CFTR super EXON constructs and uses thereof

Abstract

In some aspects, provided herein are methods, compositions, and kits relating to insertion of a super exon into genome for expression of a wildtype, or at least partially functional cystic fibrosis transmembrane regulator (CFTR) protein. In some aspects, provided herein are compositions, methods, and kits for treatment of cystic fibrosis.

Description

CFTR SUPER EXON CONSTRUCTS AND USES THEREOFCROSS REFERENCE

[0001] This application claims the benefits of U.S. Provisional Application No. 63 / 625,480, filed on January 26, 2024, U.S. Provisional Application No. 63 / 558,805, filed on February 28, 2024, and U.S. Provisional Application No. 63 / 725,298, filed on November 26, 2024, each of which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Cystic fibrosis (CF) is a life-shortening autosomal recessive disease most common among populations of Northern European descent, with a frequency of 1 in 2000 to 3000 live births, but the disease occurs with lower incidence rates in essentially all populations and ethnicities. Despite progress in the treatment of CF, there is no cure. Cystic fibrosis is caused by pathogenic mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, which comprises -189 kilobases of genomic sequence that encodes a protein that is comprised of 1480 amino acids in its mature state and is part of the ABC transporter protein superfamily. The CFTR protein functions as an anion channel that primarily mediates the flow of chloride and bicarbonate ions across the plasma membrane of epithelial cells.

[0003] Over 1000 CFTR mutations are known or predicted to cause CF. These mutations can result in reduced protein expression, poorly functional and / or non-functional protein product. These CF-causing mutations may be located throughout the CFTR gene and over 99% of CF- causing mutations are found downstream of exon 1. There is great need for approaches that can rectify CFTR mutations to treat CF.SUMMARY

[0004] Provided herein is a recombinant nucleic acid sequence comprising: a first nucleic acid sequence encoding a truncated cystic fibrosis transmembrane regulator (CFTR) protein that comprises amino acid residues encoded by at least exons 10-27 of a CFTR gene, and (a) a splice acceptor site that is immediately upstream of the first nucleic acid sequence; or (b) a second nucleic acid sequence that has at least 80% identity to a 3’ untranslated region (3’ UTR) of a gene different than the CFTR gene.

[0005] In some embodiments of the recombinant nucleic acid sequence provided herein, the first nucleic acid sequence comprises exon 2 to exon 27 of the CFTR gene. In some of these embodiments, exons of the first nucleic acid sequence are codon optimized for expression in human. In some embodiments, the first nucleic acid sequence has at least 70% identify to SEQ ID NO: 36. In some embodiments, the first nucleic acid sequence has at least 80% identify toSEQ ID NO: 36. In some embodiments, the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 37. In some embodiments, the first nucleic acid sequence has at least 80% identity to SEQ ID NO: 37. In some embodiments, the first nucleic acid sequence is free of introns. In some embodiments, the first nucleic acid sequence comprises at least one intron. In some embodiments, the at least one intron comprises an intron that is exogenous to the CFTR gene. In some embodiments, the at least one intron comprises a synthetic / chimeric intron. In some embodiments, each of the at least one intron is less than 600 nucleotides in length. In some embodiments, the splice acceptor site is exogenous to a 5 ’ end of the first nucleic acid sequence. In some embodiments, the splice acceptor site comprises a sequence identical to the sequence of an endogenous splice acceptor site immediately upstream of exon 11 of the CFTR gene. In some embodiments, the splice acceptor site comprises a nucleic acid sequence chosen from the group consisting of the sequences set forth in SEQ ID NOs: 1-3. In some embodiments, the recombinant nucleic acid sequence is configured to be inserted into a target site of an endogenous CFTR gene in a genome of a cell. In some embodiments, the target site of the endogenous CFTR gene is within an intron of the endogenous CFTR gene. In some embodiments, the intron is upstream of exon 10 of the endogenous CFTR gene. In some embodiments, the target site of the endogenous CFTR gene is within intron 1 of the endogenous CFTR gene. In some embodiments, the target site of the endogenous CFTR gene is about 325 nucleotides downstream of exon 1 of the endogenous CFTR gene. In some embodiments, the target site of the endogenous CFTR gene is about 79 nucleotides downstream of exon 1 of the endogenous CFTR gene. In some embodiments, the target site of the endogenous CFTR gene is located within a region from hg38 chr7: 117.480,148 to hg38 chr7: 117,498.307. In some embodiments, the recombinant nucleic acid sequence further comprises a 5’ homology arm upstream of the first nucleic acid sequence and a 3 ’ homology arm downstream of the first nucleic acid sequence. In some embodiments, the 5‘ homology7arm is homologous to a nucleic acid sequence upstream of the target site of the endogenous CFTR gene and the 3 ’ homology arm is homologous to a nucleic acid sequence downstream of the target site of the endogenous CFTR gene. In some embodiments, the 5’ homology arm and the 3’ homology arm comprise nucleic acid sequences that are at least 80% identical to nucleic acid sequences selected from the group consisting of: SEQ ID NO: 38 and SEQ ID NO: 39, respectively, SEQ ID NO: 38 and SEQ ID NO: 41, respectively, SEQ ID NO: 38 and SEQ ID NO: 43, respectively, SEQ ID NO: 40 and SEQ ID NO: 39, respectively, SEQ ID NO: 40 and SEQ ID NO: 41, respectively, SEQ ID NO: 40 and SEQ ID NO: 43, respectively, SEQ ID NO: 42 and SEQ ID NO: 39, respectively, SEQ ID NO: 42 SEQ ID NO: 41, respectively, and SEQ ID NO: 42 and SEQ ID NO: 43, respectively. In some embodiments, the second nucleic acid sequence has at least 90%identity to a 3’ untranslated region (3’ UTR) of a gene different than the CFTR gene. In some embodiments, the 3’ UTR of a gene is a 3’ UTR of a eukaryotic gene. In some embodiments, the 3’ UTR of a gene is a 3’ UTR of a human gene. In some embodiments, the 3’ UTR of a gene is a 3’ UTR of a viral gene. In some embodiments, the 3’ UTR of a gene is a simian virus (SV) 40 gene. In some embodiments, the human gene is selected from the group consisting of genes listed in Table 1. In some embodiments, the recombinant nucleic acid sequence is configured to fit within a vector. In some embodiments, the vector comprises a linear nucleic acid sequence, a plasmid, a viral particle or a synthetic nanoparticle. In some embodiments, the viral particle comprises an adeno-associated viral (AAV) particle.

[0006] Also provided herein is a recombinant nucleic acid sequence comprising: a first nucleic acid sequence encoding a truncated cystic fibrosis transmembrane regulator (CFTR) protein, wherein the first nucleic acid sequence comprises, between two exons of the first nucleic acid sequence, an intron that is exogenous to a CFTR gene.

[0007] In some embodiments of the recombinant nucleic acid sequence provided herein, the truncated CFTR protein comprises amino acid residues encoded by at least exons 10-27 of the CFTR gene. In some of these embodiments, the first nucleic acid sequence comprises exon 10 to exon 27 of the CFTR gene. In some embodiments, the first nucleic acid sequence comprises exon 2 to exon 27 of the CFTR gene. In some embodiments, the first nucleic acid sequence is codon optimized for expression in human. In some embodiments, the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 36. In some embodiments, the first nucleic acid sequence has at least 80% identity to SEQ ID NO: 36. In some embodiments, the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 37. In some embodiments, the first nucleic acid sequence has at least 80% identity to SEQ ID NO: 37. In some embodiments, the intron that is exogenous to the CFTR gene comprises a synthetic / chimeric intron. In some embodiments, the intron is less than 600 nucleotides in length. In some embodiments, the recombinant nucleic acid sequence further comprises a splice acceptor site immediately upstream of the first nucleic acid sequence. In some embodiments, the splice acceptor site is exogenous to a 5’ end of the first nucleic acid sequence. In some embodiments, the splice acceptor site comprises a sequence identical to the sequence of an endogenous splice acceptor site immediately upstream of exon 11 of the CFTR gene. In some embodiments, the splice acceptor site comprises a nucleic acid sequence chosen from the group consisting of the sequences set forth in SEQ ID NOs: 1-3. In some embodiments, the recombinant nucleic acid sequence is configured to be inserted into a target site of an endogenous CFTR gene in a genome of a cell. In some embodiments, the target site of the endogenous CFTR gene is within an intron of the endogenous CFTR gene. In some embodiments, the target site of the endogenous CFTRgene is within intron 1 of the endogenous CFTR gene. In some embodiments, the target site of the endogenous CFTR gene is about 325 nucleotides dow nstream of exon 1 of the endogenous CFTR gene. In some embodiments, the target site of the endogenous CFTR gene is about 79 nucleotides downstream of exon 1 of the endogenous CFTR gene. In some embodiments, the target site of the endogenous CFTR gene is located within a region from hg38 chr7: 117,480.148 to hg38 chr7:l 17,498,307. In some embodiments, the recombinant nucleic acid sequence further comprises a 5’ homology arm upstream of the first nucleic acid sequence and a 3’ homology' arm downstream of the first nucleic acid sequence. In some embodiments, the 5’ homology arm is homologous to a nucleic acid sequence upstream of the target site of the CFTR gene and the 3’ homology arm is homologous to a nucleic acid sequence downstream of the target site of the CFTR gene. In some embodiments, 5’ homology arm and the 3’ homology arm comprise nucleic acid sequences that are at least 80% identical to nucleic acid sequences selected from the group consisting of: In some embodiments, the 5’ homology arm and the 3‘ homology arm comprise nucleic acid sequences that are at least 80% identical to nucleic acid sequences selected from the group consisting of: SEQ ID NO: 38 and SEQ ID NO: 39, respectively, SEQ ID NO: 38 and SEQ ID NO: 41, respectively, SEQ ID NO: 38 and SEQ ID NO: 43, respectively, SEQ ID NO: 40 and SEQ ID NO: 39, respectively, SEQ ID NO: 40 and SEQ ID NO: 41, respectively. SEQ ID NO: 40 and SEQ ID NO: 43. respectively, SEQ ID NO: 42 and SEQ ID NO: 39, respectively, SEQ ID NO: 42 SEQ ID NO: 41, respectively, and SEQ ID NO: 42 and SEQ ID NO: 43, respectively. In some embodiments, the recombinant nucleic acid sequence comprises a second nucleic acid sequence comprising a 3’ untranslated region (3’ UTR). In some embodiments, the second nucleic acid sequence has at least 80% identity to a 3' UTR of a gene different than the CFTR gene. In some embodiments, the second nucleic acid sequence has at least 90% identity to a 3’ UTR of a gene different than the CFTR gene. In some embodiments, the 3’ UTR of a gene is a 3’ UTR of a eukary otic gene. In some embodiments, the 3’ UTR of a gene is a 3‘ UTR of a human gene. In some embodiments, the 3’ UTR of a gene is a 3‘ UTR of a viral gene. In some embodiments, the 3’ UTR of a gene is a simian virus (SV) 40 gene. In some embodiments, the human gene is selected from the group consisting of the genes listed in Table 1. In some embodiments, the recombinant nucleic acid sequence is configured to fit within a vector. In some embodiments, the vector comprises a linear nucleic acid sequence, a plasmid, a viral particle or a synthetic nanoparticle. In some embodiments, the viral particle comprises an adeno-associated viral (AAV) particle.

[0008] Also provided herein is a recombinant nucleic acid sequence comprising: (a) a first nucleic acid sequence encoding a truncated cystic fibrosis transmembrane regulator (CFTR) protein that comprises amino acid residues encoded by at least exons 10-27 of a CFTR gene; (b)a 5' homology arm upstream of the first nucleic acid sequence, and (c) a 3' homology arm downstream of the first nucleic acid sequence; wherein the 5' homology arm is homologous to a sequence that is upstream of a genomic site within an intron of an endogenous CFTR gene in a genome of a cell, wherein the 3' homolog}’ arm is homologous to a sequence that is downstream of the genomic site, and wherein the intron of the endogenous CFTR gene is upstream of exon 10 of the endogenous CFTR gene in a genome of a cell.

[0009] In some embodiments of the recombinant nucleic acid sequence provided herein, the first nucleic acid sequence comprises exon 2 to exon 27 of the CFTR gene. In some of these embodiments, exons of the first nucleic acid sequence are codon optimized for expression in human. In some embodiments, the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 36. In some embodiments, the first nucleic acid sequence has at least 80% identity to SEQ ID NO: 36. In some embodiments, the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 37. In some embodiments, the first nucleic acid sequence has at least 80% identity to SEQ ID NO: 37. In some embodiments, the first nucleic acid sequence is free of introns. In some embodiments, the first nucleic acid sequence comprises at least one intron. In some embodiments, the at least one intron comprises an intron that is exogenous to the CFTR gene. In some embodiments, the at least one intron comprises a synthetic / chimeric intron. In some embodiments, each of the at least one intron is less than 600 nucleotides in length. In some embodiments, the recombinant nucleic acid sequence further comprises a splice acceptor site. In some embodiments, the splice acceptor site is exogenous to a 5’ end of the first nucleic acid sequence. In some embodiments, the splice acceptor site comprises a sequence identical to the sequence of an endogenous splice acceptor site immediately upstream of exon 11 of the CFTR gene. In some embodiments, the splice acceptor site comprises a nucleic acid sequence chosen from the group consisting of the sequences set forth in SEQ ID NOs: 1-3. In some embodiments, the recombinant nucleic acid sequence is configured to be inserted into the genomic site of the endogenous CFTR gene. In some embodiments, the genomic site of the endogenous CFTR gene is within intron 1 of the endogenous CFTR gene. In some embodiments, the genomic site of the endogenous CFTR gene is about 325 nucleotides dow nstream of exon 1 of the endogenous CFTR gene. In some embodiments, the genomic site of the endogenous CFTR gene is about 79 nucleotides dow nstream of exon 1 of the endogenous CFTR gene. In some embodiments, the genomic site of the endogenous CFTR gene is located within a region from hg38 chr7: 117,480,148 to hg38 chr7: 117,498,307. In some embodiments, the 5’ homolog}’ arm and the 3’ homology arm comprise nucleic acid sequences that are at least 80% identical to nucleic acid sequences selected from the group consisting of: SEQ ID NO: 38 and SEQ ID NO: 39, respectively, SEQ ID NO: 38 and SEQ ID NO: 41, respectively, SEQ ID NO: 38 and SEQ IDNO: 43, respectively, SEQ ID NO: 40 and SEQ ID NO: 39, respectively, SEQ ID NO: 40 and SEQ ID NO: 41, respectively, SEQ ID NO: 40 and SEQ ID NO: 43, respectively, SEQ ID NO: 42 and SEQ ID NO: 39, respectively, SEQ ID NO: 42 SEQ ID NO: 41, respectively, and SEQ ID NO: 42 and SEQ ID NO: 43, respectively. In some embodiments, the recombinant nucleic acid sequence further comprises a second nucleic acid sequence comprising a 3’ untranslated region (3’ UTR). In some embodiments, the second nucleic acid sequence has at least 90% identity to a 3’ UTR of a eukaryotic gene different than the CFTR gene. In some embodiments, the 3’ UTR of a gene is a 3’ UTR of a eukaryotic gene. In some embodiments, the 3‘ UTR of a gene is a 3’ UTR of a human gene. In some embodiments, the 3’ UTR of a gene is a 3’ UTR of a viral gene. In some embodiments, the 3’ UTR of a gene is a simian virus (SV) 40 gene. In some embodiments, the human gene is selected from the group consisting of the genes listed in Table 1. In some embodiments, the recombinant nucleic acid sequence is configured to fit within a vector. In some embodiments, the vector comprises a linear nucleic acid sequence, a plasmid, a viral particle or a synthetic nanoparticle. In some embodiments, the viral particle comprises an adeno-associated viral (AAV) particle.

[0010] Also provided herein is a gene editing system, the gene editing system comprising: a) a first nucleic acid sequence encoding a truncated cystic fibrosis transmembrane regulator (CFTR) protein, and b) a targeting moiety or a nucleic acid sequence encoding the targeting moiety; wherein the targeting moiety’ directs insertion of the first nucleic acid sequence into a genomic site within an intron of an endogenous CFTR gene of a genome of a cell, and wherein the intron is upstream of exon 10 of the endogenous CFTR gene. In some embodiments, the targeting moiety comprises a guide RNA (gRNA), a zinc finger nuclease, or a transcription activator-like (TAL) effector nuclease. In some embodiments, the gene editing system further comprises a gene editing enzyme or a nucleic acid sequence encoding the gene editing enzy me. In some embodiments, the gene editing system results in integration of one or two copies of the first nucleic acid sequence. In some embodiments, the truncated CFTR protein comprises amino acid residues encoded by at least exons 10-27 of a CFTR gene. In some embodiments, the first nucleic acid sequence comprises exon 2 to exon 27 of the CFTR gene. In some embodiments, exons of the first nucleic acid sequence are codon optimized for expression in human. In some embodiments, the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 36. In some embodiments, the first nucleic acid sequence has at least 80% identity to SEQ ID NO: 36. In some embodiments, the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 37. In some embodiments, the first nucleic acid sequence has at least 80% identity to SEQ ID NO: 37. In some embodiments, the first nucleic acid sequence is free of introns. In some embodiments, the first nucleic acid sequence comprises at least one intron. In someembodiments, the at least one intron comprises an intron that is exogenous to the CFTR gene. In some embodiments, the at least one intron comprises a synthetic / chimeric intron. In some embodiments, each of the at least one intron is less than 600 nucleotides in length. In some embodiments, the first nucleic acid sequence comprises a splice acceptor site. In some embodiments, the splice acceptor site is exogenous to a 5?end of the first nucleic acid sequence. In some embodiments, the splice acceptor site comprises a sequence identical to the sequence of an endogenous splice acceptor site immediately upstream of exon 11 of the CFTR gene. In some embodiments, the splice acceptor site comprises a nucleic acid sequence chosen from the group consisting of the sequences set forth in SEQ ID NOs: 1-3. In some embodiments, the genomic site is within intron 1 of the endogenous CFTR gene. In some embodiments, the genomic site is about 325 nucleotides downstream of exon 1 of the endogenous CFTR gene. In some embodiments, the genomic site is about 79 nucleotides downstream of exon 1 of the endogenous CFTR gene. In some embodiments, the genomic site is located within a region from hg38 chr7: 117,480,148 to hg38 chr7: 117.498.307. In some embodiments, the gene editing system comprises a gRNA and a class 2 CRISPR / Cas endonuclease. In some embodiments, the gene editing system comprises a gRNA and a Cas9 enzyme. In some embodiments, the gRNA comprises a sequence selected from the group consisting of the sequences provided in Table 3. In some embodiments, the first nucleic acid sequence comprises a 5’ homology arm upstream of the first nucleic acid sequence and a 3’ homology arm downstream of the first nucleic acid sequence. In some embodiments, the 5 ’ homology7arm is homologous to a nucleic acid sequence upstream of the genomic site within the intron of the endogenous CFTR gene and the 3 ’ homology arm is homologous to a nucleic acid sequence downstream of the genomic site. In some embodiments, the 5’ homology arm and the 3’ homology arm comprise nucleic acid sequences that are at least 80% identical to nucleic acid sequences selected from the group consisting of: SEQ ID NO: 38 and SEQ ID NO: 39, respectively, SEQ ID NO: 38 and SEQ ID NO: 41, respectively, SEQ ID NO: 38 and SEQ ID NO: 43, respectively, SEQ ID NO: 40 and SEQ ID NO: 39. respectively, SEQ ID NO: 40 and SEQ ID NO: 41, respectively, SEQ ID NO: 40 and SEQ ID NO: 43, respectively, SEQ ID NO: 42 and SEQ ID NO: 39, respectively, SEQ ID NO: 42 SEQ ID NO: 41, respectively, and SEQ ID NO: 42 and SEQ ID NO: 43, respectively. In some embodiments, the gene editing system further comprises a second nucleic acid sequence. In some embodiments, the second nucleic acid sequence has at least 80% identity to a 3’ untranslated region (3’ UTR) of a gene different than the CFTR gene. In some embodiments, the second nucleic acid sequence has at least 90% identity to a 3’ UTR of a gene different than the CFTR gene. In some embodiments, the 3’ UTR of a gene is a 3’ UTR of a eukaryotic gene. In some embodiments, the 3' UTR of a gene is a 3’ UTR of a human gene. Insome embodiments, the 3’ UTR of a gene is a 3’ UTR of a viral gene. In some embodiments, the 3’ UTR of a gene is a simian virus (SV) 40 gene. In some embodiments, the human gene is selected from the group consisting of the genes listed in Table 1. In some embodiments, the gene editing system is configured to fit within one vector. In some embodiments, the gene editing system is configured to fit in two or more vectors. In some embodiments, the vector comprises a linear nucleic acid sequence, a plasmid, a viral particle or a synthetic nanoparticle. In some embodiments, the viral particle comprises an adeno-associated viral (AAV) particle.

[0011] Also provided herein is a gene editing system, the system comprising: a) a recombinant nucleic acid sequence provided herein, and b) a targeting moiety or a nucleic acid sequence encoding the targeting moiety; wherein the targeting moiety directs insertion of the first nucleic acid sequence into a genomic site within an intron of an endogenous CFTR gene of a genome of a cell, and wherein the intron is upstream of exon 10 of the endogenous CFTR gene.

[0012] In some embodiments of the gene editing system provided herein, the targeting moiety comprises a guide RNA (gRNA), a zinc finger nuclease, or a transcription activator-like (TAL) effector nuclease. In some embodiments, the gene editing system further comprises a gene editing enzyme or a nucleic acid sequence encoding the gene editing enzyme. In some embodiments, the gene editing system results in integration of one or two copies of the first nucleic acid sequence. In some embodiments, the genomic site is within intron 1 of the endogenous CFTR gene. In some embodiments, the genomic site is about 325 nucleotides downstream of exon 1 of the endogenous CFTR gene. In some embodiments, the genomic site is about 79 nucleotides downstream of exon 1 of the endogenous CFTR gene. In some embodiments, the genomic site is located within a region from hg38 chr7: 117,480,148 to hg38 chr7: 117,498,307. In some embodiments, the targeting moiety is a CRISPR / Cas nickase. In some embodiments, the gene editing system further comprises a recombinase. In some embodiments, the recombinase is a domain of the CRISPR / Cas nickase. In some embodiments, the recombinase is separate from the CRISPR / Cas nickase. In some embodiments, the gene editing system further comprises a reverse transcriptase. In some embodiments, the recombinant nucleic acid sequence is flanked by a sequence recognized by a recombinase. In some embodiments, the gene editing system further comprises a gRNA encoding a sequence that is recognized by a recombinase. In some embodiments, the sequence that is recognized by a recombinase is integrated into the genomic site. In some embodiments, the recombinase directs the insertion of the recombinant nucleic acid sequence into the genomic site. In some embodiments, the gene editing system comprises a gRNA and a class 2 CRISPR / Cas endonuclease. In some embodiments, the gene editing system comprises a gRNA and a Cas9 enzyme. In some embodiments, the gRNA comprises a sequence selected from the groupconsisting of the sequences provided in Table 3. In some embodiments, the gene editing system is configured to fit within one vector. In some embodiments, the gene editing system is configured to fit in two or more vectors. In some embodiments, the vector comprises a linear nucleic acid sequence, a plasmid, a viral particle or a synthetic nanoparticle. In some embodiments, the viral particle comprises an adeno-associated viral (AAV) particle.

[0013] Also provided herein is a pharmaceutical composition, the pharmaceutical composition comprising: a) a pharmaceutically acceptable excipient or carrier; and b) a recombinant nucleic acid sequence provided herein or a gene editing system provided herein.

[0014] In some embodiments, the pharmaceutical composition provided herein is formulated for intranasal administration, intratracheal administration, or intravenous injection. In some embodiments, the pharmaceutical composition is formulated for pulmonary' administration. In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, second therapeutic agent comprises a modulator of CFTR protein that potentiates chloride conductance of CFTR protein in the 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, optionally wherein the mucolytic agent is selected from the group consisting of acetylcysteine, ambroxol, bromhexine, carbocisteine, erdosteine, mecysteine, domase alfa, hypertonic saline, and mannitol. In some embodiments, the second therapeutic agent comprises a bronchodilator, optionally wherein the bronchodilator is albuterol. In some embodiments, the second therapeutic agent comprises an immunosuppressive agent. In some embodiments, the immunosuppressive agent is a corticosteroid selected from the group consisting of beclomethasone, budesonide, budesonide, formoterol, ciclesonide, fluticasone, salmeterol, umeclidinium, vilanterol, mometasone, or mometasone, prednisone, and methylprednisolone, and pharmaceutically acceptable salts thereof. In some embodiments, the immunosuppressive agent is a non-steroidal immunosuppressive agent selected from the group consisting of polyclonal anti-lymphocyte antibodies, monoclonal anti-lymphocyte antibodies, interleukin-2 (IL-2) receptor antagonists, calcineurin inhibitors, cell cycle inhibitors, mammalian target of rapamycin (mTOR) inhibitors, methotrexate, cyclophosphamide, anthracy cline, and taxane.

[0015] Also provided herein is a vector comprising a recombinant nucleic acid sequence provided herein, or a gene editing system provided herein. In some embodiments, the vector is a viral vector, optionally a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a herpes simplex viral vector. In some embodiments, the vector is a non- viral vector, optionally a lipid nanoparticle.

[0016] Also provided herein is a virus comprising a recombinant nucleic acid sequence provided herein or a gene editing system provided herein. In some embodiments, the virus is an adeno- associated viral (AAV) vector, optionally a retroviral vector, a lentiviral vector, an adenoviral vector, or a herpes simplex viral vector.

[0017] Also provided herein is a method for modifying a cell, comprising contacting an agent or a vector encoding the agent to the cell, wherein the agent modifies a genome of the cell, thereby inserting an exogenous nucleic acid sequence into a genomic site of an intron of an endogenous cystic fibrosis transmembrane regulator (CFTR)' gene of the cell, and wherein the intron is upstream of exon 10 of the endogenous CFTR gene of the cell.

[0018] In some embodiments of the method provided herein, the exogenous nucleic acid sequence comprises a recombinant nucleic acid sequence encoding a truncated CFTR protein that is encoded by at least exons 10-27 of the CFTR gene. In some embodiments, one or two copies of the exogenous nucleic acid are inserted into the genome of the cell. In some embodiments, expression of CFTR mRNA or CFTR protein is increased by 50%, 60%, 70%, 80%, 90%, 100% or more in the cell compared to an otherwise same cell not receiving insertion of the exogenous nucleic acid sequence. In some embodiments, the cell exhibits a 50%, 60%, 70%, 80%, 90%, 100% or more increase in CFTR activity compared to an otherwise same cell not receiving insertion of the exogenous nucleic acid sequence. In some embodiments, the CFTR activity comprises CFTR-mediated chloride channel conductivity measured by electrophysiology. In some embodiments, the recombinant nucleic acid sequence comprises a first nucleic acid sequence which comprises exon 2 to exon 27 of the CFTR gene. In some embodiments, exons of the first nucleic acid sequence are codon optimized for expression in human. In some embodiments, the first nucleic acid sequence has at least 70% identify to SEQ ID NO: 36. In some embodiments, the first nucleic acid sequence has at least 80% identity to SEQ ID NO: 36. In some embodiments, the first nucleic acid sequence has at least 70% identify to SEQ ID NO: 37. In some embodiments, the first nucleic acid sequence has at least 80% identify7to SEQ ID NO: 37. In some embodiments, the first nucleic acid sequence is free of introns. In some embodiments, the first nucleic acid sequence comprises at least one intron. In some embodiments, the at least one intron comprises an intron that is exogenous to the CFTR gene. In some embodiments, the at least one intron comprises a synthetic / chimeric intron. In some embodiments, each of the at least one intron is less than 600 nucleotides in length. In some embodiments, the first nucleic acid sequence of the recombinant nucleic acid sequence comprises a splice acceptor site that is exogenous to a 5’ end of the first nucleic acid sequence.In some embodiments, the splice acceptor site comprises a sequence identical to the sequence of an endogenous splice acceptor site immediately upstream of exon 11 of the CFTR gene. In some embodiments, the splice acceptor site comprises a nucleic acid sequence chosen from the group consisting of the sequences set forth in SEQ ID NOs: 1-3. In some embodiments, the genomic site of the intron of the endogenous CFTR gene is upstream of exon 10 of the endogenous CFTR gene. In some embodiments, the genomic site of the intron of the endogenous CFTR gene is within intron 1 of the endogenous CFTR gene. In some embodiments, the genomic site is about 325 nucleotides downstream of exon 1 of the endogenous CFTR gene. In some embodiments, the genomic site is about 79 nucleotides downstream of exon 1 of the endogenous CFTR gene. In some embodiments, the genomic site of the endogenous CFTR gene is located within a region from hg38 chr7: 117,480, 148 to hg38 chr7: 117,498,307. In some embodiments, the targeting moiety is a CRISPR / Cas nickase. In some embodiments, the gene editing system further comprises a recombinase. In some embodiments, the recombinase is a domain of the CRISPR / Cas nickase. In some embodiments, the recombinase is separate from the CRISPR / Cas nickase. In some embodiments, the gene editing system further comprises a reverse transcriptase. In some embodiments, the recombinant nucleic acid sequence is flanked by a sequence recognized by a recombinase. In some embodiments, the gene editing system further comprises a gRNA encoding a sequence that is recognized by a recombinase. In some embodiments, the sequence that is recognized by a recombinase is integrated into the genomic site. In some embodiments, the recombinase directs the insertion of the recombinant nucleic acid sequence into the genomic site. In some embodiments, the recombinant nucleic acid sequence further comprises a 5' homology arm upstream of the first nucleic acid sequence and a 3’ homology arm downstream of the first nucleic acid sequence. In some embodiments, the 5’ homology arm is homologous to a nucleic acid sequence upstream of the genomic site of the endogenous CFTR gene and the 3‘ homology7arm is homologous to a nucleic acid sequence downstream of the genomic site of the endogenous CFTR gene. In some embodiments, the 5’ homology arm and the 3‘ homology arm comprise nucleic acid sequences that are at least 80% identical to nucleic acid sequences selected from the group consisting of: SEQ ID NO: 38 and SEQ ID NO: 39, respectively, SEQ ID NO: 38 and SEQ ID NO: 41, respectively, SEQ ID NO: 38 and SEQ ID NO: 43, respectively, SEQ ID NO: 40 and SEQ ID NO: 39, respectively. SEQ ID NO: 40 and SEQ ID NO: 41, respectively, SEQ ID NO: 40 and SEQ ID NO: 43, respectively, SEQ ID NO: 42 and SEQ ID NO: 39, respectively, SEQ ID NO: 42 SEQ ID NO: 41, respectively, and SEQ ID NO: 42 and SEQ ID NO: 43, respectively. In some embodiments, the recombinant nucleic acid sequence further comprises a second nucleic acid sequence has at least 80% identity to a 3' untranslated region (3’ UTR) of a gene different than the CFTR gene.In some embodiments, the second nucleic acid sequence has at least 90% identify to 3’ UTR of a gene different than the CFTR gene. In some embodiments, the 3’ UTR of a gene is a 3' UTR of a eukary otic gene. In some embodiments, the 3’ UTR of a gene is a 3’ UTR of a human gene. In some embodiments, the 3’ UTR of a gene is a 3’ UTR of a viral gene. In some embodiments, the 3?UTR of a gene is a simian virus (SV) 40 gene. In some embodiments, the human gene is selected from the group consisting of the genes listed in Table 1. In some embodiments, the vector encoding the agent comprises a linear nucleic acid sequence, a plasmid, a viral particle or a synthetic nanoparticle. In some embodiments, the viral particle is an adeno-associated viral (AAV) particle. In some embodiments, the method further comprises contacting the cell with a second agent. In some embodiments, the second agent comprises a modulator of CFTR protein that potentiates chloride conductance of CFTR protein in the 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 agent comprises a targeting moiety, wherein the targeting moiety directs insertion of the exogenous nucleic acid sequence into a genomic site within an intron of an endogenous CFTR gene of a genome of a cell, and wherein the intron is upstream of exon 10 of the endogenous CFTR gene. In some embodiments, the targeting moiety comprises a guide RNA (gRNA), a zinc finger nuclease, or a transcription activator-like (TAL) effector nuclease. In some embodiments, the agent further comprises a gene editing enzyme or a nucleic acid sequence encoding the gene editing enzyme. In some embodiments, the agent comprises a gRNA and a class 2 CRISPR / Cas endonuclease. In some embodiments, the class 2 CRISPR / Cas endonuclease is a Cas9 enzyme. In some embodiments, the gRNA comprises a sequence selected from the group consisting of the sequences provided in Table 3. In some embodiments, the agent or a vector encoding the agent is delivered by intranasal administration, intratracheal administration, or intravenous injection. In some embodiments, the cell comprises a human cell.

