Gain of function CFTR variants to enhance the efficacy of genetic therapies treat cystic fibrosis

Abstract

Disclosed herein are methods and compositions for treating cystic fibrosis using gain-of- function CFTR variants inserted into the native CFTR locus via genome editing. The modified CFTR polypeptides contain at least one mutation selected from deletion of amino acids 404-435, 1539T, G550E, or E1371S. Methods include ex vivo editing of airway basal stem cells and prime / base editing approaches for correction and enhancement.

Description

CROSS-REFERENCE TO RELATED PATENT APPLICATION

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication No. 63 / 771,915, filed March 14, 2025, the entire contents of which areincorporated herein by reference.BACKGROUND

[0002] Cystic fibrosis (CF) is a genetic disorder caused by mutations in the cystic fibrosistransmembrane conductance regulator (CFTR) gene. These mutations lead to the productionof thick and sticky mucus that can clog the lungs and obstruct the pancreas. CF is anautosomal recessive disease, meaning that a person must inherit two defective copies of theCFTR gene, one from each parent, to develop the condition.

[0003] In the United States, approximately 40,000 children and adults are living with cysticfibrosis. The prevalence of CF is higher among people of Northern European descent.Current treatments focus on managing symptoms and improving quality of life. Theseinclude medications that target the underlying cause of CF, such as CFTR modulators, whichhelp improve the function of the defective protein. Gene therapy attempts have so far failedto achieve desired results. Thus, there is a great need for effective therapies for CF.SUMMARY OF THE DISCLOSURE

[0004] An aspect of the disclosure is directed to a method for treating cystic fibrosis in asubject in need thereof comprising, or alternatively consisting essentially of, or yet furtherconsisting of administering to the subject a gene editing construct comprising, or alternativelyconsisting essentially of, or yet further consisting of a nucleic acid encoding a modified cysticfibrosis transmembrane conductance regulator (CFTR) polypeptide or a functional fragmentthereof, wherein the modified CFTR polypeptide or the functional fragment thereof differsfrom a wild-type human CFTR protein of SEQ ID NO: 1 by the presence of at least onemutation selected from a deletion of amino acids at positions 404-435, I539T, G550E, S495P,or R553K, wherein the gene editing construct is configured to insert the nucleic acidencoding the modified CFTR polypeptide or functional fragment thereof into the nativeCFTR locus of a cell of the subject.

[0005] In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, or alternatively consists essentially of, or yet further consists of any one ofSEQ ID NO: 2-5.

[0006] In some embodiments, the gene editing construct is delivered within a nanoparticle.

[0007] In some embodiments, the nanoparticle is a lipid nanoparticle or a polymernanoparticle.

[0008] In some embodiments, the nanoparticle comprises, or alternatively consists essentiallyof, or yet further consists of one or more of 1,2-Distearoyl-sn-glycero-3-phosphorylcholine(DSPC), Dioleoylphosphatidylethanolamine (DOPE), Polyethylene glycol lipid (PEG-lipid),1,2-dioleoyl-3-trimethylammonium propane (DOTAP), or Dilinoleyl-methyl-4-dimethylaminobutyrate, MC3 (D-Lin-MC3).

[0009] In some embodiments, the gene editing construct is delivered by a viral vectorselected from an adeno-associated virus (AAV) vector, a bocavirus vector, or a herpesvirusvector.

[0010] In some embodiments, the AAV vector is selected from AAV1, AAV2, AAV4,AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV PHP.B,AAV rh74, AAV 2.5T, or a variant of each thereof.

[0011] In some embodiments, the gene editing construct comprises, or alternatively consistsessentially of, or yet further consists of a CRISPR-Cas system, a base editing system, a primeediting system, a Zinc Finger Nuclease system, or a TALEN system.

[0012] In some embodiments, the CRISPR-Cas system comprises, or alternatively consistsessentially of, or yet further consists of a Cas9 ribonucleoprotein (RNP) and a single guideRNA (sgRNA).

[0013] In some embodiments, the gene editing construct is delivered to airway basal stemcells of the subject ex vivo, and wherein the method further comprises, or alternativelyconsists essentially of, or yet further consists of transplanting the edited airway basal stemcells to the subject's airways.

[0014] In some embodiments, the airway basal stem cells are P63+, KRT5+, and NGFR+.

[0015] In some embodiments, the method further comprises, or alternatively consistsessentially of, or yet further consists of conditioning the subject's airways to remove diseasedairway epithelial cells prior to transplanting the edited airway basal stem cells.

[0016] In some embodiments, the conditioning comprises, or alternatively consistsessentially of, or yet further consists of introducing an acute airway injury to the subject'sairways to remove the diseased airway epithelial cells.

[0017] In some embodiments, the acute airway injury is a chemical injury induced bypolidocanol (PDOC) or a mechanical injury, wherein the mechanical injury is optionallyinduced by an endoscopic device.

[0018] In some embodiments, the conditioning comprises, or alternatively consistsessentially of, or yet further consists of removing at least 30% (e.g., at least 30, 40, 50, 60,70, 75, 80, 85, 90, 95 or 99%) of diseased airway epithelial cells. In some embodiments, theconditioning comprises, or alternatively consists essentially of, or yet further consists ofremoving all (100%) of diseased airway epithelial cells.

[0019] In some embodiments, the subject is mammal, optionally a human.

[0020] Another aspect of the disclosure is directed to a method for treating cystic fibrosis in asubject in need thereof comprising, or alternatively consisting essentially of, or yet furtherconsisting of (a) modifying airway basal stem cells from the subject ex vivo to express amodified cystic fibrosis transmembrane conductance regulator (CFTR) polypeptide or afunctional fragment thereof, wherein the modified CFTR polypeptide or the functionalfragment thereof differs from a wild-type human CFTR protein of SEQ ID NO: 1 by thepresence of at least one mutation selected from a deletion of amino acids at positions 404-435, 1539T, G550E, S495P, or R553K (or optionally E1371S), (b) conditioning the subject'sairways to remove diseased airway epithelial cells, and (c) transplanting the airway basalstem cells to the subject's airways, wherein the modifying in (a) is achieved by a gene editingsystem that inserts a nucleic acid encoding the modified CFTR polypeptide or functionalfragment thereof into the native CFTR locus of the airway basal stem cells.

[0021] Also provided is a method for treating cystic fibrosis in a conditioned subject in needthereof comprising, or alternatively consisting essentially of, or yet further consisting ofadministering to the conditioned subject autologous airway basal stem cells that have beenmodified express a modified cystic fibrosis transmembrane conductance regulator (CFTR)polypeptide or a functional fragment thereof, wherein the modified CFTR polypeptide or thefunctional fragment thereof differs from a wild-type human CFTR protein of SEQ ID NO: 1by the presence of at least one mutation selected from a deletion of amino acids at positions404-435, 1539T, G550E, S495P, or R553K (or optionally E1371S). In one aspect, thesubject's airways have been pre-conditioned prior to the therapy to remove diseased airwayepithelial cells. In a further aspect, the administration of the modified CFTR is by a methodcomprising transplanting the airway basal stem cells to the subject's airways. In a furtheraspect, the modified cells are prepared by a gene editing system that inserts a nucleic acidencoding the modified CFTR polypeptide or functional fragment thereof into the nativeCFTR locus of the airway basal stem cells ex vivo.

[0022] In some embodiments, the airway basal stem cells are P63+, KRT5+, and NGFR+.

[0023] In some embodiments, the conditioning of the subject's airways is by a method thatcomprises, or alternatively consists essentially of, or yet further consists of introducing anacute airway injury to the subject's airways.

[0024] In some embodiments, the acute airway injury a chemical injury induced bypolidocanol (PDOC) or a mechanical injury induced by an endoscopic device.

[0025] In some embodiments, the conditioning comprises, or alternatively consistsessentially of, or yet further consists of removing at least 30% (e.g., at least 30, 40, 50, 60,70, 75, 80, 85, 90, 95 or 99%) of diseased airway epithelial cells. In some embodiments, theconditioning comprises, or alternatively consists essentially of, or yet further consists ofremoving all (100%) of diseased airway epithelial cells.

[0026] In some embodiments, the gene editing construct comprises, or alternatively consistsessentially of, or yet further consists of a CRISPR-Cas system, a base editing system, a primeediting system, a Zinc Finger Nuclease system, or a TALEN system.

[0027] In some embodiments, the gene editing system comprises, or alternatively consistsessentially of, or yet further consists of a Cas9 ribonucleoprotein (RNP) and a single guideRNA (sgRNA).

[0028] In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, or alternatively consists essentially of, or yet further consists of any one ofSEQ ID NO: 2-5.

[0029] In some embodiments, the gene editing system is delivered within a nanoparticle.

[0030] In some embodiments, the nanoparticle is a lipid nanoparticle or a polymernanoparticle.

[0031] In some embodiments, the nanoparticle comprises, or alternatively consists essentiallyof, or yet further consists of one or more of 1,2-Distearoyl-sn-glycero-3-phosphorylcholine(DSPC), Dioleoylphosphatidylethanolamine (DOPE), Polyethylene glycol lipid (PEG-lipid),1,2-dioleoyl-3-trimethylammonium propane (DOTAP), or Dilinoleyl-methyl-4-dimethylaminobutyrate, MC3 (D-Lin-MC3).

[0032] In some embodiments, the gene editing system is delivered by a viral vector selectedfrom an adeno-associated virus (AAV) vector, a bocavirus vector, or a herpesvirus vector.

[0033] In some embodiments, the AAV vector is selected from AAV1, AAV2, AAV4,AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV PHP.B,AAV rh74, AAV 2.5T, or a variant of each thereof.

[0034] In some embodiments, the subject is mammal, optionally a human.

[0035] Another aspect of the disclosure is directed to a composition comprising, oralternatively consisting essentially of, or yet further consisting of a gene editing constructcomprising, or alternatively consisting essentially of, or yet further consisting of a nucleicacid encoding a modified cystic fibrosis transmembrane conductance regulator (CFTR)polypeptide or a functional fragment thereof, wherein the modified CFTR polypeptide or thefunctional fragment thereof differs from a wild-type human CFTR protein of SEQ ID NO: 1by the presence of at least one mutation selected from a deletion of amino acids at positions404-435, 1539T, G550E, S495P, or R553K (or optionally E1371S), wherein the gene editingconstruct is configured to insert the nucleic acid encoding the modified CFTR polypeptide orfunctional fragment thereof into the native CFTR locus of a cell.

[0036] In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, or alternatively consists essentially of, or yet further consists of any one ofSEQ ID NO: 2-5.

[0037] In some embodiments, the gene editing construct is contained within a nanoparticle.

[0038] In some embodiments, the nanoparticle is a lipid nanoparticle or a polymernanoparticle.

[0039] In some embodiments, the nanoparticle comprises, or alternatively consists essentiallyof, or yet further consists of one or more of 1,2-Distearoyl-sn-glycero-3-phosphorylcholine(DSPC), Dioleoylphosphatidylethanolamine (DOPE), Polyethylene glycol lipid (PEG-lipid),1,2-dioleoyl-3-trimethylammonium propane (DOTAP), or Dilinoleyl-methyl-4-dimethylaminobutyrate, MC3 (D-Lin-MC3).

[0040] In some embodiments, the gene editing construct is contained within a viral vectorselected from an adeno-associated virus (AAV) vector, a bocavirus vector, or a herpesvirusvector.

[0041] In some embodiments, the AAV vector is selected from AAV1, AAV2, AAV4,AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV PHP.B,AAV rh74, AAV 2.5T, or a variant thereof.

[0042] In some embodiments, the gene editing construct comprises, or alternatively consistsessentially of, or yet further consists of a CRISPR-Cas system, a base editing system, a primeediting system, a Zinc Finger Nuclease system, or a TALEN system.

[0043] In some embodiments, the CRISPR-Cas system comprises, or alternatively consistsessentially of, or yet further consists of a Cas9 ribonucleoprotein (RNP) and a single guideRNA (sgRNA).

[0044] In some embodiments, the composition further comprises, or alternatively consistsessentially of, or yet further consists of a pharmaceutically acceptable carrier.

[0045] Another aspect of the disclosure is directed to a method for treating cystic fibrosis in asubject having a base-editable mutation or a prime-editable mutation in the CFTR gene, themethod comprising: administering to the subject a gene editing system selected from a baseediting system or a prime editing system, the gene editing system comprising: a base editor ora prime editor; and a first single guide RNA (sgRNA) that targets the base-editable mutationor the prime-editable mutation; and a second sgRNA to install a gain-of-function mutation inthe native CFTR locus, wherein the second sgRNA targets one of (i) codon 1539, (ii) codonG550, (iii) codon S495, or (iv) codon R553 (or optionally (v) codon E1371) with respect toSEQ ID NO: 1, wherein the base editing system or the prime editing system is configured tocorrect the base-editable mutation or the prime-editable mutation and introduce the gain-of-function mutation in the native CFTR locus of a cell of the subject.

[0046] Another aspect of the disclosure is directed to a method for treating cystic fibrosis in asubject having an R553X mutation in the CFTR gene, the method comprising, oralternatively consisting essentially of, or yet further consisting of administering to the subjecta base editing system comprising, or alternatively consisting essentially of, or yet furtherconsisting of an adenosine base editor (ABE), and a first single guide RNA (sgRNA) thattargets the R553X mutation to revert a premature stop codon to an arginine codon; and asecond sgRNA to install a gain-of-function mutation in the native CFTR locus, wherein thesecond sgRNA targets one of (i) codon 1539, (ii) codon G550, (iii) codon S495P or (iv) codonS495 with respect to SEQ ID NO: 1, wherein the base editing system is configured to correctthe R553X mutation and introduce the gain-of-function mutation in the native CFTR locus ofa cell of the subject. In some embodiments, the gain-of function mutation is an I539Tmutation, a G550E mutation, a S495P mutation, or a R553K mutation with respect to SEQ IDNO: 1.

[0047] In some embodiments, the first sgRNA and the second sgRNA are deliveredsimultaneously with the adenosine base editor (ABE).

[0048] In some embodiments, the base editing system is delivered to airway epithelial cellsof the subject.

[0049] In some embodiments, the base editing system is delivered by a nebulizer.

[0050] In some embodiments, the adenosine base editor (ABE) is delivered as mRNA.

[0051] In some embodiments, the base editing system is delivered within a nanoparticle.

[0052] In some embodiments, the nanoparticle is a lipid nanoparticle or a polymernanoparticle.

[0053] In some embodiments, the nanoparticle comprises, or alternatively consists essentiallyof, or yet further consists of one or more of 1,2-Distearoyl-sn-glycero-3-phosphorylcholine(DSPC), Dioleoylphosphatidylethanolamine (DOPE), Polyethylene glycol lipid (PEG-lipid),1,2-dioleoyl-3-trimethylammonium propane (DOTAP), or Dilinoleyl-methyl-4-dimethylaminobutyrate, MC3 (D-Lin-MC3).

[0054] In some embodiments, the base editing system is delivered by a viral vector selectedfrom an adeno-associated virus (AAV) vector, a bocavirus vector, or a herpesvirus vector.

[0055] In some embodiments, the AAV vector is selected from AAV1, AAV2, AAV4,AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV PHP.B,AAV rh74, AAV 2.5T, or a variant thereof.

[0056] In some embodiments, the base editing system is delivered to airway basal stem cellsof the subject ex vivo, and wherein the method further comprises, or alternatively consistsessentially of, or yet further consists of transplanting the edited airway basal stem cells to thesubject's airways.

[0057] In some embodiments, the airway basal stem cells are P63+, KRT5+, and NGFR+.

[0058] In some embodiments, the method further comprises, or alternatively consistsessentially of, or yet further consists of conditioning the subject's airways to remove diseasedairway epithelial cells prior to transplanting the edited airway basal stem cells.

[0059] In some embodiments, the conditioning comprises, or alternatively consistsessentially of, or yet further consists of introducing an acute airway injury to the subject'sairways.

[0060] In some embodiments, the acute airway injury is a chemical injury induced bypolidocanol (PDOC) or a mechanical injury induced by an endoscopic device.

[0061] In some embodiments, the conditioning comprises, or alternatively consistsessentially of, or yet further consists of removing at least 30% (e.g., at least 30, 40, 50, 60,70, 75, 80, 85, 90, 95 or 99%) of diseased airway epithelial cells. In some embodiments, theconditioning comprises, or alternatively consists essentially of, or yet further consists ofremoving all (100%) of diseased airway epithelial cells.

[0062] In some embodiments, the subject is mammal, optionally a human.

[0063] Another aspect of the disclosure is directed to a method for treating cystic fibrosis in asubject having a base-editable mutation or a prime-editable mutation in the CFTR gene, themethod comprising: administering to the subject a gene editing system selected from a baseediting system or a prime editing system, the gene editing system comprising: a base editor ora prime editor; and a first single guide RNA (sgRNA) that targets the base-editable mutationor the prime-editable mutation; and a second sgRNA to install a gain-of-function mutation inthe native CFTR locus, wherein the second sgRNA targets one of (i) codon 1539, (ii) codonG550, (iii) codon S495, or (iv) codon R553 (or optionally (v) codon E1371) with respect toSEQ ID NO: 1, wherein the base editing system or the prime editing system is configured tocorrect the base-editable mutation or the prime-editable mutation and introduce the gain-of-function mutation in the native CFTR locus of a cell of the subject.

[0064] Another aspect of the disclosure is directed to a method for treating cystic fibrosis in asubject having an R553X mutation in the CFTR gene, the method comprising, oralternatively consisting essentially of, or yet further consisting of (a) modifying airway basalstem cells from the subject ex vivo using a base editing system comprising, or alternativelyconsisting essentially of, or yet further consisting of a base editing system, and a first singleguide RNA (sgRNA) that targets the R553X mutation to revert a premature stop codon to anarginine codon; and a second sgRNA to install a gain-of-function mutation in the nativeCFTR locus, wherein the second sgRNA targets one of (i) codon 1539, (ii) codon G550, or(iii) codon E1371 with respect to SEQ ID NO: 1, wherein the base editing system isconfigured to correct the R553X mutation and introduce the gain-of-function mutation in thenative CFTR locus of an airway basal stem cell of the subject, and (b) transplanting themodified airway basal stem cells to the subject's airways. In some embodiments, the methodcomprises conditioning the subject's airways to remove diseased airway epithelial cells beforetransplanting the modified airway basal stem cells to the subject's airways. In someembodiments, the gain-of function mutation is an I539T mutation, a G550E mutation, aS495P mutation, or a R553K mutation with respect to SEQ ID NO: 1.

[0065] In some embodiments, the airway basal stem cells are P63+, KRT5+, and NGFR+.

[0066] In some embodiments, the conditioning in (b) comprises, or alternatively consistsessentially of, or yet further consists of introducing an acute airway injury to the subject'sairways.

[0067] In some embodiments, the acute airway injury is a chemical injury induced bypolidocanol (PDOC) or a mechanical injury induced by an endoscopic device.

