequencing (eRNAseq) was performed using multiple genetically modified 16HBE cell lines (human bronchial epithelial cell lines). As shown in Figure 2A, low sequence coverage of the 3'-terminal exons of the CFTR mRNA transcript (e.g., exons 23-27) was found in 16HBEge-W1282X and 16HBEge-R1162X cells, suggesting that the W1282X and R1162X repertoires contain 3'-truncated CFTR mRNA transcripts. All sequence counts were normalized to wild-type 16HBE cells, and sequence coverage data for the CFTR exon 7-27 region are shown in the figure. Sequencing results from the 16HBEge-Y122X cell line showed a similar coverage profile to wild-type cells, due to the lack of expected splicing propensity. Both R553X and G542X showed reduced coverage in exon 12, consistent with previous experiments.

[0192] As depicted in Figure 2B, further sequence analysis revealed that the 16HBEge-W1282X CFTR mRNA transcript is presented with a normal 5' end but terminates within intron 22 with an approximately 140-bp extension into the intron on the 3' end. Furthermore, as shown in Figure 3, the 16HBEge-W1282X transcript terminates with a series of nonaligned adenosine nucleotides, indicating post-transcriptional modification of the mRNA. Consistent with this finding, a consensus alternative polyadenylation (ApA) motif was identified within intron 22 near the polyadenylation site described above, indicating ApA usage during processing of the CFTR pre-mRNA into the mature mRNA transcript in 16HBEge-W1282X and R1162X cells. Based on the sequences of the 3'-truncated mRNAs with alternative polyadenylation found in 16HBEge-W1282X and R1162X cells, it is predicted that a truncated CFTR protein of approximately 1239 amino acids can be translated, as depicted in Figure 4. The truncated CFTR mRNA sequence is approximately 3857 base pairs (bp) long and includes the first 22 exons and approximately 140 bp of intron 22 sequence. The first 27 bp of the intron 22 sequence encodes 9 amino acids in frame, followed by a stop codon and an alternative 3' untranslated region (UTR). Thus, the truncated CFTR protein generated from exon 22-truncated mRNA-ApA may contain the first 1239 amino acids of WT CFTR plus several additional amino acids (~9) encoded by intronic sequences within intron 22.

[0193] We detected novel exon 22-truncated mRNA isoforms in 16HBE14o cells using 3' RACE with a 5' primer targeting exon 8 in combination with long-read sequencing including exons 8-22. Exon 22-truncated CFTR transcripts contain post-transcriptionally added 10-30 adenosine residues that align with the putative consensus hexanucleotide alternative polyadenylation site and dinucleotide cleavage site, indicating the use of the intron 22 alternative polyadenylation site (Figure 2C, top). We characterized the exon 22-truncated isoforms using CFTR-specific cDNA long-read isoform sequencing with 5' UTR and 3' intron 22-targeted primers, identifying novel exon 22-truncated CFTR transcripts containing all 22 exons. These alternatively polyadenylated CFTR mRNA isoforms extending into intron 22 are referred to as "e22 truncated (e22 truncated)" in the following discussion. The E22 truncated mRNA contains the first 22 CFTR exons, followed by approximately 140 bp of intron 22, which encodes nine alternative amino acid residues and an alternative 3'UTR (Figure 2C, bottom). Translation of the E22 truncated mRNA results in a truncated CFTR protein that retains transmembrane domains 1 and 2, nucleotide-binding domain 1 (NBD1), and the R domain, but lacks the complete NBD2 domain, and contains nine alternative intron 22 amino acid residues (VRFEHCLLC) followed by a stop codon.

[0194] To test whether E22 truncated mRNA is NMD-insensitive, E22 truncated mRNA expression levels were assayed in airway and intestinal tissues of wild-type and CF-associated W1282X genotypes. The absolute copies of E22 truncated and full-length mRNA were assessed using RT-ddPCR, and the quantitative fraction relative to WT full-length CFTR was calculated in 16HBE14o (7.9%), WT IO (6.9%), and WT HBE from two donors at ALI (9.3% and 6.5%) (Figure 3), demonstrating constitutive low-level E22 truncated mRNA expression. As expected, E22 truncated mRNA expression was detected in W1282X airway and intestinal cells at almost the same absolute levels and low frequencies (5.7–12.5% ​​WT full-length CFTR) as in wild-type, suggesting NMD insensitivity.

[0195] Example 2: Stable exon 22-truncated CFTR mRNA transcript levels were elevated in 16HBEge-W1282X and R1162X cells. The level of exon 22-truncated CFTR mRNA transcripts was elevated at steady state in 16HBEge-W1282X and R1162X cells, likely due to nonsense-mediated decay (NMD) escape. To test this hypothesis, the level of exon 22-truncated CFTR mRNA transcripts was measured in multiple 16HBE-derived cell lines in the presence of an NMD inhibitor (SMG-1 inhibitor) via 3' RACE long-read sequencing. All sequencing results were normalized to full-length CFTR transcripts ("FL CFTR"), and the fraction of exon 22-truncated CFTR mRNA transcripts was compared. As depicted in Figure 5, a higher fraction of exon 22-truncated CFTR mRNA transcripts over FL CFTR mRNA transcripts was observed in 16HBEge-W1282X and R1162X cells compared to that in wild-type (WT) cells, and addition of SMG-1 inhibitor significantly reduced the fraction in 16HBEge-W1282X and R1162X cells. As expected, low levels of exon 22-truncated CFTR mRNA transcripts were observed in 16HBEge-G542X and N1303K cells, comparable to WT cells.

[0196] These data suggest that NMD escape may contribute to the increased proportion of exon 22-truncated / FL CFTR mRNA transcripts observed in W1282X and R1162X cells. The presence of a premature stop codon (PTC) in an mRNA transcript can normally trigger transcript degradation via NMD. FL CFTR transcripts produced in both 16HBEge-W1282X and R1162X cells contain premature stop codons in exon 23 and exon 22, respectively, and can be degraded by NMD. Blockade of NMD with SMG-1 inhibitor significantly increased the level of FL CFTR transcripts, resulting in a decrease in the fraction of truncated mRNA exon 22 transcripts. This observation suggests that exon 22-truncated CFTR mRNA transcripts were not affected by NMD inhibition, indicating NMD escape during processing of exon 22-truncated CFTR mRNA transcripts.

[0197] The stability of exon 22-truncated CFTR mRNA (e.g., E22-truncated mRNA) was quantified. To this end, a time course of actinomycin D was used to block transcription in 16HBE14o- and CFF-16HBEge-W1282X cells, and the % of remaining CFTR isoforms (WT FL CFTR, W1282X FL CFTR, and E22-truncated mRNA) was measured using ddPCR at t0, t2, t4, t6, t8, and t10 hours (Figure 6A-6B). To measure the half-life of exon 22-truncated CFTR mRNA transcripts, the W1282X-I22-SAd cell line was generated from the 16HBEge-W1282X line using cloning techniques, as shown in Figure 6A. Briefly, the splice acceptor site of intron 22 in 16HBEge-W1282X cells was disrupted in W1282X-I22-SAd cells. The resulting W1282X-I22-SAd cells produced exon 22-truncated CFTR mRNA transcripts, full-length CFTR mRNA containing the PTC ("FL-W1282X transcript"), and exon 23-skipped CFTR mRNA. The exon 22-truncated CFTR mRNA transcript was found to be present in W1282X-I22-SAd cells at levels more than four-fold higher than those in the W1282X parent strain. W1282X-I22-SAd cells were treated with actinomycin D to inhibit transcription, and RNA was collected at various time points as shown in Figure 6A. Droplet digital PCR (ddPCR) assays for the exon 25 / 26 junction and the exon 22 / intron 22 junction were used to evaluate full-length and exon 22-truncated CFTR mRNA, respectively. The amount of each CFTR mRNA species evaluated at the first time point (t = 0) was set to 1 (100%), and the proportion of remaining mRNA was measured by ddPCR. mRNA half-life was calculated from exponential decay. As shown in Figure 6B, the t of the exon 22-truncated CFTR mRNA was estimated to be approximately 4.24 hours, significantly longer than that of the FL-W1282X transcript (<<2 hours). The exon 23-skipped mRNA transcript exhibited a longer half-life (t ) equivalent to that of the wild-type CFTR transcript, as shown in Figure 6B. 1 / 2 = 10.61 hours).

[0198] The steady-state mRNA expression of exon 22-truncated CFTR mRNA is relatively short compared with that of exon 23-skipped CFTR transcripts. 1 / 2 Nevertheless, it remained highest in 16HBEge-W1282X-I22 SAd (SAd = splice acceptor disrupted) cells. This observation may be due to a faster generation rate or mRNA processing of the mature exon 22-truncated CFTR mRNA compared with that of the mature exon 23-skipped CFTR transcript, considering that these two transcripts are derived from the same nascent mRNA. Consistent with this finding, it was also observed that the splice donor site in CFTR intron 22 is relatively weak, which may contribute to the slower processing of the exon 23-skipped CFTR transcript.