[0019] Also provided herein is a method for modifying a cell, the method comprising contacting an agent or a vector encoding the agent to the cell, wherein the agent modifies a genome of the cell, thereby preventing expression of an endogenous CFTR gene in the cell after intron 1.

[0020] In some embodiments of the method provided herein, transcription of an endogenous CFTR gene in the cell after intron 1 is prevented. In some embodiments, the method comprises inserting an exogenous nucleic acid sequence into a genomic site of an intron of an endogenous cystic fibrosis transmembrane regulator (CFTR) gene of the cell, and wherein the intron is upstream of exon 10 of the endogenous CFTR gene of the cell. In some embodiments, the agent comprises a recombinant nucleic acid sequence encoding a truncated CFTR protein that isencoded by at least exons 10-27 of the CFTR gene. In some embodiments, the method increases expression of CFTR mRNA or CFTR protein by 50%, 60%, 70%, 80%, 90%, 100% or more in the cell compared to an otherwise same cell not receiving the agent. In some embodiments, the cell exhibits a 50%, 60%, 70%, 80%, 90%, 100% or more increase in CFTR activity compared to an otherwise same cell not receiving the agent. In some embodiments, the CFTR activity comprises CFTR-mediated chloride channel conductivity measured by electrophysiology. In some embodiments, the agent comprises a first nucleic acid sequence which comprises exon 2 to exon 27 of the CFTR gene. In some embodiments, exons of the first nucleic acid sequence are codon optimized for expression in human. In some embodiments, the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 36. In some embodiments, the first nucleic acid sequence has at least 80% identity to SEQ ID NO: 36. In some embodiments, the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 37. In some embodiments, first nucleic acid sequence has at least 80% identity to SEQ ID NO: 37. In some embodiments, the first nucleic acid sequence is free of introns. In some embodiments, the first nucleic acid sequence comprises at least one intron. In some embodiments, the at least one intron comprises an intron that is exogenous to the CFTR gene. In some embodiments, the at least one intron comprises a synthetic / chimeric intron. In some embodiments, each of the at least one intron is less than 600 nucleotides in length. In some embodiments, the first nucleic acid sequence of the agent comprises a splice acceptor site that is exogenous to a 5’ end of the first nucleic acid sequence. In some embodiments, the splice acceptor site comprises a sequence identical to the sequence of an endogenous splice acceptor site immediately upstream of exon 11 of the CFTR gene. In some embodiments, the splice acceptor site comprises a nucleic acid sequence chosen from the group consisting of the sequences set forth in SEQ ID NOs: 1-3. In some embodiments, the genomic site of the intron of the endogenous CFTR gene is upstream of exon 10 of the endogenous CFTR gene. In some embodiments, the genomic site of the intron of the endogenous CFTR gene is within intron 1 of the endogenous CFTR gene. In some embodiments, the genomic site is about 325 nucleotides downstream of exon 1 of the endogenous CFTR gene. In some embodiments, the genomic site is about 79 nucleotides downstream of exon 1 of the endogenous CFTR gene. In some embodiments, the genomic site of the endogenous CFTR gene is located within a region from hg38 chr7: 117,480, 148 to hg38 chr7: 117,498,307. In some embodiments, the recombinant nucleic acid sequence further comprises a 5?homology arm upstream of the first nucleic acid sequence and a 3’ homology arm downstream of the first nucleic acid sequence. In some embodiments, 5’ homology7arm is homologous to a nucleic acid sequence upstream of the genomic site of the endogenous CFTR gene and the 3 ’ homology7arm is homologous to a nucleic acid sequence downstream of the genomic site of the endogenousCFTR gene. In some embodiments, the 5’ homology arm and the 3’ homology arm comprise nucleic acid sequences that are at least 80% identical to nucleic acid sequences selected from the group consisting of: SEQ ID NO: 38 and SEQ ID NO: 39, respectively, SEQ ID NO: 38 and SEQ ID NO: 41, respectively, SEQ ID NO: 38 and SEQ ID NO: 43, respectively, SEQ ID NO: 40 and SEQ ID NO: 39, respectively, SEQ ID NO: 40 and SEQ ID NO: 41, respectively. SEQ ID NO: 40 and SEQ ID NO: 43, respectively, SEQ ID NO: 42 and SEQ ID NO: 39, respectively, SEQ ID NO: 42 SEQ ID NO: 41, respectively, and SEQ ID NO: 42 and SEQ ID NO: 43, respectively. In some embodiments, the recombinant nucleic acid sequence further comprises a second nucleic acid sequence has at least 80% identity to a 3?untranslated region (3’ UTR) of a gene different than the CFTR gene. In some embodiments, the second nucleic acid sequence has at least 90% identity to a 3’ UTR of a gene different than the CFTR gene. In some embodiments, the 3’ UTR of a gene is a 3’ UTR of a eukaryotic gene. In some embodiments, the 3’ UTR of a gene is a 3’ UTR of a human gene. In some embodiments, the 3’ UTR of a gene is a 3‘ UTR of a viral gene. In some embodiments, the 3’ UTR of a gene is a simian virus (SV) 40 gene. In some embodiments, the human gene is selected from the group consisting of the genes listed in Table 1. In some embodiments, the vector encoding the agent comprises a linear nucleic acid sequence, a plasmid, a viral particle or a synthetic nanoparticle. In some embodiments, the viral particle is an adeno-associated viral (AAV) particle. In some embodiments, the method further comprises contacting the cell with a second agent. In some embodiments, the second agent comprises a modulator of CFTR protein that potentiates chloride conductance of CFTR protein in the 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 agent comprises a targeting moiety, wherein the targeting moiety directs insertion of the exogenous nucleic acid sequence into a genomic site within an intron of an endogenous CFTR gene of a genome of a cell, and wherein the intron is upstream of exon 10 of the endogenous CFTR gene. In some embodiments, the targeting moiety comprises a guide RNA (gRNA), a zinc finger nuclease, or a transcription activator-like (TAL) effector nuclease. In some embodiments, the agent further comprises a gene editing enzy me or a nucleic acid sequence encoding the gene editing enzyme. In some embodiments, the agent comprises a gRNA and a class 2 CRISPR / Cas endonuclease. In some embodiments, the class 2 CRISPR / Cas endonuclease is a Cas9 enzyme. In some embodiments, the gRNA comprises a sequence selected from the group consisting of the sequences provided in Table 3. In some embodiments, the agent or a vector encoding the agent is delivered by intranasal administration, intratracheal administration, or intravenous injection. In some embodiments, the cell comprises a human cell.

[0021] Also proved herein is a method comprising contacting an agent or a vector encoding the agent to the cell, wherein the agent comprises a recombinant nucleic acid sequence provided herein, a gene editing system provided herein, or a pharmaceutical composition provided herein.

[0022] Also provided herein is a method for treating a subject in need thereof, comprising contacting cells of the subject with a recombinant nucleic acid sequence provided herein, a gene editing system provided herein, or a pharmaceutical composition provided herein.

[0023] Also provided herein is a recombinant nucleic acid sequence comprising: a first nucleic acid sequence encoding a N-terminal truncated cystic fibrosis transmembrane regulator (CFTR) protein that is truncated at a position downstream of amino acid residues encoded by exon 11 of a CFTR gene, and a) a splice acceptor site that is immediately upstream of the first nucleic acid sequence; or b) a second nucleic acid sequence that has at least 80% identity to a 3’ untranslated region (3’ UTR) of a gene different than the CFTR gene. In some embodiments, the first nucleic acid sequence comprises exon 22 to exon 27 of the CFTR gene.

[0024] Also provided herein is a recombinant nucleic acid sequence comprising: a first nucleic acid sequence encoding a truncated cystic fibrosis transmembrane regulator (CFTR) protein that consists of amino acid residues encoded by exons 22-27 of a CFTR gene, and a) a splice acceptor site that is immediately upstream of the first nucleic acid sequence; or b) a second nucleic acid sequence that has at least 80% identity to a 3’ untranslated region (3?UTR) of a gene different than the CFTR gene. In some embodiments, the recombinant nucleic acid sequence further comprises a 5’ homology7arm upstream of the first nucleic acid sequence and a 3’ homology arm downstream of the first nucleic acid sequence. In some embodiments, the 5‘ homology arm is homologous to a nucleic acid sequence upstream of the target site of the endogenous CFTR gene and the 3' homology arm is homologous to a nucleic acid sequence downstream of the target site of the endogenous CFTR gene.

[0025] Also provided herein is a recombinant nucleic acid sequence comprising: a) a first nucleic acid sequence encoding a truncated cystic fibrosis transmembrane regulator (CFTR) protein that comprises amino acid residues encoded by exons of a CFTR gene; b) a 5' homology arm upstream of the first nucleic acid sequence, and c) a 3' homology7arm downstream of the first nucleic acid sequence; wherein the 5' homology7arm is homologous to a sequence that is upstream of a genomic site within an intron of an endogenous CFTR gene in a genome of a cell, wherein the 3' homology arm is homologous to a sequence that is downstream of the genomic site, and wherein the intron of the endogenous CFTR gene is dow nstream of intron 10 of the endogenous CFTR gene in a genome of a cell.

[0026] In some embodiments of the recombinant nucleic acid sequence provided herein, exons of the first nucleic acid sequence are codon optimized for expression in human. In someembodiments, the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 45. In some embodiments, the first nucleic acid sequence has at least 80% identity to SEQ ID NO: 45. In some embodiments, the first nucleic acid sequence is free of introns. In some embodiments, the first nucleic acid sequence comprises at least one intron. In some embodiments, the at least one intron comprises an intron that is exogenous to the CFTR gene. In some embodiments, the at least one intron comprises a synthetic / chimeric intron. In some embodiments, each of the at least one intron is less than 600 nucleotides in length. In some embodiments, the splice acceptor site is exogenous to a 5’ end of the first nucleic acid sequence. In some embodiments, the splice acceptor site comprises a nucleic acid sequence chosen from the group consisting of the sequences set forth in SEQ ID NOs: 1-3. In some embodiments, the recombinant nucleic acid sequence is configured to be inserted into a target site of an endogenous CFTR gene in a genome of a cell. In some embodiments, the target site of the endogenous CFTR gene is within an intron of the endogenous CFTR gene. In some embodiments, the intron is downstream of intron 10 of the endogenous CFTR gene. In some embodiments, the second nucleic acid sequence has at least 90% identity to a 3’ untranslated region (3’ UTR) of a gene different than the CFTR gene. In some embodiments, the 3’ UTR of a gene is a 3’ UTR of a eukaryotic gene. In some embodiments, the 3’ UTR of a gene is a 3’ UTR of a human gene. In some embodiments, the 3‘ UTR of a gene is a 3?UTR of a viral gene. In some embodiments, the 3’ UTR of a gene is a simian virus (SV) 40 gene. In some embodiments, the human gene is selected from the group consisting of genes listed in Table 1. In some embodiments, wherein the recombinant nucleic acid sequence is configured to fit within a vector. In some embodiments, the vector comprises a linear nucleic acid sequence, a plasmid, a viral particle or a synthetic nanoparticle. In some embodiments, the viral particle comprises an adeno-associated viral (AAV) particle.

[0027] Also provided herein is a gene editing system, comprising: (a) a first nucleic acid sequence encoding a truncated cystic fibrosis transmembrane regulator (CFTR) protein, and (b) a targeting moiety or a nucleic acid sequence encoding the targeting moiety; wherein the targeting moiety directs insertion of the first nucleic acid sequence into a genomic site within an intron of an endogenous CFTR gene of a genome of a cell, and wherein the intron is dow nstream of intron 10 of the endogenous CFTR gene of the cell.

[0028] Also provided herein is a gene editing system, comprising: (a) a recombinant nucleic acid sequence provided herein, and (b) a targeting moiety or a nucleic acid sequence encoding the targeting moiety; wherein the targeting moiety directs insertion of the first nucleic acid sequence into a genomic site within an intron of an endogenous CFTR gene of a genome of a cell, and wherein the intron is downstream of intron 10 of the endogenous CFTR gene of the cell.

[0029] In some embodiments of the gene editing system provided herein, the targeting moiety comprises a guide RNA (gRNA), a zinc finger nuclease, or a transcription activator-like (TAL) effector nuclease. In some embodiments, the gene editing system further comprises a gene editing enzyme or a nucleic acid sequence encoding the gene editing enzyme. In some embodiments, the gene editing system results in integration of one or two copies of the first nucleic acid sequence. In some embodiments, the targeting moiety is a CRISPR / Cas nickase. In some embodiments, the gene editing system further comprises a recombinase. In some embodiments, the recombinase is a domain of the CRISPR / Cas nickase. In some embodiments, the recombinase is separate from the CRISPR / Cas nickase. In some embodiments, the gene editing system further comprises a reverse transcriptase. In some embodiments, the recombinant nucleic acid sequence is flanked by a sequence recognized by a recombinase. In some embodiments, the gene editing system further comprises a gRNA encoding a sequence that is recognized by a recombinase. In some embodiments, wherein the sequence that is recognized by a recombinase is integrated into the genomic site. In some embodiments, the recombinase directs the insertion of the recombinant nucleic acid sequence into the genomic site. In some embodiments, the gene editing system comprises a gRNA and a class 2 CRISPR / Cas endonuclease. In some embodiments, the gene editing system comprises a gRNA and a Cas9 enzyme. In some embodiments, the gRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 46-55. In some embodiments, the gene editing system is configured to fit within one vector. In some embodiments, the gene editing system is configured to fit in two or more vectors. In some embodiments, the vector comprises a linear nucleic acid sequence, a plasmid, a viral particle or a synthetic nanoparticle. In some embodiments, the viral particle comprises an adeno-associated viral (AAV) particle.

[0030] Also provided herein is a pharmaceutical composition, the pharmaceutical composition comprising: a) a pharmaceutically acceptable excipient or carrier; and b) a recombinant nucleic acid sequence provided herein or a gene editing system provided herein.

[0031] In some embodiments, the pharmaceutical composition is formulated for intranasal administration, intratracheal administration, or intravenous injection. In some embodiments, the pharmaceutical composition is formulated for pulmonary administration. 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 potentiates chloride conductance of CFTR protein in the 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, wherein the second therapeuticagent comprises a mucolytic agent, optionally wherein the mucolytic agent is selected from the group consisting of: acetylcysteine, ambroxol, bromhexine, carbocisteine, erdosteine, mecysteine, domase alfa, hypertonic saline, and mannitol. In some embodiments, the second therapeutic agent comprises a bronchodilator, optionally wherein the bronchodilator is albuterol. In some embodiments, the second therapeutic agent comprises an immunosuppressive agent. In some embodiments, wherein the immunosuppressive agent is a corticosteroid selected from the group consisting of beclomethasone, budesonide, budesonide, formoterol, ciclesonide, fluticasone, salmeterol, umeclidinium, vilanterol, mometasone, or mometasone, prednisone, and methylprednisolone, and pharmaceutically acceptable salts thereof. In some embodiments, the immunosuppressive agent is a non-steroidal immunosuppressive agent selected from the group consisting of polyclonal anti -lymphocyte antibodies, monoclonal anti-lymphocyte antibodies, interleukin-2 (IL-2) receptor antagonists, calcineurin inhibitors, cell cycle inhibitors, mammalian target of rapamycin (mTOR) inhibitors, methotrexate, cyclophosphamide, anthracy cline, and taxane.

[0032] Also provided herein is a method for modifying a cell, comprising contacting an agent or a vector encoding the agent to the cell, wherein the agent modifies a genome of the cell, thereby inserting an exogenous nucleic acid sequence into a genomic site of an intron of an endogenous cystic fibrosis transmembrane regulator (CFTR) gene of the cell, and wherein the intron is downstream of intron 10 of the endogenous CFTR gene of the cell.

[0033] In some embodiments of the method provided herein, the agent comprises a recombinant nucleic acid sequence encoding a truncated CFTR protein that is encoded by at least exons 22- 27 of the CFTR gene. In some embodiments, the method increases expression of CFTR mRNA or CFTR protein by 50%, 60%, 70%, 80%, 90%, 100% or more in the cell compared to an otherwise same cell not receiving the agent. In some embodiments, the cell exhibits a 50%, 60%, 70%, 80%, 90%, 100% or more increase in CFTR activity compared to an otherwise same cell not receiving the agent. In some embodiments, the CFTR activity comprises CFTR-mediated chloride channel conductivity measured by electrophysiology. In some embodiments, the recombinant nucleic acid sequence is codon optimized for expression in human. In some embodiments, the recombinant nucleic acid sequence has at least 70% identify to SEQ ID NO: 45. In some embodiments, wherein the recombinant nucleic acid sequence has at least 80% identify to SEQ ID NO: 45. In some embodiments, the recombinant nucleic acid sequence is free of introns. In some embodiments, the recombinant nucleic acid sequence comprises at least one intron. In some embodiments, the at least one intron comprises an intron that is exogenous to the CFTR gene. In some embodiments, the at least one intron comprises a synthetic / chimeric intron. In some embodiments, each of the at least one intron is less than 600 nucleotides inlength. In some embodiments, the recombinant nucleic acid sequence comprises a splice acceptor site that is exogenous to a 5 ’ end of the first nucleic acid sequence. In some embodiments, the splice acceptor site comprises a nucleic acid sequence chosen from the group consisting of the sequences set forth in SEQ ID NOs: 1-3. In some embodiments, the recombinant nucleic acid sequence further comprises a 5’ homology arm upstream of the first nucleic acid sequence and a 3’ homology arm downstream of the first nucleic acid sequence. In some embodiments, the 5 ’ homology7arm is homologous to a nucleic acid sequence upstream of the genomic site of the endogenous CFTR gene and the 3’ homology arm is homologous to a nucleic acid sequence downstream of the genomic site of the endogenous CFTR gene. In some embodiments, the recombinant nucleic acid sequence further comprises a second nucleic acid sequence has at least 80% identity to a 3’ untranslated region (3’ UTR) of a gene different than the CFTR gene. In some embodiments, the second nucleic acid sequence has at least 90% identity to a 3' UTR of a gene different than the CFTR gene. In some embodiments, the 3' UTR of a gene is a 3’ UTR of a eukaryotic gene. In some embodiments, the 3’ UTR of a gene is a 3?UTR of a human gene. In some embodiments, the 3’ UTR of a gene is a 3’ UTR of a viral gene. In some embodiments, the 3’ UTR of a gene is a simian virus (SV) 40 gene. In some embodiments, the human gene is selected from the group consisting of the genes listed in Table 1. In some embodiments, the vector encoding the agent comprises a linear nucleic acid sequence, a plasmid, a viral particle or a synthetic nanoparticle. In some embodiments, the viral particle is an adeno-associated viral (AAV) particle. In some embodiments, the method further comprises contacting the cell with a second agent. In some embodiments, the second agent comprises a modulator of CFTR protein that potentiates chloride conductance of CFTR protein in the 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 method comprises contacting the cell with a CRISPR / Cas nickase. In some embodiments, the method further comprises contacting the cell with a recombinase. In some embodiments, the recombinase is a domain of the CRISPR / Cas nickase. In some embodiments, the recombinase is separate from the CRISPR / Cas nickase. In some embodiments, the method further comprises contacting the cell with a reverse transcriptase. In some embodiments, the exogenous nucleic acid sequence is flanked by a sequence recognized by a recombinase. In some embodiments, the method further comprises contacting the cell with a gRNA encoding a sequence that is recognized by a recombinase. In some embodiments, the sequence that is recognized by a recombinase is integrated into the genomic site. In some embodiments, the recombinase directs the insertion of the recombinant nucleic acid sequence into the genomic site. In someembodiments, the agent comprises a targeting moiety, wherein the targeting moiety directs insertion of the exogenous nucleic acid sequence into a genomic site within an intron of an endogenous CFTR gene of a genome of a cell, and wherein the intron is dow nstream of intron 10 of the endogenous CFTR gene. In some embodiments, the targeting moiety comprises a guide RNA (gRNA). a zinc finger nuclease, or a transcription activator-like (TAL) effector nuclease. In some embodiments, the agent further comprises a gene editing enzyme or a nucleic acid sequence encoding the gene editing enzyme. In some embodiments, the agent comprises a gRNA and a class 2 CRISPR / Cas endonuclease. In some embodiments, the class 2 CRISPR / Cas endonuclease is a Cas9 enzyme. In some embodiments, the gRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 46-55. In some embodiments, the agent or a vector encoding the agent is delivered by intranasal administration, intratracheal administration, or intravenous injection. In some embodiments, the cell comprises a human cell.

[0034] Also provided herein is a cell comprising a recombinant nucleic acid sequence provided herein, a gene editing system provided herein, or a pharmaceutical composition provided herein. In some embodiments, the cell is a stem cell. In some embodiments, the cell is a human cell.

[0035] Also provided herein is a kit comprising: a) a recombinant nucleic acid sequence provided herein, a gene editing system provided herein, or a pharmaceutical composition provided herein; and b) instructions for use of the recombinant nucleic acid sequence, the gene editing system, or the pharmaceutical composition.

[0036] Also provided herein is a method comprising contacting an agent or a vector encoding the agent to the cell, wherein the agent comprises a recombinant nucleic acid sequence provided herein, a gene editing system provided herein, or a pharmaceutical composition provided herein. In some embodiments, the vector is a viral vector, optionally a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a herpes simplex viral vector.

[0037] Also provided herein is a method for treating a subject in need thereof, comprising contacting cells of the subject with a recombinant nucleic acid sequence provided herein, a gene editing system provided herein, or a pharmaceutical composition provided herein.INCORPORATION BY REFERENCE

[0038] 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. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0040] FIG. 1 shows a model construct of a CFTR super exon including exons 2-27 and its insertion in intron 1 of a native CFTR gene.

[0041] FIG. 2A shows a model construct of a CFTR super exon including exons 23-27 and its insertion in intron 22 of a native CFTR gene.

[0042] FIG. 2B shows two constructs of a CFTR super exon including exons 23-27 with two different 3‘ UTR / poly A constructs.

[0043] FIG. 2C shows results of a membrane potential assay comparing the CFTR function of super exon CFTRs with different 3’ UTR / poly A constructs.

[0044] FIGs. 3A-3F show results of testing efficacy of super exon integration using a GFP reporter construct inserted in intron 1 of CFTR (FIG. 3A). Integration was tested using GFP reporter constructs inserted at different locations within intron 1 (FIG. 3B). Different 3’ UTR regions and splice acceptors were also tested in constructs targeting the 3?end of intron 1 (i.e.. constructs III and IV) (FIG. 3C). “HA” stands for homology arm. Integration efficiency was measured by measuring the percentage of cells that were GFP+by flow cytometry (FIG. 3D). Further flow cytometric analyses were performed to determine intensity of GFP expression with each construct (FIGs. 3E-3F).

[0045] FIGs. 4A-4B show results of testing efficacy of super exon integration using aNanoLuc luciferase reporter construct inserted in intron 1 of CFTR (FIG. 4A). Intensity of NanoLuc activity measured by Nano-Gio assay was measured and different insertion sites in intron 1 were compared (FIG. 4B).

[0046] FIGs. 5A-5C show a schematic of an experimental process testing variations in insertion site, presence of mini-introns, different splice acceptor sites, and different 3’ UTR / polyA regions using 16HBE14o-WT human bronchial epithelial cells (FIG. 5A). Results as measured by NanoLuc activity viaNano-Glo assay are shown in FIGs. 5B-5C.

[0047] FIGs. 6A-6D show an experimental process utilizing functional CFTR super exon constructs bearing exons 2-27 in 16HBEge human bronchial epithelial cells that are deficient in CFTR due to a G542X mutation. As shown in FIG. 6A, cells were administered the gene construct via an adenovirus and subsequently were delivered via nucleofection a ribonucleoprotein to guide insertion of the construct before further functional and genomicanalyses. FIG. 6B shows results of Western blotting detecting CFTR protein expression in various clones. FIG. 6C shows results of qPCR for mRNA levels of the CFTR gene. FIG. 6D shows results of transepithelial current clamp (TECC) electrophysiological analysis of CFTR activity in various clones.

[0048] FIGs. 7A-7B depict results of testing efficacy of super exon integration and expression using constructs presented in FIG. 7A. FIG. 7B shows western blotting results detecting expression of CFTR protein.

[0049] FIG. 8 shows a model construct of a CFTR super exon including exons 22-27 and its insertion in intron 21 of a native CFTR gene.

[0050] FIGs. 9A-9B depict results of an electrophysiology experiment measuring CFTR transport activity. FIG. 9A depicts individual traces obtained from human bronchial epithelial cells deficient in CFTR transfected with adeno-associated viruses bearing an exon 22-27 super exon immediately following nucleofection with Cas9 / gRNA RNP. FIG. 9B depicts a summary of these results.

[0051] FIGs. 10A-10B depict results of an electrophysiology experiment measuring CFTR transport activity. FIG. 10A depicts individual traces obtained from human bronchial epithelial cells deficient in CFTR transfected with adeno-associated viruses bearing an exon 22-27 super exon immediately following nucleofection with Cas9 / gRNA RNP. FIG. 10B summarizes these results.

[0052] FIGs. 11A-11B depict results measuring Nanoluc activity' in an experiment testing different 3‘ UTR sequences. FIG. 11A depicts results using 3’ UTR sequences derived from SV40, RPS27, FLII, TRAP, RPL36. ZDHHC12, NDUFB6. IDH3B, PMC5, NDUFB10, APTR, GRN RPS21, TMEM208, HSD17B10, PSMB7, GPS2, ATRAID, SNRPG, HSP90AB1, PFDN2, LAMTOR4, FKBP2, EEF1G, and RPS11. FIG. 11B depicts results using 3’ UTR sequences derived from NDUFB4, FBL, RPS16, LGALS1, NDUFB1, PTTG1, ATP5PD, SQQR, PFN1, TMSB10. SOD1, COPS3, ATP6V0B, RPN2, ENO1, GSS, and CCT2. Cells were transfected with constructs containing a splice acceptor, a peptide linker, a Nanoluc cassette with a mini-intron, and a 3’ UTR. 41 of the human 3’ UTR sequences from the genes listed in Table 1 were tested.DETAILED DESCRIPTION

[0053] Mutations in the CFTR gene are among the most prevalent causes of CF. The present disclosure relates to, inter alia, nucleic acid constructs that encode truncated CFTR proteins comprising amino acids encoded by a plurality of CFTR exons (e g., a “super exon” construct disclosed herein). Also provided herein are gene editing systems to direct insertion of nucleicacids constructs and methods for modifying cells to insert these constructs into the genomes of cells. In some cases, upon insertion of a super exon construct into the genome of a cell, the cell expresses a functional CFTR protein, which comprises a N-terminal fraction that is encoded by a portion of the endogenous CFTR gene, and C-terminal fraction that is encoded by the super exon insertion. In some cases, expression of the full-length endogenous CFTR gene is prevented as a result of the insertion of the super exon, thus preventing expression of one or more mutations that the endogenous CFTR gene may harbor. In some cases, the endogenous CFTR gene harbors one or more mutations that reduce expression of the CFTR protein in the cell, and the insertion of a super exon disclosed herein into the genome of the cell increases the level of the CFTR protein in the cell. In some cases, the endogenous CFTR gene harbors one or more mutations that affects the normal function (e.g., reducing one or more activity) of the CFTR protein in the cell, and the insertion of a super exon disclosed herein into the genome of the cell results in expression of more functional CFTR proteins in the cell, thus restoring one or more of the CFTR functions in the cell.