[0068] In some embodiments, the conditioning comprises, or alternatively consistsessentially of, or yet further consists of removing at least 30% (e.g., at least 30, 40, 50, 60,70, 75, 80, 85, 90, 95 or 99%) of diseased airway epithelial cells. In some embodiments, theconditioning comprises, or alternatively consists essentially of, or yet further consists ofremoving all (100%) of diseased airway epithelial cells.

[0069] In some embodiments, the first sgRNA and the second sgRNA are deliveredsimultaneously with the adenosine base editor (ABE).

[0070] In some embodiments, the adenosine base editor (ABE) is delivered as mRNA.

[0071] In some embodiments, the base editing system is delivered by a nebulizer.

[0072] In some embodiments, the base editing system is delivered within a nanoparticle.

[0073] In some embodiments, the nanoparticle is a lipid nanoparticle or a polymernanoparticle.

[0074] In some embodiments, the nanoparticle comprises, or alternatively consists essentiallyof, or yet further consists of one or more of 1,2-Distearoyl-sn-glycero-3-phosphorylcholine(DSPC), Dioleoylphosphatidylethanolamine (DOPE), Polyethylene glycol lipid (PEG-lipid),1,2-dioleoyl-3-trimethylammonium propane (DOTAP), or Dilinoleyl-methyl-4-dimethylaminobutyrate, MC3 (D-Lin-MC3).

[0075] In some embodiments, the base editing system is delivered by a viral vector selectedfrom an adeno-associated virus (AAV) vector, a bocavirus vector, or a herpesvirus vector.

[0076] In some embodiments, the AAV vector is selected from AAV1, AAV2, AAV4,AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV PHP.B,AAV rh74, AAV 2.5T, or a variant of each thereof.

[0077] In some embodiments, the subject is mammal, optionally a human.

[0078] An aspect of the disclosure is directed to a method for treating cystic fibrosis in asubject in need thereof comprising, or alternatively consisting essentially of, or yet furtherconsisting of administering to the subject an mRNA encoding a modified cystic fibrosistransmembrane conductance regulator (CFTR) polypeptide or a functional fragment thereof,wherein the modified CFTR polypeptide or the functional fragment thereof differs from awild-type human CFTR protein as shown by SEQ ID NO:1 or a functional fragment thereofby the presence of at least one mutation selected from (i) deletion of amino acids at position404-435, or a mutation selected from one or more of (ii) 1539T, (iii) G550E, (iv) S495P, or(v) R553K with respect to SEQ ID NO: 1.

[0079] In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, consists of, or alternatively consists essentially of, or yet further consistsof SEQ ID NO: 2. In some embodiments, the modified CFTR polypeptide or the functionalfragment thereof comprises, consists of, or alternatively consists essentially of, or yet furtherconsists of SEQ ID NO: 3. In some embodiments, the modified CFTR polypeptide or thefunctional fragment thereof comprises, consists of, or alternatively consists essentially of, oryet further consists of SEQ ID NO: 4. In some embodiments, the modified CFTRpolypeptide or the functional fragment thereof comprises, consists of, or alternatively consistsessentially of, or yet further consists of SEQ ID NO: 5.

[0080] In some embodiments, the mRNA is administered within a nanoparticle, optionally alipid nanoparticle or a polymer nanoparticle.

[0081] In some embodiments, the nanoparticle comprises, consists of, or alternativelyconsists essentially of, or yet further consists of one or more of 1,2-Distearoyl-sn-glycero-3-phosphorylcholine (DSPC), Dioleoylphosphatidylethanolamine (DOPE), Polyethylene glycollipid (PEG-lipid), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), or Dilinoleyl-methyl-4-dimethylaminobutyrate, MC3 (D-Lin-MC3).

[0082] In some embodiments, a cDNA encoding the mRNA is administered to the subject byan adeno-associated virus (AAV) vector, a bocavirus vector or a herpesvirus vector. In someembodiments, the AAV vector is selected from AAV1, AAV2, AAV4, AAV5, AAV6,AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV PHP.B, AAV rh74, AAV2.5T or variants of each thereof.

[0083] In some embodiments, the mRNA is administered to the subject once every day,every two days, every three days, every four days, every five days, every six days, once aweek, once every two weeks, once every three weeks or once a month.BRIEF DESCRIPTION OF THE FIGURES

[0084] FIGS. 1A-1B show that between 30-80% of cells were genetically modified toexpress the tested CFTR variants. (A) Percent cells expressing wild type (WT) and fourCFTR mutants that are believed to be stability enhancing. (B) Percent cells expressing wildtype (WT) and CFTR mutants that are believed to increase probability of CFTR channelopening.

[0085] FIGS. 2A-2B show that Delta-RI (dRI – which is a variant that is missing aminoacids 404-435 of SEQ ID NO: 1 (WT CFTR protein)) and 1539T Stability EnhancingVariants improve CFTR function relative to WT CFTR cDNA. (A) CFTR Function wasmeasured using a Ussing Chamber. (B) Two CFTR variants (dRI and 1539T) showedimproved function relative to WT (even before adjusting for gene editing efficiencies).

[0086] FIGS. 3A-3D show that G550E and E1371S Conductivity Enhancing Variantsimprove CFTR function relative to WT CFTR cDNA (SEQ ID NO: 1). (A) CFTR Functionwas measured using a Ussing Chamber. (B) Two CFTR variants (G550E and E1371S)showed improved function relative to WT (even before adjusting for gene editingefficiencies). (C) Comparison of the stability enhancing mutants showed that dRI and I539Tvariants show the most current change fold difference from the WT when adjusted for geneediting efficiency. (D) Comparison of the conductivity enhancing mutants showed thatG550E and E1371S variants show the most current change fold difference from the WT whenadjusted for gene editing efficiency.

[0087] FIGS. 4A-4B show testing CFTR variants for improved levels of Cl- transport. Inexperiments using ABCs from multiple donors, populations of edited ABCs were obtainedwith 30-70% tCD19+ cells for (A) both stability and (B) conductivity enhancing variants.

[0088] FIGS. 5A-5C show the Cl- ion transfer activity of the CFTR variants in an Ussingchamber as measured by the change in short circuit current (ΔΙsc) following treatment withCFTR-inh172. Three variants show improved function relative to WT when we measuredCFTR function using the Ussing chamber assay. (A) Delta-RI (dRI – which is a variant thatis missing amino acids 404-435 of SEQ ID NO: 1 (WT CFTR protein)) and 1539T StabilityEnhancing Variants improve CFTR function relative to WT CFTR cDNA (SEQ ID NO:1).(B) The I539T variant show the most current change fold difference. (C) Tested conductivity-improving mutants did not show improved function relative to WT. Statistical significancewas assessed using one-way analysis of variance (ANOVA) followed by the Dunnett's test,using the WT-cDNA-corrected cells as the comparison group. ** and * represent P < 0.01and P < 0.05, respectively.

[0089] FIGS. 6A-6B show that variants I539T, G550E and dRI show improved CFTRfunction relative to WT cDNA. (A) CFTR function as measured via Ussing chamber,compared to the editing measured in each sample. Median CFTR function was determinedfrom non-CF samples across multiple experiments and is displayed by the vertical dashedline. Using linear regression, the 95% confidence interval was used to determine thepercentage of edited cells that were required to reach median CFTR function. (B) Similarlinear regressions were plotted for each CFTR variant. From this analysis, the 95%confidence interval for reaching median CFTR function was determined for each insertedCFTR variant and compared to the interval for WT-cDNA.

[0090] FIGS. 7A-7C show that variants I539T, G550E and dRI show improved CFTRfunction relative to WT cDNA (SEQ ID NO: 1). (A) 1539T (B) G550E and (C) dRI CFTRfunction compared to the editing measured in each sample. Each graph shows the functionaldata observed with the best performing CFTR variants, for comparison to cells edited withWT-CFTR. The median CFTR response observed in non-CF cells is shown by the horizontaldashed line, while the amount of editing required for each variant to reach this level offunction is shown by the vertical dashed lines. Correction with WT cDNA reaches mediannon-CF function when 75% cells are corrected. In contrast the variants reach median non-CFfunction when 25%-35% cells are corrected.

[0091] FIGS. 8A-8B show that variants I539T, G550E and dRI also show improved HCO3-transport. (A) A representative trace from an Ussing chamber assay measuring the HCO3 iontransfer by each of the best performing CFTR variants. (B) The HCO3 ion transfer activity ofthe best performing CFTR variants, as measured by the AIsc following treatment with CFTR-inh172. n=4.

[0092] FIGS. 9A-9B show that variants I539T, G550E and dRI variants show improvedprotein expression. (A)-(B) Immunoblots showing restored CFTR expression in airway cellscorrected using the sequential gene replacement approach. dRI and I539T variants show moreCFTR expression relative to cells corrected using the WT cDNA variant (SEQ ID NO: 1).Surprisingly, variant G550E also shows more mature CFTR even though it was thought of asa conductivity enhancing variant.

[0093] FIGS. 10A-10B show that gain of function variants are more effective whenexpressed from the native locus. (A) Percent transfection of differentiated airway cells asmeasured by the detection of fluorescent cells via flow cytometry. n=2. EGTA treatmenthelped improve LNP transfection of ALI cultures. (B) The indicated CFTR mRNA waspackaged into LNPs then used to transfect differentiated ALI cultures with or without priorEGTA treatment of the cells. Ion transport by the treated cells was measured in an Ussingchamber 24 hours after treatment. CFTR function was determined by measuring the changein ion current after addition of CFTRinh172 in the Ussing chamber. A significant differencebetween I539T and WT-CFTR mRNA was not observed. 1539T CFTR did not improverestoration of CFTR function relative to WT CFTR.

[0094] FIGS. 11A-11B show a schematic of base editor mediated installation of a CFTR-enhancing mutation. (A) An adenosine base editor (ABE) with an R553X-targeting sgRNAcan revert the stop codon back to an arginine codon. An I539-targeting sgRNA can install the1539T mutation to enhance CFTR function. Simultaneous delivery of both of these sgRNAswith an ABE to a person with CF caused by the R553X mutation has the potential to correctthe R553X allele, while also enhancing activity in treated cells by installing the I539Tmutation. (B) An experimental workflow for assessing base editor effectiveness at improvingCFTR function.

[0095] FIGS. 12A-12C show that base editing can introduce I539T variant and correctR553X, which improve Cl transfer. (A) Percent cells containing the indicated edited R553Xallele. (B) Response to CFTRinh. 3 days after a nebulizer, an aliquot of treated HBECs werelysed with quick extract solution. PCR amplicons were generated for exon 12 and sequencedusing Oxford Nanopore sequencing. The percentage of reads displaying a WT-CFTR allele ora functional CFTR allele with the I539T mutation were quantified to determine the number ofcells expressing functional CFTR. Simultaneously, a second aliquot of each sample was usedto seed transwell membrane plates. Once these cells produced a confluent monolayer, apicalmedia was removed and the cells were differentiated at an air-liquid interface for 4 weeks.Cells were then placed in an Ussing Chamber to measure Cl- transport through them. Thechange in current following treatment with CFTRinh172 was measured to determine CFTRfunction. A 29% improvement was observed in this response in cells that received bothgRNAs, compared to the cells that received only the R553X-targeting sgRNA. (C) Variantsfor which percent of edited cells to reach median non-CF CFTR function is lower comparedto the WT CDNA. K978C and K1250A appear blank because they never reach median non-CF CFTR function.

[0096] FIGS. 13A-13B show alternative therapeutic approaches for treating CF. (A) Ex-vivo autologous cell therapy. (B) In vivo correction of mutation (e.g., a premature stopcodon) combined with introducing a CFTR gain of function (GF) variant. Base or primeeditor is to simultaneously correct a pathogenic variant and introduce a gain of functionvariant.DETAILED DESCRIPTIONDefinitions

[0097] As it would be understood, the section or subsection headings as used herein is fororganizational purposes only and are not to be construed as limiting and / or separating thesubject matter described.

[0098] Unless defined otherwise, all technical and scientific terms used herein have the samemeanings as commonly understood by one of ordinary skill in the art to which this disclosurebelongs. Although any methods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present disclosure, the preferred methods, devices,and materials are now described. All technical and patent publications cited herein areincorporated herein by reference in their entirety. Nothing herein is to be construed as anadmission that the disclosure is not entitled to antedate such disclosure by virtue of priordisclosure.

[0099] The practice of the present disclosure will employ, unless otherwise indicated,conventional techniques of tissue culture, immunology, molecular biology, microbiology, cellbiology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook andRussell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubelet al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology(Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRLPress at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach;Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture ofAnimal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) OligonucleotideSynthesis; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic AcidHybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984)Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal(1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene TransferVectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) GeneTransfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987)Immunochemical Methods in Cell and Molecular Biology (Academic Press, London);Herzenberg et al. eds (1996) Weir's Handbook of Experimental Immunology; Manipulatingthe Mouse Embryo: A Laboratory Manual, 3rd edition (Cold Spring Harbor Laboratory Press(2002)); Sohail (ed.) (2004) Gene Silencing by RNA Interference: Technology andApplication (CRC Press).

[0100] As used in the specification and claims, the singular form “a,” “an” and “the” includeplural references unless the context clearly dictates otherwise. For example, the term "a cell”includes a plurality of cells, including mixtures thereof.

[0101] As used herein, the term "comprising" is intended to mean that the compounds,agents, compositions and methods include the recited elements, but not exclude others."Consisting essentially of" when used to define compounds, agents, compositions andmethods, shall mean excluding other elements of any essential significance to thecombination. Thus, a composition consisting essentially of the elements as defined hereinwould not exclude trace contaminants, e.g., from the isolation and purification method andpharmaceutically acceptable carriers, preservatives, and the like. "Consisting of” shall meanexcluding more than trace elements of other ingredients. Embodiments defined by each ofthese transition terms are within the scope of this technology.

[0102] All numerical designations, e.g., pH, temperature, time, concentration, and molecularweight, including ranges, are approximations which are varied (+) or (-) by increments of 1,5, or 10%. It is to be understood, although not always explicitly stated that all numericaldesignations are preceded by the term “about.” It also is to be understood, although notalways explicitly stated, that the reagents described herein are merely exemplary and thatequivalents of such are known in the art.

[0103] The term "about,” as used herein when referring to a measurable value such as anamount or concentration and the like, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0104] As used herein, comparative terms as used herein, such as high, low, increase,decrease, reduce, or any grammatical variation thereof, can refer to certain variation from thereference. In some embodiments, such variation can refer to about 10%, or about 20%, orabout 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about90%, or about 1 fold, or about 2 folds, or about 3 folds, or about 4 folds, or about 5 folds, orabout 6 folds, or about 7 folds, or about 8 folds, or about 9 folds, or about 10 folds, or about20 folds, or about 30 folds, or about 40 folds, or about 50 folds, or about 60 folds, or about 70folds, or about 80 folds, or about 90 folds, or about 100 folds or more higher than thereference. In some embodiments, such variation can refer to about 1%, or about 2%, or about3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 0%, or about10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%,or about 75%, or about 80%, or about 85%, or about 90%, or about 95%, or about 96%, orabout 97%, or about 98%, or about 99% of the reference.

[0105] As will be understood by one skilled in the art, for any and all purposes, all rangesdisclosed herein also encompass any and all possible subranges and combinations ofsubranges thereof. Furthermore, as will be understood by one skilled in the art, a rangeincludes each individual member.

[0106] "Optional" or "optionally" means that the subsequently described circumstance mayor may not occur, so that the description includes instances where the circumstance occursand instances where it does not.

[0107] As used herein, “and / or" refers to and encompasses any and all possible combinationsof one or more of the associated listed items, as well as the lack of combinations wheninterpreted in the alternative (“or”).

[0108] "Substantially” or “essentially” means nearly totally or completely, for instance, 95%or greater of some given quantity. In some embodiments, “substantially” or “essentially”means 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0109] The terms or "acceptable," "effective,” or “sufficient" when used to describe theselection of any components, ranges, dose forms, etc. disclosed herein intend that saidcomponent, range, dose form, etc. is suitable for the disclosed purpose.

[0110] A "composition" is intended to mean a combination of active agent and anothercompound or composition, inert (for example, a detectable agent or label) or active, such asan adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative,adjuvant or the like and include pharmaceutically acceptable carriers.

[0111] Carriers also include pharmaceutical excipients and additives proteins, peptides,amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri, tetra-oligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids,esterified sugars and the like; and polysaccharides or sugar polymers), which can be presentsingly or in combination, comprising alone or in combination 1-99.99% by weight or volume.Exemplary protein excipients include serum albumin such as human serum albumin (HSA),recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acidcomponents, which can also function in a buffering capacity, include alanine, arginine,glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine,isoleucine, valine, methionine, phenylalanine, aspartame, and the like. Carbohydrateexcipients are also intended within the scope of this technology, examples of which includebut are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose,and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches,and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol)and myoinositol.

[0112] A composition as disclosed herein can be a pharmaceutical composition. A"pharmaceutical composition" is intended to include the combination of an active agent witha carrier, inert or active, making the composition suitable for diagnostic or therapeutic use invitro, in vivo or ex vivo.

[0113] “Pharmaceutically acceptable carriers" refers to any diluents, excipients, or carriersthat may be used in the compositions disclosed herein. Pharmaceutically acceptable carriersinclude ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassiumsorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogenphosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-blockpolymers, polyethylene glycol and wool fat. Suitable pharmaceutical carriers are described inRemington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference textin this field. They may be selected with respect to the intended form of administration, that is,oral tablets, capsules, elixirs, syrups and the like, and consistent with conventionalpharmaceutical practices.

[0114] The compositions used in accordance with the disclosure can be packaged in dosageunit form for ease of administration and uniformity of dosage. The term "unit dose" or"dosage" refers to physically discrete units suitable for use in a subject, each unit containing apredetermined quantity of the composition calculated to produce the desired responses inassociation with its administration, i.e., the appropriate route and regimen. The quantity to beadministered, both according to number of treatments and unit dose, depends on the resultand / or protection desired. Precise amounts of the composition also depend on the judgmentof the practitioner and are peculiar to each individual. Factors affecting dose include physicaland clinical state of the subject, route of administration, intended goal of treatment(alleviation of symptoms versus cure), and potency, stability, and toxicity of the particularcomposition. Upon formulation, solutions are administered in a manner compatible with thedosage formulation and in such amount as is therapeutically or prophylactically effective.The formulations are easily administered in a variety of dosage forms, such as the type ofinjectable solutions described herein.

[0115] A combination as used herein intends that the individual active ingredients of thecompositions are separately formulated for use in combination and can be separatelypackaged with or without specific dosages. The active ingredients of the combination can beadministered concurrently or sequentially.

[0116] An "effective amount” is an amount sufficient to effect beneficial or desired results.An effective amount can be administered in one or more administrations, applications, ordosages. Such delivery is dependent on a number of variables including the time period forwhich the individual dosage unit is to be used, the bioavailability of the therapeutic agent, theroute of administration, etc. It is understood, however, that specific dose levels of thetherapeutic agents disclosed herein for any particular subject depends upon a variety offactors including the activity of the specific agent employed, bioavailability of the agent, theroute of administration, the age of the animal and its body weight, general health, sex, the dietof the animal, the time of administration, the rate of excretion, the drug combination, and theseverity of the particular disorder being treated and form of administration. In general, onewill desire to administer an amount of the agent that is effective to achieve a serum levelcommensurate with the concentrations found to be effective in vivo. These considerations, aswell as effective formulations and administration procedures are well known in the art andare described in standard textbooks.