[0199] Example 3: Exon 22-truncated CFTR protein was responsive to VX-661 / 445 / 770 (Trikafta) enhancement / correction. The TECC-24 assay was used to measure the function of CFTR variants. Briefly, cDNAs for wild-type, exon 22-truncated, and F508del CFTR proteins were expressed in Fischer Rat Thyroid (FRT) cells. Cells expressing exon 22-truncated and F508del CFTR proteins were incubated with DMSO or VX661 / 445 prior to assay. As shown in Figure 7A, compared with the untreated group, F508del CFTR function was enhanced in vitro by the addition of VX770, as indicated by an increase in the area under the curve (AUC) of CFTR-mediated chloride current. Similarly, exon 22-truncated CFTR function was also enhanced by Trikafta. Trikafta was observed to restore F508del CFTR function to approximately 35% of wild-type and enhance exon 22 truncated CFTR function to approximately 15% of wild-type. Treatment with Orkambi, the FDA-approved lumacaftar / ivacaftar combination therapy, restored F508del CFTR function to approximately 68% of wild-type.

[0200] E22 truncated cDNA was overexpressed in Fischer rat thyroid (FRT) cells and subjected to Ussing chamber analysis with 48 hours of pre-assay treatment with VX445 / VX661 (3 / 3.5 μM) 96 hours (+ / -) post-transfection, and acute addition of forskolin (10 μM) and VX-770 (1 μM) to the assay. As shown in Figure 7B, E22 truncated transfection and DMSO (0.002%) vehicle treatment after addition of VX-770 (1 μM) did not result in detectable changes in CFTR C1 currents. Conversely, 48 hours of pre-treatment with VX-445 / VX-661 (3 / 3.5 μM) and acute VX-770 (1 μM) resulted in a 0.42 AUC / min Gt (mS / cm 2) resulted in an increase in CFTR C1- current, which was reduced to pre-VX-770 levels by addition of the CFTR inhibitors CFTRinh-172 (20 μM) + GlyH-101 (20 μM). These data demonstrate that the E22 truncated protein has partial CFTR function when treated with modulators.

[0201] Example 4: Deletion of CFTR exons 23-27 promoted intron 22 alternative polyadenylation usage. A Δ23-27 (or Del23-27) gene editing model was established using CRISPR gene editing technology, as shown in Figure 8. This approximately 27 kb genomic deletion leaves all 11 putative consensus hexanucleotide alternative polyadenylation and dinucleotide cleavage sites at the 5' end of the remaining 13.5 kb of intron 22. Briefly, using a 5' guide ~13.5 kilobase pairs (kb) to intron 22 and a 3' guide ~159 bp immediately downstream of the 3' UTR of the CFTR gene, the region spanning the 5' portion of intron 22 through the post-3' UTR was deleted from the genome of 16HBE14o cells. The resulting cell genome contains exons 1-22, followed by approximately 13.5 kb of intron 22, then the intergenic region upstream of CTTNBP2, as shown in Figure 8. Three exemplary Δ23-27 gene-edited model cell lines were generated: 2-H07, 3-B09, and 3-D01.

[0202] Exon 22-truncated CFTR mRNA transcripts (black bars) from three Del23-27 clonal lines (2-H07, 3-B09, 3-D01) and 16HBE14o cells were assayed using droplet digital PCR (ddPCR) as shown in Figure 9. Full-length CFTR transcripts were measured using ddPCR from 16HBE14o cells (gray bars). The fold change in exon 22-truncated CFTR mRNA was calculated for each clone relative to the exon 22-truncated CFTR mRNA level in 16HBE14o cells. As shown in Figure 9, all three Del23-27 cell lines contained significantly higher levels of exon 22-truncated CFTR mRNA compared to the parental 16HBE14o line, approximately 12-fold the amount in parental cells. The levels of exon 22-truncated CFTR mRNA in all three Del23-27 cells were also higher than the levels of full-length WT CFTR transcripts in the parental 16HBE14o cells. The absolute copies of E22-truncated mRNA from the D23-27 clonal lines; 2-H07 (132,894 copies / 40 ng total RNA + / - 1,775), 3-B09 (120,237 copies / 40 ng total RNA + / - 4,839), and 3-D01 (121,891 copies / 40 ng total RNA + / - 6,122) were 12.6-, 11.4-, and 11.6-fold higher than the parental level (10,505 copies / 40 ng total RNA + / - 1,172) and comparable to the parental full-length CFTR mRNA level (Figure 3B), suggesting near-complete induction of intron 22ApA usage.

[0203] Example 5: Del23-27 cells expressed a truncated CFTR protein whose function was significantly enhanced by enhancers / correctors. Next, Western blot analysis was used to characterize the effect of using the forced intron 22ApA on CFTR protein expression. Three D23-27 clonal lines, 2-H07, 3-B09, and 3-D01, and the WT16HBE14o-parental line were treated with or without 3 / 3 μM VX-445 / VX-661 for 48 hours. Control lysates from WT (diluted 1:2) and HEK293 E22 truncated cDNA overexpression (diluted 1:100) were run to compare full-length and truncated Band B and Band C proteins, respectively. Blots were probed with a-CFTR UNC596 (epitope: 1204-1211aa; Ex22) to detect both FL and truncated CFTR proteins. Lysates 2-H07, 3-B09, and 3-D01 yielded 150 kDa and 110 kDa bands that aligned with the HEK293 overexpression E22 cleavage bands B and C, indicating expression of E22 cleavage proteins. Furthermore, treatment with VX-445 / VX-661 (3 / 3 μM) for 48 hours resulted in a roughly two-fold increase in cleavage bands B and C, indicating improved transport of E22 cleavage proteins by the modulators. As shown in Figure 10, the truncated forms of CFTR protein were detected in the Del23-27 clone lines (2-H07, 3-B09, 3-D01) using the α-CFTR UNC596 antibody (raised against the epitope 1204-1211aa in Ex22). Reactivity to exon 22 was detected in two bands (bands C and B in Figure 10) in all Del23-27 cells and aligned with the bands in the positive control HEK293 cells overexpressing exon 22-truncated CFTR cDNA. Na / K-ATPase was blotted as a loading control. Cells were treated with VX-445 / VX-661 (3 / 3 μM), but this did not significantly affect the levels of truncated CFTR protein, as shown by bands C and B. 16HBE14o-WT was included for comparison, as shown in Figure 10.

[0204] To assess the functional consequences of the forced intron 22ApA use, D23-27 clonal lines 2-H07, 3-B09, and 3-D01 were treated with vehicle control (0.002% DMSO) or VX-445 / VX-661 (3 / 3 μM) for 48 hours, with acute addition of VX-770 (1 μM), and CFTR function was assessed using transepithelial current clamp (TECC) conductance assays. As shown in Figure 11A, the enhancer / corrector trikafta restored the function of Del23-27 CFTR protein to a significant fraction of WT function. Briefly, transepithelial chloride conductance assays (TECC-24 assays) were performed on WT16HBE14o- (treated with vehicle), 16HBEge-F508Del (treated with VX-809), and Del23-27 clones (treated with VX-445 / 661) using sequential treatment with forskolin, VX-770, and CFTR inhibitor 172. Representative traces from the TECC-24 assay are shown in Figure 11A. In the presence of VX-445 / VX-661 / VX-770 (Trikafta), Del23-27 CFTR protein function was restored, as indicated by chloride conductance induced by VX-770 and inhibited by CFTR(inh)-172. CFTR function can be assayed by comparing the area under the induced chloride conductance curve (AUC) of the utilized cell lines. Unlike the results of E22 truncated cDNA FRT overexpression, VX-770 treatment alone did not significantly increase Cl - Vehicle treatment of 2-H07, 3-B09, and 3-D01 cells resulted in an increase in current of 11.4 + / - 2.1 FSK + VX-770 AUC / min [μA / cm 2 ], 6.5+ / -0.9FSK+VX-770AUC / min[μA / cm 2 ], and 8.3 + / - 1.3 FSK + VX - 770 AUC / min [μA / cm 2 Treatment with 3 / 3 μM VX-445 / VX-661 for 48 hours increased Cl- currents by 27.8 + / - 3.9 AUC / min [μA / cm] in 2-H07, 3-B09, and 3-D01 cells, respectively, compared to vehicle treatment.2 ](2.4X), 19.2+ / -0.5FSK+VX-770AUC / min[μA / cm 2 ] (3.0X), and 21.2 + / - 1.1 FSK + VX-770 AUC / min [μA / cm 2 ](2.6X) increase.

[0205] The AUCs of various mutant cell lines (e.g., 16HBEge-W1282X cells) were normalized to the AUCs obtained from cells expressing WT CFTR protein, with or without treatment, and can be expressed as either a percentage of WT CFTR (%WT CFTR) or a ratio to WT CFTR (variant / WT CFTR). As shown in Figure 11B, CFTR function in the three Del23-27 cell lines, 2-H07, 3-B09, and 3-D01, was restored to approximately 25%, 17%, and 18% of WT function, respectively, much higher than that of F508Del, which was enhanced by VX-809 / VX-770 (approximately 5% of WT).

[0206] As shown in Figure 11C, a dose-escalation study was performed using VX-770 (0.1-10 μM) in the 2-H07 clonal line. All concentrations tested resulted in a significant increase in CFTR chloride conductance compared to vehicle control (+ / -) VX-445 / VX-661 (3 / 3 μM) (p=<0.005, ANOVA). VX-770 treatment significantly increased CFTR chloride conductance compared to C1 - VX-445 / VX-661 (3 / 3 μM) produced a dose-dependent increase in current, with a 50.6 FSK + VX-770 AUC / min [μA / cm 2 Maximal CFTR Cl- currents were achieved with 3.0 μM (+)VX-445 / VX-661 / VX-770 (3 / 3 / 3 μM). This level of Cl- current is equivalent to approximately 42% of the WT 16HBE14o- function (FSK only).