[0054] In some aspects, provided herein is a recombinant nucleic acid sequence (e.g, a construct, a plasmid, a vector, or a viral vector) that comprises a nucleic acid sequence encoding a truncated CFTR protein that comprises amino acid residues encoded by at least exons 10-27 of a CFTR gene. In some cases, recombinant nucleic acid sequence further comprises a splice acceptor site that is immediately upstream of the nucleic acid sequence encoding the truncated CFTR protein. Alternatively or additionally, the recombinant nucleic acid sequence further comprises another nucleic acid sequence that has at least 80% identify to a 3‘ untranslated region (3’ UTR) of a gene different than the CFTR gene.

[0055] In some aspects, provided herein is a recombinant nucleic acid sequence comprising a nucleic acid sequence encoding a truncated CFTR protein, wherein the nucleic acid sequence comprises, between two exons of the nucleic acid sequence, an intron that is exogenous to a CFTR gene.

[0056] In some aspects, provided herein is a recombinant nucleic acid sequence comprising (a) a nucleic acid sequence encoding a truncated CFTR protein that comprises amino acid residues encoded by at least exons 10-27 of a CFTR gene; (b) a 5' homology7arm upstream of the nucleic acid sequence, and (c) a 3' homology arm downstream of the nucleic acid sequence. The two homology arms, in some cases, are designed to be homologous to genomic sequences within endogenous CFTR gene in the cell. For instance, the 5' homology arm can be homologous to a sequence that is upstream of a genomic site within an intron of an endogenous CFTR gene in a genome of a cell, and the 3' homology arm can be homologous to a sequence that is dow nstreamof the genomic site. In some cases, the intron of the endogenous CFTR gene is upstream of exon 10 of the endogenous CFTR gene in a genome of a cell.

[0057] In some aspects, provided herein is a gene editing system comprising a) a first nucleic acid sequence encoding a truncated CFTR protein, and b) a targeting moiety or a nucleic acid sequence encoding the targeting moiety. In some cases, the targeting moiety directs insertion of the first nucleic acid sequence into a genomic site within an intron of an endogenous CFTR gene of a genome of a cell, and wherein the intron is upstream of exon 10 of the endogenous CFTR gene.

[0058] In some aspects, provided herein is a gene editing system comprising: a) a recombinant nucleic acid sequence disclosed herein, and b) a targeting moiety or a nucleic acid sequence encoding the targeting moiety; wherein the targeting moiety directs insertion of the first nucleic acid sequence into a genomic site within an intron of an endogenous CFTR gene of a genome of a cell, and wherein the intron is upstream of exon 10 of the endogenous CFTR gene.

[0059] In some aspects, provided herein is a pharmaceutical composition comprising: a) a pharmaceutically acceptable excipient or carrier; and b) a recombinant nucleic acid sequence disclosed herein or a gene editing system disclosed herein.

[0060] In some aspects, provided herein is a method for modifying a cell comprising contacting an agent or a vector encoding the agent to the cell, wherein the agent modifies a genome of the cell, thereby inserting an exogenous nucleic acid sequence into a genomic site of an intron of an endogenous CFTR gene of the cell, and wherein the intron is upstream of exon 10 of the endogenous CFTR gene of the cell.

[0061] In some aspects, provided herein is a method for modifying a cell, the method comprising contacting an agent or a vector encoding the agent to the cell, wherein the agent modifies a genome of the cell, thereby preventing expression of an endogenous CFTR gene in the cell after intron 1.

[0062] In some aspects, provided herein is a method comprising contacting an agent or a vector encoding the agent to the cell, wherein the agent comprises a recombinant nucleic acid sequence disclosed herein, a gene editing system disclosed herein, or a pharmaceutical composition disclosed herein.

[0063] Furthermore, some aspects of the present disclosure relate to method of treating a subject. The method can comprise contacting cells of the subject with a recombinant nucleic acid sequence disclosed herein, a gene editing system disclosed herein, or a pharmaceutical composition disclosed herein.

[0064] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way ofexample only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0065] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0066] Whenever the term “no more than.” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0067] Certain inventive embodiments herein contemplate numerical ranges. When ranges are present, the ranges include the range endpoints. Additionally, every sub range and value within the range is present as if explicitly written out. The term “about” or “approximately” may mean within an acceptable error range for the particular value, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value may be assumed.CFTR

[0068] The CFTR gene can encode cy stic fibrosis transmembrane conductance regulator (CFTR protein), which is a member of the ATP-binding cassette (ABC) transporter superfamily. ABC proteins can transport various molecules across extra- and intra-cellular membranes. Generally, ABC genes are divided into seven distinct subfamilies (ABC1, MDR / TAP, MRP, ALD, OABP, GCN20, White). CFTR protein is a member of the MRP subfamily that can be involved in multi-drug resistance. CFTR protein can function as a chloride channel and control the regulation of other transport pathways. In some cases, mutations in CFTR gene are associated with the 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 can result from mutations in this gene.

[0069] Human (Homo sapiens) CFTR gene (Gene ID: 1080) is located on chromosome 7, and can be defined by chromosomal coordinates GRCh38.pl4 / hg38: chr7: 117,480,025 to GRCh38.pl4 / hg38: chr7: 117,668,665. CFTR is also designated as Ensembl:ENSG00000001626; MIM:602421; AllianceGenome:HGNC: 1884. CFTR is also known 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: NMJ100492, and Ensembl: ENST00000003084.i l. Human CFTR protein is designated as NCBI Reference Sequence: NP 000483.3.

[0070] The CFTR protein can form a cell membrane-spanning chloride channel whose function can be regulated by phosphorylation mediated by cAMP-dependent phosphokinases. Phosphorylation of CFTR expressed on the membrane of a cell in the presence of adenosine triphosphate (ATP) can trigger channel opening to allow outflux of chloride ions from the cell through the channel formed by the CFTR protein, about 10 chloride ions every minute. Certain CFTR gene mutations can lead to generation of defective CFTR proteins that cannot be processed normally by the endoplasmic reticulum for effective transport to the cell membrane. The few mutated CFTR protein molecules that do reach the cell membrane can be dysfunctional and thus cannot carry out chloride ion transportation, leading to accumulation of chloride ions and associated water molecules in epithelial cells and lack of hydration of extracellular mucus and secretions.

[0071] CFTR mutations can be categorized according to the abnormalities they can lead to, including dysfunctional protein translation, cell processing, or CFTR channel gating. Missense (single amino acid substitution) mutations account for 38.74% of CFTR mutants, frameshift (insertion or deletion) mutations account for 16.25%, splicing (incorrect intron splicing) mutations account for 10.93%, and nonsense (early termination codon) mutations account for 8.41% of all known CFTR mutations detected worldwide. Mutations of the CFTR gene can fall into six different classes that roughly correspond to specific types of CFTR dysfunction. In general, mutations in classes I to III can cause more severe disease than those in classes IV to VI. Clinical manifestations of CF caused by any combination of mutations can vary, perhaps due to effects of gene modifiers. For example, g enotype- henotype correlations are weak for CF associated with pulmonary disease but can be stronger for CF ty pes associated with pancreatic insufficiency. Characterization of mutations can be useful to guide initial therapy for some patients, as several new therapies have been recently developed that target CF disease caused by specific classes of CFTR mutations.

[0072] There can be different ty pes of CFTR mutations. Class I mutations can refer to mutations that may cause defective protein production. This type of defect can be caused by nonsense,frameshift, or splice-site mutations, leading to premature termination of messenger RNA (mRNA) transcripts and absence of full length CFTR protein. Non-limiting examples include G542X, R1162X, W1282X, R553X, 621+G>T, 1717-1G>A, 1078delT, 1154 insTC, 1525-2A > G, 1898+1G > A, 2184delA. 2184 insA. 3007delG, 3120+1G > A, 3659delC, 3876delA.3905insT, 394delTT, 4010del4, 4016insT, 4326delTC, 4374+lG > T, 441delA, 556delA, 621+1G > T, 711+1G > T, 875+1G > C, El 104X, E585X, E60X, E822X, G551D / R553X, Q493X, Q552X, Q814X, R1066C, V520F, and Y1092X. Class II mutations can refer to mutations that may cause defective protein processing. This class of mutation can cause abnormal post-translational processing of the CFTR protein, which prevents the protein from trafficking to the correct cellular location, as exemplified by the F508del mutation that is present in a homozygous state in approximately 50% of CF patients and in at least a heterozygous state in 90% of CF patients. Non-limiting examples include A559T, D979A, F508del, AI507, G480C, G85E, N1303K, S549I, S549N, and S549R. Class III mutations can refer to mutations that may cause defective regulation. These mutations cause diminished channel activity even when ATP levels are adequate. Many mutations can alter NBF ATP-binding regions (designated NBD1 and NBD2), whereby some mutants retain varying degrees of sensitivity to nucleotide binding. The mutation giving rise to CFTR substitution G551D, which can abolish ATP binding, is the most common class III mutation in Caucasian populations. Meanwhile, other CFTR mutations within the region encoding the CFTR R domain can also fall into this category. Non-limiting examples include G1244E, G1349D, G551D, G551S, G85E, H199R, I1072T, I48T, L1077P, R560T, S1255P, and S549(R75Q). Class IV mutations can refer to mutations that may cause defective conduction. CTFR protein carrying these mutations can be produced and transported correctly to the cell surface. However, the rate of ion flow and the duration of channel opening can be reduced as compared to normal CFTR protein even though chloride currents are generated in response to cAMP stimulation. A mutation that induces a CFTR protein amino acid substitution (R117H) is the most common class IV mutation in Caucasian populations. Non-limiting examples include A800G, D1152H, D1154G. D614G, delMl 140, E822K, G314E. G576A, G622D, G85E, H620Q, Il 139V, I1234V, L1335P, Ml 137V, P67L, R1 17C, R117P, R117H, R334W, R347H, R347P, R347P / R347H, R792G, S1251N, and V232D. Class V mutations can refer to mutations that may cause reduced amounts of functional CFTR protein. It includes several mutations that can alter mRNA stability and other t pes of mutations that can alter stability of the mature CFTR protein. Non-limiting examples include 2789+5G > A, 3120G > A, 3272-26A > G, 3849+lOkbC > T, 5T variant, 621+3A > G, 711+3A > G, A445E, A455E, IVS8 poly T, and P574H. Class VI mutations can refer to mutations which can cause decreased CFTRstability. This class can cause substantial plasma membrane instability and includes, but is not limited to, Phe508del when rescued by most correctors (rPhe508del).

[0073] In some embodiments, the expression levels of CFTR gene can be measured via next generation sequencing or by quantitative assays (e.g., quantitative PCR (qPCR)) known to a person skilled in the art. In some embodiments, the expression levels of CFTR protein can be measured by Western blot known to a person skilled in the art.

[0074] In some embodiments, the function of various CFTR proteins can be assessed by assays (e.g., electrophysiology assays) known to a person skilled in the art. Exemplary electrophysiology assays can include transepithelial chloride conductance assay (e.g., TECC-24 assay). In some embodiments, the function of various CFTR proteins can be assessed by comparing the area under curve (AUC) of the induced chloride conductance obtained in the electrophysiology assays. The AUC of various mutant cell lines (e.g., 16HBEge-W1282X cells), with or without treatment, can be normalized to the AUC obtained from cells expressing WT CFTR protein 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 assess the function of CFTR proteins. Variations of human bronchial epithelial (HBE) cell lines including wildtype, mutants, primary' and immortalized cell lines can be used in the electrophysiology assays to assess CFTR channel function.

[0075] In the classic form of CF, a patient can demonstrate clinical disease in one or more organ systems (as described below) and can have elevated sweat chloride (>60 mmol / L). Most of these patients can have disease manifestations in multiple organ systems (pancreas, upper and lower respiratory tract, and male reproductive tract). A CFTR-related disorder (CFTR-RD) can refer to a clinical disease limited to only one organ system associated with some evidence of CFTR dysfunction that does not meet full genetic or functional criteria for a CF diagnosis. CFTR-RDs comprise disorders of the lungs, such as disseminated bronchiectasis. CFTR-RDs comprise disorders of the gastrointestinal tract, including CF-related pancreatic insufficiency, CF-related pancreatitis, CF-related diabetes, CF-related liver disease and gallbladder disease. CFTR-RDs comprise disorders of the reproductive tract, including congenital bilateral absence of the vas deferens (CBAVD). Clinical manifestations can include isolated obstructive azoospermia, chronic sinusitis, chronic pancreatitis, or pulmonary disease in adulthood. In cystic fibrosis, deranged transport of chloride and other CFTR-affected ions, such as sodium and bicarbonate, can lead to thick, viscous secretions in the lungs, pancreas, liver, intestine, or reproductive tract, or any combination thereof. Alternatively, or additionally, deranged transport of chloride and other CFTR-affected ions can lead to increased salt content in sweat gland secretions. Symptoms of CF can vary in different age groups of individuals. Infants and children can presentrespiratory symptoms, meconium ileus, failure to thrive, or any combination thereof. Typical respiratory manifestations of CF can include a persistent productive cough, hyperinflation of the lung fields on chest radiograph, and pulmonary7function tests that are consistent with obstructive airway disease. Other clinical manifestations of pulmonary disease can include microorganism infection, bronchiectasis, airway hyperreactivity, allergic bronchopulmonary aspergillosis, obstructive sleep apnea, and pulmonary hypertension. Patients presenting with CF later in life are more likely to have aty pical symptoms, including sinus disease, pancreatic disease, infertility, musculoskeletal disorders, recurrent venous thrombosis, anemia, electrolyte abnormalities, nephrolithiasis, and aquagenic wrinkling.Super Exons

[0076] In some aspects, provided herein are compositions, methods, gene editing systems, kits, and pharmaceutical compositions involving super exon constructs that encode a plurality of functional exons of CFTR. “Super exon” as used herein refers to a construct that comprises, from 5’ to 3’, a splice acceptor site, functional exonic sequences of a plurality of exons with or without introns, and an untranslated region (UTR) / polyA. Constructs may further comprise homology7arms flanking both ends to direct insertion into the target cell genome. When these constructs are integrated into the genome, spliced by the spliceosome and translated, they will generate a functional CFTR protein that lacks the endogenous mutation(s) present in the CFTR gene.

[0077] As shown in FIG. 1, an exemplary7super exon construct may encode for exons 2-27 and be inserted within intron 1. Other constructs may encode for exons 3-27, 4-27, 5-27, 6-27, 7-27, 8-27, 9-27, 10-27, 11-27, 12-27, 13-27, 14-27, 15-27, 16-27, 17-27, 18-27, 19-27, 20-27, 21-27, 22-27, 23-27, 24-27, 25-27, or 26-27.

[0078] Super exon constructs may7be inserted at any region within an intron. As shown in FIGs. 3B and 3C, constructs could be engineered to be inserted at the 5’ end of an intron, at the 3’ end of an intron, or any region between. By flanking constructs with homology arms (also referred to as “homologous arm” herein) that are complementary w ith the targeted insertion site, the construct can be directed to insert and integrate at a specific site. In some embodiments, the homology7arms comprise a nucleotide sequence of about 50 bp, about lOObp, about 150bp, about 200bp, about 250bp, about 300bp. about 350bp, about 400bp, about 450bp, about 500bp, about 550bp. about 600bp. about 650bp. about 700bp, about 750bp, about 800bp, about 900bp, about lOOObp, about 1250bp, about 1500bp, about 1750bp, or about 2000bp in length. In some embodiments, the homology7arms comprise a nucleotide sequence of at least 50 bp, at least lOObp, at least 150bp, at least 200bp, at least 250bp, at least 300bp, at least 350bp, at least 400bp, at least 450bp, at least 500bp, at least 550bp, at least 600bp. at least 650bp, at least700bp, at least 750bp, at least 800bp, at least 900bp, at least lOOObp, at least 1250bp, at least 1500bp, at least 1750bp, or at least 2000bp in length. In some embodiments, the homology arms comprise a nucleotide sequence of at most 50 bp, at most lOObp, at most 150bp, at most 200bp, at most 250bp, at most 300bp, at most 350bp, at most 400bp, at most 450bp, at most 500bp, at most 550bp, at most 600bp, at most 650bp, at most 700bp, at most 750bp, at most 800bp, at most 900bp, at most lOOObp, at most 1250bp, at most 1500bp, at most 1750bp, or at most 2000bp in length. In some embodiments, constructs comprise a 5’ homology arm upstream of the construct and a 3’ homology arm downstream of the construct. In some embodiments, the 5’ homology arm is homologous to a nucleic acid sequence upstream of the target site of the endogenous CFTR gene and the 3’ homology arm is homologous to a nucleic acid sequence downstream of the target site of the endogenous CFTR gene. In some embodiments, the 5' homology arm is homologous to a sequence that is upstream of a genomic site within an intron of an endogenous CFTR gene in a genome of a cell, wherein the 3' homology arm is homologous to a sequence that is downstream of the genomic site. In some embodiments, the 3’ homology arm comprises a nucleic acid sequence having at least 60%, at least 61% at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 39, 41, and 43. In some embodiments, the 5’ homology arm comprises a sequence having at least 60%, at least 61% at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%. at least 90%. at least 91%. at least 92%. at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 38, 40, or 42. In some embodiments, the homology arms comprise (a) a 3’ homology7arm comprising a nucleic acid sequence having at least 60%, at least 61% at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% identity to any one of SEQ ID NOs: 39, 41, and 43; and (b) a 5’ homology arm comprising a nucleic acid sequence having at least 60%, at least 61% at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least77%. at least 78%. at least 79%. at least 80%. at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 38, 40, or 42.

[0079] Target sites in the endogenous CFTR gene can include any site downstream of exon 1. In some embodiments, the target site is within intron 1 of CFTR. In some embodiments, the target site is about 79 nucleotides downstream of exon 1 of CFTR. In some embodiments, the target site is about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 50, about 60. about 70, about 80, about 100. about 200, about 300, about 400, about 500, about 1000, about 2000, about 3000. about 4000, about 5000. about 10000. about 15000. or about 20000 nucleotides dow nstream of exon 1 of CFTR. In some embodiments, the target site is located within a region from hg38 chr7: 117,480,148 to hg38 chr7: 117,498,307.

[0080] In some embodiments, the target site is within intron 21 of CFTR. In some embodiments, the target site is about 5, about 10. about 15, about 20, about 25, about 30, about 35, about 40, about 50, about 60, about 70, about 80, about 100, about 200, about 300, about 400, about 500, about 1000, about 2000, about 3000, about 4000, about 5000, about 10000, about 15000, or about 20000 nucleotides downstream of exon 21 of CFTR. In some embodiments, the target site is located within a region from hg38 chr7: 117,614,714 to hg38 chr7: 117,627,521.

[0081] The term '‘downstream’’ can refer to a nucleic acid position that, relative to a reference position or region, is closer to the 3’ end of a nucleic acid molecule. The term ‘'upstream” can refer to a nucleic acid position that, relative to a reference position or region, is closer to the 5’ end of a nucleic acid molecule. When referring to sequences in a double stranded DNA molecule, the terms “upstream”, “downstream”. “3’ end”, and “5’ end” are used with reference to the spatial relationship within the sense strand of the double stranded DNA molecule.

[0082] Various splice acceptor sites could be utilized in these constructs at the 5’ end, including those derived from CFTR. In some embodiments, the splice acceptor site is derived from exon 11 of CFTR, as shown in FIG. 5B. In some embodiments, the splice acceptor site is derived from exon 23 of CFTR. In some embodiments, the splice acceptor site comprises a sequence having at least 60%, at least 61% at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least Tl%, at least 78%, at least 79%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 3. In some embodiments, the splice acceptor site is a synthetic splice acceptor. In some embodiments, the synthetic splice acceptor comprises sequence having at least 60%, at least 61% at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1 .

[0083] Various 3’ UTR sequences could be utilized in these constructs. In some embodiments, the 3‘ UTR sequence is naturally occurring. In some embodiments, the 3‘ UTR sequence is derived from the simian virus 40 (SV40) polyA sequence. In some embodiments, the 3’ UTR sequence is a combination of the SV40 polyA sequence and the EPH-like kinase 5 (EK5) sequence. In some embodiments, the 3’ UTR sequence is derived from a human gene. In some embodiments, the 3’ UTR sequence is an annotated sequence for a known gene. Preferably, candidate human 3’ UTR sequences are less than 400bp in length, are highly expressed in bronchial epithelial cells, and demonstrate a strong transcription termination signal as measured by RNA sequencing. Candidate 3’ UTR sequences can be derived from human genes such as those found in Table 1.Table 1. Candidate 3’ UTR Source Genes

[0084] In some embodiments, super exon constructs may include one or more intronic sequences. In some embodiments, the one or more intronic sequences are less than lOOObp, less than 950bp, less than 900bp. less than 850bp, less than 800bp, less than 750bp. less than 700bp, less than 650bp, less than 600bp, less than 550bp, less than 500bp, less than 450bp, less than 400bp, less than 350bp, less than 300bp, less than 250bp, less than 200bp, less than 150bp, less than lOObp, or less than 50bp in length. In some embodiments, the one or more intronic sequences are about lOOObp. about 950bp, about 900bp, about 850bp, about 800bp, about 750bp, about 700bp. about 650bp. about 600bp. about 550bp. about 500bp. about 450bp, about 400bp, about 350bp, about 300bp, about 250bp, about 200bp, about 150bp, about l OObp, orabout 5Obp in length. In some embodiments, the one or more intronic sequences are at least lOOObp, at least 950bp, at least 900bp, at least 850bp, at least 800bp, at least 75Obp, at least 700bp, at least 650bp, at least 600bp, at least 55Obp, at least 5OObp, at least 450bp, at least 400bp, at least 35Obp, at least 3OObp, at least 250bp, at least 200bp. at least 150bp, at least lOObp, or at least 5Obp in length. In some embodiments, the one or more intronic sequences comprise a sequence having at least 60%, at least 61% at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity7to the sequence set forth in SEQ ID NO: 4. In some embodiments, a super exon construct comprises a sequence having at least 60%, at least 61% at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of the sequences presented in Table 2. In some embodiments, a super exon construct comprises a sequence having at least 60%, at least 61% at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to anyone of the sequences presented in Table 4.Methods to Deliver Super Exon Constructs

[0085] In some embodiments, super exon constructs are inserted into the genome of a cell using any gene editing system known in the art. Gene editing systems can utilize nuclease enzy mes to introduce breaks in genomic DNA at specific sites using guide (RNAs). As the break is introduced, endogenous repair mechanisms are capable of integrating donor templates, including a super exon construct. Gene editing systems can include clustered regularly interspaced short palindromic repeats Cas endonuclease ribonuclear protein (CRISPR-Cas RNP) systems. Guide RNAs (gRNAs) can be used to direct insertion of super exon constructs at any intronic region ofthe endogenous CFTR gene. In some embodiments, a class 2 Cas endonuclease is used. In some embodiments, a Cas9 endonuclease is used. In some embodiments, a gRNA specific for a site 325bp downstream of exon 1 is utilized. In some embodiments, a gRNA specific for a site 79bp downstream of exon 1 is utilized. In some embodiments, the gRNA comprises a sequence having at least 60%, at least 61% at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%. at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence set forth in any one of SEQ ID NOs: 11-34 is used. In some embodiments, the gRNA comprises a sequence having at least 60%, at least 61% at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one sequence set forth in Table 3. In some embodiments, super exon constructs are inserted into the genome of a cell using zinc finger nucleases (ZFNs) or transcription activator-like (TAL) effector nuclease, with comprise DNA binding domains that can be specifically targeted to intronic sites.

[0086] Super exon constructs may be packaged with a CRISPR-Cas9 RNP and gRNA. In some embodiments, super exon constructs are packaged with gRNA and a nucleic acid encoding the CRISPR-Cas9. In some embodiments, super exon constructs are packaged with a ZFN protein. In some embodiments, super exon constructs are packaged with a nucleic acid encoding a ZFN. In some embodiments, super exon constructs are packaged with a TAL effector nuclease protein. In some embodiments, super exon constructs are packaged with a nucleic acid encoding a TAL effector nuclease.

[0087] Super exon constructs can be inserted into the genome using a prime editing system. Prime editing is a platform for genome editing that directly writes new genetic information into a specified DNA site using a nucleic acid programmable DNA binding protein C’napDN Abp”) w orking in association with a polymerase (i.e., in the form of a fusion protein or otherwise provided in trans with the napDNAbp), wherein the prime editing system is programmed with a prime editing (PE) guide RNA (“pegRNA”) that both specifies the target site and templates the synthesis of the desired edit in the form of a replacement DNA strand by way of an extension(either DNA or RNA) engineered onto a guide RNA (e.g., at the 5' or 3' end, or at an internal portion of a guide RNA). The replacement strand containing the desired edit shares the same (or is homologous to) sequence as the endogenous strand (immediately dow nstream of the nick site) of the target site to be edited (with the exception that it includes the desired edit). Through DNA repair and / or replication machinery, the endogenous strand downstream of the nick site is replaced by the newly synthesized replacement strand containing the desired edit. The mechanism of target-primed reverse transcription (TPRT) or “prime editing” can be leveraged or adapted for conducting precision CRISPR / Cas-based genome editing with high efficiency and genetic flexibility. Cas protein-reverse transcriptase fusions or related systems can be used to target a specific DNA sequence with a guide RNA, generate a single strand nick at the target site, and use the nicked DNA as a primer for reverse transcription of an engineered reverse transcriptase template that is integrated with the guide RNA. How ever, while the concept begins with prime editors that use reverse transcriptase as the DNA polymerase component, the prime editors are not limited to reverse transcriptases but may include the use of virtually any DNA polymerase. Thus, wherever the specification mentions a “reverse transcriptase,” the person having ordinary skill in the art should appreciate that any suitable DNA polymerase may be used in place of the reverse transcriptase. Thus, in one aspect, the prime editors may comprise Cas9 (or an equivalent napDNAbp) which is programmed to target a DNA sequence by associating it with a specialized guide RNA (i.e., pegRNA) containing a spacer sequence that anneals to a complementary protospacer in the target DNA. The specialized guide RNA also contains new genetic information in the form of an extension that encodes a replacement strand of DNA containing a desired genetic alteration which is used to replace a corresponding endogenous DNA strand at the target site.

[0088] In some embodiments, the super exon construct comprises at least exons 2 to 27. In some embodiments, the super exon comprises at least exons 4 to 27. In some embodiments, the super exon comprises at least exons 6 to 27. In some embodiments, the super exon comprises at least exons 8 to 27. In some embodiments, the super exon comprises at least exons 10 to 27. In some embodiments, the super exon comprises at least exons 12 to 27. In some embodiments, the super exon comprises at least exons 14 to 27. In some embodiments, the super exon comprises at least exons 16 to 27. In some embodiments, the super exon comprises at least exons 18 to 27. In some embodiments, the super exon comprises at least exons 20 to 27. In some embodiments, the super exon comprises at least exons 22 to 27. In some embodiments, the super exon comprises at least exons 23 to 27. In some embodiments, a cas9 nickase and prime-editing gRNAs encoding the recombinase binding site can introduce a recombinase sequence into the target site of the genome. In some embodiments, the super exon construct is flanked with recombinase bindingsites and is delivered with a recombinase. In some embodiments the recombinase mediates integration of the super exon construct into the genome via the recombinase binding sites. Recombinase systems include, but are not limited to, Cre / LoxP and attP / attB.