[0117] "Therapeutically effective amount of an agent refers to an amount of the agent thatis an amount sufficient to obtain a pharmacological response; or alternatively, is an amount ofthe agent that, when administered to a patient with a specified disorder or disease, issufficient to have the intended effect, e.g., treatment, alleviation, amelioration, palliation orelimination of one or more manifestations of the specified disorder or disease in the patient.A therapeutic effect does not necessarily occur by administration of one dose, and may occuronly after administration of a series of doses. Thus, a therapeutically effective amount may beadministered in one or more administrations.

[0118] As used herein, the phrase “derived from” means isolated from, purified from, orengineered from, or any combination thereof.

[0119] As used herein, "treating" or "treatment" of a disease in a subject refers to (1)preventing the symptoms or disease from occurring in a subject that is predisposed or doesnot yet display symptoms of the disease; (2) inhibiting the disease or arresting itsdevelopment; or (3) ameliorating or causing regression of the disease or the symptoms of thedisease. As understood in the art, "treatment" is an approach for obtaining beneficial ordesired results, including clinical results. For the purposes of the present technology,beneficial or desired results can include one or more, but are not limited to, alleviation oramelioration of one or more symptoms, diminishment of extent of a condition (including adisease), stabilized (i.e., not worsening) state of a condition (including disease), delay orslowing of condition (including disease), progression, amelioration or palliation of thecondition (including disease), states and remission (whether partial or total), whetherdetectable or undetectable. In one aspect, treatment excludes prophylaxis.

[0120] As used herein, the term "subject" is used interchangeably with “patient," andindicates a mammal, in particular a human, equine, bovine, porcine, feline, canine, murine,rat, or non-human primate. In preferred embodiments, the subject is a human.

[0121] In one embodiment, the term "disease” or “disorder" as used herein refers to cysticfibrosis.

[0122] The term “contacting” means direct or indirect binding or interaction between two ormore. A particular example of direct interaction is binding. A particular example of anindirect interaction is where one entity acts upon an intermediary molecule, which in turn actsupon the second referenced entity. Contacting as used herein includes in solution, in solidphase, in cell culture, in vitro, ex vivo, in a cell and in vivo. Contacting in vivo can bereferred to as administering, or administration.

[0123] "Administration” or “delivery” of an oncolytic virus or a composition containingsame can be performed in one dose, continuously or intermittently throughout the course oftreatment. Methods of determining the most effective means and dosage of administration areknown to those of skill in the art and will vary with the composition used for therapy, thepurpose of the therapy, the target cell being treated, and the subject being treated. Single ormultiple administrations can be carried out with the dose level and pattern being selected bythe treating physician or in the case of animals, by the treating veterinarian. Suitable dosageformulations and methods of administering the agents are known in the art. Route ofadministration can also be determined and method of determining the most effective route ofadministration are known to those of skill in the art and will vary with the composition usedfor treatment, the purpose of the treatment, the health condition or disease stage of the subjectbeing treated, and target cell or tissue. Non-limiting examples of route of administrationinclude oral administration, intraperitoneal, infusion, nasal administration, inhalation,injection, and topical application. In some embodiments, administering or a grammaticalvariation thereof also refers to more than one doses with certain interval. In someembodiments, the interval is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year orlonger. In some embodiments, one dose is repeated for once, twice, three times, four times,five times, six times, seven times, eight times, nine times, ten times or more.

[0124] The term administration shall include without limitation, administration by oral,parenteral (e.g., intramuscular, intraperitoneal, intravenous, intravascular, intraperitoneal,intracerebroventricular (ICV), intrathecal, intracisternal injection or infusion, intracranial,ocular, intradermally, percutaneously, subcutaneous injection, or implant), intratumorally, byinhalation spray nasal, intratracheal, vaginal, rectal, sublingual, urethral (e.g., urethralsuppository) or topical routes of administration (e.g., gel, ointment, cream, aerosol, etc.) andcan be formulated, alone or together, in suitable dosage unit formulations containingconventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, andvehicles appropriate for each route of administration. The disclosure is not limited by theroute of administration, the formulation or dosing schedule. In one aspect, administrationcomprises inhalation. In one aspect, administration comprises an intravenous route.

[0125] An agent of the present disclosure can be administered for therapy by any suitableroute of administration. It will also be appreciated that the optimal route will vary with thecondition and age of the recipient, and the disease being treated.

[0126] Administration or treatment in “combination" refers to administering two agents suchthat their pharmacological effects are manifest at the same time. Combination does notrequire administration at the same time or substantially the same time, although combinationcan include such administrations.

[0127] The terms "oligonucleotide" or "polynucleotide" or "portion," or "segment" thereofrefer to a stretch of polynucleotide residues which is long enough to use in PCR or varioushybridization procedures to identify or amplify identical or related parts of mRNA or DNAmolecules. The polynucleotide compositions of this invention include RNA, cDNA, genomicDNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may bechemically or biochemically modified or may contain non-natural or derivatized nucleotidebases, as will be readily appreciated by those skilled in the art. Such modifications include,for example, labels, methylation, substitution of one or more of the naturally occurringnucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages(e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides),intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g.,alpha anomeric nucleic acids, etc.). Also included are synthetic molecules that mimicpolynucleotides in their ability to bind to a designated sequence via hydrogen bonding andother chemical interactions. Such molecules are known in the art and include, for example,those in which peptide linkages substitute for phosphate linkages in the backbone of themolecule.

[0128] As used herein, the term “purified" does not require absolute purity; rather, it isintended as a relative term. Thus, for example, a purified nucleic acid, peptide, protein,biological complexes, cell, virus or other active compound is one that is isolated in whole orin part from proteins or other contaminants. Generally, substantially purified peptides,proteins, biological complexes, cell, virus or other active compounds for use within thedisclosure comprise more than 80% of all macromolecular species present in a preparationprior to admixture or formulation of the peptide, protein, biological complex, cell, virus orother active compound with a pharmaceutical carrier, excipient, buffer, absorption enhancingagent, stabilizer, preservative, adjuvant or other co-ingredient in a complete pharmaceuticalformulation for therapeutic administration. More typically, the peptide, protein, biologicalcomplex, cell, virus or other active compound is purified to represent greater than 90%, oftengreater than 95% of all macromolecular species present in a purified preparation prior toadmixture with other formulation ingredients. In other cases, the purified preparation may beessentially homogeneous, wherein other macromolecular species are not detectable byconventional techniques.

[0129] In some embodiments, the term “engineered” or “recombinant” refers to having atleast one modification not normally found in a naturally occurring protein, polypeptide,polynucleotide, strain, wild-type strain or the parental host strain of the referenced species. Insome embodiments, the term “engineered” or “recombinant” refers to being synthetized byhuman intervention.

[0130] As used herein, the term "gain-of-function mutation" intends refers to a change in thenucleotide sequence of the CFTR gene or the amino acid sequence of the CFTR polypeptidethat increases one or more functional properties of CFTR relative to the wild-type CFTRprotein (SEQ ID NO: 1). Such functional properties include, but are not limited to, increasedchannel opening probability, increased ion conductivity (e.g., chloride, bicarbonatetransport), increased protein stability, increased expression of mature CFTR at the cellsurface, or increased half-life of functional protein. Gain-of-function mutations may benaturally occurring or engineered, and in the context of the present disclosure are exemplifiedby mutations selected from deletion of amino acids 404-435 (delta-RI), 1539T, G550E,E1371S, S495P, R553K, or combinations thereof.

[0131] As used herein, the term “native CFTR locus" refers to the genomic location of theendogenous cystic fibrosis transmembrane conductance regulator (CFTR) gene within thechromosomal DNA of a cell, including associated regulatory elements such as promoters,untranslated regions (UTRs), introns, and exons. In a human cell, the native CFTR locus islocated on chromosome 7 at position q31.2. Gene editing into the native CFTR locus refers toinsertion, replacement, or modification of CFTR coding or regulatory sequences directlywithin the endogenous CFTR locus, rather than expression from an exogenous site (e.g.,random integration, episomal expression).

[0132] As used herein, the term "functional fragment" refers to a portion of a CFTRpolypeptide that retains full or partial CFTR channel function, including chloride andbicarbonate transport, relative to the wild-type CFTR protein. A functional fragment maymaintain structural domains sufficient for ion channel activity and regulation despitedeletions, insertions, or substitutions elsewhere in the molecule. Exemplary functionalfragments include the delta-RI variant (deletion of amino acids at positions 404–435 fromSEQ ID NO: 1) and variants thereof with additional gain-of-function mutations.

[0133] As used herein, the term “acute airway injury" refers to a temporary disruption orremoval of airway epithelial cells to facilitate engraftment of transplanted or corrected airwaybasal stem cells. Acute airway injury may be chemical, mechanical, thermal, or induced byother methods. Chemical injury is exemplified by administration of polidocanol (PDOC) tothe airway surface; mechanical injury may be induced using endoscopic or bronchoscopicdevices to physically debride or scrape the airway epithelium. The injury is performed underconditions that allow regeneration of airway epithelium from transplanted cells whileminimizing long-term airway damage.

[0134] As used herein, “CFTRinh-172" refers to 3-[2-(3-carbamoylpyridin-5-yl)ethyl]benzoic acid, a small molecule inhibitor of CFTR chloride channel activity,commercially available from Sigma-Aldrich (catalog no. C2992). CFTRinh-172 is used inUssing chamber assays to block CFTR-mediated ion transport for purposes of functionalmeasurement.

[0135] As used herein, the term "deletion of amino acids at positions 404-435" refers toremoval of the contiguous amino acids in the wild-type human CFTR protein (SEQ ID NO:1) corresponding to positions 404 through 435 inclusive. This deletion is referred to hereininterchangeably as "delta-RI" or "ARI” and results in increased stability and functionalactivity of CFTR when inserted at the native CFTR locus.

[0136] As used herein, the term "configured" in the context of phrases such as "configured toinsert," "configured to correct," or "configured to deliver," refers to the design, preparation,or adaptation of a therapeutic construct, composition, or device so that, under the conditionsof intended use, it is capable of performing or perform the recited function. When the geneediting construct is configured to insert a nucleic acid encoding a modified CFTRpolypeptide or functional fragment into the native CFTR locus, "configured" encompassesinclusion of targeting elements such as one or more guide RNAs designed to hybridize to theCFTR genomic sequence at or near the desired insertion site, inclusion of an insertion orrepair template containing the CFTR coding sequence or variant thereof flanked byappropriate homology arms, selection of delivery vehicles such as adeno-associated virus(e.g., AAV2, AAV6, AAV 2.5T), lipid nanoparticles containing DSPC, DOPE, PEG-lipid,DOTAP, or MC3, or polymeric nanoparticles, and optimization of delivery conditionsincluding concentration of components, timing, and use of enhancer molecules. In the contextof "configured to correct," the term includes selection of nucleic acid editing systems such asbase editors with specific guide RNAs targeting a disease-causing mutation in CFTR, primeeditors with pegRNAs encoding the corrective sequence, or CRISPR-Cas nucleases withdonor templates, together with delivery vehicles and protocols suited to the relevant cell type.In the context of "configured to deliver,” the term includes preparation of the formulation orassembly within a carrier that enables introduction into target cells or tissues, such aspackaging into AAV capsids, encapsulation in nanoparticles, or aerosolization for inhalation.Examples provided herein include electroporation of Cas9 RNP into human bronchialepithelial cells followed by immediate transduction with AAV carrying the two halves ofCFTR CDNA, use of AAV6 vectors to deliver HDR templates, and EGTA pretreatment ofairway cultures to enhance lipid nanoparticle transfection. Examples of functional outcomesinclude insertion, correction, or delivery of the designated CFTR sequence, as confirmed byassays such as flow cytometry for marker expression, amplicon sequencing of the CFTRlocus, and Ussing chamber measurements of chloride and bicarbonate transport.

[0137] As used herein, "a CRISPR-Cas system" refers to a clustered regularly interspacedshort palindromic repeats (CRISPR) associated (Cas) nuclease system used for genomeediting. A CRISPR-Cas system typically comprises a Cas nuclease protein (such as Cas9,Cas12a, Cas12b, Cas13, or variants thereof) and one or more guide RNAs (gRNAs orsgRNAs) that direct the Cas nuclease to a specific genomic locus by complementary basepairing with a target DNA sequence adjacent to a protospacer adjacent motif (PAM). Uponbinding to the target locus, the Cas nuclease induces a double-strand break (DSB) or single-strand break in the genomic DNA, which can be repaired by cellular DNA repair pathwayssuch as non-homologous end joining (NHEJ) or homology-directed repair (HDR). In thecontext of the present disclosure, the CRISPR-Cas system may be used to insert a nucleicacid encoding a modified CFTR polypeptide into the native CFTR locus. The CRISPR-Cassystem may be delivered as a ribonucleoprotein (RNP) complex comprising pre-assembledCas protein and guide RNA, as mRNA encoding the Cas protein together with guide RNA, asDNA encoding the Cas protein and guide RNA, or within a viral or non-viral deliveryvehicle. In some embodiments, the CRISPR-Cas system further comprises a donor DNAtemplate that provides the nucleic acid sequence encoding the modified CFTR polypeptide orfunctional fragment thereof for insertion into the cleaved genomic locus via homology-directed repair.

[0138] As used herein, "a base editing system" refers to a gene editing platform that performsprecise nucleotide substitutions in genomic DNA without requiring double-strand breaks ordonor DNA templates. A base editing system typically comprises a catalytically impaired orinactive DNA-targeting protein (such as a catalytically dead Cas9 (dCas9) or nickase Cas9(nCas9)) fused to a deaminase enzyme that chemically modifies a target nucleotide base.Base editing systems include, but are not limited to, cytosine base editors (CBEs) that convertcytosine (C) to thymine (T) (or guanine (G) to adenine (A) on the complementary strand) andadenosine base editors (ABEs) that convert adenine (A) to guanine (G) (or thymine (T) tocytosine (C) on the complementary strand). The base editing system is directed to a specificgenomic locus by one or more guide RNAs (such as single guide RNAs (sgRNAs)) thathybridize to a target sequence adjacent to a protospacer adjacent motif (PAM). Base editingsystems may further include additional components such as uracil glycosylase inhibitors(UGI) to enhance editing efficiency and reduce undesired byproducts. In some embodiments,the base editing system is delivered as a ribonucleoprotein complex, as mRNA encoding thebase editor components, as DNA encoding the base editor components, or within a viral ornon-viral delivery vehicle.

[0139] As used herein, "a base-editable mutation" refers to a mutation in a nucleic acidsequence that can be corrected or modified using a base editing system. A base-editablemutation is typically a point mutation (a single nucleotide substitution) that falls within theediting window of a base editor and is amenable to conversion by the deaminase enzymecomponent of the base editing system. For cytosine base editors (CBEs), a base-editablemutation includes mutations where a C-to-T (or G-to-A) conversion would restore wild-typesequence or install a desired mutation. For adenosine base editors (ABEs), a base-editablemutation includes mutations where an A-to-G (or T-to-C) conversion would restore wild-typesequence or install a desired mutation. The mutation must be located within an accessiblegenomic region that can be targeted by a guide RNA and must fall within the base editor'sactivity window (typically positions 4-8 or 4-9 of the protospacer sequence, depending on thespecific base editor architecture). In the context of the present disclosure, base-editablemutations in the CFTR gene include, but are not limited to, the R553X nonsense mutation(which can be reverted to wild-type arginine using an adenosine base editor) and wild-typecodons that can be modified to install gain-of-function mutations such as 1539T (which canbe installed by converting the isoleucine codon to a threonine codon using an adenosine baseeditor).

[0140] As used herein, "a Zinc Finger Nuclease system" or "ZFN system" refers to a geneediting platform comprising an engineered zinc finger DNA-binding domain fused to a FokInuclease domain. Zinc finger nucleases recognize and bind to specific DNA sequencesthrough an array of zinc finger motifs, each of which typically recognizes three base pairs ofDNA. Multiple zinc finger motifs are assembled in tandem to create a zinc finger protein thatrecognizes an extended target sequence of approximately 9-18 base pairs. The FokI nucleasedomain, which functions as a dimer, cleaves the DNA at the target locus when two ZFNmonomers bind to adjacent target sequences in the proper orientation and spacing. Theresulting double-strand break can be repaired by cellular DNA repair mechanisms such asnon-homologous end joining (NHEJ) or homology-directed repair (HDR). In the context ofthe present disclosure, a Zinc Finger Nuclease system may be used to introduce a double-strand break at or near the native CFTR locus to facilitate insertion of a nucleic acid encodinga modified CFTR polypeptide or functional fragment thereof. The ZFN system may bedelivered as protein, as mRNA encoding the ZFN components, as DNA encoding the ZFNcomponents, or within a viral or non-viral delivery vehicle. In some embodiments, the ZFNsystem is delivered together with a donor DNA template encoding the modified CFTRpolypeptide to enable homology-directed repair-mediated insertion at the native CFTR locus.

[0141] As used herein, "a TALEN system" refers to a transcription activator-like effectornuclease system used for genome editing. A TALEN system comprises an engineered DNA-binding domain derived from transcription activator-like effectors (TALEs) fused to a FokInuclease domain. The TALE DNA-binding domain consists of tandem repeats ofapproximately 34 amino acid modules, each of which recognizes a single base pair of DNAthrough two hypervariable residues known as repeat-variable di-residues (RVDs). MultipleTALE repeat modules are assembled to create a DNA-binding domain that recognizes anextended target sequence, typically 15-20 base pairs in length. Similar to Zinc FingerNucleases, the FokI nuclease domain functions as a dimer and cleaves DNA when twoTALEN monomers bind to adjacent target sequences with appropriate spacing andorientation. The resulting double-strand break can be repaired by non-homologous endjoining (NHEJ) or homology-directed repair (HDR) pathways. In the context of the presentdisclosure, a TALEN system may be used to cleave the genomic DNA at or near the nativeCFTR locus to facilitate insertion of a nucleic acid encoding a modified CFTR polypeptide orfunctional fragment thereof. The TALEN system may be delivered as protein, as mRNAencoding the TALEN components, as DNA encoding the TALEN components, or within aviral or non-viral delivery vehicle. In some embodiments, the TALEN system is delivered incombination with” a donor DNA template that provides the nucleic acid sequence encodingthe modified CFTR polypeptide for integration at the cleaved locus via homology-directedrepair.