[0207] Example 6: Modulation of CFTR mRNA processing by intron 22-targeted ASO in 16HBE14-WT cells. Steric blocking ASOs were designed via a 10-step, one-nucleotide “walk” tiling scheme to target the intron 22 donor site (black) or acceptor site (gray bars), as depicted in Figure 12A.

[0208] 16HBE14o-WT cells were treated with exemplary ASOs according to some embodiments of the present disclosure, and exon 22-truncated CFTR mRNA was assayed using ddPCR. As shown in Figure 12B, most candidate ASOs increased exon 22-truncated CFTR mRNA levels in WT16HBE14o- cells. Among them, exemplary ASOs SD10 (top black bar) induced exon 22-truncated CFTR mRNA to a level that was approximately 37% of the FL CFTR level in WT cells, and SA8 (bottom black bar) induced exon 22-truncated CFTR mRNA to approximately 25% of the FL CFTR level in WT cells. Scrambled ASOs, off-target ASOs (ASOs targeting CEP290 mRNA), and untreated cells were included in the experiment as controls.

[0209] Example 7: Modulation of CFTR mRNA processing and restoration of CFTR channel function by intron 22-targeted ASO in 16HBEge-W1282X cells. 16HBEge-W1282X cells were treated with exemplary ASOs according to some embodiments of the present disclosure. In some cases, the exemplary ASOs were administered alone at various doses, in combination with other ASOs at various doses, or co-administered with Trikafta, and the effects of ASO treatment on 16HBEge-W1282X cells were evaluated using multiple assays.

[0210] Exon 22-truncated CFTR mRNA was assayed using ddPCR, as shown in Figures 13A-13B. 16HBEge-W1282X cells were treated with various doses of SA08 alone, SD10 alone, or SA08 in combination with SD10 in the presence of DMSO vehicle control or Trikafta. As depicted in Figure 13A, exon 22-truncated CFTR mRNA increased in all groups treated with the exemplary ASO compared to no ASO treatment. Separate treatment with SA08 and SD10 alone enhanced exon 22-truncated CFTR mRNA expression by approximately 100% and over 200%, from less than 20,000 copies per 40 ng of RNA to approximately 30,000 copies and approximately 65,000 copies per 40 ng of RNA, respectively. The combination of SA08 and SD10 further enhanced exon 22-truncated CFTR mRNA expression to approximately 80,000 copies per 40 ng of RNA. In all groups in this experiment, Trikafta treatment did not significantly change the expression of exon 22 truncated CFTR mRNA. Full-length CFTR W1282X mRNA levels were unchanged in SD-10 and SA-08 or combined treatment compared with untreated or (-) ASO (100 μM) controls. (-) ASO (100 μM) E22 truncated mRNA levels also remained unchanged compared with untreated cells. As shown in Figure 13B, SD-10 and SA-08 ASO treatment significantly increased E22 truncated mRNA levels. SD-10 and SA-08 or combined treatment induced 4.5X, 5.1X, 6.6X, and 7.3X fold changes in E22 truncated mRNA levels relative to (-)ASO (100 μM) by SD-10 (10 μM), SD-10 (100 μM), SD-10 (10 μM), and SA-08 (10 μM), and SD-10 (100 μM) and SA-08 (10 μM), respectively.

[0211] Exon 22-truncated CFTR protein expression was assayed using Western blotting, as shown in Figure 14. 16HBEge-W1282X cells were treated with exemplary ASOs alone or in combination (e.g., SD10; SD10 and SA08) in the presence of drug vehicle or corrector VX-445 / 661. Prior to electrophoresis and Western blotting, 16HBEge-W1282X cell extracts were treated with PNGase F to remove glycosylation. The cDNAs of four CFTR variants, including WT CFTR (1480 aa), DelEx23 CFTR (1428 aa), W1282X CFTR (1281 aa), and Ex22-truncated CFTR (1248 aa), were transiently transfected into HEK293 cells. HEK293 cell extracts from these transfections were also treated with PNGase F and used as a Western blot control. As shown in Figure 14, the α-CFTR UNC596 antibody (raised against epitope 1204-1211aa in Ex22) recognizes the deglycosylated exon 22-truncated CFTR protein as a single band at approximately 130 kDa. Western blotting results indicate that the expression of exon 22-truncated CFTR protein was increased by both treatment with SD10 alone and treatment with the combination of SD10 and SA08, whereas treatment with the corrector VX-445 / VX-661 did not significantly affect the expression of exon 22-truncated CFTR protein. Actin (ACTB) was blotted as a loading control, demonstrating consistent loading of protein within each group.

[0212] The function of exon 22-truncated CFTR protein was assayed by transepithelial chloride conductance assay (TECC-24 assay), as shown in Figures 15A and 15B. 16HBEge-W1282X cells were incubated in 0.02% DMSO (vehicle) or VX445 / VX661 prior to TECC assay. The characteristic CFTR responses to forskolin, potentiator (VX-770), and CFTR inhibitor (Inh-172) were recorded in the presence and absence of various doses and ASO treatment combinations. Representative traces from the TECC-24 assay are shown in Figure 15A. As shown in the figure, chloride channel conductance increased with trikafta treatment, suggesting that trikafta treatment restored CFTR transport to the plasma membrane and induced chloride channel conductance of exon 22-truncated CFTR. ASO treatment also induced a greater chloride channel conductance compared to no ASO treatment.

[0213] The function of various CFTR proteins in cells can be assessed by comparing the area under the curve (AUC) of induced chloride conductance obtained in electrophysiology assays. The AUC of various mutant cell lines (e.g., 16HBEge-W1282X cells) was normalized to the AUC obtained from cells expressing WT CFTR protein with or without treatment and can be expressed as either a percentage of WT CFTR (%WT CFTR) or a ratio to WT CFTR (variant / WT CFTR). Figure 15B shows the enhancement of chloride conductance AUC (percentage of WT CFTR AUC) of 16HBEge-W1282X cells normalized to WT CFTR after treatment with exemplary ASOs with or without Trikafta. Briefly, 48 hours prior to assay, 16HBEge-W1282X cells were treated with either drug vehicle or 3 / 3 μM VX-661 / 445. At this time point, cells were also administered various dosage regimens of ASO, including 0.1 μM, 1 μM, and 2 μM SA08, 10 μM SD10, and a combination of SA08 and SD10. After 48 hours of incubation, 16HBEge-W1282X cells were administered acute sequential treatment with 10 μM forskolin, 3 μM VX-770, and 10 μM Inh172. The combined treatment with VX-661 / 445 and VX-770 is designated Trikafta (++) in Figure 15B. In the control group, 16HBE14o-WT cells were treated with 10 μM forskolin without any other drugs or ASOs. Trikafta treatment can restore Ex22-truncated CFTR function to approximately 3% of that of WT CFTR. All ASO treatments increased Ex22-truncated CFTR function from approximately 1% to approximately 2.9% of WT in a dose-dependent manner. Treatment of cells with ASO and trikafta further enhanced Ex22-truncated CFTR function from approximately 3.1% to approximately 13.4% of WT. In the trikafta-treated group, 0.1 μM SA08 enhanced the chloride conductance AUC of 16HBEge-W1282X cells to approximately 3.1% of WT. The effect of ASO increased with increasing doses of SA08.1 μM SA08 enhanced the AUC to approximately 4.9% of WT, and 2 μM SA08 enhanced the AUC to approximately 6.2% of WT. Furthermore, with trikafta treatment, 10 μM SD10 enhanced the chloride conductance AUC of 16HBEge-W1282X cells to approximately 9% of WT, compared to a 2.9% increase in AUC without trikafta treatment. In the presence of trikafta, the use of two ASOs enhanced Ex22-truncated CFTR function more than either ASO alone. Treatment of 16HBEge-W1282X cells with 0.1 μM SA8 / 10 μM SD10 ASOs with trikafta increased the chloride conductance AUC of 16HBEge-W1282X cells to approximately 10% of WT. Similarly, 1 μM SA8 / 10 μM SD10 ASO treatment with trikafta increased the AUC to approximately 12.6% of WT, and 2 μM SA8 / 10 μM SD10 ASO treatment with trikafta increased the AUC to approximately 13.4% of WT.