[0089] Super exon construct can also be inserted using site specific targeting elements for recombination. This method comprises the addition of an integration site into a target genome followed by the insertion of one or more nucleic acid sequences of interest (e.g., a super exon construct) at the site. This process can be done as one or more reactions in a cell. The addition of the integration site into the target genome is done using gene editing technologies that include for example, without limitation, prime editing, recombinant adeno-associated virus (rAAV)- mediated nucleic acid integration, transcription activator-like effector nucleases (TALENS), and zinc finger nucleases (ZFNs). The integration of the transgene at the integration site is done using integrase technologies that include for example, without limitation, integrases, recombinases and reverse transcriptases. The necessary components for the site-specific genetic engineering disclosed herein comprise at least one or more nucleases, one or more gRNA, one or more integration enzymes, and one or more sequences that are complementary or associated to the integration site and linked to the one or more genes of interest or one or more nucleic acid sequences of interest to be inserted into the cell genome. This method uses a catalytically- impaired Cas endonuclease (e.g., Cas9) that is fused to an engineered reverse transcriptase (RT) and programmed with a prime-editing guide RNA (pegRNA). The skilled person in the art would appreciate that the pegRNA both specifies the target site and encodes the desired edit. The catalytically-impaired Cas9 endonuclease also comprises a Cas9 nickase that is fused to the reverse transcriptase. During genetic editing, the Cas9 nickase part of the protein is guided to the DNA target site by the pegRNA. The reverse transcriptase domain then uses the pegRNA to template reverse transcription of the desired edit (e.g., an integrase-specific recognition sequence), directly polymerizing DNA onto the nicked target DNA strand. The edited DNA strand replaces the original DNA strand, creating a heteroduplex containing one edited strand and one unedited strand. Afterward, the prime editor (PE) guides resolution of the heteroduplex to favor copying the edit onto the unedited strand, completing the process. The insertion of the nucleic acid sequence of interest is directed by an integrase that recognizes the integrase-specific recognition sequence added to the genome. The integration enzy me can be an integrase that incorporates the genome or nucleic acid of interest into the cell genome at the integration site by integration. The integration enzyme can be a recombinase that incorporates the genome or nucleic acid of interest into the cell genome at the integration site by recombination. The integration enzy me can be a reverse transcriptase that incorporates the genome or nucleic acid of interest into the cell genome at the integration site by reverse transcription. The integrationenzyme can be a retrotransposase that incorporates the genome or nucleic acid of interest into the cell genome at the integration site by retrotransposition. In some embodiments, the integrase is a separate protein from the Cas endonuclease. In some embodiments, the integrase is a domain of the same protein as the Cas endonuclease and / or the reverse transcriptase. In some embodiments, the integrase is a serine integrase. In some embodiments, the integrase is a ere recombinase.

[0090] In some embodiments, the super exon construct comprises at least exons 2 to 27. In some embodiments, the super exon comprises at least exons 4 to 27. In some embodiments, the super exon comprises at least exons 6 to 27. In some embodiments, the super exon comprises at least exons 8 to 27. In some embodiments, the super exon comprises at least exons 10 to 27. In some embodiments, the super exon comprises at least exons 12 to 27. In some embodiments, the super exon comprises at least exons 14 to 27. In some embodiments, the super exon comprises at least exons 16 to 27. In some embodiments, the super exon comprises at least exons 18 to 27. In some embodiments, the super exon comprises at least exons 20 to 27. In some embodiments, the super exon comprises at least exons 22 to 27. In some embodiments, the super exon comprises at least exons 23 to 27. In some embodiments, a cas9 nickase and prime-editing gRNAs encoding the integrase-specific binding site can introduce a recombinase sequence into the target site of the genome. In some embodiments, the super exon construct is flanked with integrase binding sites and is delivered with an integrase. In some embodiments the integrase mediates integration of the super exon construct into the genome via the recombinase binding sites. Recombinase systems include, but are not limited to, Cre / LoxP and attP / attB.

[0091] Super exon constructs and gene editing systems may be delivered using any suitable delivery vehicle. Vehicles may include but are not limited to lipid nanoparticles (LNPs), exosomes, extracellular vesicles, viral vectors (e.g., adenoviral vectors, lenti viral vectors) and virus-like particles (VLPs). The super exon constructs provided herein can be relatively small in size and can be delivered by viral vectors (e g., AAV vectors) suitable for delivering small DNA fragments. In some embodiments, the super exon construct comprises at least exons 2 to 27. In some embodiments, the super exon comprises at least exons 4 to 27. In some embodiments, the super exon comprises at least exons 6 to 27. In some embodiments, the super exon comprises at least exons 8 to 27. In some embodiments, the super exon comprises at least exons 10 to 27. In some embodiments, the super exon comprises at least exons 12 to 27. In some embodiments, the super exon comprises at least exons 14 to 27. In some embodiments, the super exon comprises at least exons 16 to 27. In some embodiments, the super exon comprises at least exons 18 to 27. In some embodiments, the super exon comprises at least exons 20 to 27. In some embodiments, the super exon comprises at least exons 22 to 27. In some embodiments, the super exon comprises atleast exons 23 to 27. In some embodiments, the super exon construct is transfected into a target cell using an AAV vector. In some embodiments, the super exon construct comprises at least exons 22 to 27 and is transfected into a target cell using an AAV vector.

[0092] The term “vectors” refers to a nucleic acid molecule capable of transporting or mediating expression of a heterologous nucleic acid. A plasmid is a species of the genus encompassed by the term “vector.” A vector typically refers to a nucleic acid sequence containing an origin of replication and other entities necessary for replication and / or maintenance in a host cell. Vectors capable of directing the expression of genes and / or nucleic acid sequence to which they are operatively linked are referred to herein as “expression vectors”. In general, expression vectors of utility are often in the form of “plasmids” which refer to circular double stranded DNA molecules which, in their vector form are not bound to the chromosome, and typically comprise entities for stable or transient expression or the encoded DNA. Other expression vectors that can be used in the methods as disclosed herein include, but are not limited to plasmids, episomes, bactenal artificial chromosomes, yeast artificial chromosomes, bacteriophages or viral vectors, and such vectors can integrate into the host's genome or replicate autonomously in the cell. A vector can be a DNA or RNA vector. Other forms of expression vectors know n by those skilled in the art which serve the equivalent functions can also be used, for example, self-replicating extrachromosomal vectors or vectors capable of integrating into a host genome. Exemplary- vectors are those capable of autonomous replication and / or expression of nucleic acids to which they are linked.

[0093] Lipid nanoparticles (LNP) may comprise a polar and or a nonpolar lipid. In some embodiments cholesterol is present in the LNPs for efficient delivery. LNPs are 100-300 nm in diameter provide efficient means of mRNA delivery' to various cell types, including macrophages. In some embodiments, LNP may be used to introduce the recombinant nucleic acids into a cell in in vitro cell culture. In some embodiments, the LNP encapsulates the nucleic acid wherein the nucleic acid is a naked DNA molecule. In some embodiments, the LNP encapsulates the nucleic acid wherein the nucleic acid is an mRNA molecule. In some embodiments, the LNP encapsulates the nucleic acid wherein the nucleic acid is inserted in a vector, such as a plasmid vector. In some embodiments, the LNP encapsulates the nucleic acid wherein the nucleic acid is a circRNA molecule.

[0094] Virus-like particles (VLPs) refer to the spontaneous organization of coat proteins into the three-dimensional capsid structure of a particular virus. Like active viruses, these particles are in the 20-500 nm size range, but they are devoid of the virus nucleic acid.Measurement of Effective Integration

[0095] In some embodiments, effective construct integration is measured by protein expression. In some embodiments, effective construct integration is measured by mRNA abundance. Super exon constructs can be developed with both single and double copies of the CFTR gene. In some embodiments, a western blot is used to detect the level of CFTR protein levels from the construct. As shown in FIG. 6B, a western blot can be used to visualize protein expression from integrated CFTR super exon constructs.Functional Constructs

[0096] A DNA construct is an artificially constructed segment of nucleic acid which is transplanted into a target cell. The DNA insert contains a full or partial gene sequence encoding a protein of interest. Here, the protein of interest is CFTR. The cell constructs were examined using both functional and genomic analyses. In some embodiments, cell constructs were selected by flow cytometry. Flow cytometry technology rapidly analyzes single cells as they flow past single or multiple layers while suspended in buffered salt-based solution. Each cell is analyzed for visible light scatter and one or multiple fluorescence parameters. Super exon integration was demonstrated by a gDNA PCR screen. gDNA PCR screen is broadly used to detect CFTR expression in chromosomal DNA. The cell constructs w ere then characterized for cell expansion and banking, targeted NGS of the CFTR locus, CFTR protein expression, CFTR mRNA abundance, and electrophysiology.Method of screening

[0097] Green fluorescent protein, also known as GFP, is a marker used to detect and quantify protein expression in cells. A GFP CFTR super exon reporter system can be developed to test and measure expression efficiency of a super exon construct, where insertion and expression of the GFP super exon results in a CFTR exon-1 GFP fusion protein. Flow cytometry analysis or other means can be used to demonstrate CFTR super exon sensitivity using GFP signal.

[0098] The NanoLuc reporter system is an alternative system that can provide a more sensitive mechanism to evaluate super exon insertion and expression. The NanoLuc reporter system arises from a CFTR exon 1 -NanoLuc fusion protein where NanoLuc is functional. In some embodiments, the cells are stained with Alamar blue prior to measurement in order to read cell density normalization. In some embodiments, the Nano-Gio live cell assay is used to measure insertion frequency. In some embodiments, the Nano-Luc reporter system is used to measure the expression of the transgene. The insertion site can affect NanoLuc expression.

[0099] In some embodiments, the level of CFTR super exon expression in the cell is increased by at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at leastabout 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, or at least about 20 times as compared to a corresponding cell that is not contacted with the agent or the vector encoding the agent.Method of Treatment

[0100] In some aspects, provided herein is a method of treating cystic fibrosis in a subject in need thereof. The method can comprise administering a therapeutic agent provided herein (e.g. , a construct comprising a super exon, or a vector comprising same) to the subject. Delivery method provided herein is applicable for the administration of the therapeutic agent, for instance, via CRISPR / Cas, ZFN, or TALEN gene editing system.

[0101] In some cases, methods provided herein comprise administration of a first therapeutic agent and second therapeutic agent. In some cases, the first agent comprises a therapeutic agent provided herein, and the second agent is another agent that is a known agent for treatment of cystic fibrosis or another diseases or conditions.

[0102] In one embodiment, the additional agent is an antibiotic or anti-infective agent. In some embodiments, the antibiotic or anti-infective agent is 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, colistimethate, linezolid, ciprofloxacin, levofloxacin, or any combination thereof.

[0103] In one embodiment, the additional agent is one or more CFTR modulators. In some cases, the CFTR modulator can improve the processing and trafficking of one or more variants of CFTR proteins, e.g., lumacaftor, tezacaftor, elexacaftor, or tezacaftor. In some cases, the CFTR modulator can potentiate chloride conductance of one or more variants of CFTR proteins on the cells, e.g., ivacaftor. In some embodiments, the one or more CFTR modulators are a combination of agents that can potentiate chloride conductance and agents that can improve the processing and trafficking of one or more variants of the CFTR proteins, e.g., ivacaftor (KALYDECO®), lumacaftor, lumacaftor / ivacaftor (ORKAMBI®), tezacaftor / ivacaftor (SYMDEKO®), or TRIKAFTA® elexacaftor / ivacaftor / tezacaftor (TRIKAFTA®). In some cases, the CFTR modulators are referred to by the names used in basic research, which can be used interchangeably herein, e.g., elexacaftor / VX-661; tezacaftor / VX-445; ivacaftor / VX-770, lumacaftor / VX-809, or Trikafta / VX-445 / VX-661 / VX-770.

[0104] In one embodiment, the additional agent is a mucolytic agent for airway clearance e.g., acetylcysteine, ambroxol, bromhexine, carbocisteine, erdosteine, mecysteine. domase alfa,hypertonic saline, or mannitol. In one embodiment, the additional agent is a bronchodilator (e.g., albuterol).

[0105] In one embodiment, the additional agent is an immunosuppressive agent includes corticosteroids (e.g., an inhaled corticosteroid (e.g., beclomethasone (QVAR®), budesonide (PULMICORT®). budesonide / formoterol (SYMBICORT®). ciclesonide (ALVESCO®), fluticasone (FLOVENT HF A®), fluticasone propionate (FLOVENT DISKUS®), fluticasone furoate (ARNUITY ELLIPTA®), fluticasone propionate / salmeterol (ADV AIR®), fluticasone furoate / umeclidinium / vilanterol (TRELEGY ELLIPTA®), mometasone furoate (ASMANEX®), or mometasone / formoterol (DULERA®), prednisone, or methylprednisolone). In one embodiment, the additional agent is a non-steroidal immunosuppressive agent, which can be biologies, including polyclonal anti-lymphocyte antibodies (e.g., anti-lymphocyte globulin (ALG) or anti-thymocyte globulin (ATG) antibodies, which may be, for example, horse- or rabbit-derived), monoclonal anti-lymphocyte antibodies (e.g.. anti-CD3 antibodies (e.g., muromonab or alemtuzumab) or anti-CD20 antibodies (e.g., rituximab)), interleukin-2 (IL-2) receptor antagonists (e g., daclizumab or basiliximab). In one embodiment, the additional agent is a non-steroidal immunosuppressive agents, which can be small molecules drugs including calcineurin inhibitors (e.g., cyclosporin A or tacrolimus), cell cycle inhibitors (e.g., azathioprine, my cophenolate mofetil (MMF), or mycophenolic acid (MPA)), mammalian target of rapamycin (mTOR) inhibitors (e g., sirolimus (rapamycin) or everolimus), methotrexate, cyclophosphamide, an anthracycline (e.g., doxorubicin, idarubicin, aclarubicin, daunorubicin, epirubicin, valrubicin, mitoxantrone, or a combination thereof), a taxane (e.g., TAXOL® (paclitaxel)), and a combination thereof (e.g., a combination of a calcineurin inhibitor, a cell cycle inhibitor, or a corticosteroid).

[0106] In one embodiment, the additional agent is a nutritional agent, such as pancrelipase (pancreatic enzyme replacement) (e.g., Pancrease®, Pancreacarb®, or Ultrase®), Creon®, Liprotamase® (formerly Trizytek®), Aquadeks®, and glutathione inhalation.

[0107] In some embodiments, the therapeutic agents disclosed in the present disclosure are used in combination with one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents can comprise a small molecule. For example, the one or more additional therapeutic agents can comprise a small molecule described in US20220241206, which is incorporated by reference herein in its entirety.

[0108] In some embodiments, the therapeutic agents disclosed herein are used in combination with one or more additional nucleic-acid-based therapeutic agents for treating cystic fibrosis. In some cases, the additional therapeutic agents comprise agents for replacement therapy (e.g., gene therapy and RNA replacement), agents for gene editing (e.g., CRISPR-Cas9, TALEN, andZinc Finger), agents for RNA interference (e.g., siRNA, miRNA), ASOs (e.g., gene silencing ASO, exon-skipping ASO, or read-through ASO), and tRNA (e.g., suppressor tRNA).Pharmaceutical Composition

[0109] In some aspects, provided herein is a pharmaceutical composition comprising a therapeutic agent provided herein. The therapeutic agent can comprise a super exon construct or a vehicle bearing the super exon construct and a gene editing system. Vehicles bearing super exon constructs and gene editing systems may be administered to a subject suffering from CF alone or in a pharmaceutical composition comprising the super exon constructs and gene editing system and a pharmaceutically acceptable excipient or carrier.

[0110] Pharmaceutical compositions or formulations comprising the agent of the described compositions and for use in any of the described methods can be prepared according to conventional techniques well know n in the pharmaceutical industry and described in the published literature. Pharmaceutically acceptable salts are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., and are commensurate with a reasonable benefit / risk ratio. (See, e.g., S. M. Berge, et al., J. Pharmaceutical Sciences, 66: 1-19 (1977), incorporated herein by reference for this purpose). The term “pharmaceutically acceptable’' refers to approved or approvable by a regulator}' agency of the federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans. The salts can be prepared in situ during the final isolation and purification of the compounds, or separately by reacting the free base form with a suitable organic acid. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other documented methodologies such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy- ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate. [OHl] Pharmaceutical compositions may be administered to a subject suffering from CF intravenously, intrathecally, orally, or via inhalation. In some embodiments, the compositions are formulated into any of many possible dosage forms such as. but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. In embodiments, the compositions are formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity' of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain stabilizers. In embodiments, a pharmaceutical formulation or composition of the present disclosure includes, but is not limited to, a solution, emulsion, microemulsion, foam or liposome-containing formulation.

[0112] The pharmaceutical composition or formulation described herein may comprise one or more penetration enhancers, carriers, excipients or other active or inactive ingredients as appropriate and well known to those of skill in the art or described in the published literature. In some embodiments, liposomes also include sterically stabilized liposomes. These specialized lipids result in liposomes with enhanced circulation lifetimes. In some embodiments, a sterically stabilized liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. In some embodiments, a surfactant is included in the pharmaceutical formulation or compositions. The use of surfactants in drug products, formulations and emulsions is well known in the art. In embodiments, the present disclosure employs a penetration enhancer to promote the efficient delivery’ of the antisense oligonucleotide, e.g., to aid diffusion across cell membranes and / or enhance the permeability of a lipophilic drug. In some embodiments, the penetration enhancers are a surfactant, fatty' acid, bile salt, chelating agent, or non-chelating nonsurfactant. In some embodiments, the pharmaceutical composition or formulation can comprise a lipid nanoparticle. In some embodiments, the pharmaceutical formulation comprises multiple antisense oligonucleotides. In embodiments, the pharmaceutical composition is administered in combination with another drug or therapeutic agent. In some embodiments, the pharmaceutical composition refers to an excipient or carrier that can be administered to a subject, together with an agent, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent.

[0113] In some cases, the pharmaceutical composition is formulated for any suitable administration method for a cystic fibrosis patient, such as parenteral administration, including intranasal administration, intratracheal administration, intravenous injection, intraperitonealinjection, 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 comprises administering pulmonary dosage forms, including forms for administration via a dry powder inhaler, a metered dose inhaler, or a nebulizer either in conventional or nanoparticles and microparticles form.Kit

[0114] In one aspect, provided herein, is a kit comprising one or more vials containing a therapeutic agent provided herein or a pharmaceutical composition provided herein. For instance, the vials can contain vehicles bearing super exon constructs and gene editing components. The kit disclosed herein can comprise the composition or the pharmaceutical composition disclosed herein, and instructions for use of the composition or the pharmaceutical composition. The kit can comprise the composition or the pharmaceutical composition disclosed herein, and a second therapeutic agent (or an additional agent). In some cases, the kit further comprises one or more additional reagents, such as an immunosuppressive agent as described above, or one or more agents for treating CF as described herein. Kits provided herein typically include a label indicating the intended use of the contents of the kit. The term label includes any writing, or recorded material supplied on or with the kit. or which otherwise accompanies the kit.Table 2. Nucleotide sequencesTable 3: gRNAs UsedTable 4: Full Length Super Exon ConstructsNUMBERED EMBODIMENTS

[0115] Embodiment 1. A recombinant nucleic acid sequence comprising: a first nucleic acid sequence encoding a truncated cystic fibrosis transmembrane regulator (CFTR) protein that comprises amino acid residues encoded by at least exons 10-27 of a CFTR gene, and (a) a splice acceptor site that is immediately upstream of the first nucleic acid sequence; or (b) a second nucleic acid sequence that has at least 80% identity to a 3‘ untranslated region (3?UTR) of a gene different than the CFTR gene.

[0116] Embodiment 2. The recombinant nucleic acid sequence of embodiment 1, wherein the first nucleic acid sequence comprises exon 2 to exon 27 of the CFTR gene.

[0117] Embodiment 3. The recombinant nucleic acid sequence of embodiment 1 or 2, wherein exons of the first nucleic acid sequence are codon optimized for expression in human.

[0118] Embodiment 4. The recombinant nucleic acid sequence of any one of embodiments 1-3, wherein the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 36.

[0119] Embodiment 5. The recombinant nucleic acid sequence of any one of embodiments 1-4, wherein the first nucleic acid sequence has at least 80% identity to SEQ ID NO: 36.

[0120] Embodiment 6. The recombinant nucleic acid sequence of any one of embodiments 1-3, wherein the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 37.

[0121] Embodiment 7. The recombinant nucleic acid sequence of embodiment 6, wherein the first nucleic acid sequence has at least 80% identity to SEQ ID NO: 37.

[0122] Embodiment 8. The recombinant nucleic acid sequence of any one of embodiments 1-7, wherein the first nucleic acid sequence is free of introns.

[0123] Embodiment 9. The recombinant nucleic acid sequence of any one of embodiments 1-7, wherein the first nucleic acid sequence comprises at least one intron.

[0124] Embodiment 10. The recombinant nucleic acid sequence of embodiment 9, wherein the at least one intron comprises an intron that is exogenous to the CFTR gene.

[0125] Embodiment 11. The recombinant nucleic acid sequence of embodiment 9, wherein the at least one intron comprises a synthetic / chimeric intron.

[0126] Embodiment 12. The recombinant nucleic acid sequence of any one of embodiments 9-10, wherein each of the at least one intron is less than 600 nucleotides in length.

[0127] Embodiment 13. The recombinant nucleic acid sequence of any one of embodiments 1-12, wherein the splice acceptor site is exogenous to a 5?end of the first nucleic acid sequence.

[0128] Embodiment 14. The recombinant nucleic acid sequence of any one of embodiments 1-12, wherein the splice acceptor site comprises a sequence identical to the sequence of an endogenous splice acceptor site immediately upstream of exon 11 of the CFTR gene.

[0129] Embodiment 15. The recombinant nucleic acid sequence of any one of embodiments 1-12, wherein the splice acceptor site comprises a nucleic acid sequence chosen from the group consisting of the sequences set forth in SEQ ID NOs: 1-3.

[0130] Embodiment 16. The recombinant nucleic acid sequence of any one of embodiments 1-15, wherein the recombinant nucleic acid sequence is configured to be inserted into a target site of an endogenous CFTR gene in a genome of a cell.

[0131] Embodiment 17. The recombinant nucleic acid sequence of embodiment 16, wherein the target site of the endogenous CFTR gene is within an intron of the endogenous CFTR gene.

[0132] Embodiment 18. The recombinant nucleic acid sequence of embodiment 17, wherein the intron is upstream of exon 10 of the endogenous CFTR gene.

[0133] Embodiment 19. The recombinant nucleic acid sequence of any one of embodiments 16-18, wherein the target site of the endogenous CFTR gene is within intron 1 of the endogenous CFTR gene.

[0134] Embodiment 20. The recombinant nucleic acid sequence of any one of embodiments 16-19, wherein the target site of the endogenous CFTR gene is about 325 nucleotides downstream of exon 1 of the endogenous CFTR gene.

[0135] Embodiment 21. The recombinant nucleic acid sequence of any one of embodiments 16-19, wherein the target site of the endogenous CFTR gene is about 79 nucleotides downstream of exon 1 of the endogenous CFTR gene.

[0136] Embodiment 22. The recombinant nucleic acid sequence of any one of embodiments 16-19, wherein the target site of the endogenous CFTR gene is located within a region from hg38 chr7: 117,480, 148 to hg38 chr7: 117,498,307.

[0137] Embodiment 23. The recombinant nucleic acid sequence of any one of embodiments 16-22, further comprising a 5’ homology arm upstream of the first nucleic acid sequence and a 3’ homology arm downstream of the first nucleic acid sequence.

[0138] Embodiment 24. The recombinant nucleic acid sequence of embodiment 23, wherein the 5 ’ homology' arm is homologous to a nucleic acid sequence upstream of the target site of the endogenous CFTR gene and the 3 ’ homology arm is homologous to a nucleic acid sequence downstream of the target site of the endogenous CFTR gene.

[0139] Embodiment 25. The recombinant nucleic acid sequence of any one of embodiments 23-24, wherein the 5’ homology' arm and the 3’ homology arm comprise nucleic acid sequences that are at least 80% identical to nucleic acid sequences selected from the group consisting of: (a) SEQ ID NO: 38 and SEQ ID NO: 39, respectively; (b) SEQ ID NO: 38 and SEQ ID NO: 41, respectively; (c) SEQ ID NO: 38 and SEQ ID NO: 43, respectively; (d) SEQ ID NO: 40 and SEQ ID NO: 39, respectively; (e) SEQ ID NO: 40 and SEQ ID NO: 41, respectively; (f) SEQ ID NO: 40 and SEQ ID NO: 43, respectively; (g) SEQ ID NO: 42 andSEQ ID NO: 39, respectively; (h) SEQ ID NO: 42 and SEQ ID NO: 41, respectively; and (i) SEQ ID NO: 42 and SEQ ID NO: 43, respectively.

[0140] Embodiment 26. The recombinant nucleic acid sequence of any one of embodiments 1-25, wherein the second nucleic acid sequence has at least 90% identity to a 3’ untranslated region (3’ UTR) of a gene different than the CFTR gene.

[0141] Embodiment 27. The recombinant nucleic acid sequence of embodiment 26, wherein the 3’ UTR of a gene is a 3’ UTR of a eukary otic gene.

[0142] Embodiment 28. The recombinant nucleic acid sequence of embodiments 26 or 27, wherein the 3’ UTR of a gene is a 3’ UTR of a human gene.

[0143] Embodiment 29. The recombinant nucleic acid sequence of embodiments 26, wherein the 3’ UTR of a gene is a 3’ UTR of a viral gene.

[0144] Embodiment 30. The recombinant nucleic acid sequence of embodiment 29, wherein the 3’ UTR of a gene is a simian virus (SV) 40 gene.

[0145] Embodiment 31. The recombinant nucleic acid sequence of embodiment 28, wherein the human gene is selected from the group consisting of genes listed in Table 1.

[0146] Embodiment 32. The recombinant nucleic acid sequence of embodiment 31, wherein the 3’ UTR is selected from the group of genes consisting of RPS27, FLII, TRAP, RPL36, ZDHHC12, NDUFB6, IDH3B, PMC5, NDUFB10. APTR. GRN, RPS21, TMEM208. HSD17B10, PSMB7, GPS2, ATRAID, SNRPG, HSP90AB1, PFDN2, LAMTOR4, FKBP2, EEF1G, RPS11, NDUFB4, FBL, RPS16, LGALS1, NDUFB1, PTTG1, ATP5PD, SQQR, PFN1, TMSB10, SOD1, COPS3, ATP6V0B, RPN2, ENO1, GSS, and CCT2.

[0147] Embodiment 33. The recombinant nucleic acid sequence of embodiment 32, wherein the 3’ UTR is selected from the group of genes consisting of NDUFB6, HSD17B10, PTTG1, and SOD 1.

[0148] Embodiment 34. The recombinant nucleic acid sequence of any one of embodiment 1-33, wherein the recombinant nucleic acid sequence is configured to fit within a vector.

[0149] Embodiment 35. The recombinant nucleic acid sequence of embodiment 34, wherein the vector comprises a linear nucleic acid sequence, a plasmid, a viral particle or a synthetic nanoparticle.

[0150] Embodiment 36. The recombinant nucleic acid sequence of embodiment 35, wherein the viral particle comprises an adeno-associated viral (AAV) particle.

[0151] Embodiment 37. A recombinant nucleic acid sequence comprising: a first nucleic acid sequence encoding a truncated cystic fibrosis transmembrane regulator (CFTR) protein, wherein the first nucleic acid sequence comprises, between two exons of the first nucleic acid sequence, an intron that is exogenous to a CFTR gene.

[0152] Embodiment 38. The recombinant nucleic acid sequence of embodiment 37, wherein the truncated CFTR protein comprises amino acid residues encoded by at least exons 10-27 of the CFTR gene.

[0153] Embodiment 39. The recombinant nucleic acid sequence of embodiment 37 or 38, wherein the first nucleic acid sequence comprises exon 10 to exon 27 of the CFTR gene.

[0154] Embodiment 40. The recombinant nucleic acid sequence of embodiment 37 or 38, wherein the first nucleic acid sequence comprises exon 2 to exon 27 of the CFTR gene.

[0155] Embodiment 41. The recombinant nucleic acid sequence of any one of embodiments 37-40, wherein the first nucleic acid sequence is codon optimized for expression in human.

[0156] Embodiment 42. The recombinant nucleic acid sequence of any one of embodiments 37-41, wherein the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 36.

[0157] Embodiment 43. The recombinant nucleic acid sequence of any one of embodiments 37-42, wherein the first nucleic acid sequence has at least 80% identity to SEQ ID NO: 36.

[0158] Embodiment 44. The recombinant nucleic acid sequence of any one of embodiments 37-41, wherein the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 37.

[0159] Embodiment 45. The recombinant nucleic acid sequence of embodiment 44, wherein the first nucleic acid sequence has at least 80% identity to SEQ ID NO: 37.

[0160] Embodiment 46. The recombinant nucleic acid sequence of any one of embodiments 37-45, wherein the intron that is exogenous to the CFTR gene comprises a synthetic / chimeric intron.

[0161] Embodiment 47. The recombinant nucleic acid sequence of any one of embodiments 37-46, wherein the intron is less than 600 nucleotides in length.

[0162] Embodiment 48. The recombinant nucleic acid sequence of any one of embodiments 37-47, further comprising a splice acceptor site immediately upstream of the first nucleic acid sequence.

[0163] Embodiment 49. The recombinant nucleic acid sequence of embodiment 48, wherein the splice acceptor site is exogenous to a 5’ end of the first nucleic acid sequence.

[0164] Embodiment 50. The recombinant nucleic acid sequence of embodiment 48 or 49, wherein the splice acceptor site comprises a sequence identical to the sequence of an endogenous splice acceptor site immediately upstream of exon 11 of the CFTR gene.

[0165] Embodiment 51. The recombinant nucleic acid sequence of any one of embodiments 48 or 49, wherein the splice acceptor site comprises a nucleic acid sequence chosen from the group consisting of the sequences set forth in SEQ ID NOs: 1-3.

[0166] Embodiment 52. The recombinant nucleic acid sequence of any one of embodiments 37-51. wherein the recombinant nucleic acid sequence is configured to be inserted into a target site of an endogenous CFTR gene in a genome of a cell.

[0167] Embodiment 53. The recombinant nucleic acid sequence of embodiment 52, wherein the target site of the endogenous CFTR gene is within an intron of the endogenous CFTR gene.

[0168] Embodiment 54. The recombinant nucleic acid sequence of embodiment 52, wherein the target site of the endogenous CFTR gene is within intron 1 of the endogenous CFTR gene.

[0169] Embodiment 55. The recombinant nucleic acid sequence of any one of embodiments 52-54. wherein the target site of the endogenous CFTR gene is about 325 nucleotides downstream of exon 1 of the endogenous CFTR gene.