[0142] As used herein, the term "prime editing delivery or administration method" refers toany process for introducing the components of a prime editing system into a target cell,tissue, or organism for the purpose of performing the desired genomic modification.Prime editing delivery methods may include, but are not limited to: viral vector delivery,wherein prime editing components (e.g., Cas9 nickase-reverse transcriptase fusion protein,prime editing guide RNA (pegRNA), optional nicking sgRNA) are encoded by nucleic acidspackaged into replication-deficient viral particles such as adeno-associated virus (AAV),adenovirus, lentivirus, bocavirus, or herpesvirus; non-viral delivery, wherein prime editingcomponents are delivered as mRNA, DNA, ribonucleoprotein complexes, or other formatsinside delivery vehicles such as lipid nanoparticles (LNPs), polymeric nanoparticles,liposomes, electroporation, microinjection, or physical transfection methods; and directairway administration, wherein prime editing components are delivered to airway epithelialcells or airway basal stem cells via inhalation (e.g., nebulization of LNP-encapsulatedmRNA), intratracheal instillation, intranasal administration, or aerosolized spray.The delivery method may be selected based on target cell type, desired editing efficiency,payload size constraints, and patient safety profile.

[0143] As used herein, the term “mRNA therapeutic composition" refers to a pharmaceuticalcomposition comprising one or more messenger RNA (mRNA) molecules encoding a CFTRpolypeptide or functional fragment thereof, wherein the CFTR polypeptide comprises one ormore gain-of-function mutations. The mRNA therapeutic composition may further comprise,consist of or consist essentially of: a delivery vehicle, such as a lipid nanoparticle (LNP),polymeric nanoparticle, micelle, liposome, or other carrier capable of transfecting airwayepithelial cells; pharmaceutically acceptable excipients, buffers, salts, sugars, proteins, aminoacids, preservatives and / or stabilizers to maintain mRNA integrity and facilitate delivery. Insome embodiments, the mRNA therapeutic composition is formulated for inhalation,intranasal administration, intratracheal instillation, or systemic administration, and may beadministered once or repeatedly at intervals ranging from daily to monthly. In specificembodiments, the mRNA encodes a CFTR variant selected from deletion of amino acids404-435 (delta-RI), I539T, G550E, E1371S, S495P, R553K, or combinations thereof.

[0144] As used herein, the term “nebulization" refers to the process of converting a liquidpharmaceutical composition into an aerosol for inhalation through the nose or mouth into therespiratory tract. In the context of the present disclosure, nebulization includes delivery ofmRNA therapeutic compositions, gene editing constructs, or prime / base editing systemspackaged in nanoparticles to the airway epithelium. Nebulization devices may include jetnebulizers, ultrasonic nebulizers, vibrating mesh nebulizers, or dry powder inhalers adaptedto aerosolize and deliver nucleic acid-based therapeutics to airway surfaces.

[0145] As used herein, the term “sequential gene editing" refers to a process in which two ormore genome editing events are performed in succession, either temporally separated (e.g.,days or weeks apart) or as sequential molecular reactions within the same treatment session,to achieve multiple modifications in a target gene. In the context of the present disclosure,sequential gene editing may include: correction of a CF-causing mutation (e.g., R553X,F508del) in the CFTR gene; and subsequent insertion or installation of a gain-of-functionmutation in the CFTR gene at the native locus. The sequential gene editing can be performedusing the same editing platform (e.g., base editor, prime editor, CRISPR-Cas with HDR) ordifferent platforms and may utilize distinct delivery vehicles or payload formats for eachevent.

[0146] As used herein, the term “reduced correction threshold" refers to the minimumpercentage of target airway epithelial cells that must be genetically corrected (via insertion ofa functional CFTR) to restore CFTR-mediated function to a clinically beneficial level, asmeasured by chloride and / or bicarbonate transport in vitro or in vivo. In the context of thepresent disclosure, gain-of-function CFTR variants may reduce the correction threshold fromapproximately 75% for wild-type CFTR to approximately 25–35% corrected cells, therebyincreasing feasibility of gene therapy in heterogeneous or partially accessible airway tissue.

[0147] The term “adeno-associated virus" or "AAV” as used herein refers to a member of theclass of viruses associated with this name and belonging to the genus dependoparvovirus,family Parvoviridae. Multiple serotypes of this virus are known to be suitable for genedelivery; all known serotypes can infect cells from various tissue types. Different AAVserotypes are known in the art. Non-limiting exemplary serotypes useful in the methodsdisclosed herein include any of the following serotypes, e.g., AAV2, AAV8, AAV9, orvariant or synthetic serotypes, e.g., AAV2.5T, AAV-DJ and AAV PHP.B. The AAV particlecomprises, alternatively consists essentially of, or yet further consists of three major viralproteins: VP1, VP2 and VP3. In one embodiment, the AAV refers to of the serotype AAV1,AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13,AAV PHP.B, AAV rh74, or a variant of each thereof. These vectors are commerciallyavailable or have been described in the patent or technical literature.

[0148] Recombinant AAV (i.e., infectious encapsidated rAAV particles) of the disclosurecomprise an rAAV genome. The rAAV may be purified by methods standard in the art suchas by column chromatography or cesium chloride gradients. Methods for purifying rAAVvectors from helper virus are known in the art and include methods disclosed in, for example,Clark et al., Hum. Gene Ther., 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol.Med., 69 427-443 (2002); U.S. Patent No. 6,566,118 and WO 98 / 09657.

[0149] In another embodiment, the disclosure contemplates compositions comprising, orconsisting essentially of a vector (e.g., an rAAV) of the present disclosure. Compositionsdescribed herein comprise, or consist essentially of the vector in a pharmaceuticallyacceptable carrier. In another embodiment, the disclosure contemplates compositionscomprising, or consisting essentially of a gene editing system of the present disclosure.Compositions described herein comprise, or consist essentially of the gene editing system in apharmaceutically acceptable carrier. In another embodiment, the disclosure contemplatescompositions comprising, or consisting essentially of a nanoparticle of the present disclosure.Compositions described herein comprise, or consist essentially of the nanoparticle in apharmaceutically acceptable carrier.

[0150] In one particular embodiment, the composition of this disclosure comprise, or consistessentially of Lactated Ringer's Solution (LRS). The compositions may also comprise otheringredients such as diluents and adjuvants. Acceptable carriers, diluents and adjuvants arenontoxic to recipients and are preferably inert at the dosages and concentrations employed,and include buffers such as phosphate, citrate, or other organic acids; antioxidants such asascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin,or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids suchas glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA;sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / ornonionic surfactants such as Tween, pluronics or polyethylene glycol (PEG). Thecompositions disclosed can be used for treating cystic fibrosis.

[0151] Methods of transducing a target cell with rAAV, in vivo or in vitro, are contemplatedby the disclosure. The term "transduction" is used to refer to the administration / delivery of apolynucleotide of interest (e.g., a polynucleotide sequence encoding a CFTR gain of functionmutant) to a recipient cell either in vivo or in vitro, via a replication-deficient rAAV describedresulting in expression of a modified CFTR by the recipient cell.

[0152] Some of the DNA plasmids of the disclosure comprise rAAV genomes. The DNAplasmids are transferred to cells permissible for infection with a helper virus of AAV (e.g.,adenovirus, El-deleted adenovirus or herpesvirus) for assembly of the rAAV genome intoinfectious viral particles. Techniques to produce rAAV particles, in which an AAV genometo be packaged, rep and cap genes, and helper virus functions are provided to a cell arestandard in the art. Production of rAAV requires that the following components are presentwithin a single cell (denoted herein as a packaging cell): a rAAV genome, AAV rep and capgenes separate from (i.e., not in) the rAAV genome, and helper virus functions. The AAV repand cap genes may be from any AAV serotype for which recombinant virus can be derivedand may be from a different AAV serotype than the rAAV genome ITRs, including, but notlimited to, AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,AAV9, AAV10, AAV11, AAV12, AAV13 and AAV rh.74, and variants of each thereof. Insome embodiments, the AAV serotype can transduce airway cells. In some embodiments, arAAV vector comprises the inverted terminal repeat (ITR) sequences of AAV2. Productionof pseudotyped rAAV is disclosed in, for example, WO 01 / 83692 which is incorporated byreference herein in its entirety. In certain aspects, a rAAV vector comprises the inverted ITRsequences of AAV2 and is encapsidated in a capsid of AAV rh.74 or a variant thereof.

[0153] A method of generating a packaging cell line is to create a cell line that stablyexpresses all the necessary components for AAV particle production. For example, a plasmid(or multiple plasmids) comprising a rAAV genome lacking AAV rep and cap genes, AAVrep and cap genes separate from the rAAV genome, and a selectable marker, such as aneomycin resistance gene, are integrated into the genome of a cell. AAV genomes have beenintroduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982,Proc. Natl. Acad. S6. USA, 79:2077-2081), addition of synthetic linkers containingrestriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73) or by direct,blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). Thepackaging cell line is then infected with a helper virus such as adenovirus. The advantages ofthis method are that the cells are selectable and are suitable for large-scale production ofrAAV. Other examples of suitable methods employ adenovirus or baculovirus rather thanplasmids to introduce rAAV genomes and / or rep and cap genes into packaging cells.

[0154] General principles of rAAV production are reviewed in, for example, Carter, 1992,Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics inMicrobial, and Immunol., 158:97-129). Various approaches are described in Ratschin et al.,Mol. Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984);Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J. Virol., 62: 1963 (1988);and Lebkowski et al., 1988 Mol. Cell. Biol., 7:349 (1988). Samulski et al. (1989, J. Virol.,63:3822-3828); U.S. Patent No. 5,173,414; WO 95 / 13365 and corresponding U.S. Patent No.5,658.776; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441(PCT / US96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777);WO 97 / 06243 (PCT / FR96 / 01064); WO 99 / 11764; Perrin et al. (1995) Vaccine 13: 1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark et al. (1996) Gene Therapy3: 1124-1132; U.S. Patent. No. 5,786,211; U.S. Patent No. 5,871,982; and U.S. Patent. No.6,258,595. The foregoing documents are hereby incorporated by reference in their entiretyherein, with particular emphasis on those sections of the documents relating to rAAVproduction.

[0155] The disclosure thus provides packaging cells that produce infectious rAAV. In oneembodiment packaging cells may be stably transformed cancer cells such as HeLa cells, 293cells and PerC.6 cells (a cognate 293 line). In another embodiment, packaging cells are cellsthat are not transformed cancer cells, such as low passage 293 cells (human fetal kidney cellstransformed with El of adenovirus), MRC-5 cells (human fetal fibroblasts), WI-38 cells(human fetal fibroblasts), Vero cells (monkey kidney cells) and FRhL-2 cells (rhesus fetallung cells).

[0156] In some embodiments, the rAAV genome comprises a promoter, an untranslatedregion (UTR), and / or a poly A signal. In some embodiments, the rAAV genome comprisesone or more of a truncated CMV promoter or a tg83 promoter as described inUS20070098690A1 and WO2017155973A1, both of which are incorporated herein in theirentireties. In some embodiments, the rAAV genome comprises an SV40 polyA signal (asdescribed in Salem et al. PLoS One. 2015 Dec 14;10(12):e0145019, incorporated herein in itsentirety) or a BGH polyA signal (as described in Sarrion-Perdigones A. et al., Nat Commun.2019 Dec 13;10(1):5710, incorporated herein in its entirety).

[0157] In some embodiments, the delivery vehicle is a bocavirus vector. In someembodiments, the bocavirus vector is a Human bocavirus 1 (HBoV1) vector as described inShao et al (Front Microbiol. 2021 Jun 16:12:696604) or a bocavirus / AAV chimera vector asdescribed in Fahiri et al (Mol Ther Methods Clin Dev. 2019 Jan 18;12:202–222, incorporatedherein in its entirety). In some embodiments, the delivery vehicle is a herpesvirus vector asdescribed in Marconi et al. (Pharmaceutical Biotechnology (2009): 118-144, incorporatedherein in its entirety).

[0158] As used herein, the term "mutation" refers to a change in the nucleotide sequence of agene or the amino acid sequence of a polypeptide compared to a reference sequence. Amutation may include, but is not limited to, a substitution of one or more nucleotides oramino acids, an insertion of one or more nucleotides or amino acids, a deletion of one ormore nucleotides or amino acids, or any combination thereof. In the context of the presentdisclosure, a mutation in a modified CFTR polypeptide refers to a change in the amino acidsequence (e.g., a substitution, a deletion or an insertion) relative to the wild-type humanCFTR protein of SEQ ID NO: 1.

[0159] As used herein, "a base-editable mutation" refers to a mutation in a nucleic acidsequence that can be corrected or modified using a base editing system. A base-editablemutation is typically a point mutation (a single nucleotide substitution) that falls within theediting window of a base editor and is amenable to conversion by the deaminase enzymecomponent of the base editing system. For cytosine base editors (CBEs), a base-editablemutation includes mutations where a C-to-T (or G-to-A) conversion would restore wild-typesequence or install a desired mutation. For adenosine base editors (ABEs), a base-editablemutation includes mutations where an A-to-G (or T-to-C) conversion would restore wild-typesequence or install a desired mutation. The mutation must be located within an accessiblegenomic region that can be targeted by a guide RNA and must fall within the base editor'sactivity window (typically positions 4-8 or 4-9 of the protospacer sequence, depending on thespecific base editor architecture). In the context of the present disclosure, base-editablemutations in the CFTR gene include, but are not limited to, the R553X nonsense mutation(which can be reverted to wild-type arginine using an adenosine base editor) and wild-typecodons that can be modified to install gain-of-function mutations such as 1539T (which canbe installed by converting the isoleucine codon to a threonine codon using an adenosine baseeditor).

[0160] As used herein, a "prime editing system" refers to a gene editing platform that enablesprecise insertions, deletions, and substitutions of nucleotides in genomic DNA withoutrequiring double-strand breaks or donor DNA templates. A prime editing system typicallycomprises a catalytically impaired Cas protein (such as a Cas9 nickase (nCas9)) fused to areverse transcriptase enzyme, together with a prime editing guide RNA (pegRNA). ThepegRNA serves both to direct the prime editor to the target genomic locus and to encode thedesired edit by containing a primer binding site, a reverse transcriptase template encoding thenew genetic information, and a homology region complementary to the target site. Uponbinding to the target locus, the Cas nickase creates a single-strand nick in the DNA, exposinga 3' end that serves as a primer for the reverse transcriptase. The reverse transcriptasesynthesizes a DNA strand complementary to the reverse transcriptase template sequenceencoded in the pegRNA, thereby installing the desired edit. Cellular DNA repair andreplication machinery subsequently incorporate the edited DNA strand into the genome.Prime editing systems may further include additional guide RNAs to nick the non-editedDNA strand and bias cellular repair toward the edited strand. In the context of the presentdisclosure, a prime editing system may be used to install nucleotide changes encoding gain-of-function CFTR mutations (such as I539T, G550E, S495P, R553K or E1371S) at the nativeCFTR locus, or to correct disease-causing mutations while simultaneously introducing gain-of-function mutations. The prime editing system may be delivered as a ribonucleoproteincomplex, as mRNA encoding the prime editor components together with the pegRNA, asDNA encoding the prime editor components and pegRNA, or within a viral or non-viraldelivery vehicle.

[0161] As used herein, a "prime editable mutation" refers to a mutation in a nucleic acidsequence that can be corrected, installed, or otherwise modified using a prime editing system.A prime editable mutation encompasses insertions, deletions, or substitutions of nucleotidesthat fall within the editing capability of a prime editing system, including but not limited topoint mutations, small insertions (typically up to approximately 44 base pairs), smalldeletions (typically up to approximately 80 base pairs), and combinations thereof. Unlikebase editing systems, which are limited to single nucleotide conversions (C-to-T, A-to-G, ortheir complements), prime editing systems can install a broader range of genetic changes byutilizing a reverse transcriptase template sequence encoded in the prime editing guide RNA(pegRNA). For cytosine or adenine substitutions that could theoretically be achieved byeither base editing or prime editing, the mutation may be categorized as both a base-editablemutation and a prime editable mutation. The mutation must be located within an accessiblegenomic region that can be targeted by a prime editing guide RNA (pegRNA) and must beencodable within the reverse transcriptase template sequence of the pegRNA. In the contextof the present disclosure, prime editable mutations in the CFTR gene include, but are notlimited to, insertions or deletions that install gain-of-function mutations such as the deletionof amino acids 404-435 (delta-RI), point mutations that install gain-of-function substitutionssuch as 1539T, G550E, S495P, R553K or E1371S, correction of disease-causing mutationssuch as the F508del mutation (a three-nucleotide deletion), and simultaneous correction of adisease-causing mutation while installing a gain-of-function mutation at the same locus or ata separate locus within the editing range of the prime editing system.

[0162] As used herein, a “functional fragment” refers to a portion of a polypeptide providedherein which retains full or partial molecular, physiological or biochemical function of thefull length polypeptide. For CFTR, a functional fragment retains chloride and bicarbonatetransport activity and is able to maintain the balance of salt and water on lung surfaces. Insome embodiments, "functional fragment" refers to the delta RI variant (deletion of aminoacids at positions 404-435 from SEQ ID NO: 1).

[0163] As used herein, “polidocanol” or “PDOC” (commercially available from LGMPharma, CAS No: 3055-99-0) has a chemical structure as shown below:Modes for Carrying Out the Disclosure

[0164] Cystic fibrosis (CF) is a genetic disorder caused by mutations in the cystic fibrosistransmembrane conductance regulator (CFTR) gene. These mutations lead to the productionof thick and sticky mucus that can clog the lungs and obstruct the pancreas. CF is anautosomal recessive disease, meaning that a person must inherit two defective copies of theCFTR gene, one from each parent, to develop the condition. In the United States,approximately 40,000 children and adults are living with cystic fibrosis. The prevalence ofCF is higher among people of Northern European descent.

[0165] Current treatments focus on managing symptoms and improving quality of life. Theseinclude medications that target the underlying cause of CF, such as CFTR modulators, whichhelp improve the function of the defective protein. Gene therapy attempts have so far failed toachieve desired results. Previous clinical trials using wild-type CFTR delivered via viralvectors or mRNA overexpression did not show clinical benefit. The technical problemaddressed by the present disclosure is the need for effective genetic therapies for cysticfibrosis that overcome the limitations of prior approaches.

[0166] The solution provided by the present disclosure is the use of gain-of-function CFTRvariants inserted into the native CFTR locus via gene editing. Specifically, the disclosureprovides modified CFTR polypeptides differing from wild-type human CFTR (SEQ ID NO:1) by the presence of at least one mutation selected from deletion of amino acids at positions404-435, or variants I539T, S495P, R553K, or G550E (or optionally E1371S). Thesevariants, when inserted at the native locus, demonstrate improved CFTR function comparedto wild-type CFTR and reduce the percentage of corrected cells required to achievetherapeutic benefit (e.g., increased chloride and bicarbonate transport activity) fromapproximately 75% to approximately 25-35%. Improved CFTR function can be measured byincreased chloride transport activity in an in vitro assay (e.g., an Ussing chamber assay,which is the gold-standard for predicting therapeutic benefit).