[0214] The TECC-24 assay (n=3) was used to quantify functional rescue of CFTR chloride current by ASO treatment in CFF-16HBEge-W1282X, as shown in Figure 15C. Untreated vehicle had a 0.5 + / - 0.09 FSK + VX-770 AUC / min (μA / cm 2) produced barely detectable CFTR C1-currents, which increased with VX-445 / VX-661 (3 / 3 μM) to 3.2 ± 0.06 FSK + VX-770 AUC / min (μA / cm2). These data are consistent with previously assayed functional responses of CFF-16HBEge-W1282X to VX-445 / VX-661 / VX-770 (3 / 3 / 1 μM). Treatment with (-) ASO (110 μM) was similar to vehicle alone (+ / -) VX-445 / VX-661 (3 / 3 μM), 2.7 ± 0.2, and 2.2 ± 0.1 FSK + VX-770 AUC / min (μA / cm2), respectively. All ASO treatments resulted in statistically significant increases in CFTR C1-current compared with vehicle and VX-661 / 445 (-) ASO controls, respectively (p<0.05, ANOVA with Tukey's post-hoc test). In all cases, functional responses to SD-10, SA-08, or combined ASO treatment were increased with VX-445 / VX-661 (3 / 3 μM) compared with ASO alone. The AUC / min (μA / cm2) of FSK+VX-770 for SD-10 (μM), SD-10 (100 μM), SD-10 (10 μM) and SA-08 (10 μM), and SD-10 (100 μM) and SA-08 (10 μM) were 5.5 + / - 0.7, 4.6 + / - 0.4, 9.3 + / - 0.2, and 12.1 + / - 1.1 for VX-445 / VX-661 (3 / 3 μM), and 10.4 + / - 1.2, 11.5 + / - 0.5, 18.2 + / - 1.3, and 21.9 + / - 0.7 for FSK+VX-770 AUC / min (μA / cm2) for VX-445 / VX-661 (3 / 3 μM), respectively. As observed in the mRNA response data, SD-10 (10 μM) and SA-08 (10 μM) produced a greater effect than SD-10 (100 μM) treatment.

[0215] Example 8: Modulation of mRNA processing and restoration of CFTR channel function by intron 22-targeted ASO in primary human bronchial epithelial (HBE) cells. Primary HBE cells were treated with exemplary ASOs for 2 or 3 weeks to assess the long-term effects of ASO treatment. Briefly, primary HBE cells were cultured in homozygous W1282X + / + CFTR variant (original W1282X + / + Cells were extracted from CF patients with HBE (Human Beta Cells) and propagated and differentiated in vitro according to well-known, established protocols, including culture at an air-liquid interface. The cells were then treated with either 10 μM SD10 or 4 μM SA08 / 10 μM SD10 for 2 or 3 weeks, with ASO retreatment every 2–3 days. CFTR correctors 3 / 3 μM VX-661 / 445 were added to the cells 48 h before transepithelial chloride conductance assay (TECC-24 assay), which was performed as described above in Example 7. Benzamil was added to the primary cells before forskolin activation to block epithelial sodium channel (ENaC) activity, which may interfere with the detection of CFTR chloride channel activity.

[0216] Exon 22 truncated CFTR mRNA was assayed using ddPCR, as shown in Figure 16. Treatment with the exemplary ASO for both 2 and 3 weeks significantly reduced the expression of primary W1282X + / + Increased expression of exon 22 truncated CFTR mRNA in HBE. Representative I values ​​of chloride conductance from TECC-24 assay. eq The traces are shown in Figure 17. Transepithelial electrical resistance (TEER) was consistent across all experimental groups, and no toxicity was observed with repeated treatment. eq ) were observed after 2 and 3 weeks of treatment with exemplary ASOs. An elevation in baseline and an increase in the slope of the chloride conductance curve (I eq Trace) was observed in the 3-week SD10 / SA08 treatment group. Figure 18 shows the primary W1282X cells when normalized to WT cells. + / +1 is a bar graph showing that chloride conductance AUC (percent of WT CFTR AUC) measured in HBEs increased after 2 or 3 weeks of ASO treatment. These data suggest that chronic treatment with an exemplary ASO improved Ex22-truncated CFTR function compared to no treatment.

[0217] To extend the analysis to a more patient-relevant model, we used the "gold standard" CF model: human bronchial epithelial (hBE) culture at the air / liquid interface (ALI). Unlike previous CFF-16HBEgeW1282X cultures, hBE at the ALI formed a pseudoepithelium that created a barrier to ad libitum feeding. Therefore, to achieve sufficient upregulation of E22-cleaved mRNA, we found that hBE in ALI cultures needed to be subjected to prolonged ASO exposure by initiating ASO administration in an undifferentiated state and refreshing with ASO at each feeding throughout differentiation. + / +hBE (CF290) were harvested during 28 days of differentiation with ASO refreshment every two days during feeding. ASOs were administered / maintained at various doses: SD-10 (10 μM), SD-10 (100 μM), a combination of SD-10 (10 μM) and SA-08 (10 μM), a combination of SD-10 (100 μM) and SA-08 (10 μM), and the (-) ASO TNMD (100 μM). ASO treatment was discontinued 48 h before RNA collection on day 21. To assess the effect of ASO blockade of exon 22 / 23 splicing on transcription, E22 truncated and full-length CFTR mRNA (25 / 26 assay) were assessed via ddPCR from cells subjected to functional analysis. As shown in Figure 19, CF290 cells treated with the (-) ASO control (100 μM) resulted in 495 + / - 42 and 715 + / - 36 absolute copies / 40 ng total RNA of E22-truncated and full-length CFTR mRNA, respectively. SD-10 treatment (10 μM) alone shortened E22 3.0-fold, increasing it to 1482 + / - 61 absolute copies / 40 ng total RNA, while full-length CFTR mRNA remained unchanged at 583 + / - 40 absolute copies / 40 ng total RNA. SD-10 (100 μM) alone further increased E22-truncated mRNA 5.8-fold, to 2870 + / - 145 absolute copies / 40 ng total RNA, while full-length CFTR mRNA remained largely unchanged at 682 + / - 40 absolute copies / 40 ng total RNA. SD-10 (10 μM) and SA-08 (10 μM) treatment increased E22-cleaved mRNA 4.1-fold to 2035 ± 98 absolute copies per 40 ng of total RNA; again, full-length CFTR mRNA remained unchanged at 767 ± 56 absolute copies per 40 ng of total RNA. Finally, consistent with the CFF-16HBEge-W1282X data, SD-10 (100 μM) and SA-08 (10 μM) treatment resulted in a maximal induction of E22 cleavage, 7.7-fold, and 3802 ± 88 absolute copies per 40 ng of total RNA. SD-10 (100 μM) and SA-08 (10 μM) treatment did not significantly affect full-length CFTR mRNA levels, 983 ± 78 absolute copies per 40 ng of total RNA.As in the CFF-16HBEge W1282X line, exon 22 / 23 splice-blocking ASO treatment upregulated E22-truncated mRNA levels, while FL-CFTR-W1282X levels remained largely unchanged.

[0218] Next, as shown in Figure 20A, Western blot analysis was used to measure the effect of ASO blockade of exon 22 / 23 splicing on E22 truncated protein expression (+ / -)VX-445 / VX-661 / VX-770 (3 / 3 / 3 μM). Protein lysates were collected from the assay filters used for the TECC-24 functional assessment described above, run on gels, and probed with CFTR UNC596 and ACTB loading controls. CFTR mature band C intensity and the ACTB band were quantified using densitometry. Band C intensity was normalized to ACTB and referenced to (-)ASO (100 μM)(-)VX-445 / VX-661 (3 / 3 μM). All ASO blockade treatments increased band C intensity compared to (-)ASO(-)VX-445 / VX-661 (3 / 3 μM). Furthermore, VX-445 / VX-661 increased band C intensity compared to vehicle control for all ASO treatments. As observed with E22-truncated mRNA copy number, the order of band C intensity was 5.1X, 4.9X, 3.3X, and 2.5X for VX-445 / VX-661 (3 / 3 μM) (+ / -), SD-10 (100 μM), and SA-08 (10 μM) > SD-10 (100 μM) > SD-10 (10 μM), and 3.2X, 2.4X, 2.1X, and 1.3X for SA-08 (10 μM) > SD-10 (10 μM), respectively, as shown in Figure 20B.

[0219] Finally, to assess the functional impact of blocking exon 22 / 23 splicing on the upregulated E22 truncated mRNA and protein, the TECC-24 assay was performed on fully differentiated hBE W1282X at ALI, as shown in Figure 21. + / +For the study, SD-10 (10 μM) or SD-10 (10 μM) and SA-08 (4 μM) (+ / -) 445 / VX-661 (3 / 3 μM) were used. (-) ASO control (10 μM or 100 μM) (n=2) with vehicle (0.002% DMSO) elicited negligible CFTR Cl- currents of 0.28 ± 0.06 and 0.45 ± 0.58 AUC / min FSK+POT (μA / cm 2 ), which was slightly increased by 445 / VX-661 (3 / 3 μM) to 1.1 + / - 0.37 and 1.1 (n=1) AUC / min FSK+POT (μA / cm 2 Unlike the gene-edited model and exon 22 / 23 splice-blocking ASO treatment in CFF-16HBEge-W1282X, ASO treatment in the absence of VX-445 / VX-661 (3 / 3 μM) did not significantly increase CFTR C1 mRNA levels, despite the observed increases in mRNA and protein levels. - Twenty-eight days after ALI, CFTR C1 currents were significantly increased by 48 hours of chronic treatment with VX-445 / VX-661 (3 / 3 μM) to 4.95 + / - 0.83, 8.59 + / - 0.74, 8.37 + / - 2.55, and 9.32 + / - 0.64 for SD-10 (10 μM), SD-10 (100 μM), SD-10 (10 μM) and SA-08 (10 μM), and SD-10 (100 μM) and SA-08 (10 μM), respectively (p < 0.05 compared with vehicle, ANOVA with Tukey's post-hoc test).