[0170] Embodiment 56. The recombinant nucleic acid sequence of any one of embodiments 52-54, wherein the target site of the endogenous CFTR gene is about 79 nucleotides downstream of exon 1 of the endogenous CFTR gene.

[0171] Embodiment 57. The recombinant nucleic acid sequence of any one of embodiments 52-56, wherein the target site of the endogenous CFTR gene is located within a region from hg38 chr7: 117,480,I48 to hg38 chr7: 117,498,307.

[0172] Embodiment 58. The recombinant nucleic acid sequence of any one of embodiments 37-57, further comprising a 5’ homology arm upstream of the first nucleic acid sequence and a 3’ homology arm downstream of the first nucleic acid sequence.

[0173] Embodiment 59. The recombinant nucleic acid sequence of embodiment 58, wherein the 5’ homolog}- arm is homologous to a nucleic acid sequence upstream of the target site of the CFTR gene and the 3’ homology arm is homologous to a nucleic acid sequence downstream of the target site of the CFTR gene.

[0174] Embodiment 60. The recombinant nucleic acid sequence of any one of embodiments 58-59, wherein the 5’ homology arm and the 3' homology arm comprise nucleic acid sequences that are at least 80% identical to nucleic acid sequences selected from the group consisting of: (a) SEQ ID NO: 38 and SEQ ID NO: 39, respectively; (b) SEQ ID NO: 38 and SEQ ID NO: 41, respectively; (c) SEQ ID NO: 38 and SEQ ID NO: 43, respectively; (d) SEQ ID NO: 40 and SEQ ID NO: 39, respectively; (e) SEQ ID NO: 40 and SEQ ID NO: 41, respectively; (f) SEQ ID NO: 40 and SEQ ID NO: 43, respectively; (g) SEQ ID NO: 42 andSEQ ID NO: 39, respectively; (h) SEQ ID NO: 42 and SEQ ID NO: 41, respectively; and (i) SEQ ID NO: 42 and SEQ ID NO: 43, respectively.

[0175] Embodiment 61. The recombinant nucleic acid sequence of any one of embodiments 37-60, further comprising a second nucleic acid sequence comprising a 3’ untranslated region (3’ UTR).

[0176] Embodiment 62. The recombinant nucleic acid sequence of embodiment 61, wherein the second nucleic acid sequence has at least 80% identity to a 3’ UTR of a gene different than the CFTR gene.

[0177] Embodiment 63. The recombinant nucleic acid sequence of embodiment 61, wherein the second nucleic acid sequence has at least 90% identity to a 3’ UTR of a gene different than the CFTR gene.

[0178] Embodiment 64. The recombinant nucleic acid sequence of embodiment 62 or 63, wherein the 3’ UTR of a gene is a 3' UTR of a eukaryotic gene.

[0179] Embodiment 65. The recombinant nucleic acid sequence of embodiments 64, wherein the 3’ UTR of a gene is a 3’ UTR of a human gene.

[0180] Embodiment 66. The recombinant nucleic acid sequence of embodiments 62 or 63, wherein the 3’ UTR of a gene is a 3‘ UTR of a viral gene.

[0181] Embodiment 67. The recombinant nucleic acid sequence of embodiment 66, wherein the 3’ UTR of a gene is a simian virus (SV) 40 gene.

[0182] Embodiment 68. The recombinant nucleic acid sequence of embodiment 65, wherein the human gene is selected from the group consisting of the genes listed in Table 1.

[0183] Embodiment 69. The recombinant nucleic acid sequence of embodiment 68, wherein the 3’ UTR is selected from the group of genes consisting of RPS27, FLII, TRAP, RPL36, ZDHHC12, NDUFB6, IDH3B, PMC5, NDUFB10, APTR, GRN RPS21, TMEM208, HSD17B10, PSMB7, GPS2, ATRAID, SNRPG, HSP90AB1, PFDN2, LAMTOR4, FKBP2, EEF1G, RPS11, NDUFB4, FBE, RPS16, LGALS1, NDUFB1, PTTG1, ATP5PD, SQQR, PFN1, TMSB10. SOD1. COPS3, ATP6V0B, RPN2, ENO1, GSS, and CCT2.

[0184] Embodiment 70. The recombinant nucleic acid sequence of embodiment 69, wherein the 3’ UTR is selected from the group of genes consisting of NDUFB6, HSD17B10, PTTG1, and SOD 1.

[0185] Embodiment 71. The recombinant nucleic acid sequence of any one of embodiment 37-70, wherein the recombinant nucleic acid sequence is configured to fit within a vector.

[0186] Embodiment 72. The recombinant nucleic acid sequence of embodiment 71, wherein the vector comprises a linear nucleic acid sequence, a plasmid, a viral particle or a synthetic nanoparticle.

[0187] Embodiment 73. The recombinant nucleic acid sequence of embodiment 72, wherein the viral particle comprises an adeno-associated viral (AAV) particle.

[0188] Embodiment 74. A recombinant nucleic acid sequence comprising: (a) a first nucleic acid sequence encoding a truncated cystic fibrosis transmembrane regulator (CFTR) protein that comprises amino acid residues encoded by at least exons 10-27 of a CFTR gene: (b) a 5' homology arm upstream of the first nucleic acid sequence, and (c) a 3' homology arm dow nstream of the first nucleic acid sequence; wherein the 5' homology arm is homologous to a sequence that is upstream of a genomic site within an intron of an endogenous CFTR gene in a genome of a cell, wherein the 3' homology arm is homologous to a sequence that is downstream of the genomic site, and wherein the intron of the endogenous CFTR gene is upstream of exon 10 of the endogenous CFTR gene in a genome of a cell.

[0189] Embodiment 75. The recombinant nucleic acid sequence of embodiment 74, wherein the first nucleic acid sequence comprises exon 2 to exon 27 of the CFTR gene.

[0190] Embodiment 76. The recombinant nucleic acid sequence of embodiment 74 or 75, w herein exons of the first nucleic acid sequence are codon optimized for expression in human.

[0191] Embodiment 77. The recombinant nucleic acid sequence of any one of embodiments 74-76, wherein the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 36.

[0192] Embodiment 78. The recombinant nucleic acid sequence of any one of embodiments 74-76, wherein the first nucleic acid sequence has at least 80% identity to SEQ ID NO: 36.

[0193] Embodiment 79. The recombinant nucleic acid sequence of any one of embodiments 74-76, wherein the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 37.

[0194] Embodiment 80. The recombinant nucleic acid sequence of embodiment 79, wherein the first nucleic acid sequence has at least 80% identity to SEQ ID NO: 37.

[0195] Embodiment 81. The recombinant nucleic acid sequence of any one of embodiments 74-80, w herein the first nucleic acid sequence is free of introns.

[0196] Embodiment 82. The recombinant nucleic acid sequence of any one of embodiments 74-80, wherein the first nucleic acid sequence comprises at least one intron.

[0197] Embodiment 83. The recombinant nucleic acid sequence of embodiment 82, wherein the at least one intron comprises an intron that is exogenous to the CFTR gene.

[0198] Embodiment 84. The recombinant nucleic acid sequence of embodiment 83, wherein the at least one intron comprises a synthetic / chimeric intron.

[0199] Embodiment 85. The recombinant nucleic acid sequence of any one of embodiments 82-84, wherein each of the at least one intron is less than 600 nucleotides in length.

[0200] Embodiment 86. The recombinant nucleic acid sequence of any one of embodiments 74-85. further comprising a splice acceptor site.

[0201] Embodiment 87. The recombinant nucleic acid sequence of embodiment 86, wherein the splice acceptor site is exogenous to a 5 ’ end of the first nucleic acid sequence.

[0202] Embodiment 88. The recombinant nucleic acid sequence of embodiment 86 or 87, wherein the splice acceptor site comprises a sequence identical to the sequence of an endogenous splice acceptor site immediately upstream of exon 11 of the CFTR gene.

[0203] Embodiment 89. The recombinant nucleic acid sequence of any one of embodiments 86 or 87, wherein the splice acceptor site comprises a nucleic acid sequence chosen from the group consisting of the sequences set forth in SEQ ID NOs: 1-3.

[0204] Embodiment 90. The recombinant nucleic acid sequence of any one of embodiments 74-89, wherein the recombinant nucleic acid sequence is configured to be inserted into the genomic site of the endogenous CFTR gene.

[0205] Embodiment 91. The recombinant nucleic acid sequence of any one of embodiments 74-90, wherein the genomic site of the endogenous CFTR gene is within intron 1 of the endogenous CFTR gene.

[0206] Embodiment 92. The recombinant nucleic acid sequence of embodiment 91, wherein the genomic site of the endogenous CFTR gene is about 325 nucleotides downstream of exon 1 of the endogenous CFTR gene.

[0207] Embodiment 93. The recombinant nucleic acid sequence of embodiment 91, wherein the genomic site of the endogenous CFTR gene is about 79 nucleotides downstream of exon 1 of the endogenous CFTR gene.

[0208] Embodiment 94. The recombinant nucleic acid sequence of any one of embodiments 74-93. wherein the genomic site of the endogenous CFTR gene is located within a region from hg38 chr7: l 17,480,148 to hg38 chr7: 117,498,307.

[0209] Embodiment 95. The recombinant nucleic acid sequence of any one of embodiments 74-94, wherein the 5’ homology arm and the 3' homology arm comprise nucleic acid sequences that are at least 80% identical to nucleic acid sequences selected from the group consisting of: (a) SEQ ID NO: 38 and SEQ ID NO: 39, respectively; (b) SEQ ID NO: 38 and SEQ ID NO: 41, respectively; (c) SEQ ID NO: 38 and SEQ ID NO: 43, respectively; (d) SEQ ID NO: 40 and SEQ ID NO: 39, respectively; (e) SEQ ID NO: 40 and SEQ ID NO: 41, respectively; (I) SEQ ID NO: 40 and SEQ ID NO: 43, respectively; (g) SEQ ID NO: 42 andSEQ ID NO: 39, respectively; (h) SEQ ID NO: 42 and SEQ ID NO: 41, respectively; and (i) SEQ ID NO: 42 and SEQ ID NO: 43, respectively.

[0210] Embodiment 96. The recombinant nucleic acid sequence of any one of embodiments 74-95, further comprising a second nucleic acid sequence comprising a 3’ untranslated region (3’ UTR).

[0211] Embodiment 97. The recombinant nucleic acid sequence of embodiment 96, wherein the second nucleic acid sequence has at least 90% identity to a 3’ UTR of a eukaryotic gene different than the CFTR gene.

[0212] Embodiment 98. The recombinant nucleic acid sequence of embodiment 96 or 97, wherein the 3’ UTR of a gene is a 3’ UTR of a eukaryotic gene.

[0213] Embodiment 99. The recombinant nucleic acid sequence of embodiments 98, wherein the 3’ UTR of a gene is a 3‘ UTR of a human gene.

[0214] Embodiment 100. The recombinant nucleic acid sequence of embodiments 96 or 97, wherein the 3’ UTR of a gene is a 3’ UTR of a viral gene.

[0215] Embodiment 101. The recombinant nucleic acid sequence of embodiment 100, wherein the 3’ UTR of a gene is a simian virus (SV) 40 gene.

[0216] Embodiment 102. The recombinant nucleic acid sequence of embodiment 99, wherein the human gene is selected from the group consisting of the genes listed in Table 1.

[0217] Embodiment 103. The recombinant nucleic acid sequence of embodiment 102, wherein the 3’ UTR is selected from the group of genes consisting of RPS27, FEII, TRAP, RPL36, ZDHHC12, NDUFB6, IDH3B, PMC5, NDUFB10, APTR, GRN RPS21, TMEM208, HSD17B10, PSMB7, GPS2. ATRAID, SNRPG, HSP90AB1. PFDN2, LAMTOR4, FKBP2, EEF1G, RPS11, NDUFB4, FBL, RPS16, LGALS1, NDUFB1, PTTG1, ATP5PD, SQQR, PFN1, TMSB10, SOD1, COPS3, ATP6V0B, RPN2, ENO1, GSS, and CCT2.

[0218] Embodiment 104. The recombinant nucleic acid sequence of embodiment 103, wherein the 3’ UTR is selected from the group of genes consisting of NDUFB6, HSD17B10, PTTG1, and SOD 1.

[0219] Embodiment 105. The recombinant nucleic acid sequence of any one of embodiments 74-104, wherein the recombinant nucleic acid sequence is configured to fit within a vector.

[0220] Embodiment 106. The recombinant nucleic acid sequence of embodiment 105, wherein the vector comprises a linear nucleic acid sequence, a plasmid, a viral particle or a synthetic nanoparticle.

[0221] Embodiment 107. The recombinant nucleic acid sequence of embodiment 106, wherein the viral particle comprises an adeno-associated viral (AAV) particle.

[0222] Embodiment 108. A gene editing system, the gene editing system comprising: a) a first nucleic acid sequence encoding a truncated cystic fibrosis transmembrane regulator (CFTR) protein, and b) a targeting moiety or a nucleic acid sequence encoding the targeting moiety; wherein the targeting moiety directs insertion of the first nucleic acid sequence into a genomic site within an intron of an endogenous CFTR gene of a genome of a cell, and wherein the intron is upstream of exon 10 of the endogenous CFTR gene.

[0223] Embodiment 109. The gene editing system of embodiment 108, wherein the targeting moiety comprises a guide RNA (gRNA), a zinc finger nuclease, or a transcription activator-like (TAL) effector nuclease.

[0224] Embodiment 110. The gene editing system of embodiment 108 or 109, wherein the gene editing system further comprises a gene editing enzyme or a nucleic acid sequence encoding the gene editing enzyme.

[0225] Embodiment 111. The gene editing system of any one of embodiments 108-110, wherein the gene editing system results in integration of one or two copies of the first nucleic acid sequence.

[0226] Embodiment 112. The gene editing system of any one of embodiments 108-111, wherein the truncated CFTR protein comprises amino acid residues encoded by at least exons 10-27 of a CFTR gene.

[0227] Embodiment 113. The gene editing system of embodiment 108-112, wherein the first nucleic acid sequence comprises exon 2 to exon 27 of the CFTR gene.

[0228] Embodiment 114. The gene editing system of embodiment 113, wherein exons of the first nucleic acid sequence are codon optimized for expression in human.

[0229] Embodiment 115. The gene editing system of any one of embodiments 108-114, wherein the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 36.

[0230] Embodiment 116. The gene editing system of any one of embodiments 108-115, wherein the first nucleic acid sequence has at least 80% identity to SEQ ID NO: 36.

[0231] Embodiment 117. The gene editing system of any one of embodiments 108-114. wherein the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 37.

[0232] Embodiment 118. The gene editing system of embodiment 117, wherein the first nucleic acid sequence has at least 80% identity to SEQ ID NO: 37.

[0233] Embodiment 119. The gene editing system of any one of embodiments 108-118, wherein the first nucleic acid sequence is free of introns.

[0234] Embodiment 120. The gene editing system of any one of embodiments 108-118, wherein the first nucleic acid sequence comprises at least one intron.

[0235] Embodiment 121. The gene editing system of embodiment 120, wherein the at least one intron comprises an intron that is exogenous to the CFTR gene.

[0236] Embodiment 122. The gene editing system of embodiment 121, wherein the at least one intron comprises a synthetic / chimeric intron.

[0237] Embodiment 123. The gene editing system of any one of embodiments 120-122, wherein each of the at least one intron is less than 600 nucleotides in length.

[0238] Embodiment 124. The gene editing system of any one of embodiments 108-123, wherein the first nucleic acid sequence comprises a splice acceptor site.

[0239] Embodiment 125. The gene editing system of embodiment 124. wherein the splice acceptor site is exogenous to a 5’ end of the first nucleic acid sequence.

[0240] Embodiment 126. The gene editing system of embodiment 124 or 125, wherein the splice acceptor site comprises a sequence identical to the sequence of an endogenous splice acceptor site immediately upstream of exon 11 of the CFTR gene.

[0241] Embodiment 127. The gene editing system of any one of embodiments 124 or 125. wherein the splice acceptor site comprises a nucleic acid sequence chosen from the group consisting of the sequences set forth in SEQ ID NOs: 1-3.

[0242] Embodiment 128. The gene editing system of any one of embodiments 108-127, wherein the genomic site is within intron 1 of the endogenous CFTR gene.

[0243] Embodiment 129. The gene editing system of any one of embodiments 108-128, wherein the genomic site is about 325 nucleotides dow nstream of exon 1 of the endogenous CFTR gene.

[0244] Embodiment 130. The gene editing system of any one of embodiments 108-128, w herein the genomic site is about 79 nucleotides downstream of exon 1 of the endogenous CFTR gene.

[0245] Embodiment 131. The gene editing system of any one of embodiments 108-130, wherein the genomic site is located within a region from hg38 chr7: 117,480, 148 to hg38 chr7: 117,498,307.

[0246] Embodiment 132. The gene editing system of any one of embodiments 108-131, wherein the gene editing system comprises a gRNA and a class 2 CRISPR / Cas endonuclease.

[0247] Embodiment 133. The gene editing system of embodiment 132, wherein the gene editing system comprises a gRNA and a Cas9 enzyme.

[0248] Embodiment 134. The gene editing system of embodiment 132 or 133, wherein the gRNA comprises a sequence selected from the group consisting of the sequences provided in Table 3.

[0249] Embodiment 135. The gene editing system of any one of embodiments 108-134, wherein the first nucleic acid sequence comprises a 5’ homology arm upstream of the first nucleic acid sequence and a 3’ homology arm dow nstream of the first nucleic acid sequence.

[0250] Embodiment 136. The gene editing system of embodiment 135. wherein the 5' homology arm is homologous to a nucleic acid sequence upstream of the genomic site within the intron of the endogenous CFTR gene and the 3’ homology arm is homologous to a nucleic acid sequence dow nstream of the genomic site.

[0251] Embodiment 137. The gene editing system of embodiment 135 or 136, wherein the 5’ homology arm and the 3’ homology’ arm comprise nucleic acid sequences that are at least 80% identical to nucleic acid sequences selected from the group consisting of: (a) SEQ ID NO: 38 and SEQ ID NO: 39, respectively; (b) SEQ ID NO: 38 and SEQ ID NO: 41, respectively; (c) SEQ ID NO: 38 and SEQ ID NO: 43, respectively; (d) SEQ ID NO: 40 and SEQ ID NO: 39, respectively; (e) SEQ ID NO: 40 and SEQ ID NO: 41, respectively; (f) SEQ ID NO: 40 and SEQ ID NO: 43. respectively; (g) SEQ ID NO: 42 and SEQ ID NO: 39, respectively; (h) SEQ ID NO: 42 and SEQ ID NO: 41, respectively; and (i) SEQ ID NO: 42 and SEQ ID NO: 43, respectively.

[0252] Embodiment 138. The gene editing system of any one of embodiments 108-137, wherein the gene editing system further comprises a second nucleic acid sequence.

[0253] Embodiment 139. The gene editing system of embodiment 138, wherein the second nucleic acid sequence has at least 80% identity to a 3’ untranslated region (3’ UTR) of a gene different than the CFTR gene.

[0254] Embodiment 140. The gene editing system of embodiment 138. wherein the second nucleic acid sequence has at least 90% identity to a 3’ UTR of a gene different than the CFTR gene.

[0255] Embodiment 141. The gene editing system of embodiment 139 or 140, wherein the 3’ UTR of a gene is a 3’ UTR of a eukaryotic gene.

[0256] Embodiment 142. The gene editing system of embodiments 139-141. wherein the 3’ UTR of a gene is a 3’ UTR of a human gene.

[0257] Embodiment 143. The gene editing system of embodiments 139 or 140, wherein the 3’ UTR of a gene is a 3’ UTR of a viral gene.

[0258] Embodiment 144. The gene editing system of embodiment 143. wherein the 3’ UTR of a gene is a simian virus (SV) 40 gene.

[0259] Embodiment 145. The gene editing system of embodiment 142, wherein the human gene is selected from the group consisting of the genes listed in Table 1.

[0260] Embodiment 146. The gene editing system of embodiment 145, wherein the 3’ UTR is selected from the group of genes consisting of RPS27, FLII, TRAP, RPL36, ZDHHC12, NDUFB6, IDH3B, PMC5, NDUFB10, APTR, GRN RPS21, TMEM208, HSD17B10, PSMB7, GPS2, ATRAID. SNRPG, HSP90AB1, PFDN2, LAMTOR4, FKBP2, EEF1G, RPS 11, NDUFB4, FBL, RPS16. LGALS1, NDUFB1. PTTG1, ATP5PD. SQQR. PFN1, TMSB10. SOD1, COPS3, ATP6V0B, RPN2, ENO1, GSS, and CCT2.

[0261] Embodiment 147. The gene editing system of embodiment 146, wherein the 3’ UTR is selected from the group of genes consisting of NDUFB6, HSD17B10, PTTG1. and SOD1.

[0262] Embodiment 148. The gene editing system of any one of embodiments 108-147, wherein the gene editing system is configured to fit within one vector.

[0263] Embodiment 149. The gene editing system of any one of embodiments 108-148, wherein the gene editing system is configured to fit in two or more vectors.

[0264] Embodiment 150. The gene editing system of embodiment 147 or 149, wherein the vector comprises a linear nucleic acid sequence, a plasmid, a viral particle or a synthetic nanoparticle.

[0265] Embodiment 151. The gene editing system of embodiment 150, wherein the viral particle comprises an adeno-associated viral (AAV) particle.

[0266] Embodiment 152. A gene editing system, the gene editing system comprising: a) a recombinant nucleic acid sequence of any one of embodiments 1-107, and b) a targeting moiety or a nucleic acid sequence encoding the targeting moiety; wherein the targeting moiety directs insertion of the first nucleic acid sequence into a genomic site within an intron of an endogenous CFTR gene of a genome of a cell, and wherein the intron is upstream of exon 10 of the endogenous CFTR gene.

[0267] Embodiment 153. The gene editing system of embodiment 152, wherein the targeting moiety comprises a guide RNA (gRNA), a zinc finger nuclease, or a transcription activator-like (TAL) effector nuclease.

[0268] Embodiment 154. The gene editing system of embodiment 152 or 153, wherein the gene editing system further comprises a gene editing enzyme or a nucleic acid sequence encoding the gene editing enzyme.

[0269] Embodiment 155. The gene editing system of embodiments 152-154, wherein the gene editing system results in integration of one or two copies of the first nucleic acid sequence.

[0270] Embodiment 156. The gene editing system of any one of embodiments 152-155, wherein the genomic site is within intron 1 of the endogenous CFTR gene.

[0271] Embodiment 157. The gene editing system of any one of embodiments 152-156, wherein the genomic site is about 325 nucleotides downstream of exon 1 of the endogenous CFTR gene.

[0272] Embodiment 158. The gene editing system of any one of embodiments 152-156, wherein the genomic site is about 79 nucleotides downstream of exon 1 of the endogenous CFTR gene.

[0273] Embodiment 159. The gene editing system of any one of embodiments 152-158, wherein the genomic site is located within a region from hg38 chr7: 117,480,148 to hg38 chr7: 117,498,307.

[0274] Embodiment 160. The gene editing system of any one of embodiments 152-159, wherein the targeting moiety is a CRISPR / Cas nickase.

[0275] Embodiment 161. The gene editing system of embodiment 160, wherein the gene editing system further comprises a recombinase.

[0276] Embodiment 162. The gene editing system of embodiment 161. wherein the recombinase is a domain of the CRISPR / Cas nickase.

[0277] Embodiment 163. The gene editing system of embodiment 161, wherein the recombinase is separate from the CRISPR / Cas nickase.

[0278] Embodiment 164. The gene editing system of any one of embodiments 160-163, wherein the gene editing system further comprises a reverse transcriptase.

[0279] Embodiment 165. The gene editing system of any one of embodiments 160-164, wherein the recombinant nucleic acid sequence is flanked by a sequence recognized by a recombinase.

[0280] Embodiment 166. The gene editing system of any one of embodiments 160-165, wherein the gene editing system further comprises a gRNA encoding a sequence that is recognized by a recombinase.

[0281] Embodiment 167. The gene editing system of any one of embodiments 166, wherein the sequence that is recognized by a recombinase is integrated into the genomic site.

[0282] Embodiment 168. The gene editing system of any one of embodiments 161-167, wherein the recombinase directs the insertion of the recombinant nucleic acid sequence into the genomic site.

[0283] Embodiment 169. The gene editing system of any one of embodiments 152-159, wherein the gene editing system comprises a gRNA and a class 2 CRISPR / Cas endonuclease.

[0284] Embodiment 170. The gene editing system of embodiment 169, wherein the gene editing system comprises a gRNA and a Cas9 enzy me.

[0285] Embodiment 171. The gene editing system of embodiment 169 or 170, wherein the gRNA comprises a sequence selected from the group consisting of the sequences provided in Table 3.

[0286] Embodiment 172. The gene editing system of any one of embodiments 152-171, wherein the gene editing system is configured to fit within one vector.

[0287] Embodiment 173. The gene editing system of any one of embodiments 152-171, wherein the gene editing system is configured to fit in two or more vectors.

[0288] Embodiment 174. The gene editing system of embodiment 172 or 173, wherein the vector comprises a linear nucleic acid sequence, a plasmid, a viral particle or a synthetic nanoparticle.

[0289] Embodiment 175. The gene editing system of embodiment 174, wherein the viral particle comprises an adeno-associated viral (AAV) particle.

[0290] Embodiment 176. A pharmaceutical composition, the pharmaceutical composition comprising: a) a pharmaceutically acceptable excipient or carrier; and b) the recombinant nucleic acid sequence of any one of embodiments 1-107 or the gene editing system of any one of embodiments 108-175.

[0291] Embodiment 177. The pharmaceutical composition of embodiment 176, wherein the pharmaceutical composition is formulated for intranasal administration, intratracheal administration, or intravenous injection.

[0292] Embodiment 178. The pharmaceutical composition of embodiment 176 or 177, wherein the pharmaceutical composition is formulated for pulmonary administration.

[0293] Embodiment 179. The pharmaceutical composition of any one of embodiments 176- 178, wherein the pharmaceutical composition further comprises a second therapeutic agent.

[0294] Embodiment 180. The pharmaceutical composition of embodiment 179, wherein the second therapeutic agent comprises a modulator of CFTR protein that potentiates chloride conductance of CFTR protein in the cells.

[0295] Embodiment 181. The pharmaceutical composition of embodiment 179 or 180. wherein 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.

[0296] Embodiment 182. The pharmaceutical composition of any one of embodiments 179- 181, wherein the second therapeutic agent comprises a mucolytic agent, optionally wherein the mucolytic agent is selected from the group consisting of: acetylcysteine, ambroxol, bromhexine, carbocisteine, erdosteine, mecysteine, domase alfa, hypertonic saline, and mannitol.

[0297] Embodiment 183. The pharmaceutical composition of any one of embodiments 179-182, wherein the second therapeutic agent comprises a bronchodilator, optionally wherein the bronchodilator is albuterol.

[0298] Embodiment 184. The pharmaceutical composition of any one of embodiments 179-183, wherein the second therapeutic agent comprises an immunosuppressive agent.

[0299] Embodiment 185. The pharmaceutical composition of embodiment 184, wherein the immunosuppressive agent is a corticosteroid selected from the group consisting of beclomethasone, budesonide, budesonide, formoterol. ciclesonide, fluticasone, salmeterol, umeclidinium, vilanterol, mometasone, or mometasone. prednisone, and methylprednisolone, and pharmaceutically acceptable salts thereof.

[0300] Embodiment 186. The pharmaceutical composition of embodiment 184, wherein the immunosuppressive agent is a non-steroidal immunosuppressive agent selected from the group consisting of polyclonal anti-lymphocyte antibodies, monoclonal anti-lymphocyte antibodies, interleukin-2 (IL-2) receptor antagonists, calcineurin inhibitors, cell cycle inhibitors, mammalian target of rapamycin (mTOR) inhibitors, methotrexate, cyclophosphamide, anthracy cline, and taxane.

[0301] Embodiment 187. A vector comprising the recombinant nucleic acid sequence of any one of embodiments 1-107 or the gene editing system of any one of embodiments 108-175.

[0302] Embodiment 188. The vector of embodiment 187, wherein the vector is a viral vector, optionally a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno- associated viral vector, or a herpes simplex viral vector.

[0303] Embodiment 189. The vector of embodiment 187. wherein the vector is a non- viral vector, optionally a lipid nanoparticle.

[0304] Embodiment 190. A virus comprising the recombinant nucleic acid sequence of any one of embodiments 1-107 or the gene editing system of any one of embodiments 108-175.

[0305] Embodiment 191. The virus of embodiment 190, wherein the virus is an adeno- associated viral (AAV) vector, optionally a retroviral vector, a lentiviral vector, an adenoviral vector, or a herpes simplex viral vector.

[0306] Embodiment 192. A cell comprising the recombinant nucleic acid sequence of any one of embodiments 1-107 or the gene editing system of any one of embodiments 108-175.

[0307] Embodiment 193. The cell of embodiment 192, wherein the cell is a stem cell.

[0308] Embodiment 194. The cell of embodiment 192 or 193, wherein the cell is a human cell.

[0309] Embodiment 195. A kit comprising: a) the recombinant nucleic acid sequence of any one of embodiments 1-107, the gene editing system of any one of embodiments 108-175, or thepharmaceutical composition of any one of embodiments 176-186; and b) instructions for use of the recombinant nucleic acid sequence, the gene editing system, or the pharmaceutical composition.

[0310] Embodiment 196. A method for modifying a cell, the method comprising contacting an agent or a vector encoding the agent to the cell, wherein the agent modifies a genome of the cell, thereby inserting an exogenous nucleic acid sequence into a genomic site of an intron of an endogenous cystic fibrosis transmembrane regulator (CFTR) gene of the cell, and wherein the intron is upstream of exon 10 of the endogenous CFTR gene of the cell.