[0167] The disclosed methods require fewer corrected cells to restore CFTR function (e.g.,chloride and bicarbonate transport activity) to levels observed in non-CF subjects. This isparticularly advantageous given the challenges of delivering gene editing constructs to allcells throughout the complex airway epithelium. The disclosure demonstrates that modifiedCFTR variants including deletion of amino acids 404-435 (delta-RI), or variants I539T, andG550E exhibit superior chloride transport activity compared to wild-type CFTR whenmeasured in Ussing chamber assays. These variants also show improved bicarbonatetransport (e.g., when measured in an Ussing chamber assay) and increased expression ofmature CFTR protein (e.g., when measured by Western blot).Methods for treating Cystic Fibrosis

[0168] An aspect of the disclosure is directed to a method for modifying an airway epithelialcell comprising, or alternatively consisting essentially of, or yet further consisting ofadministering to the cell a gene editing construct comprising, or alternatively consistingessentially of, or yet further consisting of a nucleic acid encoding a modified cystic fibrosistransmembrane conductance regulator (CFTR) polypeptide or a functional fragment thereof,wherein the modified CFTR polypeptide or the functional fragment thereof differs from awild-type human CFTR protein of SEQ ID NO: 1 by the presence of at least one mutationselected from a deletion of amino acids at positions 404-435, or variant 1539T, G550E,S495P, or R553K.

[0169] An aspect of the disclosure is directed to a method for treating cystic fibrosis in asubject in need thereof comprising, or alternatively consisting essentially of, or yet furtherconsisting of administering to the subject a gene editing construct comprising, or alternativelyconsisting essentially of, or yet further consisting of a nucleic acid encoding a modified cysticfibrosis transmembrane conductance regulator (CFTR) polypeptide or a functional fragmentthereof, wherein the modified CFTR polypeptide or the functional fragment thereof differsfrom a wild-type human CFTR protein of SEQ ID NO: 1 by the presence of at least onemutation selected from a deletion of amino acids at positions 404-435, or variant 1539T,G550E, S495P, or R553K, wherein the gene editing construct is configured to insert thenucleic acid encoding the modified CFTR polypeptide or functional fragment thereof into thenative CFTR locus of a cell of the subject.

[0170] In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, consists of, or alternatively consists essentially of, or yet further consistsof a deletion of amino acids at position 404-435 with respect to SEQ ID NO: 1. In someembodiments, the modified CFTR polypeptide or the functional fragment thereof comprises,consists of, or alternatively consists essentially of, or yet further consists of a I539T mutationwith respect to SEQ ID NO: 1. In some embodiments, the modified CFTR polypeptide or thefunctional fragment thereof comprises, consists of, or alternatively consists essentially of, oryet further consists of a G550E mutation with respect to SEQ ID NO: 1. In someembodiments, the modified CFTR polypeptide or the functional fragment thereof comprises,consists of, or alternatively consists essentially of, or yet further consists of a S495P mutationwith respect to SEQ ID NO: 1. In some embodiments, the modified CFTR polypeptide or thefunctional fragment thereof comprises, consists of, or alternatively consists essentially of, oryet further consists of a R553K mutation with respect to SEQ ID NO: 1. In someembodiments, the modified CFTR polypeptide or the functional fragment thereof comprises,consists of, or alternatively consists essentially of, or yet further consists of a E1371S withrespect to SEQ ID NO: 1.

[0171] In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, consists of, or alternatively consists essentially of, or yet further consistsof a deletion of amino acids at positions 404-435 and a I539T mutation with respect to SEQID NO: 1. In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, consists of, or alternatively consists essentially of, or yet further consistsof a deletion of amino acids at positions 404-435 and a G550E mutation with respect to SEQID NO: 1. In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, consists of, or alternatively consists essentially of, or yet further consistsof a deletion of amino acids at positions 404-435 and a S495P mutation with respect to SEQID NO: 1. In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, consists of, or alternatively consists essentially of, or yet further consistsof a deletion of amino acids at positions 404-435 and a R553K mutation with respect to SEQID NO: 1. In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, consists of, or alternatively consists essentially of, or yet further consistsof a deletion of amino acids at positions 404-435 and a E1371S mutation with respect to SEQID NO: 1.

[0172] In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, consists of, or alternatively consists essentially of, or yet further consistsof a I539T mutation and a G550E mutation with respect to SEQ ID NO: 1. In someembodiments, the modified CFTR polypeptide or the functional fragment thereof comprises,consists of, or alternatively consists essentially of, or yet further consists of a I539T mutationand a S495P mutation with respect to SEQ ID NO: 1. In some embodiments, the modifiedCFTR polypeptide or the functional fragment thereof comprises, consists of, or alternativelyconsists essentially of, or yet further consists of a 1539T mutation and a R553K mutation withrespect to SEQ ID NO: 1.

[0173] In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, consists of, or alternatively consists essentially of, or yet further consistsof a I539T mutation, a G550E mutation, a S495P mutation and a I539T mutation with respectto SEQ ID NO: 1.

[0174] In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, consists of, or alternatively consists essentially of, or yet further consistsof a deletion of amino acids at positions 404-435, a I539T mutation, a G550E mutation, aS495P mutation and a I539T mutation with respect to SEQ ID NO: 1.

[0175] In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, or alternatively consists essentially of, or yet further consists of any one ofSEQ ID NO: 2-5.

[0176] In some embodiments, the gene editing construct is delivered within a nanoparticle. Insome embodiments, the nanoparticle is a lipid nanoparticle or a polymer nanoparticle. Insome embodiments, the nanoparticle comprises, or alternatively consists essentially of, or yetfurther consists of one or more of 1,2-Distearoyl-sn-glycero-3-phosphorylcholine (DSPC)(commercially available from Millipore Sigma, CAS No. 816-94-4),Dioleoylphosphatidylethanolamine (DOPE) (commercially available from Millipore Sigma,CAS No. 4004-05-1), Polyethylene glycol lipid (PEG-lipid), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) (commercially available from Millipore Sigma, CASNo. 132172-61-3), or Dilinoleyl-methyl-4-dimethylaminobutyrate, MC3 (D-Lin-MC3)(commercially available from Cayman Chem, CAS No. 1224606-06-7).

[0177] In some embodiments, the gene editing construct is delivered by a viral vectorselected from an adeno-associated virus (AAV) vector, a bocavirus vector, or a herpesvirusvector. In some embodiments, the AAV vector is selected from AAV1, AAV2, AAV4,AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV PHP.B,AAV rh74, AAV 2.5T, or a variant thereof.

[0178] In some embodiments, the gene editing construct comprises, or alternatively consistsessentially of, or yet further consists of a CRISPR-Cas system, a base editor, a Zinc FingerNuclease system, or a TALEN system.

[0179] In some embodiments, the CRISPR-Cas system comprises, or alternatively consistsessentially of, or yet further consists of a Cas9 ribonucleoprotein (RNP) and a single guideRNA (sgRNA).

[0180] In some embodiments, the gene editing construct is delivered to airway basal stemcells of the subject ex vivo, and wherein the method further comprises, or alternativelyconsists essentially of, or yet further consists of transplanting the edited airway basal stemcells to the subject's airways.

[0181] In some embodiments, the airway basal stem cells are P63+, KRT5+, and NGFR+.

[0182] In some embodiments, the method further comprises, or alternatively consistsessentially of, or yet further consists of conditioning the subject's airways to remove diseasedairway epithelial cells prior to transplanting the edited airway basal stem cells.

[0183] In some embodiments, the conditioning comprises, or alternatively consistsessentially of, or yet further consists of introducing an acute airway injury to the subject'sairways.

[0184] In some embodiments, the acute airway injury is a chemical injury induced bypolidocanol (PDOC) or a mechanical injury induced by an endoscopic device.

[0185] In some embodiments, the conditioning comprises, or alternatively consistsessentially of, or yet further consists of removing at least 30% (e.g., at least 30, 40, 50, 60,70, 75, 80, 85, 90, 95 or 99%) of diseased airway epithelial cells. In some embodiments, theconditioning comprises, or alternatively consists essentially of, or yet further consists ofremoving all (100%) of diseased airway epithelial cells.

[0186] In some embodiments, the subject is a mammal, optionally a human.

[0187] An aspect of the disclosure is directed to a method for treating cystic fibrosis in asubject in need thereof comprising administering to the subject an mRNA encoding amodified cystic fibrosis transmembrane conductance regulator (CFTR) polypeptide or afunctional fragment thereof, wherein the modified CFTR polypeptide or the functionalfragment thereof differs from a wild-type human CFTR protein as shown by SEQ ID NO:1 ora functional fragment thereof by the presence of at least one mutation selected from (i)deletion of amino acids at positions 404-435, or a variant selected from (ii) 1539T, (iii)G550E, (iv) S495P, or (v) R553K (or optionally (vi) E1371S) with respect to SEQ ID NO: 1.

[0188] In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, consists of, or alternatively consists essentially of, or yet further consistsof a deletion of amino acids at position 404-435 with respect to SEQ ID NO: 1. In someembodiments, the modified CFTR polypeptide or the functional fragment thereof comprises,consists of, or alternatively consists essentially of, or yet further consists of a I539T mutationwith respect to SEQ ID NO: 1. In some embodiments, the modified CFTR polypeptide or thefunctional fragment thereof comprises, consists of, or alternatively consists essentially of, oryet further consists of a G550E mutation with respect to SEQ ID NO: 1. In someembodiments, the modified CFTR polypeptide or the functional fragment thereof comprises,consists of, or alternatively consists essentially of, or yet further consists of a S495P mutationwith respect to SEQ ID NO: 1. In some embodiments, the modified CFTR polypeptide or thefunctional fragment thereof comprises, consists of, or alternatively consists essentially of, oryet further consists of a R553K with respect to SEQ ID NO: 1.

[0189] In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, consists of, or alternatively consists essentially of, or yet further consistsof a 1539T mutation, a G550E mutation, and a S495P mutation with respect to SEQ ID NO:1.

[0190] In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, consists of, or alternatively consists essentially of, or yet further consistsof a deletion of amino acids at positions 404-435, a I539T mutation, a G550E mutation and aR553K mutation with respect to SEQ ID NO: 1.

[0191] In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, consists of, or alternatively consists essentially of, or yet further consistsof SEQ ID NO: 2. In some embodiments, the modified CFTR polypeptide or the functionalfragment thereof comprises, consists of, or alternatively consists essentially of, or yet furtherconsists of SEQ ID NO: 3. In some embodiments, the modified CFTR polypeptide or thefunctional fragment thereof comprises, consists of, or alternatively consists essentially of, oryet further consists of SEQ ID NO: 4. In some embodiments, the modified CFTRpolypeptide or the functional fragment thereof comprises, consists of, or alternatively consistsessentially of, or yet further consists of SEQ ID NO: 5.

[0192] In some embodiments, the mRNA is administered within a nanoparticle, optionally alipid nanoparticle or a polymer nanoparticle.

[0193] In some embodiments, the nanoparticle comprises, consists of, or alternativelyconsists essentially of, or yet further consists of one or more of 1,2-Distearoyl-sn-glycero-3-phosphorylcholine (DSPC), Dioleoylphosphatidylethanolamine (DOPE), Polyethylene glycollipid (PEG-lipid), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), or Dilinoleyl-methyl-4-dimethylaminobutyrate, MC3 (D-Lin-MC3). In some embodiment, thenanoparticle comprises, consists of, or alternatively consists essentially of, or yet furtherconsists of nanoparticles and lipids as described in US11648209B2, which is incorporatedherein by reference in its entirety.

[0194] In some embodiments, a cDNA encoding the mRNA is administered to the subject byan adeno-associated virus (AAV) vector, a bocavirus vector or a herpesvirus vector. In someembodiments, the AAV vector is selected from AAV1, AAV2, AAV4, AAV5, AAV6,AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV PHP.B, AAV rh74, AAV2.5T or variants of each thereof. In some embodiments, the AAV vector is selected fromAAV2, AAV 2.5T or variants of each thereof.

[0195] In some embodiments, the mRNA is administered to the subject once every day,every two days, every three days, every four days, every five days, every six, once a week,once every two weeks, once every three weeks or once a month.Autologous Airway Stem Cell Therapy

[0196] An aspect of the disclosure is directed to a method for treating cystic fibrosis in asubject in need thereof comprising, or alternatively consisting essentially of, or yet furtherconsisting of administering to the subject a gene editing construct comprising, or alternativelyconsisting essentially of, or yet further consisting of a nucleic acid encoding a modified cysticfibrosis transmembrane conductance regulator (CFTR) polypeptide or a functional fragmentthereof, wherein the modified CFTR polypeptide or the functional fragment thereof differsfrom a wild-type human CFTR protein of SEQ ID NO: 1 by the presence of at least onemutation selected from a deletion of amino acids at positions 404-435, I539T, G550E, S495Por R553K (or optionally E1371S), wherein the gene editing construct is configured to insertthe nucleic acid encoding the modified CFTR polypeptide or functional fragment thereof intothe native CFTR locus of a cell of the subject.

[0197] Another aspect of the disclosure is directed to a method for treating cystic fibrosis in asubject in need thereof comprising: (a) modifying airway basal stem cells from the subject exvivo to express a modified cystic fibrosis transmembrane conductance regulator (CFTR)polypeptide or a functional fragment thereof, wherein the modified CFTR polypeptide or thefunctional fragment thereof differs from a wild-type human CFTR protein (SEQ ID NO: 1) ora functional fragment thereof by the presence of at least one mutation selected from (i)deletion of amino acids at position 404-435, (ii) I539T, (iii) G550E, (iv) S495P or (v) R553K;(b) conditioning the subject's airways to remove diseased airway epithelial cells (e.g., removeat least 30% of diseased airway epithelial cells); and (c) transplanting the airway basal stemcells to the subject's airways.

[0198] In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, consists of, or alternatively consists essentially of, or yet further consistsof a deletion of amino acids at position 404-435 with respect to SEQ ID NO: 1. In someembodiments, the modified CFTR polypeptide or the functional fragment thereof comprises,consists of, or alternatively consists essentially of, or yet further consists of a 1539T mutationwith respect to SEQ ID NO: 1. In some embodiments, the modified CFTR polypeptide or thefunctional fragment thereof comprises, consists of, or alternatively consists essentially of, oryet further consists of a G550E mutation with respect to SEQ ID NO: 1. In someembodiments, the modified CFTR polypeptide or the functional fragment thereof comprises,consists of, or alternatively consists essentially of, or yet further consists of a S495P mutationwith respect to SEQ ID NO: 1. In some embodiments, the modified CFTR polypeptide or thefunctional fragment thereof comprises, consists of, or alternatively consists essentially of, oryet further consists of a R553K with respect to SEQ ID NO: 1.

[0199] In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, consists of, or alternatively consists essentially of, or yet further consistsof a deletion of amino acids at positions 404-435 and a I539T mutation with respect to SEQID NO: 1. In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, consists of, or alternatively consists essentially of, or yet further consistsof a deletion of amino acids at positions 404-435 and a G550E mutation with respect to SEQID NO: 1. In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, consists of, or alternatively consists essentially of, or yet further consistsof a deletion of amino acids at positions 404-435 and a S495P mutation with respect to SEQID NO: 1. In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, consists of, or alternatively consists essentially of, or yet further consistsof a deletion of amino acids at positions 404-435 and a R553K mutation with respect to SEQID NO: 1.

[0200] In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, consists of, or alternatively consists essentially of, or yet further consistsof a I539T mutation, a G550E mutation, and a S495P mutation with respect to SEQ ID NO:1.

[0201] In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, consists of, or alternatively consists essentially of, or yet further consistsof a deletion of amino acids at positions 404-435, a I539T mutation, a G550E mutation and aS495P mutation with respect to SEQ ID NO: 1.

[0202] In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, or alternatively consists essentially of, or yet further consists of any one ofSEQ ID NO: 2-5.

[0203] In some embodiments, the airway basal stem cells are P63+, KRT5+ and NGFR+.

[0204] In some embodiments, the conditioning the subject's airways in (b) comprises,consists of, or alternatively consists essentially of, or yet further consists of introducing anacute airway injury to the subject's airways.

[0205] In some embodiments, the acute airway injury is a chemical injury induced bypolidocanol (PDOC) or a mechanical injury induced by an endoscopic device.

[0206] In some embodiments, the conditioning comprises, or alternatively consistsessentially of, or yet further consists of removing at least 30% (e.g., at least 30, 40, 50, 60,70, 75, 80, 85, 90, 95 or 99%) of diseased airway epithelial cells. In some embodiments, theconditioning comprises, or alternatively consists essentially of, or yet further consists ofremoving all (100%) of diseased airway epithelial cells.

[0207] In some embodiments, the modifying in (i) is achieved by a gene editing systemselected from CRISPR-Cas, a base editing system, a prime editing system, Zinc FingerNuclease or TALEN.

[0208] In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, consists of, or alternatively consists essentially of, or yet further consistsof SEQ ID NO: 2. In some embodiments, the modified CFTR polypeptide or the functionalfragment thereof comprises, consists of, or alternatively consists essentially of, or yet furtherconsists of SEQ ID NO: 3. In some embodiments, the modified CFTR polypeptide or thefunctional fragment thereof comprises, consists of, or alternatively consists essentially of, oryet further consists of SEQ ID NO: 4. In some embodiments, the modified CFTRpolypeptide or the functional fragment thereof comprises, consists of, or alternatively consistsessentially of, or yet further consists of SEQ ID NO: 5.

[0209] In some embodiments, the gene editing system is delivered within a nanoparticle,optionally a lipid nanoparticle or a polymer nanoparticle.

[0210] In some embodiments, the nanoparticle comprises, consists of, or alternativelyconsists essentially of, or yet further consists of one or more of 1,2-Distearoyl-sn-glycero-3-phosphorylcholine (DSPC), Dioleoylphosphatidylethanolamine (DOPE), Polyethylene glycollipid (PEG-lipid), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), or Dilinoleyl-methyl-4-dimethylaminobutyrate, MC3 (D-Lin-MC3). In some embodiment, thenanoparticle comprises, consists of, or alternatively consists essentially of, or yet furtherconsists of nanoparticles and lipids as described in US11648209B2, which is incorporatedherein by reference in its entirety.

[0211] In some embodiments, a cDNA encoding the gene editing system is delivered by anAAV vector. In some embodiments, a cDNA encoding the modified CFTR polypeptide isdelivered by an AAV vector. In some embodiments, the AAV vector is selected from AAV1,AAV2, AAV2.5T, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11,AAV12, AAV13, AAV rh.74 or variants thereof. In some embodiments, the AAV vector isAAV2, or a variant of each thereof. In some embodiments, the AAV vector is AAV2.5T, ora variant of each thereof.

[0212] In some embodiments, the subject is a mammal. In some embodiments, the subject isa human CF patient.

[0213] Another aspect of the disclosure is directed to a method for treating cystic fibrosis in asubject having a base-editable mutation or a prime-editable mutation in the CFTR gene, themethod comprising: administering to the subject a gene editing system selected from a baseediting system or a prime editing system, the gene editing system comprising: a base editor ora prime editor; and a first single guide RNA (sgRNA) that targets the base-editable mutationor the prime-editable mutation; and a second sgRNA to install a gain-of-function mutation inthe native CFTR locus, wherein the second sgRNA targets one of (i) codon 1539, (ii) codonG550, (iii) codon S495, or (iv) codon R553 (or optionally (v) codon E1371) with respect toSEQ ID NO: 1, wherein the base editing system or the prime editing system is configured tocorrect the base-editable mutation or the prime-editable mutation and introduce the gain-of-function mutation in the native CFTR locus of a cell of the subject.