[0220] This data supports the therapeutic potential of modulating ApA usage in intron 22 for cystic fibrosis patients with 3'-terminal PTCs. Based on existing clinical and correlative in vitro data, there is reasonable evidence that achieving a 10% WT response in an in vitro functional assay predicts clinical benefit across the entire cystic fibrosis patient cohort. The D23-27 gene editing model suggests that the functional response of complete conversion of CFTR mRNA to E22-truncated mRNA achieves approximately 25% WT function, and with VX-770 dose escalation, up to 50%. These functional levels are potentially within the therapeutic range in which patients would expect to see benefit using a single responsive allele. Additionally, overexpression of the E22-truncated FRT in the presence of modulators results in functional measurements consistent with other FDA-approved modulator CF genotype combinations, including Cl- currents that are 1.6-fold higher than F508del overexpression in VX-770 / VX-809 (Orkambi) and approximately 80% of F508del VX-770 / VX-661 (Symdeko).

[0221] Tables 3A-3B list exemplary ASO sequences according to some embodiments of the present disclosure.

[0222] [Table 1]

[0223] [Table 2-1]

[0224] [Table 2-2]

[0225] [Table 2-3]

[0226]

Table 2-4

[0227]

Table 2-5

[0228]

Table 2-6

[0229]

Table 2-7

[0230]

Table 2-8

[0231]

Table 2-9

[0232]

Table 2-10

[0233]

Table 2-11

[0234]

Table 2-12

[0235]

Table 2-13

[0236]

Table 2-14

[0237]

Table 2-15

[0238]

Table 2-16

[0239]

Table 2-17

[0240]

Table 3-1

[0241]

Table 3-2

[0242]

Table 4-1

[0243]

Table 4-2

[0244]

Table 4-3

[0245] The ASO structures provided in Table 3B are chemically modified as indicated by the symbol for the chemical modification. 2-Methoxyethoxy RNA bases are shown in Table 3B, with "2MOEr" followed by the name of the RNA base. For example, "2MOErT" is 2-methoxyethoxythymine ribonucleotide. Phosphorothioated RNA bases are used, with "*" indicating a phosphorothioate linkage. Nucleotides are separated by " / ". The 5' and 3' ends of the ASO structure are marked with "5" and "3", respectively, before the nucleotide symbol. Internal nucleotides are marked with an "i" before the nucleotide symbol.

[0246] [Table 5]

[0247] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present disclosure. It is understood that various alternatives to the embodiments of the present disclosure may be employed in implementing the present disclosure. The following claims define the scope of the disclosure, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. 1. A method for modulating expression of a CFTR gene in a cell, the method comprising contacting the cell with an agent or a vector encoding the agent, wherein the cell contains a pre-mRNA, the pre-mRNA being transcribed from the CFTR gene and comprising a first intron that includes an alternative polyadenylation site, and the agent modifies the CFTR gene or regulates processing of the pre-mRNA, thereby increasing the level of processed mRNA that is processed from the pre-mRNA and lacks the nucleic acid sequence of the pre-mRNA downstream of the first intron.

2. The method of claim 1, wherein the cell is a human cell and the CFTR gene is a human gene.

3. 3. The method of claim 2, wherein the first intron is intron 22.

4. 3. The method of claim 2, wherein the first intron is located in the region from GRCh38.p14 / hg38:chr7:117,627,771 to GRCh38.p14 / hg38:chr7:117,642,437.

5. The method of any one of claims 1 to 4, wherein the agent removes the nucleic acid sequence of the CFTR gene downstream of the first intron from the genome of the cell.

6. 6. The method of claim 5, wherein the nucleic acid sequence removed from the genome is located at GRCh38.p14 / hg38:chr7:117,642,438 to GRCh38.p14 / hg38:chr7:117,668,665.

7. 7. The method of claim 5 or 6, wherein the agent comprises a gene editing agent based on CRISPR / Cas9, TALEN, zinc finger, or any combination thereof.

8. 7. The method of claim 5 or 6, wherein the agent comprises a pair of guide RNAs, the pair of guide RNAs comprising the sequences set forth in SEQ ID NOs: 94 and 95, respectively.

9. The method of any one of claims 1 to 8, wherein the agent removes from the pre-mRNA a nucleic acid sequence of the pre-mRNA downstream of the first intron.

10. 10. The method of claim 9, wherein the nucleic acid sequence removed from the pre-mRNA is located at GRCh38.p14 / hg38:chr7:117,642,438 to GRCh38.p14 / hg38:chr7:117,668,665.

11. The method of any one of claims 1 to 10, wherein the agent inhibits splicing out of the first intron from the pre-mRNA.

12. 1. A method for regulating expression of a CFTR gene in a cell, the method comprising contacting the cell with an agent or a vector encoding the agent, wherein the cell comprises a pre-mRNA transcribed from the CFTR gene and comprises a first intron comprising an alternative polyadenylation site, and the agent suppresses splicing-out of the first intron from the pre-mRNA during splicing of the pre-mRNA in the cell.

13. 13. The method of claim 12, wherein the cell is a human cell and the CFTR gene is a human gene.

14. 14. The method of claim 13, wherein the first intron is intron 22.

15. 14. The method of claim 13, wherein the first intron is located in the region from GRCh38.p14 / hg38:chr7:117,627,771 to GRCh38.p14 / hg38:chr7:117,642,437.

16. 16. The method of any one of claims 12 to 15, wherein the agent increases the level of processed mRNA that is processed from the pre-mRNA and lacks the nucleic acid sequence of the pre-mRNA downstream of the first intron.

17. 17. The method of claim 16, wherein the nucleic acid sequence of the pre-mRNA downstream of the first intron is located in the region from GRCh38.p14 / hg38:chr7:117,642,438 to GRCh38.p14 / hg38:chr7:117,668,665.

18. 18. The method of any one of claims 1-11, 16 or 17, wherein the level of the processed mRNA is increased in the cell by at least about 1.5 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, or at least about 20 fold compared to a corresponding cell not contacted with the agent or the vector encoding the agent.

19. 18. The method of any one of claims 1-11, 16 or 17, wherein the level of the processed mRNA is increased by at least about 10-fold in the cell compared to a corresponding cell not contacted with the agent or the vector encoding the agent.

20. 20. The method of any one of claims 1 to 11 or 16 to 19, wherein the processed mRNA comprises, in 5' to 3' order, 22 exons, an intron sequence encoding 9 amino acids, a stop codon, and an alternative 3' untranslated region.

21. 21. The method of claim 20, wherein the intron sequence encoding 9 amino acids is transcribed from a genomic sequence located at GRCh38.p14 / hg38:chr7:117,627,771 to GRCh38.p14 / hg38:chr7:117,627,797.

22. 22. The method of any one of claims 1-11 or 16-21, wherein the processed mRNA is polyadenylated at an alternative polyadenylation site in a nucleic acid sequence transcribed from a genomic sequence located between GRCh38.p14 / hg38:chr7:117,627,771 and GRCh38.p14 / hg38:chr7:117,642,437.

23. 23. The method of any one of claims 1 to 22, wherein the agent increases the level of a truncated CTFR protein in the cell that lacks an amino acid sequence expressed from an exon sequence of the CFTR gene downstream of the first intron.

24. 24. The method of claim 23, wherein the level of the truncated CFTR protein in the cell is increased by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, or at least about 20-fold compared to a corresponding cell not contacted with the agent or the vector encoding the agent.

25. 25. The method of claim 23 or 24, wherein the truncated CFTR protein in the cell has a chloride channel conductance that is less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% of the chloride channel conductance of a wild-type CFTR protein.

26. The method of any one of claims 12 to 25, further comprising contacting the cell with a second agent.

27. 27. The method of claim 26, wherein the second agent comprises a modulator of a CFTR protein that enhances chloride conductance of CFTR in the cell.

28. 28. The method of claim 26 or 27, wherein the second agent comprises ivacaftor, lumacaftor, tezacaftor, elexacaftor, a combination of lumacaftor and ivacaftor, a combination of tezacaftor and ivacaftor, or a combination of elexacaftor, ivacaftor, and tezacaftor.

29. and wherein the second agent increases the chloride channel conductance of the truncated CFTR protein by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, at least 500%, at least 510%, at least 520%, at least 530%, at least 540%, at least 550%, at least 560%, at least 570%, at least 580%, at least 590%, at least 600%, at least 610%, at least 620%, at least 630%, at least 640%, at least 650%, at least 660%, at least 670%, at least 680%, at least 690%, at least 700%, at least 710%, at least 28. The method of claim 26 or 27, wherein the method results in an improvement of 20%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 950%, at least 1000%.

30. 28. The method of claim 26 or 27, wherein the second agent restores the chloride channel conductance of the truncated CFTR protein to approximately at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more of the chloride channel conductance of the wild-type CFTR protein.

31. The drug is (a) binding to the 5' splice site of the first intron; (b) binding to the 3' splice site of the first intron; or (c) binding to the branch point of the 3' splice site of the first intron; or 31. The method of any one of claims 1 to 30, wherein (d) interfering with a splicing factor involved in splicing from the first intron.

32. 32. The method of any one of claims 1 to 31, wherein the agent comprises a polynucleotide sequence that is at least 80% complementary to at least 8 consecutive nucleic acids of a sequence selected from the group consisting of SEQ ID NOs: 6 to 36.