[0311] Embodiment 197. The method of embodiment 196, wherein the exogenous nucleic acid sequence comprises a recombinant nucleic acid sequence encoding a truncated CFTR protein that is encoded by at least exons 10-27 of the CFTR gene.

[0312] Embodiment 198. The method of embodiment 196 or 197, wherein one or two copies of the exogenous nucleic acid are inserted into the genome of the cell.

[0313] Embodiment 199. The method of any one of embodiments 196-197, wherein expression of CFTR rnRNA or CFTR protein is increased by 50%, 60%, 70%, 80%, 90%, 100% or more in the cell compared to an otherwise same cell not receiving insertion of the exogenous nucleic acid sequence.

[0314] Embodiment 200. The method of any one of embodiments 196-199, wherein the cell exhibits a 50%, 60%, 70%, 80%, 90%, 100% or more increase in CFTR activity compared to an otherwise same cell not receiving insertion of the exogenous nucleic acid sequence.

[0315] Embodiment 201. The method of embodiment 200, wherein the CFTR activity comprises CFTR-mediated chloride channel conductivity measured by electrophysiolog)’.

[0316] Embodiment 202. The method of any one of embodiments 197-201, wherein the recombinant nucleic acid sequence comprises a first nucleic acid sequence which comprises exon 2 to exon 27 of the CFTR gene.

[0317] Embodiment 203. The method of embodiment 202, wherein exons of the first nucleic acid sequence are codon optimized for expression in human.

[0318] Embodiment 204. The method of any one of embodiments 197-203, wherein the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 36.

[0319] Embodiment 205. The method of any one of embodiments 197-204, wherein the first nucleic acid sequence has at least 80% identity to SEQ ID NO: 36.

[0320] Embodiment 206. The method of any one of embodiments 197-203, wherein the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 37.

[0321] Embodiment 207. The method of embodiment 206, wherein the first nucleic acid sequence has at least 80% identity to SEQ ID NO: 37.

[0322] Embodiment 208. The method of any one of embodiments 197-207, wherein the first nucleic acid sequence is free of introns.

[0323] Embodiment 209. The method of any one of embodiments 197-207, wherein the first nucleic acid sequence comprises at least one intron.

[0324] Embodiment 210. The method of embodiment 209, wherein the at least one intron comprises an intron that is exogenous to the CFTR gene.

[0325] Embodiment 211. The method of embodiment 210, wherein the at least one intron comprises a synthetic / chimeric intron.

[0326] Embodiment 212. The method of any one of embodiments 209-211, wherein each of the at least one intron is less than 600 nucleotides in length.

[0327] Embodiment 213. The method of any one of embodiments 197-212, wherein the first nucleic acid sequence of the recombinant nucleic acid sequence comprises a splice acceptor site that is exogenous to a 5' end of the first nucleic acid sequence.

[0328] Embodiment 214. The method of any one of embodiment 213, wherein the splice acceptor site comprises a sequence identical to the sequence of an endogenous splice acceptor site immediately upstream of exon 11 of the CFTR gene.

[0329] Embodiment 215. The method of any one of embodiment 213, wherein the splice acceptor site comprises a nucleic acid sequence chosen from the group consisting of the sequences set forth in SEQ ID NOs: 1-3.

[0330] Embodiment 216. The method of any one of embodiments 196-215, wherein the genomic site of the intron of the endogenous CFTR gene is upstream of exon 10 of the endogenous CFTR gene.

[0331] Embodiment 217. The method of any one of embodiments 196-216, wherein the genomic site of the intron of the endogenous CFTR gene is within intron 1 of the endogenous CFTR gene.

[0332] Embodiment 218. The method of any one of embodiments 196-217, wherein the genomic site is about 325 nucleotides downstream of exon 1 of the endogenous CFTR gene.

[0333] Embodiment 219. The method of any one of embodiments 196-217, wherein the genomic site is about 79 nucleotides dow nstream of exon 1 of the endogenous CFTR gene.

[0334] Embodiment 220. The method of any one of embodiments 196-219, wherein the genomic site of the endogenous CFTR gene is located within a region from hg38 chr7: 117,480,148 to hg38 chr7: 117,498,307.

[0335] Embodiment 221. The method of any one of embodiments 196-220, wherein the method comprises contacting the cell with a CRISPR / Cas nickase.-Tl-

[0336] Embodiment 222. The method of embodiment 221, wherein the method further comprises contacting the cell with a recombinase.

[0337] Embodiment 223. The method of embodiment 222, wherein the recombinase is a domain of the CRISPR / Cas nickase.

[0338] Embodiment 224. The method of embodiment 222, wherein the recombinase is separate from the CRISPR / Cas nickase.

[0339] Embodiment 225. The method of any one of embodiments 221-224, wherein the method further comprises contacting the cell with a reverse transcriptase.

[0340] Embodiment 226. The method of any one of embodiments 221-225, wherein the exogenous nucleic acid sequence is flanked by a sequence recognized by a recombinase.

[0341] Embodiment 227. The method of any one of embodiments 221-226, wherein the method further comprises contacting the cell with a gRNA encoding a sequence that is recognized by a recombinase.

[0342] Embodiment 228. The method of any one of embodiments 227. wherein the sequence that is recognized by a recombinase is integrated into the genomic site.

[0343] Embodiment 229. The method of any one of embodiments 222-228, wherein the recombinase directs the insertion of the recombinant nucleic acid sequence into the genomic site.

[0344] Embodiment 230. The method of any one of embodiments 197-220, wherein the recombinant nucleic acid sequence further comprises a 5’ homology arm upstream of the first nucleic acid sequence and a 3’ homology arm dow nstream of the first nucleic acid sequence.

[0345] Embodiment 231. The method of embodiment 230, wherein the 5?homology arm is homologous to a nucleic acid sequence upstream of the genomic site of the endogenous CFTR gene and the 3’ homology arm is homologous to a nucleic acid sequence downstream of the genomic site of the endogenous CFTR gene.

[0346] Embodiment 232. The method of embodiment 230 or 231, wherein the 5 ' homology arm and the 3’ homology arm comprise nucleic acid sequences that are at least 80% identical to nucleic acid sequences selected from the group consisting of: (a) SEQ ID NO: 38 and SEQ ID NO: 39, respectively; (b) SEQ ID NO: 38 and SEQ ID NO: 41, respectively; (c) SEQ ID NO: 38 and SEQ ID NO: 43, respectively; (d) SEQ ID NO: 40 and SEQ ID NO: 39, respectively; (e) SEQ ID NO: 40 and SEQ ID NO: 41, respectively; (I) SEQ ID NO: 40 and SEQ ID NO: 43, respectively; (g) SEQ ID NO: 42 and SEQ ID NO: 39, respectively; (h) SEQ ID NO: 42 and SEQ ID NO: 41, respectively; and (i) SEQ ID NO: 42 and SEQ ID NO: 43, respectively.

[0347] Embodiment 233. The method of any one of embodiments 197-232, wherein the recombinant nucleic acid sequence further comprises a second nucleic acid sequence has at least 80% identity to a 3’ untranslated region (3’ UTR) of a gene different than the CFTR gene.

[0348] Embodiment 234. The method of embodiment 233, wherein the second nucleic acid sequence has at least 90% identity to 3’ UTR of a gene different than the CFTR gene.

[0349] Embodiment 235. The method of embodiment 233 or 234, wherein the 3’ UTR of a gene is a 3’ UTR of a eukary otic gene.

[0350] Embodiment 236. The method of embodiment 235, wherein the 3’ UTR of a gene is a 3 ’ UTR of a human gene.

[0351] Embodiment 237. The method of embodiment 233 or 234, wherein the 3’ UTR of a gene is a 3’ UTR of a viral gene.

[0352] Embodiment 238. The method of embodiment 237, wherein the 3’ UTR of a gene is a simian virus (SV) 40 gene.

[0353] Embodiment 239. The method of embodiment 236, wherein the human gene is selected from the group consisting of the genes listed in Table 1.

[0354] Embodiment 240. The method of embodiment 239, wherein the 3’ UTR is selected from the group of genes consisting of RPS27, FLII, TRAP, RPL36. ZDHHC12, NDUFB6. IDH3B, PMC5, NDUFB10, APTR, GRN RPS21. TMEM208, HSD17B10. PSMB7, GPS2, ATRAID, SNRPG, HSP90AB1, PFDN2, LAMTOR4, FKBP2, EEF1G, RPS11, NDUFB4, FBL, RPS16, LGALS1, NDUFB1, PTTG1, ATP5PD, SQQR, PFN1, TMSB10, SOD1, COPS3, ATP6V0B, RPN2, ENO1, GSS, and CCT2.

[0355] Embodiment 241. The method of embodiment 240, wherein the 3' UTR is selected from the group of genes consisting of NDUFB6, HSD17B10, PTTG1, and SOD1.

[0356] Embodiment 242. The method of any one of embodiments 196-241, wherein the vector encoding the agent comprises a linear nucleic acid sequence, a plasmid, a viral particle or a synthetic nanoparticle.

[0357] Embodiment 243. The method of embodiment 242, wherein the viral particle is an adeno-associated viral (AAV) particle.

[0358] Embodiment 244. The method of any one of embodiments 196-243, further comprising contacting the cell with a second agent.

[0359] Embodiment 245. The method of embodiment 244, wherein the second agent comprises a modulator of CFTR protein that potentiates chloride conductance of CFTR protein in the cells.

[0360] Embodiment 246. The method of embodiment 244 or 245, wherein the second agent comprises ivacaftor, lumacaftor, tezacaftor, elexacaftor. a combination of lumacaftor andivacaftor, a combination of tezacaftor and ivacaftor, or a combination of elexacaftor, ivacaftor, and tezacaftor.

[0361] Embodiment 247. The method of any one of embodiments 196-246, wherein the agent comprises a targeting moiety, wherein the targeting moiety directs insertion of the exogenous nucleic acid sequence into a genomic site within an intron of an endogenous CFTR gene of a genome of a cell, and wherein the intron is upstream of exon 10 of the endogenous CFTR gene.

[0362] Embodiment 248. The method of embodiment 247, wherein the targeting moiety comprises a guide RNA (gRNA), a zinc finger nuclease, or a transcription activator-like (TAL) effector nuclease.

[0363] Embodiment 249. The method of any one of embodiment 247-248, wherein the agent further comprises a gene editing enzy me or a nucleic acid sequence encoding the gene editing enzyme.

[0364] Embodiment 250. The method of any one of embodiments 247-249, wherein the agent comprises a gRNA and a class 2 CRISPR / Cas endonuclease.

[0365] Embodiment 251. The method of embodiment 250, wherein the class 2 CRISPR / Cas endonuclease is a Cas9 enzyme.

[0366] Embodiment 252. The method of embodiment 250 or 251, wherein the gRNA comprises a sequence selected from the group consisting of the sequences provided in Table 3.

[0367] Embodiment 253. The method of any one of embodiments 196-252, wherein the agent or a vector encoding the agent is delivered by intranasal administration, intratracheal administration, or intravenous injection.

[0368] Embodiment 254. The method of any one of embodiments 196-253, wherein the cell comprises a human cell.

[0369] Embodiment 255. A method for modifying a cell, the method comprising contacting an agent or a vector encoding the agent to the cell, wherein the agent modifies a genome of the cell, thereby preventing expression of an endogenous CFTR gene in the cell after intron 1.

[0370] Embodiment 256. The method of embodiment 255, wherein transcription of an endogenous CFTR gene in the cell after intron 1 is prevented.

[0371] Embodiment 257. The method of embodiment 255, comprising inserting an exogenous nucleic acid sequence into a genomic site of an intron of an endogenous cystic fibrosis transmembrane regulator (CFTR) gene of the cell, and wherein the intron is upstream of exon 10 of the endogenous CFTR gene of the cell.

[0372] Embodiment 258. The method of any one of embodiments 255-257, wherein the agent comprises a recombinant nucleic acid sequence encoding a truncated CFTR protein that is encoded by at least exons 10-27 of the CFTR gene.

[0373] Embodiment 259. The method of any one of embodiments 255-258, wherein the method increases expression of CFTR mRNA or CFTR protein by 50%, 60%, 70%, 80%, 90%, 100% or more in the cell compared to an otherwise same cell not receiving the agent.

[0374] Embodiment 260. The method of any one of embodiments 255-259, wherein the cell exhibits a 50%, 60%, 70%, 80%, 90%, 100% or more increase in CFTR activity compared to an otherwise same cell not receiving the agent.

[0375] Embodiment 261. The method of embodiment 260, wherein the CFTR activity comprises CFTR-mediated chloride channel conductivity measured by electrophysiology.

[0376] Embodiment 262. The method of any one of embodiments 255-261, wherein the agent comprises a first nucleic acid sequence which comprises exon 2 to exon 27 of the CFTR gene.

[0377] Embodiment 263. The method of embodiment 262, wherein exons of the first nucleic acid sequence are codon optimized for expression in human.

[0378] Embodiment 264. The method of any one of embodiments 255-263, wherein the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 36.

[0379] Embodiment 265. The method of any one of embodiments 255-264, wherein the first nucleic acid sequence has at least 80% identity to SEQ ID NO: 36.

[0380] Embodiment 266. The method of any one of embodiments 255-263, wherein the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 37.

[0381] Embodiment 267. The method of embodiment 266, wherein the first nucleic acid sequence has at least 80% identity to SEQ ID NO: 37.

[0382] Embodiment 268. The method of any one of embodiments 255-267, wherein the first nucleic acid sequence is free of introns.

[0383] Embodiment 269. The method of any one of embodiments 255-267, wherein the first nucleic acid sequence comprises at least one intron.

[0384] Embodiment 270. The method of any one of embodiment 269, wherein the at least one intron comprises an intron that is exogenous to the CFTR gene.

[0385] Embodiment 271. The method of any one of embodiment 270, wherein the at least one intron comprises a synthetic / chimeric intron.

[0386] Embodiment 272. The method of any one of embodiments 269-271, wherein each of the at least one intron is less than 600 nucleotides in length.

[0387] Embodiment 273. The method of any one of embodiments 255-272, wherein the first nucleic acid sequence of the agent comprises a splice acceptor site that is exogenous to a 5’ end of the first nucleic acid sequence.

[0388] Embodiment 274. The method of embodiment 273, wherein the splice acceptor site comprises a sequence identical to the sequence of an endogenous splice acceptor site immediately upstream of exon 11 of the CFTR gene.

[0389] Embodiment 275. The method of any one of embodiment 273 or 274, wherein the splice acceptor site comprises a nucleic acid sequence chosen from the group consisting of the sequences set forth in SEQ ID NOs: 1-3.

[0390] Embodiment 276. The method of any one of embodiments 257-275, wherein the genomic site of the intron of the endogenous CFTR gene is upstream of exon 10 of the endogenous CFTR gene.

[0391] Embodiment 277. The method of any one of embodiments 257-276, wherein the genomic site of the intron of the endogenous CFTR gene is within intron 1 of the endogenous CFTR gene.

[0392] Embodiment 278. The method of any one of embodiments 257-277, wherein the genomic site is about 325 nucleotides downstream of exon 1 of the endogenous CFTR gene.

[0393] Embodiment 279. The method of any one of embodiments 257-277, wherein the genomic site is about 79 nucleotides downstream of exon 1 of the endogenous CFTR gene.

[0394] Embodiment 280. The method of any one of embodiments 257-279, wherein the genomic site of the endogenous CFTR gene is located within a region from hg38 chr7: 117,480, 148 to hg38 chr7: 117.498,307.

[0395] Embodiment 281. The method of any one of embodiments 257-280, wherein the recombinant nucleic acid sequence further comprises a 5’ homology arm upstream of the first nucleic acid sequence and a 3’ homology arm dow nstream of the first nucleic acid sequence.

[0396] Embodiment 282. The method of embodiment 281, wherein the 5’ homology arm is homologous to a nucleic acid sequence upstream of the genomic site of the endogenous CFTR gene and the 3’ homology arm is homologous to a nucleic acid sequence downstream of the genomic site of the endogenous CFTR gene.

[0397] Embodiment 283. The method of embodiment 281 or 231, wherein the 5' homology arm and the 3’ homology’ arm comprise nucleic acid sequences that are at least 80% identical to nucleic acid sequences selected from the group consisting of: (a) SEQ ID NO: 38 and SEQ ID NO: 39, respectively; (b) SEQ ID NO: 38 and SEQ ID NO: 41, respectively; (c) SEQ ID NO: 38 and SEQ ID NO: 43, respectively; (d) SEQ ID NO: 40 and SEQ ID NO: 39, respectively; (e) SEQ ID NO: 40 and SEQ ID NO: 41, respectively; (1) SEQ ID NO: 40 and SEQ ID NO: 43,respectively; (g) SEQ ID NO: 42 and SEQ ID NO: 39, respectively; (h) SEQ ID NO: 42 and SEQ ID NO: 41, respectively; and (i) SEQ ID NO: 42 and SEQ ID NO: 43, respectively.

[0398] Embodiment 284. The method of any one of embodiments 258-283, wherein the recombinant nucleic acid sequence further comprises a second nucleic acid sequence has at least 80% identity to a 3’ untranslated region (3’ UTR) of a gene different than the CFTR gene.

[0399] Embodiment 285. The method of embodiment 284, wherein the second nucleic acid sequence has at least 90% identity to a 3’ UTR of a gene different than the CFTR gene.

[0400] Embodiment 286. The method of embodiment 284 or 285, wherein the 3' UTR of a gene is a 3’ UTR of a eukaryotic gene.

[0401] Embodiment 287. The method of embodiment 286, wherein the 3’ UTR of a gene is a 3 ’ UTR of a human gene.

[0402] Embodiment 288. The method of embodiment 284 or 285, wherein the 3' UTR of a gene is a 3' UTR of a viral gene.

[0403] Embodiment 289. The method of embodiment 288, wherein the 3’ UTR of a gene is a simian virus (SV) 40 gene.

[0404] Embodiment 290. The method of embodiment 286, wherein the human gene is selected from the group consisting of the genes listed in Table 1.

[0405] Embodiment 291. The method of embodiment 290, wherein the 3?UTR is selected from the group of genes consisting of RPS27, FLII, TRAP, RPL36, ZDHHC12, NDUFB6, IDH3B, PMC5, NDUFB10, APTR, GRN RPS21, TMEM208, HSD17B10, PSMB7, GPS2, ATRAID, SNRPG, HSP90AB1, PFDN2, LAMTOR4, FKBP2, EEF1G, RPS11, NDUFB4, FBL, RPS16, LGALS 1, NDUFB1, PTTG1, ATP5PD, SQQR, PFN1, TMSB10, SOD1, COPS3, ATP6V0B, RPN2, ENO1, GSS, and CCT2.

[0406] Embodiment 292. The method of embodiment 291, wherein the 3’ UTR is selected from the group of genes consisting of NDUFB6, HSD17B10, PTTG1, and SOD1.

[0407] Embodiment 293. The method of any one of embodiments 255-292, wherein the vector encoding the agent comprises a linear nucleic acid sequence, a plasmid, a viral particle or a synthetic nanoparticle.

[0408] Embodiment 294. The method of embodiment 293, wherein the viral particle is an adeno-associated viral (AAV) particle.

[0409] Embodiment 295. The method of any one of embodiments 255-294, further comprising contacting the cell with a second agent.

[0410] Embodiment 296. The method of embodiment 295, wherein the second agent comprises a modulator of CFTR protein that potentiates chloride conductance of CFTR protein in the cells.

[0411] Embodiment 297. The method of embodiment 295 or 296, 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.

[0412] Embodiment 298. The method of any one of embodiments 255-297, wherein the agent comprises a targeting moiety, wherein the targeting moiety directs insertion of the exogenous nucleic acid sequence into a genomic site within an intron of an endogenous CFTR gene of a genome of a cell, and wherein the intron is upstream of exon 10 of the endogenous CFTR gene.

[0413] Embodiment 299. The method of embodiment 298, wherein the targeting moiety comprises a guide RNA (gRNA), a zinc finger nuclease, or a transcription activator-like (TAL) effector nuclease.

[0414] Embodiment 300. The method of any one of embodiment 255-299, wherein the agent further comprises a gene editing enzyme or a nucleic acid sequence encoding the gene editing enzyme.

[0415] Embodiment 301. The method of any one of embodiments 255-300, wherein the agent comprises a gRNA and a class 2 CRISPR / Cas endonuclease.

[0416] Embodiment 302. The method of embodiment 301, wherein the class 2 CRISPR / Cas endonuclease is a Cas9 enzyme.

[0417] Embodiment 303. The method of embodiment 301 or 302, wherein the gRNA comprises a sequence selected from the group consisting of the sequences provided in Table 3.

[0418] Embodiment 304. The method of any one of embodiments 255-303, wherein the agent or a vector encoding the agent is delivered by intranasal administration, intratracheal administration, or intravenous injection.

[0419] Embodiment 305. The method of any one of embodiments 255-304, wherein the cell comprises a human cell.

[0420] Embodiment 306. A method comprising contacting an agent or a vector encoding the agent to the cell, wherein the agent comprises the recombinant nucleic acid sequence of any one of embodiments 1-107, the gene editing system of any one of embodiments 108-175, or the pharmaceutical composition of any one of embodiments 176-186.

[0421] Embodiment 307. A method for treating a subject in need thereof, the method comprising contacting cells of the subject with the recombinant nucleic acid sequence of any one of embodiments 1-107, the gene editing system of any one of embodiments 108-175, or the pharmaceutical composition of any one of embodiments 176-186.

[0422] Embodiment 308. A recombinant nucleic acid sequence comprising: a first nucleic acid sequence encoding a N-terminal truncated cystic fibrosis transmembrane regulator (CFTR) protein that is truncated at a position down stream of amino acid residues encoded by exon 11 of a CFTR gene, and a) a splice acceptor site that is immediately upstream of the first nucleic acid sequence; or b) a second nucleic acid sequence that has at least 80% identity to a 3?untranslated region (3’ UTR) of a gene different than the CFTR gene.

[0423] Embodiment 309. The recombinant nucleic acid sequence of embodiment 308, wherein the first nucleic acid sequence comprises exon 22 to exon 27 of the CFTR gene.

[0424] Embodiment 310. A recombinant nucleic acid sequence comprising: a first nucleic acid sequence encoding a truncated cystic fibrosis transmembrane regulator (CFTR) protein that consists of amino acid residues encoded by exons 22-27 of a CFTR gene, and a) a splice acceptor site that is immediately upstream of the first nucleic acid sequence; or b) a second nucleic acid sequence that has at least 80% identity to a 3’ untranslated region (3’ UTR) of a gene different than the CFTR gene.

[0425] Embodiment 311. The recombinant nucleic acid sequence of any one of embodiments 308-310, further comprising a 5’ homology arm upstream of the first nucleic acid sequence and a 3’ homology arm downstream of the first nucleic acid sequence.

[0426] Embodiment 312. The recombinant nucleic acid sequence of embodiment 311, w herein the 5 ’ homology arm is homologous to a nucleic acid sequence upstream of the target site of the endogenous CFTR gene and the 3’ homology7arm is homologous to a nucleic acid sequence downstream of the target site of the endogenous CFTR gene.

[0427] Embodiment 313. A recombinant nucleic acid sequence comprising: a) a first nucleic acid sequence encoding a truncated cystic fibrosis transmembrane regulator (CFTR) protein that comprises amino acid residues encoded by exons of a CFTR gene; b) a 5' homology7arm upstream of the first nucleic acid sequence, and c) a 3' homology arm downstream of the first nucleic acid sequence; wherein the 5' homology arm is homologous to a sequence that is upstream of a genomic site within an intron of an endogenous CFTR gene in a genome of a cell, w herein the 3' homology7arm is homologous to a sequence that is downstream of the genomic site, and w herein the intron of the endogenous CFTR gene is downstream of intron 10 of the endogenous CFTR gene in a genome of a cell.

[0428] Embodiment 314. The recombinant nucleic acid sequence of any one of embodiments 308-313, wherein exons of the first nucleic acid sequence are codon optimized for expression in human.

[0429] Embodiment 315. The recombinant nucleic acid sequence of any one of embodiments 308-314, wherein the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 45.

[0430] Embodiment 316. The recombinant nucleic acid sequence of any one of embodiments 308-315, wherein the first nucleic acid sequence has at least 80% identity to SEQ ID NO: 45.

[0431] Embodiment 317. The recombinant nucleic acid sequence of any one of embodiments 308-316, wherein the first nucleic acid sequence is free of introns.

[0432] Embodiment 318. The recombinant nucleic acid sequence of any one of embodiments 308-316, wherein the first nucleic acid sequence comprises at least one intron.

[0433] Embodiment 319. The recombinant nucleic acid sequence of embodiment 318, wherein the at least one intron comprises an intron that is exogenous to the CFTR gene.

[0434] Embodiment 320. The recombinant nucleic acid sequence of embodiment 319, wherein the at least one intron comprises a synthetic / chimeric intron.

[0435] Embodiment 321. The recombinant nucleic acid sequence of any one of embodiments 318-320, wherein each of the at least one intron is less than 600 nucleotides in length.

[0436] Embodiment 322. The recombinant nucleic acid sequence of any one of embodiments 308-321, wherein the splice acceptor site is exogenous to a 5’ end of the first nucleic acid sequence.

[0437] Embodiment 323. The recombinant nucleic acid sequence of any one of embodiments 308-322, wherein the splice acceptor site comprises a nucleic acid sequence chosen from the group consisting of the sequences set forth in SEQ ID NOs: 1-3.

[0438] Embodiment 324. The recombinant nucleic acid sequence of any one of embodiments 308-323, wherein the recombinant nucleic acid sequence is configured to be inserted into a target site of an endogenous CFTR gene in a genome of a cell.

[0439] Embodiment 325. The recombinant nucleic acid sequence of embodiment 324. wherein the target site of the endogenous CFTR gene is within an intron of the endogenous CFTR gene.

[0440] Embodiment 326. The recombinant nucleic acid sequence of embodiment 325, wherein the intron is downstream of intron 10 of the endogenous CFTR gene.

[0441] Embodiment 327. The recombinant nucleic acid sequence of any one of embodiments 308-326, wherein the second nucleic acid sequence has at least 90% identity to a 3’ untranslated region (3’ UTR) of a gene different than the CFTR gene.

[0442] Embodiment 328. The recombinant nucleic acid sequence of embodiment 327, wherein the 3’ UTR of a gene is a 3’ UTR of a eukaryotic gene.

[0443] Embodiment 329. The recombinant nucleic acid sequence of embodiment 327 or 328, wherein the 3’ UTR of a gene is a 3’ UTR of a human gene.

[0444] Embodiment 330. The recombinant nucleic acid sequence of embodiment 327. wherein the 3’ UTR of a gene is a 3’ UTR of a viral gene.

[0445] Embodiment 331. The recombinant nucleic acid sequence of embodiment 330, wherein the 3’ UTR of a gene is a simian virus (SV) 40 gene.

[0446] Embodiment 332. The recombinant nucleic acid sequence of embodiment 329, wherein the human gene is selected from the group consisting of genes listed in Table 1.

[0447] Embodiment 333. The recombinant nucleic acid sequence of embodiment 332, wherein the 3’ UTR is selected from the group of genes consisting of RPS27, FLII, TRAP, RPL36, ZDHHC12, NDUFB6, IDH3B, PMC5, NDUFB10. APTR. GRN RPS21, TMEM208, HSD17B10, PSMB7, GPS2. ATRAID, SNRPG, HSP90AB1. PFDN2, LAMTOR4, FKBP2. EEF1G, RPS11, NDUFB4, FBL, RPS16, LGALS1, NDUFB1, PTTG1, ATP5PD, SQQR, PFN1, TMSB10, SOD1, COPS3, ATP6V0B, RPN2, ENO1, GSS, and CCT2.

[0448] Embodiment 334. The recombinant nucleic acid sequence of embodiment 333, wherein the 3’ UTR is selected from the group of genes consisting of NDUFB6, HSD17B10, PTTG1, and SOD1.

[0449] Embodiment 335. The recombinant nucleic acid sequence of any one of embodiment 308-334, wherein the recombinant nucleic acid sequence is configured to fit within a vector.

[0450] Embodiment 336. The recombinant nucleic acid sequence of embodiment 335, wherein the vector comprises a linear nucleic acid sequence, a plasmid, a viral particle or a synthetic nanoparticle.

[0451] Embodiment 337. The recombinant nucleic acid sequence of embodiment 336, wherein the viral particle comprises an adeno-associated viral (AAV) particle.

[0452] Embodiment 338. A gene editing system, comprising: (a) a first nucleic acid sequence encoding a truncated cystic fibrosis transmembrane regulator (CFTR) protein, and (b) a targeting moiety or a nucleic acid sequence encoding the targeting moiety; wherein the targeting moiety directs insertion of the first nucleic acid sequence into a genomic site within an intron of an endogenous CFTR gene of a genome of a cell, and wherein the intron is downstream of intron 10 of the endogenous CFTR gene of the cell.

[0453] Embodiment 339. A gene editing system, comprising: (a) a recombinant nucleic acid sequence of any one of embodiments 308-337, and (b) a targeting moiety or a nucleic acid sequence encoding the targeting moiety: wherein the targeting moiety directs insertion of thefirst nucleic acid sequence into a genomic site within an intron of an endogenous CFTR gene of a genome of a cell, and wherein the intron is downstream of intron 10 of the endogenous CFTR gene of the cell.