[0214] Another aspect of the disclosure is directed to a method for treating cystic fibrosis in asubject having an R553X mutation in the CFTR gene, the method comprising, oralternatively consisting essentially of, or yet further consisting of administering to the subjecta base editing system comprising, or alternatively consisting essentially of, or yet furtherconsisting of an adenosine base editor (ABE), and a first single guide RNA (sgRNA) thattargets the R553X mutation to revert a premature stop codon to an arginine codon; and asecond sgRNA to install a gain-of-function mutation in the native CFTR locus, wherein thesecond sgRNA targets one of (i) codon 1539, (ii) codon G550, (iii) codon S495, or (iv) codonR553 (or optionally (v) codon E1371) with respect to SEQ ID NO: 1, wherein the baseediting system is configured to correct the R553X mutation and introduce the gain-of-function mutation in the native CFTR locus of a cell of the subject. In some embodiments,the gain-of function mutation is an I539T mutation, a G550E mutation, or a E1371S mutationwith respect to SEQ ID NO: 1.

[0215] In some embodiments, the first sgRNA and the second sgRNA are deliveredsimultaneously with the gene editing system.

[0216] In some embodiments, the gene editing system is delivered to airway epithelial cellsof the subject.

[0217] In some embodiments, the gene editing system is delivered by a nebulizer. In someembodiments, the gene editing system is delivered as mRNA. In some embodiments, thegene editing system is delivered within a nanoparticle. In some embodiments, thenanoparticle is a lipid nanoparticle or a polymer nanoparticle.

[0218] In some embodiments, the nanoparticle comprises, or alternatively consists essentiallyof, or yet further consists of one or more of 1,2-Distearoyl-sn-glycero-3-phosphorylcholine(DSPC), Dioleoylphosphatidylethanolamine (DOPE), Polyethylene glycol lipid (PEG-lipid),1,2-dioleoyl-3-trimethylammonium propane (DOTAP), or Dilinoleyl-methyl-4-dimethylaminobutyrate, MC3 (D-Lin-MC3).

[0219] In some embodiments, the gene editing system is delivered by a viral vector selectedfrom an adeno-associated virus (AAV) vector, a bocavirus vector, or a herpesvirus vector.

[0220] In some embodiments, the AAV vector is selected from AAV1, AAV2, AAV4,AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV PHP.B,AAV rh74, AAV 2.5T, or a variant thereof.

[0221] In some embodiments, the gene editing system is delivered to airway basal stem cellsof the subject ex vivo, and wherein the method further comprises, or alternatively consistsessentially of, or yet further consists of transplanting the edited airway basal stem cells to thesubject's airways.

[0222] In some embodiments, the airway basal stem cells are P63+, KRT5+, and NGFR+.

[0223] In some embodiments, the method further comprises, or alternatively consistsessentially of, or yet further consists of conditioning the subject's airways to remove diseasedairway epithelial cells prior to transplanting the edited airway basal stem cells.

[0224] In some embodiments, the conditioning comprises, or alternatively consistsessentially of, or yet further consists of introducing an acute airway injury to the subject'sairways.

[0225] In some embodiments, the acute airway injury is a chemical injury induced bypolidocanol (PDOC) or a mechanical injury induced by an endoscopic device.

[0226] In some embodiments, the conditioning comprises, or alternatively consistsessentially of, or yet further consists of removing at least 30% (e.g., at least 30, 40, 50, 60,70, 75, 80, 85, 90, 95 or 99%) of diseased airway epithelial cells. In some embodiments, theconditioning comprises, or alternatively consists essentially of, or yet further consists ofremoving all (100%) of diseased airway epithelial cells.

[0227] In some embodiments, the subject is a mammal, optionally a human.

[0228] Another aspect of the disclosure is directed to a method for treating cystic fibrosis in asubject having a base-editable mutation or a prime-editable mutation in the CFTR gene, themethod comprising: administering to the subject a gene editing system selected from a baseediting system or a prime editing system, the gene editing system comprising: a base editor ora prime editor; and a first single guide RNA (sgRNA) that targets the base-editable mutationor the prime-editable mutation; and a second sgRNA to install a gain-of-function mutation inthe native CFTR locus, wherein the second sgRNA targets one of (i) codon 1539, (ii) codonG550, (iii) codon S495, or (iv) codon R553 (or optionally (v) codon E1371) with respect toSEQ ID NO: 1, wherein the base editing system or the prime editing system is configured tocorrect the base-editable mutation or the prime-editable mutation and introduce the gain-of-function mutation in the native CFTR locus of a cell of the subject.

[0229] Another aspect of the disclosure is directed to a method for treating cystic fibrosis in asubject having an R553X mutation in the CFTR gene, the method comprising, oralternatively consisting essentially of, or yet further consisting of (a) modifying airway basalstem cells from the subject ex vivo using a base editing system comprising, or alternativelyconsisting essentially of, or yet further consisting of an adenosine base editor (ABE), and afirst single guide RNA (sgRNA) that targets the R553X mutation to revert a premature stopcodon to an arginine codon; and a second sgRNA to install a gain-of-function mutation in thenative CFTR locus, wherein the second sgRNA targets one of (i) codon 1539, (ii) codonG550, or (iii) codon E1371 with respect to SEQ ID NO: 1, wherein the base editing system isconfigured to correct the R553X mutation and introduce the gain-of-function mutation in thenative CFTR locus of the airway basal stem cells, (b) optionally conditioning the subject'sairways to remove diseased airway epithelial cells, and (c) transplanting the airway basalstem cells to the subject's airways.

[0230] wherein the base editing system is configured to correct the R553X mutation andintroduce a gain-of-function mutation in the native CFTR locus of a cell of the subject

[0231] In some embodiments, the airway basal stem cells are P63+, KRT5+, and NGFR+.

[0232] In some embodiments, the conditioning in (b) comprises, or alternatively consistsessentially of, or yet further consists of introducing an acute airway injury to the subject'sairways.

[0233] In some embodiments, the acute airway injury is a chemical injury induced bypolidocanol (PDOC) or a mechanical injury induced by an endoscopic device.

[0234] In some embodiments, the conditioning comprises, or alternatively consistsessentially of, or yet further consists of removing at least 30% (e.g., at least 30, 40, 50, 60,70, 75, 80, 85, 90, 95 or 99%) of diseased airway epithelial cells. In some embodiments, theconditioning comprises, or alternatively consists essentially of, or yet further consists ofremoving all (100%) of diseased airway epithelial cells.

[0235] In some embodiments, the first sgRNA and the second sgRNA are deliveredsimultaneously with the adenosine base editor (ABE).

[0236] In some embodiments, the adenosine base editor (ABE) is delivered as mRNA.

[0237] In some embodiments, the base editing system is delivered by a nebulizer. In someembodiments, the base editing system is delivered within a nanoparticle. In someembodiments, the nanoparticle is a lipid nanoparticle or a polymer nanoparticle.

[0238] In some embodiments, the nanoparticle comprises, or alternatively consists essentiallyof, or yet further consists of one or more of 1,2-Distearoyl-sn-glycero-3-phosphorylcholine(DSPC), Dioleoylphosphatidylethanolamine (DOPE), Polyethylene glycol lipid (PEG-lipid),1,2-dioleoyl-3-trimethylammonium propane (DOTAP), or Dilinoleyl-methyl-4-dimethylaminobutyrate, MC3 (D-Lin-MC3).

[0239] In some embodiments, the base editing system is delivered by a viral vector selectedfrom an adeno-associated virus (AAV) vector, a bocavirus vector, or a herpesvirus vector.

[0240] In some embodiments, the AAV vector is selected from AAV1, AAV2, AAV4,AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV PHP.B,AAV rh74, AAV 2.5T, or a variant thereof.

[0241] In some embodiments, the subject is a mammal, optionally a human.Compositions

[0242] In one aspect, provided is a composition (e.g., a pharmaceutical composition)comprising, or alternatively consisting essentially of, or yet further consisting of a geneediting construct comprising, or alternatively consisting essentially of, or yet furtherconsisting of a nucleic acid encoding a modified cystic fibrosis transmembrane conductanceregulator (CFTR) polypeptide or a functional fragment thereof, wherein the modified CFTRpolypeptide or the functional fragment thereof differs from a wild-type human CFTR proteinas shown by SEQ ID NO:1 or a functional fragment thereof by the presence of at least onemutation selected from (i) deletion of amino acids at positions 404-435, (ii) I539T, (iii)G550E, (iv) S495P or (v) R553K.

[0243] In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, consists of, or alternatively consists essentially of, or yet further consistsof a deletion of amino acids at position 404-435 with respect to SEQ ID NO: 1. In someembodiments, the modified CFTR polypeptide or the functional fragment thereof comprises,consists of, or alternatively consists essentially of, or yet further consists of a I539T mutationwith respect to SEQ ID NO: 1. In some embodiments, the modified CFTR polypeptide or thefunctional fragment thereof comprises, consists of, or alternatively consists essentially of, oryet further consists of a G550E mutation with respect to SEQ ID NO: 1. In someembodiments, the modified CFTR polypeptide or the functional fragment thereof comprises,consists of, or alternatively consists essentially of, or yet further consists of a S495P withrespect to SEQ ID NO: 1. In some embodiments, the modified CFTR polypeptide or thefunctional fragment thereof comprises, consists of, or alternatively consists essentially of, oryet further consists of a R553K with respect to SEQ ID NO: 1.

[0244] In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, consists of, or alternatively consists essentially of, or yet further consistsof a deletion of amino acids at positions 404-435 and a I539T mutation with respect to SEQID NO: 1. In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, consists of, or alternatively consists essentially of, or yet further consistsof a deletion of amino acids at positions 404-435 and a G550E mutation with respect to SEQID NO: 1. In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, consists of, or alternatively consists essentially of, or yet further consistsof a deletion of amino acids at positions 404-435 and a S595P mutation with respect to SEQID NO: 1. In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, consists of, or alternatively consists essentially of, or yet further consistsof a deletion of amino acids at positions 404-435 and a R553K mutation with respect to SEQID NO: 1.

[0245] In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, consists of, or alternatively consists essentially of, or yet further consistsof a I539T mutation, a G550E mutation, and a S495P with respect to SEQ ID NO: 1.

[0246] In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, consists of, or alternatively consists essentially of, or yet further consistsof a deletion of amino acids at positions 404-435, a I539T mutation, a G550E mutation and aR553K mutation with respect to SEQ ID NO: 1.

[0247] In some embodiments, the modified CFTR polypeptide or the functional fragmentthereof comprises, consists of, or alternatively consists essentially of, or yet further consistsof SEQ ID NO: 2. In some embodiments, the modified CFTR polypeptide or the functionalfragment thereof comprises, consists of, or alternatively consists essentially of, or yet furtherconsists of SEQ ID NO: 3. In some embodiments, the modified CFTR polypeptide or thefunctional fragment thereof comprises, consists of, or alternatively consists essentially of, oryet further consists of SEQ ID NO: 4. In some embodiments, the modified CFTRpolypeptide or the functional fragment thereof comprises, consists of, or alternatively consistsessentially of, or yet further consists of SEQ ID NO: 5.

[0248] In some embodiments, the gene editing construct is contained within a nanoparticle.

[0249] In some embodiments, the nanoparticle is a lipid nanoparticle or a polymernanoparticle.

[0250] In some embodiments, the nanoparticle comprises, or alternatively consists essentiallyof, or yet further consists of one or more of 1,2-Distearoyl-sn-glycero-3-phosphorylcholine(DSPC), Dioleoylphosphatidylethanolamine (DOPE), Polyethylene glycol lipid (PEG-lipid),1,2-dioleoyl-3-trimethylammonium propane (DOTAP), or Dilinoleyl-methyl-4-dimethylaminobutyrate, MC3 (D-Lin-MC3).

[0251] In some embodiments, the gene editing construct is contained within a viral vectorselected from an adeno-associated virus (AAV) vector, a bocavirus vector, or a herpesvirusvector.

[0252] In some embodiments, the AAV vector is selected from AAV1, AAV2, AAV4,AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV PHP.B,AAV rh74, AAV 2.5T, or a variant thereof.

[0253] In some embodiments, the gene editing construct comprises, or alternatively consistsessentially of, or yet further consists of a CRISPR-Cas system, a base editing system, a ZincFinger Nuclease system, or a TALEN system.

[0254] In some embodiments, the CRISPR-Cas system comprises, or alternatively consistsessentially of, or yet further consists of a Cas9 ribonucleoprotein (RNP) and a single guideRNA (sgRNA).

[0255] In some embodiments, the composition further comprises, or alternatively consistsessentially of, or yet further consists of a pharmaceutically acceptable carrier.

[0256] The following examples are included to demonstrate some embodiments of thedisclosure. However, those of skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodiments which are disclosedand still obtain a like or similar result without departing from the spirit and scope of theinvention.EXAMPLESExample 1: Gain of Function CFTR Variants to Enhance the Efficacy of GeneticTherapies to Treat Cystic Fibrosis

[0257] The present study focuses on the development and testing of CFTR variants toenhance the efficacy of genetic therapies for cystic fibrosis. The goal is to identify CFTRvariants that improve stability and function compared to the wild-type (WT) CFTR proteinwhich can also work in a gene therapy context. Although gain of function mutations havebeen reported before, they have never been used in or developed for gene therapies. Gain offunction mutants in CFTR come in two types: stability enhancing mutants and conductivityenhancing mutants.

[0258] Diseases that are caused by a single mutated gene are attractive targets for genetherapy. However, sometimes expressing the functional wild type gene is not enough forachieving treatment. In the case of cystic fibrosis, gene therapy attempts using the wild typegene failed numerous times due to several reasons. First, wild type CFTR gene does not havea stable transcript. Only 20% of CFTR transcripts maturate and find their way to the cellsurface where they function. In addition, wild type CFTR channels have about 50%probability of being open once on the cell membrane. Finally, lungs have a complexstructure with numerous branching airways and a large surface area, making it hard for thegene therapy vectors to reach all the target cells. For these reasons, initial CF gene therapieshave all failed to deliver enough functional CFTR gene to correct the deficiency in affectedlungs.

[0259] Applicant has developed a gene therapy strategy which overcomes these difficulties.The CFTR gain of function mutants identified herein that are shown work in a gene therapycontext will make it possible to increase CFTR function in affected lungs enough to treatcystic fibrosis.Materials and Methods

[0260] Cell Culture and Transfection: Cells were cultured and genetically modified toexpress various CFTR variants, including wild-type (WT) and mutants believed to enhancestability and increase the probability of CFTR channel opening.

[0261] Measurement of CFTR Function: CFTR function was assessed using a UssingChamber. Responses to CFTR activator (Forskolin) and inhibitor (CFTRinh) were measured.CF cells do not respond to CFTR activator (Forskolin) or inhibitor (CFTRinh), and onlyrespond to UTP (added at the end of experiment as a positive control).

[0262] Data Analysis: The percentage of cells expressing each CFTR variant wasdetermined. The stability and conductivity enhancing mutants were analyzed for their currentchange fold difference from the WT.

[0263] Human bronchial epithelial cell (HBEC) culture conditions: Cells weremaintained in PneumaCultTM Ex Plus medium (StemCell Technologies #05040) withaddition of 10 µM ROCK Inhibitor (Y-27632, MedChemExpress # HY10583), iMatrix-511(Nacalai USA #892021) and penicillin-streptomycin (ThermoFisher Scientific, 15140122).Cells were seeded at a density of 5,000 – 10,000 cells / cm2 and passaged after 3-5 days,before reaching confluency, using TrypLE according to manufacturer's instructions.

[0264] In vitro transcription: Linear transcription templates were generated via PCR withKOD multi & EpiTM polymerase (Toyobo, KME-101) according to manufacturer'sspecifications. A 120-nucleotide poly-T sequence was included in the 3' primer in order togenerate the poly-A tail. This template was purified by gel purification using the GeneJETGel Extraction Kit (Thermo Scientific, K0692). Transcription was performed usingHiScribeTM T7 High Yield RNA polymerase (New England Biolabs, E2040S), according tomanufacturer's instructions with the following changes: UTP was replaced with N1-Methylpseudouridine-5'-Triphosphate (TriLink Biotechnologies, N-1081-1) at the sameconcentration, and CleanCap® Reagent AG (TriLink Biotechnologies, N-7113) was addedwith a final concentration of 4 mM. The reaction was incubated at 37C overnight, then RNAwas purified with LiCl extraction. mRNA was diluted with nuclease-free water and stored at -80C.

[0265] AAV editing of HBECs: HBECs were edited via electroporation of Cas9 RNP,followed by immediate transduction with AAV containing templates for the sequentialinsertion of full-length CFTR cDNA. Cells were detached using TrypLE Express Enzyme(Life Technologies, 12605028), then resuspended in Opti-MEMTM Reduced Serum Medium(Thermo Fisher Scientific, 31985088) at a concentration of 1 – 5 x 107 cells / ml. 6 µg of HiFiCas9 protein was mixed with 3.2 µg of sgRNA and incubated at room temperature for 10minutes to form a ribonuclear protein complex (RNP). The Cas9 RNP solution was mixedwith the cell suspension and 5 µM HDR-enhancer protein (Integrated DNA Technologies,10029790), then transferred to a Nucleocuvette vessel for a nebulizer.

[0266] A nebulizer was performed in a Lonza 4D-NucleofectorTM with a 16-wellNucleocuvette Strip (Lonza, V4XP-3032), using program CA-137 with solution setting P3.Immediately following a nebulizer, cells were diluted with 80 µl Opti-MEM and split intoaliquots with 100,000 cells in each. Two AAVs (which are of serotype AAV6) carrying thetwo halves of the CFTR cDNA and the tCD19 enrichment cassette were added at amultiplicity of infection (MOI) of 105 genomes per cell. Cells were incubated at roomtemperature for 10 minutes, then plated in a 6-well plate and diluted with PneumaCultTM ExPlus medium (StemCell Technologies #05040) with addition of 10 µM ROCK Inhibitor (Y-27632, MedChemExpress # HY10583), iMatrix-511 (Nacalai USA #892021), penicillin-streptomycin (ThermoFisher Scientific, 15140122), and 0.5 µM AZD-7648 (MedChemExpress, HY-111783). Culture media was replaced 24 hours after a nebulizer in order toremove AZD-7648.

[0267] Measurement of AAV editing: Insertion of the full-length CFTR cDNA wasdetermined by staining edited cells for CD19 and counting the percentage of stained cells viaflow cytometry. Edited HBECs were detached with TrypLE Express Enzyme (LifeTechnologies, 12605028) 5-7 days after editing. 50,000 – 100,000 cells were suspended inOpti-MEM then stained with FITC-anti-CD19 (Biolegend; 302256). Cells were incubated at4°C for 30 minutes. Then washed 3 times with Opti-MEM to dilute out residual antibody.The percentage of CD19+ cells was quantified using an AttuneTM NxT Flow Cytometer(Invitrogen), and data was analyzed using FlowJo (BD Biosciences).