33. 33. The method of any one of claims 1 to 32, wherein the agent comprises a polynucleotide sequence that is at least 80% complementary to at least 10, 12, 14, or 16 contiguous nucleic acids of a sequence selected from the group consisting of SEQ ID NOs: 6-36.

34. 34. The method of any one of claims 1 to 33, wherein the agent comprises a polynucleotide sequence that is at least 80% complementary to at least 18 contiguous nucleic acids of a sequence selected from the group consisting of SEQ ID NOs: 6 to 36.

35. 35. The method of any one of claims 1 to 34, wherein the agent comprises a polynucleotide sequence that is 100% complementary to a sequence selected from the group consisting of SEQ ID NOs: 6 to 36.

36. 32. The method of any one of claims 1 to 31, wherein the agent comprises a polynucleotide sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 37-64.

37. 37. The method of claim 36, wherein the agent comprises a polynucleotide sequence comprising at least 8 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 37-64.

38. 1. A method comprising the step of contacting a cell with an agent or a vector encoding said agent, wherein said agent comprises a polynucleotide sequence that is at least 80% complementary to a sequence selected from the group consisting of SEQ ID NOs: 6-36.

39. 1. A method comprising the step of contacting a cell with an agent or a vector encoding said agent, wherein said agent comprises a polynucleotide sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 37-64.

40. 1. A method comprising the step of contacting a cell with an agent or a vector encoding said agent, wherein said agent comprises at least 8 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 37-64.

41. 41. The method of any one of claims 36-37 or 39-40, wherein the polynucleotide sequence of the agent has at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 37-64.

42. 41. The method of any one of claims 36-37 or 39-40, wherein the polynucleotide sequence of the agent has 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 37-64.

43. 41. The method of any one of claims 36-37 or 39-40, wherein the polynucleotide sequence of the agent comprises at least 10, 12, 14, or 16 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 37-64.

44. 44. The method of claim 43, wherein the polynucleotide sequence of the agent comprises at least 18 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 37-64.

45. 45. The method of claim 44, wherein the polynucleotide sequence of the agent comprises at least 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 37-64.

46. 46. ​​The method of any one of claims 1 to 45, wherein the agent is an antisense oligomer.

47. 47. The method of claim 46, wherein the antisense oligomer comprises a backbone modification, a modified sugar moiety, or a combination thereof.

48. 47. The method of claim 46, wherein the antisense oligomer comprises a phosphorothioate or phosphorodiamidate linkage.

49. 49. The method of any one of claims 46 to 48, wherein each internucleotide linkage of the antisense oligomer is a phosphorothioate linkage.

50. 50. The method of any one of claims 46-49, wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl moiety, a 2'-fluoro moiety, a 2'-O-methoxyethyl moiety, or a 2'-NMA moiety.

51. 51. The method of any one of claims 46-50, wherein the antisense oligomer comprises at least one modified sugar moiety.

52. 52. The method of claim 51 , wherein each nucleotide of the antisense oligomer comprises a modified sugar moiety.

53. 52. The method of claim 51, wherein each nucleotide of the antisense oligomer comprises a 2'-O-methoxyethyl moiety.

54. 54. The method of any one of claims 46 to 53, wherein the antisense oligomer comprises at least one modified nucleobase.

55. 54. The method of any one of claims 46 to 53, wherein the antisense oligomer comprises hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, or 5-hydroxymethoylcytosine.

56. The antisense oligomer may be 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, 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, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 1 56. The method of any one of claims 46-55, wherein the nucleic acid sequence is 2 to 20 nucleobases, 12 to 15 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, 15 to 19 nucleobases, 15 to 18 nucleobases, 15 to 16 nucleobases, 16 to 20 nucleobases, 16 to 19 nucleobases, 16 to 18 nucleobases, 17 to 20 nucleobases, 17 to 19 nucleobases, or 18 to 20 nucleobases in length.

57. 57. The method of any one of claims 46 to 56, wherein the antisense oligomer comprises a sequence set forth in any one of SEQ ID NOs: 66 to 93.

58. 58. The method of any one of claims 1 to 57, wherein the method comprises contacting the cell with the vector, wherein the vector comprises a viral vector encoding the agent.

59. 59. The method of claim 58, wherein the viral vector comprises an adenoviral vector, an adeno-associated viral (AAV) vector, a lentiviral vector, a herpes simplex viral (HSV) viral vector, or a retroviral vector.

60. 60. The method of any one of claims 1 to 59, wherein the CFTR gene comprises a mutation downstream of the first intron.

61. 61. The method of claim 60, wherein the mutation downstream of the first intron is a nonsense mutation.

62. 62. The method of any one of claims 1 to 61, wherein the CFTR gene comprises a mutation that results in the presence of an in-frame premature stop codon downstream of the first intron.

63. At least one allele of the CFTR gene in the cell is selected from the group consisting of c.3717G>A, c.3717+4A>G, c.3717+5G>A, c.3717+40A>G, c.3718-2477C>T, c.3718-1G>A, c.3718-3T>G, c.3719T>G, c.3731G>A, c.3737C>T, c.3744delA, c.3745G>A, c.3747delG, c.3752G>A, c.3761T>G, c.3763T>C, c.3764C>A, c.3773dupT, c.3806T>A, c. 3808delG, c. 3808G>A, c. 3822G>A, c. 3846G>A, c. [3846G>A; 3848G>T], c. 3848G>T, c. 3872A>G, c. 3873G>C, c. 3873+1G>A, c. (3873+1_3874-1)_(3963+1_3964-1) del, c. 3873+2T>C, 3876delA, c. 3883_3886delATTT, c. 3883delA, c. 3889dupT, c. 3891dupT, c. 3908delA, c. 3909C>G, c. 3929G>A, c. 3937C>T, c. (3963+1_3964-1)_(*1_?) del, c. 3964-78_4242+577del, c. 3971T>C, c. 3988C>T, c. 4004T>C, c. 4036_4042del, c. 4046G>A, c. 4077_4080delTGTTinsAA, c. 4086dupT, c. 4097T>A, c. 4111G>T, c. 4124A>C, c. 4127_4131delTGGAT, c. 4144C>T, c. 4147dupA, c.

63. The method of any one of claims 1 to 62, wherein the variant is selected from the group consisting of c.4197_4198delCT, c.4231C>T, c.4234C>T, c.4242+1G>T, c.4242+1G>A, c.4251delA, c.4300_4301dup, c.4364C>G, c.4426C>T, and c.4439T>C.

64. A composition comprising an agent or a vector encoding said agent, wherein said agent, when present in a human cell containing a pre-mRNA transcribed from the CFTR gene and including a first intron containing an alternative polyadenylation site, modifies the CFTR gene or regulates processing of the pre-mRNA, thereby increasing the level of processed mRNA that is processed from the pre-mRNA and lacks the nucleic acid sequence of the pre-mRNA downstream of the first intron.

65. 65. The composition of claim 64, wherein the cell is a human cell and the CFTR gene is a human gene.

66. 66. The composition of claim 65, wherein the first intron is intron 22.

67. 66. The composition of claim 65, wherein the first intron is located in the region from GRCh38.p14 / hg38:chr7:117,627,771 to GRCh38.p14 / hg38:chr7:117,642,437.

68. 68. The composition of any one of claims 64 to 67, wherein the agent removes the nucleic acid sequence of the CFTR gene downstream of the first intron from the genome of the cell.

69. 69. The composition of claim 68, wherein the removed nucleic acid sequence is located at GRCh38.p14 / hg38:chr7:117,642,438 to GRCh38.p14 / hg38:chr7:117,668,665.

70. 70. The composition of Claim 68 or 69, wherein the agent comprises a gene editing agent based on CRISPR / Cas9, TALEN, zinc finger, or any combination thereof.

71. 70. The composition of claim 68 or 69, wherein the agent comprises a pair of guide RNAs, the pair of guide RNAs comprising the sequences set forth in SEQ ID NOs: 94 and 95, respectively.

72. 72. The composition of any one of claims 64 to 71, wherein the agent removes from the pre-mRNA a nucleic acid sequence of the pre-mRNA downstream of the first intron.

73. 73. The composition of claim 72, wherein the removed nucleic acid sequence is located at GRCh38.p14 / hg38:chr7:117,642,438 to GRCh38.p14 / hg38:chr7:117,668,665.

74. 74. The composition of any one of claims 64 to 73, wherein the agent inhibits splicing out from the first intron.

75. A composition comprising an agent or a vector encoding said agent, wherein said agent inhibits splicing out from a first intron when said agent is present in a cell containing a pre-mRNA transcribed from a CFTR gene and containing a first intron that contains an alternative polyadenylation site.

76. 76. The composition of claim 75, wherein the cell is a human cell and the CFTR gene is a human gene.

77. 77. The composition of claim 76, wherein the first intron is intron 22.

78. 77. The composition of claim 76, wherein the first intron is located in the region of GRCh38.p14 / hg38:chr7:117,627,771 to GRCh38.p14 / hg38:chr7:117,642,437.

79. 79. The composition of claims 75-78, wherein the agent increases the level of processed mRNA that is processed from the pre-mRNA and lacks the nucleic acid sequence of the pre-mRNA downstream of the first intron.

80. 80. The composition of claim 79, wherein the nucleic acid sequence of the pre-mRNA downstream of the first intron is located in the region of GRCh38.p14 / hg38:chr7:117,642,438 to GRCh38.p14 / hg38:chr7:117,668,665.