[0454] Embodiment 340. The gene editing system of embodiment 338 or 339, wherein the targeting moiety comprises a guide RNA (gRNA), a zinc finger nuclease, or a transcription activator-like (TAL) effector nuclease.

[0455] Embodiment 341. The gene editing system of any one of embodiments 338-340, wherein the gene editing system further comprises a gene editing enzyme or a nucleic acid sequence encoding the gene editing enzyme.

[0456] Embodiment 342. The gene editing system of any one of embodiments 338-341, wherein the gene editing system results in integration of one or two copies of the first nucleic acid sequence.

[0457] Embodiment 343. The gene editing system of any one of embodiments 338-342, wherein the targeting moiety is a CRISPR / Cas nickase.

[0458] Embodiment 344. The gene editing system of any one of embodiments 343, wherein the gene editing system further comprises a recombinase.

[0459] Embodiment 345. The gene editing system of embodiment 344. wherein the recombinase is a domain of the CRISPR / Cas nickase.

[0460] Embodiment 346. The gene editing system of embodiment 345, wherein the recombinase is separate from the CRISPR / Cas nickase.

[0461] Embodiment 347. The gene editing system of any one of embodiments 338-346, wherein the gene editing system further comprises a reverse transcriptase.

[0462] Embodiment 348. The gene editing system of any one of embodiments 338-347, wherein the recombinant nucleic acid sequence is flanked by a sequence recognized by a recombinase.

[0463] Embodiment 349. The gene editing system of any one of embodiments 338-348 wherein the gene editing system further comprises a gRNA encoding a sequence that is recognized by a recombinase.

[0464] Embodiment 350. The gene editing system of any one of embodiments 349, wherein the sequence that is recognized by a recombinase is integrated into the genomic site.

[0465] Embodiment 351. The gene editing system of any one of embodiments 344-350, wherein the recombinase directs the insertion of the recombinant nucleic acid sequence into the genomic site.

[0466] Embodiment 352. The gene editing system of any one of embodiments 338-342, wherein the gene editing system comprises a gRNA and a class 2 CRISPR / Cas endonuclease.

[0467] Embodiment 353. The gene editing system of embodiment 352, wherein the gene editing system comprises a gRNA and a Cas9 enzyme.

[0468] Embodiment 354. The gene editing system of embodiment 352 or 353, wherein the gRNA comprises a sequence selected from the group consisting of the sequences provided in Table 3.

[0469] Embodiment 355. The gene editing system of any one of embodiments 338-354 wherein the gene editing system is configured to fit within one vector.

[0470] Embodiment 356. The gene editing system of any one of embodiments 338-354, wherein the gene editing system is configured to fit in two or more vectors.

[0471] Embodiment 357. The gene editing system of embodiment 355 or 356, wherein the vector comprises a linear nucleic acid sequence, a plasmid, a viral particle or a synthetic nanoparticle.

[0472] Embodiment 358. The gene editing system of embodiment 357. wherein the viral particle comprises an adeno-associated viral (AAV) particle.

[0473] Embodiment 359. A pharmaceutical composition, the pharmaceutical composition comprising: a) a pharmaceutically acceptable excipient or carrier; and b) the recombinant nucleic acid sequence of any one of embodiments 308-337 or the gene editing system of any one of embodiments 338-358.

[0474] Embodiment 360. The pharmaceutical composition of embodiment 359, wherein the pharmaceutical composition is formulated for intranasal administration, intratracheal administration, or intravenous injection.

[0475] Embodiment 361. The pharmaceutical composition of embodiment 359 or 360. wherein the pharmaceutical composition is formulated for pulmonary administration.

[0476] Embodiment 362. The pharmaceutical composition of any one of embodiments 359- 361, wherein the pharmaceutical composition further comprises a second therapeutic agent.

[0477] Embodiment 363. The pharmaceutical composition of embodiment 362, wherein the second therapeutic agent comprises a modulator of CFTR protein that potentiates chloride conductance of CFTR protein in the cells.

[0478] Embodiment 364. The pharmaceutical composition of embodiment 362 or 363, wherein 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.

[0479] Embodiment 365. The pharmaceutical composition of any one of embodiments 362- 364, wherein the second therapeutic agent comprises a mucolytic agent, optionally wherein themucolytic agent is selected from the group consisting of: acetylcysteine, ambroxol, bromhexine, carbocisteine, erdosteine, mecysteine, domase alfa, hypertonic saline, and mannitol.

[0480] Embodiment 366. The pharmaceutical composition of any one of embodiments 362-365, wherein the second agent comprises a bronchodilator, optionally wherein the bronchodilator is albuterol.

[0481] Embodiment 367. The pharmaceutical composition of any one of embodiments 362-366, wherein the second agent comprises an immunosuppressive agent.

[0482] Embodiment 368. The pharmaceutical composition of embodiment 367, wherein the immunosuppressive agent is a corticosteroid selected from the group consisting of beclomethasone, budesonide, budesonide, formoterol, ciclesonide, fluticasone, salmeterol, umeclidinium, vilanterol, mometasone, or mometasone, prednisone, and methylprednisolone, and pharmaceutically acceptable salts thereof.

[0483] Embodiment 369. The pharmaceutical composition of embodiment 367, wherein the immunosuppressive agent is a non-steroidal immunosuppressive agent selected from the group consisting of polyclonal anti -lymphocyte antibodies, monoclonal anti-lymphocyte antibodies, interleukin-2 (IL-2) receptor antagonists, calcineurin inhibitors, cell cycle inhibitors, mammalian target of rapamycin (mTOR) inhibitors, methotrexate, cyclophosphamide, anthracy cline, and taxane.

[0484] Embodiment 370. A vector comprising the recombinant nucleic acid sequence of any one of embodiments 308-337 or the gene editing system of any one of embodiments 338-358.

[0485] Embodiment 371. The vector of embodiment 370, wherein the vector is a viral vector, optionally a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno- associated viral vector, or a herpes simplex viral vector.

[0486] Embodiment 372. The vector of embodiment 370, wherein the vector is a non-viral vector, optionally a lipid nanoparticle.

[0487] Embodiment 373. A virus comprising the recombinant nucleic acid sequence of anyone of embodiments 308-337 or the gene editing system of any one of embodiments 338-358.

[0488] Embodiment 374. The virus of embodiment 373, wherein the virus is an adeno- associated viral (AAV) vector, optionally a retroviral vector, a lentiviral vector, an adenoviral vector, or a herpes simplex viral vector.

[0489] Embodiment 375. A cell comprising the recombinant nucleic acid sequence of any one of embodiments 308-337 or the gene editing system of any one of embodiments 338-358.

[0490] Embodiment 376. The cell of embodiment 375, wherein the cell is a stem cell.

[0491] Embodiment 377. The cell of embodiment 375 or 376, wherein the cell is a human cell.

[0492] Embodiment 378. A kit comprising: a) the recombinant nucleic acid sequence of any one of embodiments 308-337, the gene editing system of any one of embodiments 338-358, or the pharmaceutical composition of any one of embodiments 359-369; and b) instructions for use of the recombinant nucleic acid sequence, the gene editing system, or the pharmaceutical composition.

[0493] Embodiment 379. A method for modifying a cell, the method comprising contacting an agent or a vector encoding the agent to the cell, wherein the agent modifies a genome of the cell, thereby inserting an exogenous nucleic acid sequence into a genomic site of an intron of an endogenous cystic fibrosis transmembrane regulator (CFTR) gene of the cell, and wherein the intron is dow nstream of intron 10 of the endogenous CFTR gene of the cell.

[0494] Embodiment 380. The method of embodiment 379, wherein the agent comprises a recombinant nucleic acid sequence encoding a truncated CFTR protein that is encoded by at least exons 22-27 of the CFTR gene.

[0495] Embodiment 381. The method of embodiment 379 or 380, wherein the method increases expression of CFTR mRNA or CFTR protein by 50%, 60%, 70%, 80%, 90%, 100% or more in the cell compared to an otherwise same cell not receiving the agent.

[0496] Embodiment 382. The method of any one of embodiments 379-381, wherein the cell exhibits a 50%, 60%, 70%, 80%, 90%, 100% or more increase in CFTR activity compared to an otherwise same cell not receiving the agent.

[0497] Embodiment 383. The method of embodiment 382, wherein the CFTR activity comprises CFTR-mediated chloride channel conductivity measured by electrophysiolog}'.

[0498] Embodiment 384. The method of any one of embodiments 379-383, wherein the recombinant nucleic acid sequence is codon optimized for expression in human.

[0499] Embodiment 385. The method of any one of embodiments 379-384, wherein the recombinant nucleic acid sequence has at least 70% identity to SEQ ID NO: 45.

[0500] Embodiment 386. The method of any one of embodiments 379-385, wherein the recombinant nucleic acid sequence has at least 80% identify to SEQ ID NO: 45.

[0501] Embodiment 387. The method of any one of embodiments 379-386, wherein the recombinant nucleic acid sequence is free of introns.

[0502] Embodiment 388. The method of any one of embodiments 379-386, wherein the recombinant nucleic acid sequence comprises at least one intron.

[0503] Embodiment 389. The method of embodiment 388, wherein the at least one intron comprises an intron that is exogenous to the CFTR gene.

[0504] Embodiment 390. The method of embodiment 389, wherein the at least one intron comprises a synthetic / chimeric intron.

[0505] Embodiment 391. The method of any one of embodiments 388-390, wherein each of the at least one intron is less than 600 nucleotides in length.

[0506] Embodiment 392. The method of any one of embodiments 379-391, wherein the recombinant nucleic acid sequence comprises a splice acceptor site that is exogenous to a 5’ end of the first nucleic acid sequence.

[0507] Embodiment 393. The method of any one of embodiment 392, wherein the splice acceptor site comprises a nucleic acid sequence chosen from the group consisting of the sequences set forth in SEQ ID NOs: 1-3.

[0508] Embodiment 394. The method of any one of embodiments 379-393, wherein the recombinant nucleic acid sequence further comprises a 5’ homology arm upstream of the first nucleic acid sequence and a 3’ homology arm downstream of the first nucleic acid sequence.

[0509] Embodiment 395. The method of embodiment 394, wherein the 5’ homology arm is homologous to a nucleic acid sequence upstream of the genomic site of the endogenous CFTR gene and the 3’ homology arm is homologous to a nucleic acid sequence downstream of the genomic site of the endogenous CFTR gene.

[0510] Embodiment 396. The method of any one of embodiments 379-395, wherein the recombinant nucleic acid sequence further comprises a second nucleic acid sequence has at least 80% identity to a 3?untranslated region (3‘ UTR) of a gene different than the CFTR gene.

[0511] Embodiment 397. The method of embodiment 396, wherein the second nucleic acid sequence has at least 90% identity to a 3’ UTR of a gene different than the CFTR gene.

[0512] Embodiment 398. The method of embodiment 396 or 397, wherein the 3' UTR of a gene is a 3’ UTR of a eukaryotic gene.

[0513] Embodiment 399. The method of embodiment 398, wherein the 3’ UTR of a gene is a 3 ' UTR of a human gene.

[0514] Embodiment 400. The method of embodiment 396 or 397, wherein the 3' UTR of a gene is a 3‘ UTR of a viral gene.

[0515] Embodiment 401. The method of embodiment 400, wherein the 3’ UTR of a gene is a simian virus (SV) 40 gene.

[0516] Embodiment 402. The method of embodiment 399, wherein the human gene is selected from the group consisting of the genes listed in Table 1.

[0517] Embodiment 403. The method of embodiment 402, wherein the 3' UTR is selected from the group of genes consisting of RPS27, FLII, TRAP, RPL36, ZDHHC12, NDUFB6, IDH3B, PMC5, NDUFB10, APTR, GRN RPS21, TMEM208, HSD17B10, PSMB7, GPS2, ATRAID, SNRPG, HSP90AB1, PFDN2, LAMTOR4, FKBP2, EEF1G, RPS11, NDUFB4, FBL,RPS16, LGALS1, NDUFB1, PTTG1, ATP5PD, SQQR, PFN1, TMSB10, SOD1, COPS3, ATP6V0B, RPN2, ENO1, GSS, and CCT2.

[0518] Embodiment 404. The method of embodiment 403, wherein the 3’ UTR is selected from the group of genes consisting of NDUFB6, HSD17B10, PTTG1, and SODE

[0519] Embodiment 405. The method of any one of embodiments 379-404, wherein the vector encoding the agent comprises a linear nucleic acid sequence, a plasmid, a viral particle or a synthetic nanoparticle.

[0520] Embodiment 406. The method of embodiment 405, wherein the viral particle is an adeno-associated viral (AAV) particle.

[0521] Embodiment 407. The method of any one of embodiments 379-406, further comprising contacting the cell with a second agent.

[0522] Embodiment 408. The method of embodiment 407, wherein the second agent comprises a modulator of CFTR protein that potentiates chloride conductance of CFTR protein in the cells.

[0523] Embodiment 409. The method of embodiment 407 or 408, 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.

[0524] Embodiment 410. The method of any one of embodiments 379-409, wherein the method comprises contacting the cell with a CRISPR / Cas nickase.

[0525] Embodiment 41 E The method of embodiment 410, wherein the method further comprises contacting the cell with a recombinase.

[0526] Embodiment 412. The method of embodiment 411, wherein the recombinase is a domain of the CRISPR / Cas nickase.

[0527] Embodiment 413. The method of embodiment 411, wherein the recombinase is separate from the CRISPR / Cas nickase.

[0528] Embodiment 414. The method of any one of embodiments 379-413, wherein the method further comprises contacting the cell with a reverse transcriptase.

[0529] Embodiment 415. The method of any one of embodiments 379-414, wherein the exogenous nucleic acid sequence is flanked by a sequence recognized by a recombinase.

[0530] Embodiment 416. The method of any one of embodiments 379-415, wherein the method further comprises contacting the cell with a gRNA encoding a sequence that is recognized by a recombinase.

[0531] Embodiment 417. The method of any one of embodiments 416, wherein the sequence that is recognized by a recombinase is integrated into the genomic site.

[0532] Embodiment 418. The method of any one of embodiments 411-417, wherein the recombinase directs the insertion of the recombinant nucleic acid sequence into the genomic site.

[0533] Embodiment 419. The method of any one of embodiments 379-409, wherein the agent comprises a targeting moiety, wherein the targeting moiety directs insertion of the exogenous nucleic acid sequence into a genomic site within an intron of an endogenous CFTR gene of a genome of a cell, and wherein the intron is dow nstream of intron 10 of the endogenous CFTR gene.

[0534] Embodiment 420. The method of embodiment 419, wherein the targeting moiety comprises a guide RNA (gRNA), a zinc finger nuclease, or a transcription activator-like (TAL) effector nuclease.

[0535] Embodiment 421. The method of embodiment 419 or 420, wherein the agent further comprises a gene editing enzyme or a nucleic acid sequence encoding the gene editing enzyme.

[0536] Embodiment 422. The method of any one of embodiments 419-421, wherein the agent comprises a gRNA and a class 2 CRISPR / Cas endonuclease.

[0537] Embodiment 423. The method of embodiment 422, wherein the class 2 CRISPR / Cas endonuclease is a Cas9 enzyme.

[0538] Embodiment 424. The method of embodiment 420-423. wherein the gRNA comprises a sequence selected from the group consisting of the sequences provided in Table 3.

[0539] Embodiment 425. The method of any one of embodiments 379-424, wherein the agent or a vector encoding the agent is delivered by intranasal administration, intratracheal administration, or intravenous injection.

[0540] Embodiment 426. The method of any one of embodiments 379-425, wherein the cell comprises a human cell.

[0541] Embodiment 427. A method comprising contacting an agent or a vector encoding the agent to the cell, wherein the agent comprises the recombinant nucleic acid sequence of any one of embodiments 308-337, the gene editing system of any one of embodiments 338-358, or the pharmaceutical composition of any one of embodiments 359-369.

[0542] Embodiment 428. A method for treating a subject in need thereof, the method comprising contacting cells of the subject with the recombinant nucleic acid sequence of any one of embodiments 308-337, the gene editing system of any one of embodiments 338-358, or the pharmaceutical composition of any one of embodiments 359-369.EXAMPLESExample 1. Generation of Functional Super Exons of CFTR

[0543] Super exons were designed similar to the constructs shown in FIGs. 2A and 2B. A cas9 / gRNA and a construct comprising, from 5' to 3’, a splice acceptor site, exons 23-37, and a 3‘ UTR / polyA region was delivered into the cells via nucleofection, resulting in insertion of the super exon within intron 22 of the native CFTR gene in the genome of the cells. Different 3’ UTR / polyA regions were tested. A membrane potential assay was performed to assess the function of CFTR generated from the modified CFTR gene in the cells. In this assay, a monolayer of cells was cultured on porous transwells. Then the monolayers were then tested using a 24-channel transepithelial current clamp. The electrical current was measured which is generated by the transport of chloride ions by CFTR. CFTR function of test cells was measured relative to that of normal healthy cells. Cells bearing a nonfunctional CFTR mutant (N1303K) were used as negative controls. As shown in FIG. 2C, both super exon constructs were able to achieve some level of functional activity and one construct with a specific 3’ UTR / polyA region achieved nearly 80% of normal CFTR function. Thus, selection of different 3’ UTRs can affect functionality of these constructs. Taken together, this demonstrates that super exon constructs can be stably expressed in cells and can encode functional CFTRs that can achieve near normal levels of activity and that different 3?UTRs can affect expression.Example 2. Generation of Super Exon Reporter Constructs

[0544] In order to determine the feasibility of inserting super exons at sites further upstream in the CFTR gene, GFP reporters as exemplified in FIG. 3A were generated. The constructs comprise, from 5' to 3’, a splice acceptor site, a peptide linker, GFP, and a 3’ UTR / polyA region. Constructs were generated using various homology arms flanking the ends of the construct to direct insertion at positions throughout intron 1. Constructs were generated that are designed to be inserted at the 5’ end of intron 1, at the lOkb region, or at the 3’ end of intron 1 (FIGs. 3B and 3C). In construct IV. the native splice acceptor for CFTR exon 23 and a bovine growth hormone (BGH) 3’ UTR / polyA were used. Overall efficiency of insertion of the reporter constructs is shown in FIG. 3D. All constructs were able to integrate effectively, as measured by %GFP+cells using flow cytometry, in about 8% of cells. The construct bearing the native CFTR exon 23 splice acceptor show ed low efficiency and only expressed in about 1% of cells. When intensity of GFP expression was assessed comparing constructs inserted at the 5’ end of intron 1 vs the 3’ end, the GFP signal was found to be brighter in the constructs inserted at the 5’ end (FIGs. 3E and 3F). When the two different 3’ inserted constructs were similarly compared, construct IV, bearing the native CFTR exon 23 splice acceptor site and BGH polyA demonstrated lower intensity of GFP expression.

[0545] In order to further examine the efficacy of various super exon constructs, a more sensitive reporter system was utilized. The constructs comprise, from 5’ to 3’, a splice acceptor site, a peptide linker, aNanoLuc luciferase reporter, and a 3’ UTR / polyA region (FIG. 4A). Nucleofection of constructs inserted at the 5’ end of intron 1 yielded the highest signal and the linear construct donor was the most effective donor type (FIG. 4B). Taken together, these experiments demonstrate that super exon constructs can be efficiently expressed by cells.Example 3. Testing of CFTR Super Exon Constructs

[0546] To comprehensively assess the effects of various construct components, experiments were performed using the NanoLuc reporter system. 16HBE14o-WT human bronchial epithelial cells were nucleofected with linear constructs and a CRISPR / Cas system containing a guide RNA to direct insertion in intron 1. Summarized in FIG. 5A, cells were stained with Alamar blue prior to measurement of NanoLuc activity7using the Nano-Gio live cell assay. The components tested included the following: Insertion site-directed guide RNA (gRNA)• gRNA167 (325 bp downstream of exon 1) (SEQ ID NO: 20)• gRNA207 (79bp downstream of exon 1) (SEQ ID NO: 34) Splice Acceptor• Synthetic splice acceptor (SEQ ID NO: 1)• CFTR exon 11 native splice acceptor (SEQ ID NO: 2) Prescence of Introns• pCl mini-intron (SEQ ID NO: 4)• No introns3’ UTR / polvA signal sequence• SV40 (SEQ ID NO: 5)• eK5+SV40 (SEQ ID NO: 6)• RPS27 (natural human 3 ’ UTR, 62bp) (SEQ ID NO: 7)• NAA38 (natural human 3’ UTR. 46bp) (SEQ ID NO: 8)• CTSA (natural human 3 ’ UTR, 370bp) (SEQ ID NO: 9)Accounting for all combinations of the above, 40 different constructs were tested.

[0547] NanoLuc activity yielded by the different constructs is summarized in FIGs. 5B-5C. Two of the constructs (pCFF206 and pCFF227) outperformed the original construct that was selected based on the prior experiments. These results also indicate that addition of mini-introns may further enhance the efficacy of the constructs.Example 4. Efficient Rescue of CFTR Function using Super Exon Construct Bearing Exons 2-27 in CFTR Deficient Human Cells

[0548] To investigate the functional efficacy of a super exon construct as a therapeutic approach, 16HBEge cells which harbor a G542X mutation in CFTR gene were utilized for the experiments. As shown in FIG. 6A. 16HBEge cells were transfected with an adenovirus bearing the gene construct 24 hours prior to nucleofection-mediated delivery of the ribonucleoprotein complex for targeting the integration of the construct into the cell genome. Isolated clonal cell lines were then subjected to a genomic DNA PCR screen to identify clones that had the desired super exon insertion. Identified clonal lines were then assessed for various characterizations of CFTR expression and function including next generation sequencing of the CFTR locus, CFTR protein expression, CFTR mRNA abundance, and electrophysiology. 11 cell clones demonstrated at least partial rescue of CFTR protein expression measured via western blot (FIG. 6B). CFTR mRNA levels were also measured. Clones also demonstrated decreased mRNA expression compared to a wild type control, three of which are shown (FIG. 6C). CFTR function was then analyzed using a transepithelial current clamp (TECC-24) electrophysiology assay wherein forskolin is administered and changes in electric current are measured as CFTR activity is stimulated by forskolin (FIG. 6D). 11 cell clones demonstrated functional CFTR activity at least similar to the wild type control and many constructs demonstrated increased activity compared to the wild type control (ranging from 76% to 327% of the wild type control). Three clones were found to be homozygous for super exon insertion. In order to further investigate the integration efficiency of these constructs, next generation sequencing was performed. Shown in Table 5, most clones demonstrated effective splicing of the super exon construct (labeled as M470) and little undesired splicing to recapitulate the mutant (G542X) CFTR. Taken together, these results demonstrate that the super exon approach is capable of producing WT CFTR RNA transcripts after splicing.Table 5. Next Generation Long Read Sequencing Results of Functional CFTR Super Exon TestingExample 5. Super Exon Integration Efficiency in Human Cells Using Various Construct Designs

[0549] To investigate the effects of the components used to design super exon constructs, the constructs shown in FIG. 7A were utilized. These constructs were delivered to human 16HBEge cells which harbor a G542X mutation in CFTR gene. Constructs were delivered to the cells using a spinfection protocol, wherein cells are centrifuged with supernatant containing an adenoviral vector bearing SE construct components and a CRISPR / Cas system containing a guide RNA to direct insertion in various sites of intron 1 as depicted in FIG. 7A. Then, cells were lysed and protein was extracted to analyze CFTR expression via Western blotting. The 7 constructs tested showed variable CFTR rescue by Western blot, indicating that the components, such as the splice acceptor, 3' UTR, mini-intron, can have robust effects on super exon expression (FIG. 7B).Example 6. Rescue of CFTR function using Exon 22-27 Super Exon

[0550] The construct shown in FIG. 8 was designed. This construct contains wild-type exons 22-27 with the synthetic splice acceptor site and the SV40 3’ UTR / poly A region. This construct can mitigate the effects of mutations downstream of intron 22, including R1162X, W1282X, N1303K, and other mutations.

[0551] To test the efficacy of the Exon 22-27 super exon, human bronchial epithelial cells bearing the F508del / W1282X mutations were transfected with the exon 22-27 super exon packaged in AAVs with a multiplicity of infection (MOI) of either 100,000 or 500,000 immediately following nucleofection with Cas9 / gRNA RNP. As negative controls, some cells were untreated or treated with ribonucleoprotein alone. Electrophysiology was performed on the cells to measure CFTR activity, shown in FIG. 9A. Summarized in FIG. 9B, cells transfectedwith the super exon construct demonstrated partial rescue of CFTR function in a dose dependent manner.

[0552] To confirm these results, a human bronchial epithelial cell line was used bearing the R1162X mutation in CFTR. Similarly, these cells were transfected with the exon 22-27 super exon packaged in AVVs at a MOI of 500.000 immediately following nucleofection with Cas9 / gRNA RNP. Cells were subcloned to isolate individual clones. Electrophysiology was performed to measure CFTR function as shown in FIG. 10A. Among the 4clones tested, all demonstrated at least partial rescue of CFTR function compared to cells bearing the R1162X mutation as a negative control, as shown in FIG. 10B. These data were also normalized to the activity measured in wild type cells, which is summarized in Table 6. A transepithelial electrical resistance assay was also performed as another indicator of CFTR function. Taken together, these results demonstrate that super exon constructs can be effectively packaged in AAV vectors and can rescue function of mutated CFTR.Table 6. Exon 22-27 Super Exon CFTR Activity Normalized to Wild-TypeExample 7. Testing of Human 3’ UTR Sequences for CFTR Super Exon Constructs

[0553] NanoLuc reporter system was used to test the effect of different human 3’ UTR sequences on the delivery, integration, and expression of super exon constructs. 16HBE14o-WT human bronchial epithelial cells were transfected w ith constructs containing a splice acceptor of SEQ ID NO: 1, a peptide linker, aNanoluc cassette with the pCl mini-intron (SEQ ID NO: 4), and a 3’ UTR.

[0554] 41 constructs w ere designed, each containing a different 3’ UTR sequence of a human gene found in Table 1. The 3’UTR sequences tested included the 3’ UTR sequences of SV40, RPS27, FEII, TRAP, RPL36, ZDHHC12, NDUFB6, IDH3B, PMC5, NDUFB10, APTR, GRN RPS21, TMEM208, HSD17B10, PSMB7, GPS2, ATRAID, SNRPG, HSP90AB1, PFDN2, EAMTOR4, FKBP2, EEF1G, RPS11. NDUFB4, FBL, RPS16, LGALS1, NDUFB1. PTTG1, ATP5PD, SQQR, PFN1 , TMSB10, SOD1, COPS3, ATP6V0B, RPN2, ENO1 , GSS, and CCT2.The constructs also include homology arms specific for the insertion site targeted by gRNA167. Constructs were delivered with a Cas9 system and gRNA167 using CRISPRMax™ transfection system. Similar to Example 3, cells were stained with Alamar blue prior to measurement of NanoLuc activity using the Nano-Gio live cell assay. As summarized in FIGs. 11A-11B, 3’UTR sequences affected the activity of Nanoluc measured from the cells, with the highest activity observed using 3’ UTR sequences from NDUFB6, HSD17B10, PTTG1, and SOD1.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A recombinant nucleic acid sequence comprising: a first nucleic acid sequence encoding a truncated cystic fibrosis transmembrane regulator (CFTR) protein that comprises amino acid residues encoded by at least exons 10-27 of a CFTR gene, and(a) a splice acceptor site that is immediately upstream of the first nucleic acid sequence; or(b) a second nucleic acid sequence that has at least 80% identity to a 3’ untranslated region (3’ UTR) of a gene different than the CFTR gene.

2. The recombinant nucleic acid sequence of claim 1, wherein the first nucleic acid sequence comprises exon 2 to exon 27 of the CFTR gene.

3. The recombinant nucleic acid sequence of claim 1, wherein exons of the first nucleic acid sequence are codon optimized for expression in human.

4. The recombinant nucleic acid sequence of claim 1, wherein the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 36.

5. The recombinant nucleic acid sequence of claim 1, wherein the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 37.

6. The recombinant nucleic acid sequence of claim 1, wherein the first nucleic acid sequence is free of introns.

7. The recombinant nucleic acid sequence of claim 1, wherein the first nucleic acid sequence comprises at least one intron.

8. The recombinant nucleic acid sequence of claim 7, wherein the at least one intron comprises an intron that is exogenous to the CFTR gene.

9. The recombinant nucleic acid sequence of claim 7, wherein the at least one intron comprises a synthetic / chimeric intron.

10. The recombinant nucleic acid sequence of claim 7, wherein each of the at least one intron is less than 600 nucleotides in length.

11. The recombinant nucleic acid sequence of claim 1, wherein the splice acceptor site is exogenous to a 5’ end of the first nucleic acid sequence.

12. The recombinant nucleic acid sequence of claim 1, wherein the splice acceptor site comprises a sequence identical to the sequence of an endogenous splice acceptor site immediately upstream of exon 11 of the CFTR gene.

13. The recombinant nucleic acid sequence of claim 1, wherein the splice acceptor site comprises a nucleic acid sequence chosen from the group consisting of the sequences set forth in SEQ ID NOs: 1-3.

14. The recombinant nucleic acid sequence of claim 1, wherein the recombinant nucleic acid sequence is configured to be inserted into a target site of an endogenous CFTR gene in a genome of a cell.

15. The recombinant nucleic acid sequence of claim 14, wherein the target site of the endogenous CFTR gene is within an intron of the endogenous CFTR gene.

16. The recombinant nucleic acid sequence of claim 15, wherein the intron is upstream of exon 10 of the endogenous CFTR gene.

17. The recombinant nucleic acid sequence of claim 14, wherein the target site of the endogenous CFTR gene is within intron 1 of the endogenous CFTR gene.