[0268] Base editing of HBECs: Briefly, HBECs were electroporated with ABE mRNAproduced via in vitro transcription and sgRNAs acquired from Synthego. Cells were detachedusing TrypLE Express Enzyme (Life Technologies, 12605028), then 100,000 cells weresuspended in Opti-MEMTM Reduced Serum Medium (Thermo Fisher Scientific, 31985088)and combined with lug of ABE8e mRNA and 90 pmol of sgRNA in a final volume of 20 µl.A nebulizer was performed in a Lonza 4D-NucleofectorTM with a 16-well NucleocuvetteStrip (Lonza, V4XP-3032), using program CA-137 with solution setting P3. Immediatelyfollowing a nebulizer, cells were diluted with 80 µl Opti-MEM and allowed to recover for 10minutes at room temperature. Edited HBECs were then plated in a 6-well plate and culturedas described above.

[0269] Measurement of base editing efficiency: Three days after a nebulizer, editing ofCFTR exon 12 was determined with amplicon sequencing. Briefly, cells were detached withTrypLE Express Enzyme (Life Technologies, 12605028) and an aliquot of 100,000 cells wascentrifuged at 300 x g for 5 minutes. The supernatant was removed and 50 µl of QuickExtract DNA Extraction Solution was added (Biosearch Technologies, QE09050). Thissuspension was incubated at 65C for 6 minutes, then 98C for 10 minutes PCR. 1 µl was thenused for template in a PCR reaction using Phusion High-Fidelity polymerase (New EnglandBiolabs M0531S) with primers that anneal to the introns adjacent to exon 12 of the CFTRlocus. Amplicons were then sequenced with Oxford Nanopore sequencing to determine thepercentage of corrected or enhanced alleles.

[0270] Air-liquid interface cultures: To measure CFTR function HBECs weredifferentiated at an air-liquid interface (ALI). Briefly, HBECs were plated on the apical sideof 6.5 mm Transwell® polyester membrane inserts with 0.4 µm pores (StemCellTechnologies, 38024) in Pneumacult Ex Plus media with 10 µM ROCK Inhibitor andiMatrix-511, as above. The basolateral side of the insert received Pneumacult Ex Plus mediawith 10 µM ROCK Inhibitor. ~80,000 cells were seeded and grown to confluency over 3-4days. Then, apical media was removed to allow growth at an air-liquid interface. Basolateralchamber media was replaced with ALI medium from the University of North Carolina, whichwas replaced every 2-3 days with fresh media, while the apical side of cells remained airexposed.

[0271] Ussing chamber functional assay: Four weeks after HBEC cultures were initiallyair-exposed for differentiation, ion transfer was measured within an Ussing Chamber.Differentiated epithelial sheets were placed in U2500 Self-contained Ussing chambers(Warner Instruments, Hamden CT), controlled with the VCC MC6 Ussing chamber system(Physiologic Instruments Inc. San Diego, CA, USA). Ion transfer was allowed to equilibratein a mucosal solution on the apical side [120 mM NaGluconate, 25 mM NaHCO3, 3.3 mMKH2PO4, 0.8 mM K2HPO4, 4 mM Ca (Gluconate)2, 1.2 mM Mg (Gluconate)2, 10 mMmannitol] and a serosal solution on the basolateral side [120 mM NaCl, 25 mM NaHCO3, 3.3mM KH2PO4, 0.8 mM K2HPO4, 1.2 mM CaCl2, 1.2 mM MgCl2, 10 mM glucose].Following equilibration, ion channel activators and inhibitors were added in the followingorder, with time provided for ion transport to equilibrate again after addition of each: 10 µMamiloride apically to inhibit Epithelial Sodium Channel (ENaC), 10 µM forskolin bilaterally(Abcam #ab120058) to activate CFTR channels via activation of adenylyl cyclaseconcentrations, 10 μM CFTRinh-172 (Sigma Millipore, #C2992) apically to inhibit CFTRchannel, and 100 μ Muridine-5' -triphosphate trisodium salt (Thermo Scientific Chemicals#AAJ63427MC) apically to stimulate ion transfer again.Results

[0272] Between 30-80% of cells were genetically modified to express the tested CFTRvariants. The expression levels of WT and four CFTR mutants believed to enhance stabilitywere compared (FIGS. 1A-1B).

[0273] Surprisingly, not all variants previously reported to improve stability of CFTR (e.g.,R553K) improved CFTR mediated ion transport in a gene therapy / editing context. Rather,the dRI and 1539T variants showed improved CFTR function relative to WT CFTR CDNA(FIGS. 2A-2B & 3C).

[0274] Similarly, not all variants previously reported to improve CFTR channel conductivity(e.g., E1371Q, H1402A, K978C, and K1250A) improved CFTR mediated ion transport in agene therapy / editing context. Only, the G550E and E1371S variants showed improved CFTRfunction relative to WT CFTR cDNA. (FIGS. 3A-3B & 3D).Example 2: mRNA therapy delivered using lipid or polymeric nanoparticles

[0275] Previous clinical trials using WT-CFTR did not show clinical benefit. Expressing anew CFTR variant that shows greater function than the WT may provide clinical benefit topatients. mRNA expressing one of the identified gain of function CFTR variants (dRI andI539T, G550E, E1371S, or a CFTR variant having a combination of these mutations) arepackaged in lipid or polymeric nanoparticles.

[0276] Lipid nanoparticles are composed of a combination of 1,2-Distearoyl-sn-glycero-3-phosphorylcholine (DSPC), Dioleoylphosphatidylethanolamine (DOPE), Polyethylene glycollipid (PEG-lipid), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), or Dilinoleyl-methyl-4-dimethylaminobutyrate, MC3 (D-Lin-MC3) or other lipids shown to enable in vivotransfection of the airway epithelium.

[0277] In some embodiments, the mRNA is administered by nebulization into the airways ofan individual with CF once every day, once every 2 days, once every 3 days, once every 4days, once every 5 days, once every 6 days, once a week, once every two weeks, once everythree weeks or once a month. Therapy is to be continued for the lifetime of a person with CF.Example 3: Autologous airway stem cell therapy to treat cystic fibrosis

[0278] An airway stem cell therapy is likely to be effective if it replaces at least 20% of theairway epithelium. However, many small airways, which form the bulk of the lungs, areinaccessible. Applicant has hypothesized that expressing a gain-of-function variant of CFTRmay reduce the fraction of the epithelium that needs to be corrected to provide therapeuticbenefit.

[0279] Airway basal stem cells expressing P63, KRT5, and NGFR are obtained from aperson with CF. Expression of the marker genes was determined by FACS. These cells arethen edited using CRISPR / Cas9, zinc finger nuclease, or TALEN to insert the full-lengthCFTR CDNA expressing one of the gain-of-function variants. AAV or single-stranded DNA-based templates are used in conjunction with CRISPR / Cas9 or other nucleases to insert theCFTR CDNA.

[0280] The patient's airways are conditioned to remove the resident diseased airway stemcells. The edited airway basal stem cells are then transplanted into the airways. This a one-time treatment for CF airway disease.Example 4: Adeno-associated virus based gene therapy for CF

[0281] Existing AAV gene therapies for CF use a truncated variant of CFTR, known as thedelta-R variant, which retains 80-100% of the function of the wild-type (WT) CFTR asdetermined by an Ussing Chamber assay. However, there is very little room remaining toinclude a strong promoter and polyA tail within the AAV vector. The delta-RI variant, whichis different from the delta-R variant, shows 2-3 times the function of WT CFTR.

[0282] The delta-RI variant can be packaged into an AAV along with a small promoter andpolyA tail. AAV serotypes can include AAV 2, AAV 2.5T, or other serotypes designed totransduce airway cells more effectively. AAV gene therapy may need to be administeredrepeatedly, at least once a year if not more frequently.Example 5 - Testing CFTR Variants for Improved Chloride Transport

[0283] Airway basal cells (ABCs) obtained from multiple donors were edited to introduceCFTR variants designed to enhance either protein stability or conductivity. Edited cellpopulations were assessed for the presence of the intended edits by quantifying tCD19-positive cells via flow cytometry. Populations containing approximately 30–70% tCD19+cells were generated for both stability-enhancing variants (FIG. 4A) and conductivity-enhancing variants (FIG. 4B). These edited cells were subsequently subjected to functionalassays measuring chloride ion transport.Example 6 - Ussing Chamber Assessment of Chloride Ion Transport by CFTR Variants

[0284] Edited ABCs containing various CFTR variants were cultured at an air-liquidinterface and evaluated for chloride ion transport using Ussing chamber electrophysiology.CFTR function was measured as the change in short-circuit current (ΔΙsc) upon addition ofCFTR-inh172.

[0285] As shown in FIG. 5A, the ARI variant (deletion of amino acids 404–435 of SEQ IDNO: 1) and the I539T variant, both classified as stability-enhancing, demonstrated improvedchloride transport compared to WT CFTR cDNA.As shown in FIG. 5B, the I539T variant exhibited the greatest fold-change in current relativeto WT CFTR.As shown in FIG. 5C, conductivity-enhancing mutants tested did not produce significantimprovements in chloride transport relative to WT CFTR.

[0286] Statistical analysis was performed using one-way ANOVA followed by Dunnett'stest, with WT-cDNA corrected cells as the reference group. Significance thresholds were P <0.05 (*) and P < 0.01 (**).Example 7 – Determination of Editing Thresholds for CFTR Function Improvement

[0287] ABCs edited with CFTR variants 1539T, G550E, or ARI were assessed for chloridetransport activity using Ussing chamber assays (FIG. 6A). Median CFTR function wasdetermined from non-CF donor samples across multiple experiments, and is indicated by thevertical dashed line on the plots.

[0288] Linear regression analysis (FIG. 6B) was performed to determine the percentage ofedited cells required to reach the median CFTR function observed in non-CF donor cells. The95% confidence intervals were calculated for each variant and compared to WT CFTRcDNA, showing that these variants required fewer edited cells to achieve median function.Example 8 – CFTR Variants Achieve Median Non-CF Function at Lower CorrectionLevels

[0289] Ussing chamber assays were performed on ABC cultures edited with WT CFTRCDNA or CFTR variants I539T (FIG. 7A), G550E (FIG. 7B), or ARI (FIG. 7C). The medianCFTR response observed in non-CF cultures is indicated by the horizontal dashed line;vertical dashed lines indicate the percentage of corrected cells required to reach this function.

[0290] WT CFTR-cDNA corrected cultures required 75% corrected cells to achieve mediannon-CF response. In contrast, the CFTR variants achieved median non-CF chloride transportfunction when only 25-35% of cells were corrected.Example 9 – Bicarbonate Transport by CFTR Variants

[0291] Differentiated airway epithelial cells edited with CFTR variants 1539T, G550E, orARI were evaluated for bicarbonate ion transport in an Ussing chamber assay.

[0292] A representative trace of bicarbonate transport activity is shown in FIG. 8A.Quantitation of bicarbonate transport, based on AIsc following CFTRinh172 treatment, isshown in FIG. 8B (n = 4 replicates). All three variants demonstrated improved bicarbonatetransport relative to WT CFTR.Example 10 - Improved CFTR Protein Expression in Edited Airway Cells

[0293] Sequential gene replacement was used to correct CFTR in airway epithelial cells witheither WT CDNA or CFTR variants I539T, G550E, or ARI. Immunoblot analyses (FIG. 9A,FIG. 9B) revealed restored CFTR expression in edited cells.

[0294] The ARI and 1539T variants produced higher levels of mature CFTR protein than WTCDNA. Unexpectedly, the G550E variant, originally classified as conductivity-enhancing,also produced more mature CFTR than WT CFTR.Example 11 - Effect of Native Locus Expression on CFTR Variant Function

[0295] Differentiated airway cultures were transfected with CFTR mRNA packaged in lipidnanoparticles (LNPs – LipidLaunch™™, Cayman Chemical, Item No. 702620) either with orwithout EGTA pretreatment to improve transfection (FIG. 10A).

[0296] Ion transport activity was assessed in Ussing chambers 24 hours post-transfection(FIG. 10B). ΔΙsc changes following CFTRinh172 treatment were recorded. No significantdifference in restored chloride transport was observed between I539T CFTR mRNA and WTCFTR mRNA under these transient expression conditions.

[0297] Importantly, these results establish that the gain-of-function benefit is morepronounced in native locus expression achieved through genome editing. Experiments withmRNA overexpression of gain-of-function variants did not demonstrate the same functionalimprovement observed with genome editing approaches.Example 12 - Base Editor-Mediated Installation of an Enhancing CFTR Mutation

[0298] An adenosine base editor (ABE) was combined with two single guide RNAs(sgRNAs): (1) an sgRNA targeting R553X to revert a premature stop codon to arginine (FIG.11A), and (2) an sgRNA targeting 1539 to install the I539T mutation.

[0299] Simultaneous delivery of both sgRNAs and ABE enables correction of the R553Xallele and enhancement of CFTR function by installing the I539T mutation.

[0300] The experimental workflow for evaluating base editor efficacy in improving CFTRfunction is shown schematically in FIG. 11B.Example 13 - Dual Base Editing Improves Chloride Transport

[0301] Human bronchial epithelial cells (HBECs) with the R553X CFTR mutation wereelectroporated with ABE and either one or both sgRNAs described in Example 12. Threedays post-a nebulizer, aliquots were lysed for genomic sequencing (FIG. 12A), PCRamplification of exon 12, and Oxford Nanopore sequencing. The percentage of readscontaining WT CFTR or the functional I539T CFTR allele was quantified.

[0302] Parallel cultures were seeded onto transwell membranes and differentiated at an air-liquid interface for 4 weeks. Ion transport activity was measured in Ussing chambers (FIG.12B) by recording AIsc changes following CFTRinh172 treatment. A 29% improvement inchloride transport was observed in cells edited with both sgRNAs compared to cells editedonly with the R553X-targeting sgRNA.

[0303] Variants for which percent of edited cells to reach median non-CF CFTR function islower compared to the WT cDNA (FIG. 12C). K978C and K1250A appear blank becausethey never reach median non-CF CFTR function.Listing of Sequences

[0304] SEQ ID NO: 1, Protein, Human, wild type CFTR proteinMQRSPLEKASVVSKLFFSWTRPILRKGYRQRLELSDIYQIPSVDSADNLSEKLEREWDRELASKKNPKLINALRRCFFWRFMFYGIFLYLGEVTKAVQPLLLGRIIASYDPDNKEERSIAIYLGIGLCLLFIVRTLLLHPAIFGLHHIGMQMRIAMFSLIYKKTLKLSSRVLDKISIGQLVSLLSNNLNKFDEGLALAHFVWIAPLQVALLMGLIWELLQASAFCGLGFLIVLALFQAGLGRMMMKYRDQRAGKISERLVITSEMIENIQSVKAYCWEEAMEKMIENLRQTELKLTRKAAYVRYFNSSAFFFSGFFVVFLSVLPYALIKGIILRKIFTTISFCIVLRMAVTRQFPWAVQTWYDSLGAINKIQDFLQKQEYKTLEYNLTTTEVVMENVTAFWEEGFGELFEKAKQNNNNRKTSNGDDSLFFSNFSLLGTPVLKDINFKIERGQLLAVAGSTGAGKTSLLMVIMGELEPSEGKIKHSGRISFCSQFSWIMPGTIKENIIFGVSYDEYRYRSVIKACQLEEDISKFAEKDNIVLGEGGITLSGGQRARISLARAVYKDADLYLLDSPFGYLDVLTEKEIFESCVCKLMANKTRILVTSKMEHLKKADKILILHEGSSYFYGTFSELQNLQPDFSSKLMGCDSFDQFSAERRNSILTETLHRFSLEGDAPVSWTETKKQSFKQTGEFGEKRKNSILNPINSIRKFSIVQKTPLQMNGIEEDSDEPLERRLSLVPDSEQGEAILPRISVISTGPTLQARRRQSVLNLMTHSVNQGQNIHRKTTASTRKVSLAPQANLTELDIYSRRLSQETGLEISEEINEEDLKECFFDDMESIPAVTTWNTYLRYITVHKSLIFVLIWCLVIFLAEVAASLVVLWLLGNTPLQDKGNSTHSRNNSYAVIITSTSSYYVFYIYVGVADTLLAMGFFRGLPLVHTLITVSKILHHKMLHSVLQAPMSTLNTLKAGGILNRFSKDIAILDDLLPLTIFDFIQLLLIVIGAIAVVAVLQPYIFVATVPVIVAFIMLRAYFLQTSQQLKQLESEGRSPIFTHLVTSLKGLWTLRAFGRQPYFETLFHKALNLHTANWFLYLSTLRWFQMRIEMIFVIFFIAVTFISILTTGEGEGRVGIILTLAMNIMSTLQWAVNSSIDVDSLMRSVSRVFKFIDMPTEGKPTKSTKPYKNGQLSKVMIIENSHVKKDDIWPSGGQMTVKDLTAKYTEGGNAILENISFSISPGQRVGLLGRTGSGKSTLLSAFLRLLNTEGEIQIDGVSWDSITLQQWRKAFGVIPQKVFIFSGTFRKNLDPYEQWSDQEIWKVADEVGLRSVIEQFPGKLDFVLVDGGCVLSHGHKQLMCLARSVLSKAKILLLDEPSAHLDPVTYQIIRRTLKQAFADCTVILCEHRIEAMLECQQFLVIEENKVRQYDSIQKLLNERSLFRQAISPSDRVKLFPHRNSSKCKSKPQIAALKEETEEEVQDTRL