81. 81. The composition of any one of claims 64-75, 79, or 80, wherein the level of processed mRNA is increased in the cells by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, or at least about 20-fold, compared to a corresponding cell not contacted with the agent or the vector encoding the agent.

82. 81. The composition of any one of claims 64-75, 79, or 80, wherein the level of processed mRNA is increased by at least about 10-fold in the cell compared to a corresponding cell not contacted with the agent or the vector encoding the agent.

83. 83. The composition of any one of claims 64-75 or 79-82, wherein the processed mRNA comprises, in 5' to 3' order, 22 exons, an intron sequence encoding 9 amino acids, a stop codon, and an alternative 3' untranslated region.

84. 84. The composition of claim 83, wherein the intron sequence encoding 9 amino acids is transcribed from a genomic sequence located at GRCh38.pl4 / hg38:chr7:117,627,771 to GRCh38.pl4 / hg38:chr7:117,627,797.

85. 85. The composition of any one of claims 64-75 or 79-84, wherein the processed mRNA is polyadenylated at an alternative polyadenylation site in a nucleic acid sequence transcribed from a genomic sequence located between GRCh38.p14 / hg38:chr7:117,627,771 and GRCh38.p14 / hg38:chr7:117,642,437.

86. 86. The composition of any one of claims 64-85, wherein the agent increases the level of a truncated CTFR protein in the cell lacking an amino acid sequence expressed from an exon sequence of the CFTR gene downstream of the first intron.

87. 87. The composition of claim 86, wherein the level of the truncated CFTR protein in the cells is increased by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, or at least about 20-fold compared to corresponding cells not contacted with the agent or the vector encoding the agent.

88. 88. The composition of claim 87, wherein the truncated CFTR protein in the cell has a chloride channel conductance that is less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% of the chloride channel conductance of a wild-type CFTR protein.

89. 89. The composition of any one of claims 64 to 88, further comprising a second agent.

90. 90. The composition of claim 89, wherein the second agent comprises a modulator of a CFTR protein that enhances chloride conductance of CFTR in the cell.

91. 91. The composition of claim 89 or 90, wherein the second agent comprises ivacaftor, lumacaftor, tezacaftor, elexacaftor, a combination of lumacaftor and ivacaftor, a combination of tezacaftor and ivacaftor, or a combination of elexacaftor, ivacaftor, and tezacaftor.

92. and wherein the second agent increases the chloride channel conductance of the truncated CFTR protein by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, at least 500%, at least 510%, at least 520%, at least 530%, at least 540%, at least 550%, at least 560%, at least 570%, at least 580%, at least 590%, at least 600%, at least 610%, at least 620%, at least 630%, at least 640%, at least 650%, at least 660%, at least 670%, at least 680%, at least 690%, at least 700%, at least 710%, at least 92. The composition of any one of claims 89 to 91, wherein the composition improves the saturation of ...

93. 93. The composition of any one of claims 89-92, wherein the second agent restores the chloride channel conductance of the truncated CFTR protein to about at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more of the chloride channel conductance of the wild-type CFTR protein.

94. The drug is (a) binding to the 5' splice site of the first intron; (b) binding to the 3' splice site of the first intron; or (c) binding to the branch point of the 3' splice site of the first intron; or (d) the composition of any one of claims 64 to 93, which interferes with a splicing factor involved in splicing from the first intron.

95. 95. The composition of any one of claims 64 to 94, wherein the agent comprises a polynucleotide sequence that is at least 80% complementary to at least 8 consecutive nucleic acids of a sequence selected from the group consisting of SEQ ID NOs: 6 to 36.

96. 96. The composition of any one of claims 64 to 95, wherein the agent comprises a polynucleotide sequence that is at least 80% complementary to at least 10, 12, 14, or 16 contiguous nucleic acids of a sequence selected from the group consisting of SEQ ID NOs: 6-36.

97. 97. The composition of any one of claims 64 to 96, wherein the agent comprises a polynucleotide sequence that is at least 80% complementary to at least 18 contiguous nucleic acids of a sequence selected from the group consisting of SEQ ID NOs: 6 to 36.

98. 98. The composition of any one of claims 64 to 97, wherein the agent comprises a polynucleotide sequence that is 100% complementary to a sequence selected from the group consisting of SEQ ID NOs: 6-36.

99. 95. The composition of any one of claims 64 to 94, wherein the agent comprises a polynucleotide sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 37 to 64.

100. 100. The composition of claim 99, wherein the agent comprises a polynucleotide sequence comprising at least 8 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 37-64.

101. A composition comprising an agent or a vector encoding said agent, wherein said agent comprises a polynucleotide sequence that is at least 80% complementary to a sequence selected from the group consisting of SEQ ID NOs: 6-36.

102. A composition comprising an agent or a vector encoding said agent, wherein said agent comprises a polynucleotide sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 37-64.

103. A composition comprising an agent or a vector encoding said agent, wherein said agent comprises at least 8 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 37-64.

104. 104. The composition of any one of claims 99-100 or 102-103, wherein the polynucleotide sequence of the agent has at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 37-64.

105. 104. The composition of any one of claims 99-100 or 102-103, wherein the polynucleotide sequence of the agent has 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 37-64.

106. 104. The composition of any one of claims 99-100 or 102-103, wherein the polynucleotide sequence of the agent comprises at least 10, 12, 14, or 16 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 37-64.

107. 104. The composition of any one of claims 99-100 or 102-103, wherein the polynucleotide sequence of the agent comprises at least 18 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 37-64.

108. 104. The composition of any one of claims 99-100 or 102-103, wherein the polynucleotide sequence of the agent comprises at least 20 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 37-64.

109. 109. The composition of any one of claims 101 to 108, wherein the polynucleotide sequence of the agent is at least 80% complementary to at least 8 consecutive nucleic acids of a sequence selected from the group consisting of SEQ ID NOs: 6 to 36.

110. 109. The composition of any one of claims 101-108, wherein the polynucleotide sequence of the agent is at least 80% complementary to at least 10, 12, 14, or 16 contiguous nucleic acids of a sequence selected from the group consisting of SEQ ID NOs: 6-36.

111. 109. The composition of any one of claims 101 to 108, wherein the polynucleotide sequence of the agent is at least 80% complementary to at least 18 contiguous nucleic acids of a sequence selected from the group consisting of SEQ ID NOs: 6 to 36.

112. 109. The composition of any one of claims 101 to 108, wherein the polynucleotide sequence of the agent is 100% complementary to a sequence selected from the group consisting of SEQ ID NOs: 6 to 36.

113. The composition of any one of claims 64 to 112, wherein the agent is an antisense oligomer.

114. 114. The composition of claim 113, wherein the antisense oligomer comprises a backbone modification, a modified sugar moiety, or a combination thereof.

115. The composition of claim 113, wherein the antisense oligomer comprises a phosphorothioate or phosphorodiamidate linkage.

116. 116. The composition of any one of claims 113 to 115, wherein each internucleotide linkage of the antisense oligomer is a phosphorothioate linkage.

117. The composition of any one of claims 113-116, wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl moiety, a 2'-fluoro moiety, a 2'-O-methoxyethyl moiety, or a 2'-NMA moiety.

118. 118. The composition of any one of claims 113-117, wherein the antisense oligomer comprises at least one modified sugar moiety.

119. 119. The composition of claim 118, wherein each nucleotide of the antisense oligomer comprises a modified sugar moiety.

120. 120. The composition of claim 119, wherein each nucleotide of said antisense oligomer comprises a 2'-O-methoxyethyl moiety.

121. 121. The composition of any one of claims 113 to 120, wherein the antisense oligomer comprises at least one modified nucleobase.

122. 122. The composition of any one of claims 113-121, wherein the antisense oligomer comprises hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, or 5-hydroxymethoylcytosine.

123. The antisense oligomer may be 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, 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, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 2 ...

123. The composition of any one of claims 113-122, which is 20 nucleobases, 12 to 15 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, 15 to 19 nucleobases, 15 to 18 nucleobases, 15 to 16 nucleobases, 16 to 20 nucleobases, 16 to 19 nucleobases, 16 to 18 nucleobases, 17 to 20 nucleobases, 17 to 19 nucleobases, or 18 to 20 nucleobases in length.

124. The composition of any one of claims 113 to 123, wherein the antisense oligomer comprises a sequence set forth in any one of SEQ ID NOs: 66 to 93.

125. 113. The composition of any one of claims 64 to 112, wherein the composition comprises the vector, and the vector comprises a viral vector encoding the agent.

126. 126. The composition of claim 125, wherein the viral vector comprises an adenoviral vector, an adeno-associated viral (AAV) vector, a lentiviral vector, a herpes simplex viral (HSV) viral vector, or a retroviral vector.

127. 127. A method of treating a subject in need thereof, said method comprising contacting cells of said subject with a composition according to any one of claims 64 to 126.

128. 128. The method of claim 127, wherein the cells are ex vivo.

129. 128. The method of claim 127, wherein the cell is in vivo.

130. 128. The method of claim 127, comprising administering the composition to the subject via intranasal administration, intratracheal administration, intravenous injection, intraperitoneal injection, intramuscular injection, intrathecal injection, subcutaneous injection, oral administration, synovial injection, intraventricular injection, intravitreal administration, subretinal injection, topical application, or implantation.