18. The recombinant nucleic acid sequence of claim 14, wherein the target site of the endogenous CFTR gene is located within a region from hg38 chr7: 117,480, 148 to hg38 chr7: 117,498,307.

19. The recombinant nucleic acid sequence of claim 14, further comprising a 5’ homology arm upstream of the first nucleic acid sequence and a 3’ homology arm downstream of the first nucleic acid sequence.

20. The recombinant nucleic acid sequence of claim 19, wherein the 5’ homology arm and the 3’ homology arm comprise nucleic acid sequences that are at least 80% identical to nucleic acid sequences selected from the group consisting of:(a) SEQ ID NO: 38 and SEQ ID NO: 39, respectively;(b) SEQ ID NO: 38 and SEQ ID NO: 41, respectively;(c) SEQ ID NO: 38 and SEQ ID NO: 43, respectively;(d) SEQ ID NO: 40 and SEQ ID NO: 39, respectively;(e) SEQ ID NO: 40 and SEQ ID NO: 41, respectively;(1) SEQ ID NO: 40 and SEQ ID NO: 43, respectively;(g) SEQ ID NO: 42 and SEQ ID NO: 39, respectively;(h) SEQ ID NO: 42 and SEQ ID NO: 41, respectively; and(i) SEQ ID NO: 42 and SEQ ID NO: 43, respectively.

21. The recombinant nucleic acid sequence of claim 1, wherein the second nucleic acid sequence has at least 90% identity to a 3’ untranslated region (3’ UTR) of a gene different than the CFTR gene.

22. The recombinant nucleic acid sequence of claim 21, wherein the 3‘ UTR of a gene is a 3’ UTR of a eukaryotic gene.

23. The recombinant nucleic acid sequence of claims 21, wherein the 3’ UTR of a gene is a 3’ UTR of a human gene.

24. The recombinant nucleic acid sequence of claim 23, wherein the human gene is selected from the group consisting of genes listed in Table 1.

25. The recombinant nucleic acid sequence of claim 24, wherein the 3’ UTR is selected from the group of genes consisting of RPS27, FLII, TRAP, RPL36, ZDHHC12, NDUFB6, IDH3B, PMC5, NDUFB10, APTR, GRN, RPS21, TMEM208, HSD17B10, PSMB7, GPS2, ATRAID, SNRPG, HSP90AB1, PFDN2, LAMTOR4, FKBP2, EEF1G, RPS11, NDUFB4. FBL. RPS16, LGALS1, NDUFB1, PTTG1, ATP5PD, SQQR, PFN1. TMSB10, SOD1, COPS3, ATP6V0B, RPN2, ENO1, GSS, and CCT2.

26. The recombinant nucleic acid sequence of claim 25, wherein the 3’ UTR is selected from the group of genes consisting ofNDUFB6, HSD17B10, PTTG1, and SOD1.

27. The recombinant nucleic acid sequence of claim 1, wherein the recombinant nucleic acid sequence is configured to fit within a vector.

28. The recombinant nucleic acid sequence of claim 27, wherein the vector comprises a linear nucleic acid sequence, a plasmid, a viral particle or a synthetic nanoparticle.

29. The recombinant nucleic acid sequence of claim 28, wherein the viral particle comprises an adeno-associated viral (AAV) particle.

30. A recombinant nucleic acid sequence comprising: a first nucleic acid sequence encoding a truncated cystic fibrosis transmembrane regulator (CFTR) protein, wherein the first nucleic acid sequence comprises, between two exons of the first nucleic acid sequence, an intron that is exogenous to a CFTR gene.

31. The recombinant nucleic acid sequence of claim 30, wherein the truncated CFTR protein comprises amino acid residues encoded by at least exons 10-27 of the CFTR gene.

32. The recombinant nucleic acid sequence of claim 30, wherein the first nucleic acid sequence comprises exon 10 to exon 27 of the CFTR gene.

33. The recombinant nucleic acid sequence of claim 30, wherein the first nucleic acid sequence comprises exon 2 to exon 27 of the CFTR gene.

34. The recombinant nucleic acid sequence of claim 30, wherein the first nucleic acid sequence is codon optimized for expression in human.

35. The recombinant nucleic acid sequence of claim 30, wherein the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 36 or 37.

36. The recombinant nucleic acid sequence of claim 30, further comprising a splice acceptor site immediately upstream of the first nucleic acid sequence.

37. The recombinant nucleic acid sequence of claim 36, wherein the splice acceptor site is exogenous to a 5’ end of the first nucleic acid sequence, optionally, wherein the splice acceptor site comprises a sequence identical to the sequence of an endogenous splice acceptor site immediately upstream of exon 11 of the CFTR gene, optionally wherein the splice acceptor site comprises a nucleic acid sequence chosen from the group consisting of the sequences set forth in SEQ ID NOs: 1-3.

38. The recombinant nucleic acid sequence of claim 30, wherein the recombinant nucleic acid sequence is configured to be inserted into a target site of an endogenous CFTR gene in a genome of a cell, optionally wherein the target site of the endogenous CFTR gene is within an intron of the endogenous CFTR gene, optionally wherein the target site of the endogenous CFTR gene is within intron 1 of the endogenous CFTR gene, optionally wherein the target site of the endogenous CFTR gene is located within a region from hg38 chr7: 117,480,148 to hg38 chr7: 117,498,307.

39. The recombinant nucleic acid sequence of claim 38, further comprising a 5’ homology arm upstream of the first nucleic acid sequence and a 3’ homology arm downstream of the first nucleic acid sequence.

40. The recombinant nucleic acid sequence of claim 39, wherein the 5’ homology arm and the 3’ homology arm comprise nucleic acid sequences that are at least 80% identical to nucleic acid sequences selected from the group consisting of:(a) SEQ ID NO: 38 and SEQ ID NO: 39, respectively;(b) SEQ ID NO: 38 and SEQ ID NO: 41, respectively;(c) SEQ ID NO: 38 and SEQ ID NO: 43, respectively;(d) SEQ ID NO: 40 and SEQ ID NO: 39, respectively;(e) SEQ ID NO: 40 and SEQ ID NO: 41, respectively;(1) SEQ ID NO: 40 and SEQ ID NO: 43, respectively;(g) SEQ ID NO: 42 and SEQ ID NO: 39, respectively;(h) SEQ ID NO: 42 and SEQ ID NO: 41, respectively; and(i) SEQ ID NO: 42 and SEQ ID NO: 43, respectively.

41. The recombinant nucleic acid sequence of claim 30, further comprising a second nucleic acid sequence comprising a 3’ untranslated region (3’ UTR), optionally wherein the second nucleic acid sequence has at least 80% identity to a 3’ UTR of a gene different than the CFTR gene, optionally wherein the second nucleic acid sequence has at least 90% identity to a 3’ UTR of a gene different than the CFTR gene, optionally whereinthe 3’ UTR of a gene is a 3’ UTR of a eukaryotic gene, optionally wherein the 3’ UTR of a gene is a 3’ UTR of a human gene.

42. The recombinant nucleic acid sequence of claim 41, wherein the human gene is selected from the group consisting of the genes listed in Table 1.

43. The recombinant nucleic acid sequence of claim 42, wherein the 3’ UTR is selected from the group of genes consisting of RPS27, FLII, TRAP, RPL36, ZDHHC12, NDUFB6, IDH3B, PMC5, NDUFB10, APTR, GRN RPS21, TMEM208, HSD17B10, PSMB7, GPS2, ATRAID, SNRPG, HSP90AB1, PFDN2, LAMTOR4, FKBP2, EEF1G, RPS11, NDUFB4. FBL. RPS16, LGALS1, NDUFB1, PTTG1, ATP5PD, SQQR, PFN1. TMSB10, SOD1, COPS3, ATP6V0B, RPN2, ENO1, GSS, and CCT2.

44. The recombinant nucleic acid sequence of claim 30, wherein the recombinant nucleic acid sequence is configured to fit within a vector.

45. The recombinant nucleic acid sequence of claim 44, wherein the vector comprises a linear nucleic acid sequence, a plasmid, a viral particle or a synthetic nanoparticle.

46. The recombinant nucleic acid sequence of claim 45, wherein the viral particle comprises an adeno-associated viral (AAV) particle.

47. A recombinant nucleic acid sequence comprising:(a) a first nucleic acid sequence encoding a truncated cystic fibrosis transmembrane regulator (CFTR) protein that comprises amino acid residues encoded by at least exons 10-27 of a CFTR gene;(b) a 5' homology arm upstream of the first nucleic acid sequence, and(c) a 3' homology arm downstream of the first nucleic acid sequence; wherein the 5' homology arm is homologous to a sequence that is upstream of a genomic site within an intron of an endogenous CFTR gene in a genome of a cell, wherein the 3' homology arm is homologous to a sequence that is dow nstream of the genomic site, and wherein the intron of the endogenous CFTR gene is upstream of exon 10 of the endogenous CFTR gene in a genome of a cell.

48. The recombinant nucleic acid sequence of claim 47, wherein the first nucleic acid sequence comprises exon 2 to exon 27 of the CFTR gene.

49. The recombinant nucleic acid sequence of claim 47, wherein exons of the first nucleic acid sequence are codon optimized for expression in human.

50. The recombinant nucleic acid sequence of claim 47, wherein the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 36 or 37.

51. The recombinant nucleic acid sequence of claim 47, wherein the first nucleic acid sequence is free of introns.

52. The recombinant nucleic acid sequence of claim 47, wherein the first nucleic acid sequence comprises at least one intron, optionally wherein the at least one intron comprises an intron that is exogenous to the CFTR gene, optionally wherein the at least one intron comprises a synthetic / chimeric intron, optionally wherein each of the at least one intron is less than 600 nucleotides in length.

53. The recombinant nucleic acid sequence of claim 47, further comprising a splice acceptor site, optionally wherein the splice acceptor site is exogenous to a 5’ end of the first nucleic acid sequence, optionally wherein the splice acceptor site comprises a sequence identical to the sequence of an endogenous splice acceptor site immediately upstream of exon 11 of the CFTR gene, optionally wherein the splice acceptor site comprises a nucleic acid sequence chosen from the group consisting of the sequences set forth in SEQ ID NOs: 1-3.

54. The recombinant nucleic acid sequence of claim 47, wherein the recombinant nucleic acid sequence is configured to be inserted into the genomic site of the endogenous CFTR gene, optionally wherein the genomic site of the endogenous CFTR gene is within intron 1 of the endogenous CFTR gene.

55. The recombinant nucleic acid sequence of claim 47, wherein the 5’ homology arm and the 3’ homology arm comprise nucleic acid sequences that are at least 80% identical to nucleic acid sequences selected from the group consisting of:(a) SEQ ID NO: 38 and SEQ ID NO: 39, respectively;(b) SEQ ID NO: 38 and SEQ ID NO: 41, respectively;(c) SEQ ID NO: 38 and SEQ ID NO: 43, respectively;(d) SEQ ID NO: 40 and SEQ ID NO: 39, respectively;(e) SEQ ID NO: 40 and SEQ ID NO: 41, respectively;(1) SEQ ID NO: 40 and SEQ ID NO: 43, respectively;(g) SEQ ID NO: 42 and SEQ ID NO: 39, respectively;(h) SEQ ID NO: 42 and SEQ ID NO: 41, respectively; and(i) SEQ ID NO: 42 and SEQ ID NO: 43, respectively.

56. The recombinant nucleic acid sequence of claim 47, further comprising a second nucleic acid sequence comprising a 3’ untranslated region (3’ UTR), optionally wherein the second nucleic acid sequence has at least 90% identity to a 3’ UTR of a eukaryotic gene different than the CFTR gene, optionally wherein the 3’ UTR of a gene is a 3’ UTR of a eukaryotic gene, optionally wherein the 3’ UTR of a gene is a 3’ UTR of a human gene.

57. The recombinant nucleic acid sequence of claim 56, wherein the human gene is selected from the group consisting of the genes listed in Table 1.

58. The recombinant nucleic acid sequence of claim 57, wherein the 3‘ UTR is selected from the group of genes consisting of RPS27. FLII, TRAP, RPL36, ZDHHC12, NDUFB6, IDH3B, PMC5, NDUFB10, APTR, GRN RPS21, TMEM208, HSD17B10. PSMB7, GPS2, ATRAID, SNRPG, HSP90AB1, PFDN2, LAMTOR4, FKBP2, EEF1G, RPS11, NDUFB4, FBL, RPS16, LGALS1, NDUFB1, PTTG1, ATP5PD, SQQR, PFN1, TMSB10, SOD1, COPS3, ATP6V0B. RPN2, ENO1, GSS, and CCT2.

59. The recombinant nucleic acid sequence of claim 47, wherein the recombinant nucleic acid sequence is configured to fit within a vector.

60. The recombinant nucleic acid sequence of claim 59, wherein the vector comprises a linear nucleic acid sequence, a plasmid, a viral particle or a synthetic nanoparticle.

61. The recombinant nucleic acid sequence of claim 60, wherein the viral particle comprises an adeno-associated viral (AAV) particle.

62. A recombinant nucleic acid sequence comprising: a first nucleic acid sequence encoding a N-terminal truncated cystic fibrosis transmembrane regulator (CFTR) protein that is truncated at a position downstream of amino acid residues encoded by exon 11 of a CFTR gene, and a) a splice acceptor site that is immediately upstream of the first nucleic acid sequence; or b) a second nucleic acid sequence that has at least 80% identity to a 3’ untranslated region (3‘ UTR) of a gene different than the CFTR gene.

63. The recombinant nucleic acid sequence of claim 62, wherein the first nucleic acid sequence comprises exon 22 to exon 27 of the CFTR gene.

64. A recombinant nucleic acid sequence comprising: a first nucleic acid sequence encoding a truncated cystic fibrosis transmembrane regulator (CFTR) protein that consists of amino acid residues encoded by exons 22-27 of a CFTR gene, and a) a splice acceptor site that is immediately upstream of the first nucleic acid sequence: or b) a second nucleic acid sequence that has at least 80% identity to a 3’ untranslated region (3’ UTR) of a gene different than the CFTR gene.

65. The recombinant nucleic acid sequence of claim 64, further comprising a 5’ homology' arm upstream of the first nucleic acid sequence and a 3’ homology arm downstream of the first nucleic acid sequence.

66. The recombinant nucleic acid sequence of claim 64, wherein the 5’ homology arm is homologous to a nucleic acid sequence upstream of the target site of the endogenous CFTR gene and the 3’ homology' arm is homologous to a nucleic acid sequence downstream of the target site of the endogenous CFTR gene.

67. A recombinant nucleic acid sequence comprising: a) a first nucleic acid sequence encoding a truncated cystic fibrosis transmembrane regulator (CFTR) protein that comprises amino acid residues encoded by exons of a CFTR gene; b) a 5' homologyarm upstream of the first nucleic acid sequence, and c) a 3' homology- arm downstream of the first nucleic acid sequence; wherein the 5' homology arm is homologous to a sequence that is upstream of a genomic site within an intron of an endogenous CFTR gene in a genome of a cell, wherein the 3' homology- arm is homologous to a sequence that is downstream of the genome site, and wherein the intron of the endogenous CFTR gene is downstream of intron 10 of the endogenous CFTR gene in a genome of a cell.

68. The recombinant nucleic acid sequence of claim 67, wherein exons of the first nucleic acid sequence are codon optimized for expression in human.

69. The recombinant nucleic acid sequence of claim 67, wherein the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 45.

70. The recombinant nucleic acid sequence of claim 67, wherein the first nucleic acid sequence has at least 80% identity to SEQ ID NO: 45.

71. The recombinant nucleic acid sequence of claim 67, wherein the first nucleic acid sequence is free of introns.

72. The recombinant nucleic acid sequence of claim 67, wherein the first nucleic acid sequence comprises at least one intron.

73. A gene editing system, comprising: a) a first nucleic acid sequence encoding a truncated cystic fibrosis transmembrane regulator (CFTR) protein, and b) a targeting moiety or a nucleic acid sequence encoding the targeting moiety; wherein the targeting moiety directs insertion of the first nucleic acid sequence into a genomic site within an intron of an endogenous CFTR gene of a genome of a cell, and wherein the intron is upstream of exon 10 of the endogenous CFTR gene.

74. The gene editing system of claim 73, wherein the targeting moiety comprises a guide RNA (gRNA), a zinc finger nuclease, or a transcription activator-like (TAL) effector nuclease.

75. The gene editing system of claim 73, wherein the gene editing system further comprises a gene editing enzyme or a nucleic acid sequence encoding the gene editing enzyme.

76. The gene editing system of claim 73, wherein the gene editing system results in integration of one or two copies of the first nucleic acid sequence.

77. The gene editing system of claim 73, wherein the truncated CFTR protein comprises amino acid residues encoded by at least exons 10-27 of a CFTR gene.

78. The gene editing system of claim 73, wherein the first nucleic acid sequence comprises exon 2 to exon 27 of the CFTR gene.

79. The gene editing system of claim 78, wherein exons of the first nucleic acid sequence are codon optimized for expression in human.

80. The gene editing system of claim 73, wherein the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 36 or 37.

81. The gene editing system of claim 73, wherein the first nucleic acid sequence is free of introns.

82. The gene editing system of claim 73, wherein the first nucleic acid sequence comprises at least one intron.

83. The gene editing system of claim 82, wherein the at least one intron comprises an intron that is exogenous to the CFTR gene.

84. The gene editing system of claim 83, wherein the at least one intron comprises a synthetic / chimeric intron.

85. The gene editing system of claim 82, wherein each of the at least one intron is less than 600 nucleotides in length.

86. The gene editing system of claim 73, wherein the first nucleic acid sequence comprises a splice acceptor site.

87. The gene editing system of claim 86, wherein the splice acceptor site is exogenous to a 5’ end of the first nucleic acid sequence.

88. The gene editing system of claim 86, wherein the splice acceptor site comprises a sequence identical to the sequence of an endogenous splice acceptor site immediately upstream of exon 11 of the CFTR gene.

89. The gene editing system of claim 86, wherein the splice acceptor site comprises a nucleic acid sequence chosen from the group consisting of the sequences set forth in SEQ ID NOs: 1-3.

90. The gene editing system of claim 73, wherein the genomic site is within intron 1 of the endogenous CFTR gene.

91. The gene editing system of claim 73, wherein the genomic site is about 325 nucleotides down stream of exon 1 of the endogenous CFTR gene.

92. The gene editing system of claim 73, wherein the genomic site is about 79 nucleotides downstream of exon 1 of the endogenous CFTR gene.

93. The gene editing system of claim 73, wherein the genomic site is located within a region from hg38 chr7: 117,480, 148 to hg38 chr7: 117,498,307.

94. The gene editing system of claim 73, wherein the gene editing system comprises a gRNA and a class 2 CRISPR / Cas endonuclease.

95. The gene editing system of claim 94, wherein the gene editing system comprises a gRNA and a Cas9 enzyme.

96. The gene editing system of claim 94, wherein the gRNA comprises a sequence selected from the group consisting of the sequences provided in Table 3.

97. The gene editing system of claim 73, wherein the first nucleic acid sequence comprises a 5’ homology arm upstream of the first nucleic acid sequence and a 3‘ homology arm downstream of the first nucleic acid sequence.

98. The gene editing system of claim 97, wherein the 5’ homology arm is homologous to a nucleic acid sequence upstream of the genomic site within the intron of the endogenous CFTR gene and the 3’ homology' arm is homologous to a nucleic acid sequence downstream of the genomic site.

99. The gene editing system of claim 97, wherein the 5’ homology arm and the 3’ homology’ arm comprise nucleic acid sequences that are at least 80% identical to nucleic acid sequences selected from the group consisting of:(a) SEQ ID NO: 38 and SEQ ID NO: 39, respectively;(b) SEQ ID NO: 38 and SEQ ID NO: 41, respectively;(c) SEQ ID NO: 38 and SEQ ID NO: 43, respectively;(d) SEQ ID NO: 40 and SEQ ID NO: 39, respectively;(e) SEQ ID NO: 40 and SEQ ID NO: 41, respectively;(I) SEQ ID NO: 40 and SEQ ID NO: 43, respectively;(g) SEQ ID NO: 42 and SEQ ID NO: 39, respectively;(h) SEQ ID NO: 42 and SEQ ID NO: 41, respectively; and(i) SEQ ID NO: 42 and SEQ ID NO: 43, respectively.

100. The gene editing system of claim 73, wherein the gene editing system further comprises a second nucleic acid sequence.

101. The gene editing system of claim 100, yvherein the second nucleic acid sequence has at least 80% identity to a 3’ untranslated region (3’ UTR) of a gene different than the CFTR gene.

102. The gene editing system of claim 100, wherein the second nucleic acid sequence has at least 90% identity to a 3’ UTR of a gene different than the CFTR gene.

103. The gene editing system of claim 101, yvherein the 3’ UTR of a gene is a 3’ UTR of a eukaryotic gene.-HO-104. The gene editing system of claim 101, wherein the 3’ UTR of a gene is a 3’ UTR of a human gene.

105. The gene editing system of claim 104, wherein the human gene is selected from the group consisting of the genes listed in Table 1.

106. The gene editing system of claim 105, wherein the 3’ UTR is selected from the group of genes consisting of RPS27, FLII, TRAP, RPL36, ZDHHC12, NDUFB6, IDH3B, PMC5, NDUFB10, APTR, GRN RPS21, TMEM208, HSD17B10, PSMB7, GPS2, ATRAID, SNRPG, HSP90AB1, PFDN2, LAMTOR4, FKBP2, EEF1G, RPS11, NDUFB4, FBL, RPS16, LGALS1, NDUFB1, PTTG1. ATP5PD, SQQR, PFN1. TMSB10, SOD1, COPS3, ATP6V0B, RPN2, ENO1, GSS, and CCT2.

107. The gene editing system of claim 106, wherein the 3’ UTR is selected from the group of genes consisting ofNDUFB6, HSD17B10, PTTG1, and SOD1.

108. The gene editing system of claim 73, wherein the gene editing system is configured to fit within one vector.

109. The gene editing system of claim 73, wherein the gene editing system is configured to fit in two or more vectors.

110. The gene editing system of claim 108, wherein the vector comprises a linear nucleic acid sequence, a plasmid, a viral particle or a synthetic nanoparticle.1 11. The gene editing system of claim 110, wherein the viral particle comprises an adeno- associated viral (AAV) particle.

112. A gene editing system, comprising: a) a recombinant nucleic acid sequence of any one of claims 1-72, and b) a targeting moiety or a nucleic acid sequence encoding the targeting moiety; wherein the targeting moiety directs insertion of the first nucleic acid sequence into a genomic site within an intron of an endogenous CFTR gene of a genome of a cell, and wherein the intron is upstream of exon 10 of the endogenous CFTR gene.

113. The gene editing system of claim 112, wherein the targeting moiety comprises a guide RNA (gRNA), a zinc finger nuclease, or a transcription activator-like (TAL) effector nuclease.

114. The gene editing system of claim 112, wherein the gene editing system further comprises a gene editing enzyme or a nucleic acid sequence encoding the gene editing enzyme.

115. The gene editing system of claim 112, wherein the gene editing system results in integration of one or two copies of the first nucleic acid sequence.1 16. The gene editing system of claim 112, wherein the genomic site is within intron 1 of the endogenous CFTR gene.

117. The gene editing system of claim 112, wherein the genomic site is about 325 nucleotides downstream of exon 1 of the endogenous CFTR gene.

118. The gene editing system of claim 112, wherein the genomic site is about 79 nucleotides downstream of exon 1 of the endogenous CFTR gene.

119. The gene editing system of claim 112, wherein the genomic site is located within a region from hg38 chr7: 117,480, 148 to hg38 chr7:l 17,498,307.

120. The gene editing system of claim 112, wherein the targeting moiety is a CRISPR / Cas nickase.

121. The gene editing system of claim 120, wherein the gene editing system further comprises a recombinase.

122. The gene editing system of claim 121, wherein the recombinase is a domain of the CRISPR / Cas nickase.

123. The gene editing system of claim 121, wherein the recombinase is separate from the CRISPR / Cas nickase.

124. The gene editing system of claim 120, wherein the gene editing system further comprises a reverse transcriptase.

125. The gene editing system of claim 120, wherein the recombinant nucleic acid sequence is flanked by a sequence recognized by a recombinase.

126. The gene editing system of claim 120, wherein the gene editing system further comprises a gRNA encoding a sequence that is recognized by a recombinase.

127. The gene editing system of any one of claims 126, wherein the sequence that is recognized by a recombinase is integrated into the genomic site.

128. The gene editing system of claim 121, wherein the recombinase directs the insertion of the recombinant nucleic acid sequence into the genomic site.

129. The gene editing system of claim 112, wherein the gene editing system comprises a gRNA and a class 2 CRISPR / Cas endonuclease.

130. The gene editing system of claim 129, wherein the gRNA comprises a sequence selected from the group consisting of the sequences provided in Table 3.

131. A gene editing system, comprising: (a) a first nucleic acid sequence encoding a truncated cystic fibrosis transmembrane regulator (CFTR) protein, and (b) a targeting moiety or a nucleic acid sequence encoding the targeting moiety; wherein the targeting moiety directs insertion of the first nucleic acid sequence into a genomic site within anintron of an endogenous CFTR gene of a genome of a cell, and wherein the intron is downstream of intron 10 of the endogenous CFTR gene of the cell.

132. The gene editing system of claim 131, wherein the targeting moiety comprises a guide RNA (gRNA). a zinc finger nuclease, or a transcription activator-like (TAL) effector nuclease.

133. The gene editing system of claim 131, wherein the gene editing system comprises a gRNA and a class 2 CRISPR / Cas endonuclease.

134. The gene editing system of claim 133, wherein the gRNA comprises a sequence selected from the group consisting of the sequences provided in Table 3.

135. A pharmaceutical composition, comprising: a) a pharmaceutically acceptable excipient or carrier; and b) the recombinant nucleic acid sequence of any one of claims 1-72 or the gene editing system of any one of claims 73-134.

136. The pharmaceutical composition of claim 135, wherein the pharmaceutical composition is formulated for intranasal administration, intratracheal administration, or intravenous injection.

137. The pharmaceutical composition of claim 135, wherein the pharmaceutical composition is formulated for pulmonary administration.

138. The pharmaceutical composition of claim 135, wherein the pharmaceutical composition further comprises a second therapeutic agent, optionally wherein the second therapeutic agent comprises a modulator of CFTR protein that potentiates chloride conductance of CFTR protein in the cells, optionally wherein 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.

139. A vector comprising the recombinant nucleic acid sequence of any one of claims 1-72 or the gene editing system of any one of claims 73-134.

140. The vector of claim 139, wherein the vector is a viral vector, optionally a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a herpes simplex viral vector.

141. The vector of claim 139, wherein the vector is a non-viral vector, optionally a lipid nanoparticle.

142. A virus comprising the recombinant nucleic acid sequence of any one of claims 1-72 or the gene editing system of any one of claims 73-134.

143. The virus of claim 142, wherein the virus is an adeno-associated viral (AAV) vector, optionally a retroviral vector, a lentiviral vector, an adenoviral vector, or a herpes simplex viral vector.

144. A cell comprising the recombinant nucleic acid sequence of any one of claims 1-72 or the gene editing system of any one of claims 73-134.

145. The cell of claim 144, wherein the cell is a stem cell.

146. The cell of claim 144, wherein the cell is a human cell.

147. A kit comprising: a) the recombinant nucleic acid sequence of any one of claims 1-72. the gene editing system of any one of claims 73-134, or the pharmaceutical composition of any one of claims 135-138; and b) instructions for use of the recombinant nucleic acid sequence, the gene editing system, or the pharmaceutical composition.

148. A method for modifying a cell, comprising contacting to the cell (a) the recombinant nucleic acid sequence of any one of claims 1-72; (b) the gene editing system of any one of claims 73-134; or (c) the kit of claim 147.

149. A method for modifying a cell, the method comprising contacting an agent or a vector encoding the agent to the cell, wherein the agent modifies a genome of the cell, thereby inserting an exogenous nucleic acid sequence into a genomic site of an intron of an endogenous cystic fibrosis transmembrane regulator (CFTR) gene of the cell, and wherein the intron is downstream of intron 10 of the endogenous CFTR gene of the cell.

150. The method of claim 149, wherein the agent comprises a recombinant nucleic acid sequence encoding a truncated CFTR protein that is encoded by at least exons 22-27 of the CFTR gene.

151. The method of claim 149, wherein the recombinant nucleic acid sequence has at least 70% identity to SEQ ID NO: 45.

152. A method for modifying a cell, the method comprising contacting an agent or a vector encoding the agent to the cell, wherein the agent modifies a genome of the cell, thereby preventing expression of an endogenous CFTR gene in the cell after intron 1.

153. The method of claim 152, wherein the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 36.

154. The method of claim 152, wherein the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 37.

155. A method for modifying a cell, the method comprising contacting an agent or a vector encoding the agent to the cell, wherein the agent modifies a genome of the cell, therebyinserting an exogenous nucleic acid sequence into a genomic site of an intron of an endogenous cystic fibrosis transmembrane regulator (CFTR) gene of the cell, and wherein the intron is upstream of exon 10 of the endogenous CFTR gene of the cell.

156. The method of claim 155, wherein the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 36.

157. The method of claim 155, wherein the first nucleic acid sequence has at least 70% identity to SEQ ID NO: 37.

158. A method of treating a subject in need thereof, comprising administering to the subject a therapeutically effective amount of (a) the recombinant nucleic acid sequence of any one of claims 1-72; (b) the pharmaceutical composition of any one of claims 135-138;(c) the vector of any one of claims 140-141; (d) the virus of claim 142 or 143; or (e) the cell of any one of claims 144-146.

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