131. 128. The method of claim 127, comprising administering the composition to the subject via the respiratory route.

132. 132. The method of any one of claims 127 to 131, further comprising administering to the subject a second agent.

133. 133. The method of claim 132, wherein the second agent comprises a modulator of a CFTR protein that enhances chloride conductance of CFTR in the cell.

134. 134. The method of claim 132 or 133, wherein the second agent comprises ivacaftor, lumacaftor, tezacaftor, elexacaftor, a combination of lumacaftor and ivacaftor, a combination of tezacaftor and ivacaftor, or a combination of elexacaftor, ivacaftor, and tezacaftor.

135. 135. The method of any one of claims 132-134, wherein the second agent comprises a mucolytic agent for airway clearance, optionally wherein the mucolytic agent for airway clearance is selected from the group consisting of acetylcysteine, ambroxol, bromhexine, carbocysteine, erdosteine, mecysteine, dornase alfa, hypertonic saline, and mannitol.

136. 136. The method of any one of claims 132-135, wherein the second medicament comprises a bronchodilator, and optionally, the bronchodilator is albuterol.

137. 137. The method of any one of claims 132-136, wherein the second agent comprises an immunosuppressant.

138. 138. The method of claim 137, wherein the immunosuppressant is a corticosteroid selected from the group consisting of beclomethasone, budesonide, formoterol, ciclesonide, fluticasone, salmeterol, umeclidinium, vilanterol, mometasone, or mometasone, prednisone, and methylprednisolone, and pharmaceutically acceptable salts thereof.

139. 138. The method of claim 137, wherein the immunosuppressant is a nonsteroidal immunosuppressant selected from the group consisting of polyclonal antilymphocyte antibodies, monoclonal antilymphocyte antibodies, interleukin-2 (IL-2) receptor antagonists, calcineurin inhibitors, cell cycle inhibitors, mammalian target of rapamycin (mTOR) inhibitors, methotrexate, cyclophosphamide, anthracyclines, and taxanes.

140. 140. The method of any one of claims 127 to 139, wherein the method treats a disease or disorder caused by a mutation in the CFTR gene in the subject.

141. 141. The method of any one of claims 127 to 140, wherein the CFTR gene comprises a mutation downstream of the first intron.

142. 142. The method of claim 141, wherein the mutation downstream of the first intron is a nonsense mutation.

143. 141. The method of any one of claims 127-140, wherein the CFTR gene comprises a mutation that results in the presence of an in-frame premature stop codon downstream of the first intron.

144. and wherein at least one allele of the CFTR gene in said cells of said subject is selected from the group consisting of c.3717G>A, c.3717+4A>G, c.3717+5G>A, c.3717+40A>G, c.3718-2477C>T, c.3718-1G>A, c.3718-3T>G, c.3719T>G, c.3731G>A, c.3737C>T, c.3744delA, c.3745G>A, c.3747delG, c.3752G>A, c.3761T>G, c.3763T>C, c.3764C>A, c.3773dupT, c. 3806T>A, c. 3808delG, c. 3808G>A, c. 3822G>A, c. 3846G>A, c. [3846G>A, 3848G>T], c. 3848G>T, c. 3872A>G, c. 3873G>C, c. 3873+1G>A, c. (3873+1_3874-1)_(3963+1_3964-1) del, c. 3873+2T>C, c. 3883_3886delATTT, c. 3883delA, c. 3889dupT, c. 3891dupT, c. 3908delA, c. 3909C>G, c. 3929G>A, c. 3937C>T, c. (3963+1_3964-1)_(*1_?) del, c. 3964-78_4242+577del, c. 3971T>C, c. 3988C>T, c. 4004T>C, c. 4036_4042del, c. 4046G>A, c. 4077_4080delTGTTinsAA, c. 4086dupT, c. 4097T>A, c. 4111G>T, c. 4124A>C, c. 4127_4131delTGGAT, c. 4144C>T, c. 4147dupA, c.

141. The method of any one of claims 127 to 140, wherein the mutant is selected from the group consisting of c.4197_4198delCT, c.4231C>T, c.4234C>T, c.4242+1G>T, c.4242+1G>A, c.4251delA, c.4300_4301dup, c.4364C>G, c.4426C>T, and c.4439T>C.

145. 145. The method of any one of claims 127 to 144, wherein one or more symptoms associated with cystic fibrosis are ameliorated or prevented.

146. 1. A pharmaceutical composition comprising: (a) a pharmaceutically acceptable excipient or carrier; and (b) a composition according to any one of claims 64 to 126; A pharmaceutical composition comprising:

147. 147. The pharmaceutical composition of claim 146, formulated for intranasal administration, intratracheal administration, intravenous injection, intraperitoneal injection, intramuscular injection, intrathecal injection, subcutaneous injection, oral administration, synovial injection, intraventricular injection, intravitreal administration, subretinal injection, topical application, or implantation.

148. 147. The pharmaceutical composition of claim 146, formulated for administration via the respiratory route.

149. 149. The pharmaceutical composition of any one of claims 146 to 148, further comprising a second therapeutic agent.

150. 150. The pharmaceutical composition of claim 149, wherein the second therapeutic agent comprises a modulator of a CFTR protein that enhances chloride conductance of CFTR in a cell.

151. 151. The pharmaceutical composition of claim 149 or 150, wherein the second agent comprises ivacaftor, lumacaftor, tezacaftor, elexacaftor, a combination of lumacaftor and ivacaftor, a combination of tezacaftor and ivacaftor, or a combination of elexacaftor, ivacaftor, and tezacaftor.

152. 152. The pharmaceutical composition of any one of claims 149-151, wherein the second agent comprises a mucolytic agent for airway clearance, optionally wherein the mucolytic agent for airway clearance is selected from the group consisting of acetylcysteine, ambroxol, bromhexine, carbocysteine, erdosteine, mecysteine, dornase alfa, hypertonic saline, and mannitol.

153. 153. The pharmaceutical composition of any one of claims 149-152, wherein the second agent comprises a bronchodilator, and optionally, the bronchodilator is albuterol.

154. 154. The pharmaceutical composition of any one of claims 149-153, wherein the second agent comprises an immunosuppressant.

155. 155. The pharmaceutical composition of claim 154, wherein the immunosuppressant is a corticosteroid selected from the group consisting of beclomethasone, budesonide, formoterol, ciclesonide, fluticasone, salmeterol, umeclidinium, vilanterol, mometasone, or mometasone, prednisone, and methylprednisolone, and pharmaceutically acceptable salts thereof.

156. 155. The pharmaceutical composition of claim 154, wherein the immunosuppressant is a nonsteroidal immunosuppressant selected from the group consisting of polyclonal antilymphocyte antibodies, monoclonal antilymphocyte antibodies, interleukin-2 (IL-2) receptor antagonists, calcineurin inhibitors, cell cycle inhibitors, mammalian target of rapamycin (mTOR) inhibitors, methotrexate, cyclophosphamide, anthracyclines, and taxanes.

157. A kit comprising: (a) a composition according to any one of claims 64 to 126 or a pharmaceutical composition according to any one of claims 146 to 156; (b) instructions for use of said composition or said pharmaceutical composition; Includes a kit.

158. A kit comprising: (a) a composition according to any one of claims 64 to 126 or a pharmaceutical composition according to any one of claims 146 to 156; (b) a second therapeutic agent; and Includes a kit.

159. 159. The kit of claim 158, wherein the second therapeutic agent comprises a modulator of a CFTR protein that enhances chloride conductance of CFTR in a cell.

160. 160. The kit of claim 158 or 159, wherein the second agent comprises ivacaftor, lumacaftor, tezacaftor, elexacaftor, a combination of lumacaftor and ivacaftor, a combination of tezacaftor and ivacaftor, or a combination of elexacaftor, ivacaftor, and tezacaftor.

161. 161. The kit of any one of claims 158-160, wherein the second agent comprises a mucolytic agent for airway clearance, optionally wherein the mucolytic agent for airway clearance is selected from the group consisting of acetylcysteine, ambroxol, bromhexine, carbocysteine, erdosteine, mecysteine, dornase alfa, hypertonic saline, and mannitol.

162. 162. The kit of any one of claims 158-161, wherein the second medicament comprises a bronchodilator, and optionally, the bronchodilator is albuterol.

163. 163. The kit of any one of claims 158-162, wherein the second agent comprises an immunosuppressant.

164. The kit of claim 163, wherein the immunosuppressant is a corticosteroid selected from the group consisting of beclomethasone, budesonide, formoterol, ciclesonide, fluticasone, salmeterol, umeclidinium, vilanterol, mometasone, or mometasone, prednisone, and methylprednisolone, and pharmaceutically acceptable salts thereof.

165. 164. The kit of claim 163, wherein the immunosuppressant is a nonsteroidal immunosuppressant selected from the group consisting of polyclonal antilymphocyte antibodies, monoclonal antilymphocyte antibodies, interleukin-2 (IL-2) receptor antagonists, calcineurin inhibitors, cell cycle inhibitors, mammalian target of rapamycin (mTOR) inhibitors, methotrexate, cyclophosphamide, anthracyclines, and taxanes.

166. 166. The kit of any one of claims 158-165, further comprising instructions for use of the composition or pharmaceutical composition and instructions for use of the second therapeutic agent.

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