[0305] SEQ ID NO: 2, Protein, Human, CFTR delta404-435(dRI) mutantMQRSPLEKASVVSKLFFSWTRPILRKGYRQRLELSDIYQIPSVDSADNLSEKLEREWDRELASKKNPKLINALRRCFFWRFMFYGIFLYLGEVTKAVQPLLLGRIIASYDPDNKEERSIAIYLGIGLCLLFIVRTLLLHPAIFGLHHIGMQMRIAMFSLIYKKTLKLSSRVLDKISIGQLVSLLSNNLNKFDEGLALAHFVWIAPLQVALLMGLIWELLQASAFCGLGFLIVLALFQAGLGRMMMKYRDQRAGKISERLVITSEMIENIQSVKAYCWEEAMEKMIENLRQTELKLTRKAAYVRYFNSSAFFFSGFFVVFLSVLPYALIKGIILRKIFTTISFCIVLRMAVTRQFPWAVQTWYDSLGAINKIQDFLQKQEYKTLEYNLTTTEVVMENVTAFWEELGTPVLKDINFKIERGQLLAVAGSTGAGKTSLLMVIMGELEPSEGKIKHSGRISFCSQFSWIMPGTIKENIIFGVSYDEYRYRSVIKACQLEEDISKFAEKDNIVLGEGGITLSGGQRARISLARAVYKDADLYLLDSPFGYLDVLTEKEIFESCVCKLMANKTRILVTSKMEHLKKADKILILHEGSSYFYGTFSELQNLQPDFSSKLMGCDSFDQFSAERRNSILTETLHRFSLEGDAPVSWTETKKQSFKQTGEFGEKRKNSILNPINSIRKFSIVQKTPLQMNGIEEDSDEPLERRLSLVPDSEQGEAILPRISVISTGPTLQARRRQSVLNLMTHSVNQGQNIHRKTTASTRKVSLAPQANLTELDIYSRRLSQETGLEISEEINEEDLKECFFDDMESIPAVTTWNTYLRYITVHKSLIFVLIWCLVIFLAEVAASLVVLWLLGNTPLQDKGNSTHSRNNSYAVIITSTSSYYVFYIYVGVADTLLAMGFFRGLPLVHTLITVSKILHHKMLHSVLQAPMSTLNTLKAGGILNRFSKDIAILDDLLPLTIFDFIQLLLIVIGAIAVVAVLQPYIFVATVPVIVAFIMLRAYFLQTSQQLKQLESEGRSPIFTHLVTSLKGLWTLRAFGRQPYFETLFHKALNLHTANWFLYLSTLRWFQMRIEMIFVIFFIAVTFISILTTGEGEGRVGIILTLAMNIMSTLQWAVNSSIDVDSLMRSVSRVFKFIDMPTEGKPTKSTKPYKNGQLSKVMIIENSHVKKDDIWPSGGQMTVKDLTAKYTEGGNAILENISFSISPGQRVGLLGRTGSGKSTLLSAFLRLLNTEGEIQIDGVSWDSITLQQWRKAFGVIPQKVFIFSGTFRKNLDPYEQWSDQEIWKVADEVGLRSVIEQFPGKLDFVLVDGGCVLSHGHKQLMCLARSVLSKAKILLLDEPSAHLDPVTYQIIRRTLKQAFADCTVILCEHRIEAMLECQQFLVIEENKVRQYDSIQKLLNERSLFRQAISPSDRVKLFPHRNSSKCKSKPQIAALKEETEEEVQDTRL

[0306] SEQ ID NO: 3, Protein, Human, CFTR 1539T mutantMQRSPLEKASVVSKLFFSWTRPILRKGYRQRLELSDIYQIPSVDSADNLSEKLEREWDRELASKKNPKLINALRRCFFWRFMFYGIFLYLGEVTKAVQPLLLGRIIASYDPDNKEERSIAIYLGIGLCLLFIVRTLLLHPAIFGLHHIGMQMRIAMFSLIYKKTLKLSSRVLDKISIGQLVSLLSNNLNKFDEGLALAHFVWIAPLQVALLMGLIWELLQASAFCGLGFLIVLALFQAGLGRMMMKYRDQRAGKISERLVITSEMIENIQSVKAYCWEEAMEKMIENLRQTELKLTRKAAYVRYFNSSAFFFSGFFVVFLSVLPYALIKGIILRKIFTTISFCIVLRMAVTRQFPWAVQTWYDSLGAINKIQDFLQKQEYKTLEYNLTTTEVVMENVTAFWEEGFGELFEKAKQNNNNRKTSNGDDSLFFSNFSLLGTPVLKDINFKIERGQLLAVAGSTGAGKTSLLMVIMGELEPSEGKIKHSGRISFCSQFSWIMPGTIKENIIFGVSYDEYRYRSVIKACQLEEDISKFAEKDNTVLGEGGITLSGGQRARISLARAVYKDADLYLLDSPFGYLDVLTEKEIFESCVCKLMANKTRILVTSKMEHLKKADKILILHEGSSYFYGTFSELQNLQPDFSSKLMGCDSFDQFSAERRNSILTETLHRFSLEGDAPVSWTETKKQSFKQTGEFGEKRKNSILNPINSIRKFSIVQKTPLQMNGIEEDSDEPLERRLSLVPDSEQGEAILPRISVISTGPTLQARRRQSVLNLMTHSVNQGQNIHRKTTASTRKVSLAPQANLTELDIYSRRLSQETGLEISEEINEEDLKECFFDDMESIPAVTTWNTYLRYITVHKSLIFVLIWCLVIFLAEVAASLVVLWLLGNTPLQDKGNSTHSRNNSYAVIITSTSSYYVFYIYVGVADTLLAMGFFRGLPLVHTLITVSKILHHKMLHSVLQAPMSTLNTLKAGGILNRFSKDIAILDDLLPLTIFDFIQLLLIVIGAIAVVAVLQPYIFVATVPVIVAFIMLRAYFLQTSQQLKQLESEGRSPIFTHLVTSLKGLWTLRAFGRQPYFETLFHKALNLHTANWFLYLSTLRWFQMRIEMIFVIFFIAVTFISILTTGEGEGRVGIILTLAMNIMSTLQWAVNSSIDVDSLMRSVSRVFKFIDMPTEGKPTKSTKPYKNGQLSKVMIIENSHVKKDDIWPSGGQMTVKDLTAKYTEGGNAILENISFSISPGQRVGLLGRTGSGKSTLLSAFLRLLNTEGEIQIDGVSWDSITLQQWRKAFGVIPQKVFIFSGTFRKNLDPYEQWSDQEIWKVADEVGLRSVIEQFPGKLDFVLVDGGCVLSHGHKQLMCLARSVLSKAKILLLDEPSAHLDPVTYQIIRRTLKQAFADCTVILCEHRIEAMLECQQFLVIEENKVRQYDSIQKLLNERSLFRQAISPSDRVKLFPHRNSSKCKSKPQIAALKEETEEEVQDTRL

[0307] SEQ ID NO: 4, Protein, Human, CFTR G550E mutantMQRSPLEKASVVSKLFFSWTRPILRKGYRQRLELSDIYQIPSVDSADNLSEKLEREWDRELASKKNPKLINALRRCFFWRFMFYGIFLYLGEVTKAVQPLLLGRIIASYDPDNKEERSIAIYLGIGLCLLFIVRTLLLHPAIFGLHHIGMQMRIAMFSLIYKKTLKLSSRVLDKISIGQLVSLLSNNLNKFDEGLALAHFVWIAPLQVALLMGLIWELLQASAFCGLGFLIVLALFQAGLGRMMMKYRDQRAGKISERLVITSEMIENIQSVKAYCWEEAMEKMIENLRQTELKLTRKAAYVRYFNSSAFFFSGFFVVFLSVLPYALIKGIILRKIFTTISFCIVLRMAVTRQFPWAVQTWYDSLGAINKIQDFLQKQEYKTLEYNLTTTEVVMENVTAFWEEGFGELFEKAKQNNNNRKTSNGDDSLFFSNFSLLGTPVLKDINFKIERGQLLAVAGSTGAGKTSLLMVIMGELEPSEGKIKHSGRISFCSQFSWIMPGTIKENIIFGVSYDEYRYRSVIKACQLEEDISKFAEKDNIVLGEGGITLSEGQRARISLARAVYKDADLYLLDSPFGYLDVLTEKEIFESCVCKLMANKTRILVTSKMEHLKKADKILILHEGSSYFYGTFSELQNLQPDFSSKLMGCDSFDQFSAERRNSILTETLHRFSLEGDAPVSWTETKKQSFKQTGEFGEKRKNSILNPINSIRKFSIVQKTPLQMNGIEEDSDEPLERRLSLVPDSEQGEAILPRISVISTGPTLQARRRQSVLNLMTHSVNQGQNIHRKTTASTRKVSLAPQANLTELDIYSRRLSQETGLEISEEINEEDLKECFFDDMESIPAVTTWNTYLRYITVHKSLIFVLIWCLVIFLAEVAASLVVLWLLGNTPLQDKGNSTHSRNNSYAVIITSTSSYYVFYIYVGVADTLLAMGFFRGLPLVHTLITVSKILHHKMLHSVLQAPMSTLNTLKAGGILNRFSKDIAILDDLLPLTIFDFIQLLLIVIGAIAVVAVLQPYIFVATVPVIVAFIMLRAYFLQTSQQLKQLESEGRSPIFTHLVTSLKGLWTLRAFGRQPYFETLFHKALNLHTANWFLYLSTLRWFQMRIEMIFVIFFIAVTFISILTTGEGEGRVGIILTLAMNIMSTLQWAVNSSIDVDSLMRSVSRVFKFIDMPTEGKPTKSTKPYKNGQLSKVMIIENSHVKKDDIWPSGGQMTVKDLTAKYTEGGNAILENISFSISPGQRVGLLGRTGSGKSTLLSAFLRLLNTEGEIQIDGVSWDSITLQQWRKAFGVIPQKVFIFSGTFRKNLDPYEQWSDQEIWKVADEVGLRSVIEQFPGKLDFVLVDGGCVLSHGHKQLMCLARSVLSKAKILLLDEPSAHLDPVTYQIIRRTLKQAFADCTVILCEHRIEAMLECQQFLVIEENKVRQYDSIQKLLNERSLFRQAISPSDRVKLFPHRNSSKCKSKPQIAALKEETEEEVQDTRL

[0308] SEQ ID NO: 5, Protein, Human, E1371S mutantMQRSPLEKASVVSKLFFSWTRPILRKGYRQRLELSDIYQIPSVDSADNLSEKLEREWDRELASKKNPKLINALRRCFFWRFMFYGIFLYLGEVTKAVQPLLLGRIIASYDPDNKEERSIAIYLGIGLCLLFIVRTLLLHPAIFGLHHIGMQMRIAMFSLIYKKTLKLSSRVLDKISIGQLVSLLSNNLNKFDEGLALAHFVWIAPLQVALLMGLIWELLQASAFCGLGFLIVLALFQAGLGRMMMKYRDQRAGKISERLVITSEMIENIQSVKAYCWEEAMEKMIENLRQTELKLTRKAAYVRYFNSSAFFFSGFFVVFLSVLPYALIKGIILRKIFTTISFCIVLRMAVTRQFPWAVQTWYDSLGAINKIQDFLQKQEYKTLEYNLTTTEVVMENVTAFWEEGFGELFEKAKQNNNNRKTSNGDDSLFFSNFSLLGTPVLKDINFKIERGQLLAVAGSTGAGKTSLLMVIMGELEPSEGKIKHSGRISFCSQFSWIMPGTIKENIIFGVSYDEYRYRSVIKACQLEEDISKFAEKDNIVLGEGGITLSGGQRARISLARAVYKDADLYLLDSPFGYLDVLTEKEIFESCVCKLMANKTRILVTSKMEHLKKADKILILHEGSSYFYGTFSELQNLQPDFSSKLMGCDSFDQFSAERRNSILTETLHRFSLEGDAPVSWTETKKQSFKQTGEFGEKRKNSILNPINSIRKFSIVQKTPLQMNGIEEDSDEPLERRLSLVPDSEQGEAILPRISVISTGPTLQARRRQSVLNLMTHSVNQGQNIHRKTTASTRKVSLAPQANLTELDIYSRRLSQETGLEISEEINEEDLKECFFDDMESIPAVTTWNTYLRYITVHKSLIFVLIWCLVIFLAEVAASLVVLWLLGNTPLQDKGNSTHSRNNSYAVIITSTSSYYVFYIYVGVADTLLAMGFFRGLPLVHTLITVSKILHHKMLHSVLQAPMSTLNTLKAGGILNRFSKDIAILDDLLPLTIFDFIQLLLIVIGAIAVVAVLQPYIFVATVPVIVAFIMLRAYFLQTSQQLKQLESEGRSPIFTHLVTSLKGLWTLRAFGRQPYFETLFHKALNLHTANWFLYLSTLRWFQMRIEMIFVIFFIAVTFISILTTGEGEGRVGIILTLAMNIMSTLQWAVNSSIDVDSLMRSVSRVFKFIDMPTEGKPTKSTKPYKNGQLSKVMIIENSHVKKDDIWPSGGQMTVKDLTAKYTEGGNAILENISFSISPGQRVGLLGRTGSGKSTLLSAFLRLLNTEGEIQIDGVSWDSITLQQWRKAFGVIPQKVFIFSGTFRKNLDPYEQWSDQEIWKVADEVGLRSVIEQFPGKLDFVLVDGGCVLSHGHKQLMCLARSVLSKAKILLLDSPSAHLDPVTYQIIRRTLKQAFADCTVILCEHRIEAMLECQQFLVIEENKVRQYDSIQKLLNERSLFRQAISPSDRVKLFPHRNSSKCKSKPQIAALKEETEEEVQDTRLEquivalents

[0309] Unless otherwise defined, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art to which this technologybelongs.

[0310] The present technology illustratively described herein may suitably be practiced in theabsence of any element or elements, limitation or limitations, not specifically disclosedherein. Thus, for example, the terms "comprising,” “including,” “containing," etc. shall beread expansively and without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, and there is no intentionin the use of such terms and expressions of excluding any equivalents of the features shownand described or portions thereof, but it is recognized that various modifications are possiblewithin the scope of the present technology claimed.

[0311] Thus, it should be understood that the materials, methods, and examples providedhere are representative of preferred aspects, are exemplary, and are not intended aslimitations on the scope of the present technology.

[0312] It should be understood that although the present invention has been specificallydisclosed by certain aspects, embodiments, and optional features, modification, improvementand variation of such aspects, embodiments, and optional features can be resorted to by thoseskilled in the art, and that such modifications, improvements and variations are considered tobe within the scope of this disclosure.

[0313] The present technology has been described broadly and generically herein. Each ofthe narrower species and sub-generic groupings falling within the generic disclosure alsoform part of the present technology. This includes the generic description of the presenttechnology with a proviso or negative limitation removing any subject matter from the genus,regardless of whether or not the excised material is specifically recited herein.

[0314] In addition, where features or aspects of the present technology are described in termsof Markush groups, those skilled in the art will recognize that the present technology is alsothereby described in terms of any individual member or subgroup of members of the Markushgroup.

[0315] All publications, patent applications, patents, and other references mentioned hereinare expressly incorporated by reference in their entirety, to the same extent as if each wereincorporated by reference individually. In case of conflict, the present specification,including definitions, will control.

[0316] Other aspects are set forth within the following claims.

Claims

WHAT IS CLAIMED IS:1.A method for treating cystic fibrosis in a subject in need thereof comprising:administering to the subject a gene editing construct comprising a nucleic acid encoding amodified cystic fibrosis transmembrane conductance regulator (CFTR) polypeptide or afunctional fragment thereof, wherein the modified CFTR polypeptide or the functionalfragment thereof differs from a wild-type human CFTR protein of SEQ ID NO: 1 by thepresence of at least one mutation selected from:a deletion of amino acids at positions 404-435, I539T, G550E, S495P or R553K,wherein the gene editing construct is configured to insert the nucleic acid encoding themodified CFTR polypeptide or functional fragment thereof into the native CFTR locus of acell of the subject.2.The method of claim 1, wherein the modified CFTR polypeptide or the functionalfragment thereof comprises any one of SEQ ID NO: 2-5.

3. The method of claim 1 or claim 2, wherein the gene editing construct is deliveredwithin a nanoparticle.4.The method of claim 3, wherein the nanoparticle is a lipid nanoparticle or a polymernanoparticle.5.The method of any one of claims 3 or 4, wherein the nanoparticle comprises one ormore of 1,2-Distearoyl-sn-glycero-3-phosphorylcholine (DSPC),Dioleoylphosphatidylethanolamine (DOPE), Polyethylene glycol lipid (PEG-lipid), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), or Dilinoleyl-methyl-4-dimethylaminobutyrate, MC3 (D-Lin-MC3).

6. The method of any one of claims 1-5, wherein the gene editing construct furthercomprises a viral vector selected from an adeno-associated virus (AAV) vector, a bocavirusvector, or a herpesvirus vector.7.The method of claim 6, wherein the AAV vector is selected from AAV1, AAV2,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVPHP.B, AAV rh74, AAV 2.5T, or a variant of each thereof.

8. The method of any one of claims 1-7, wherein the gene editing construct furthercomprises a CRISPR-Cas system, a base editing system, a prime editing system, a ZincFinger Nuclease system, or a TALEN system.

9. The method of claim 8, wherein the CRISPR-Cas system comprises a Cas9ribonucleoprotein (RNP) and a single guide RNA (sgRNA).

10. The method of any one of claims 1-9, wherein the gene editing construct is deliveredto airway basal stem cells of the subject ex vivo, and wherein the method further comprisestransplanting the edited airway basal stem cells to the subject's airways.

11. The method of claim 10, wherein the airway basal stem cells are P63+, KRT5+, andNGFR+.

12. The method of claim 10 or claim 11, further comprising conditioning the subject'sairways to remove diseased airway epithelial cells prior to transplanting the edited airwaybasal stem cells.

13. The method of claim 12, wherein the conditioning comprises introducing an acuteairway injury to the subject's airways.

14. The method of claim 13, wherein the acute airway injury is a chemical injury inducedby polidocanol (PDOC) or a mechanical injury induced by an endoscopic device.

15. The method of any one of claims 12-14, wherein the conditioning comprisesremoving between at least 30% and 99% of diseased airway epithelial cells.

16. The method of any one of claims 1-15, wherein the subject is a human patient.17.A method for treating cystic fibrosis in a subject in need thereof comprising:(a) modifying airway basal stem cells from the subject ex vivo to express a modified cysticfibrosis transmembrane conductance regulator (CFTR) polypeptide or a functional fragmentthereof, wherein the modified CFTR polypeptide or the functional fragment thereof differsfrom a wild-type human CFTR protein of SEQ ID NO: 1 by the presence of at least onemutation selected from:a deletion of amino acids at positions 404-435, I539T, G550E, S495P or R553K;and(b) transplanting the airway basal stem cells to the subject's airways,wherein the modifying in (a) is achieved by a gene editing system that inserts a nucleic acidencoding the modified CFTR polypeptide or functional fragment thereof into the nativeCFTR locus of the airway basal stem cells.

18. The method of claim 17, wherein the airway basal stem cells are P63+, KRT5+, andNGFR+.

19. The method of claim 17 or claim 18, further comprising conditioning the subject'sairways to remove diseased airway epithelial cells before (b), optionally wherein theconditioning of the airways comprises introducing an acute airway injury to the subject'sairways.

20. The method of claim 19, wherein the acute airway injury is a chemical injury inducedby polidocanol (PDOC) or a mechanical injury induced by an endoscopic device.

21. The method of any one of claims 17-20, wherein the conditioning comprisesremoving between at least 30% and 99% of diseased airway epithelial cells.

22. The method of any one of claims 17-21, wherein the gene editing system is selectedfrom CRISPR-Cas, Zinc Finger Nuclease, or TALEN.

23. The method of claim 22, wherein the gene editing system comprises a Cas9ribonucleoprotein (RNP) and a single guide RNA (sgRNA).

24. The method of any one of claims 17-23, wherein the modified CFTR polypeptide orthe functional fragment thereof comprises any one of SEQ ID NO: 2-5.25.The method of any one of claims 17-24, wherein the gene editing system is deliveredwithin a nanoparticle.

26. The method of claim 25, wherein the nanoparticle is a lipid nanoparticle or a polymernanoparticle.27.The method of claim 25 or claim 26, wherein the nanoparticle comprises one or moreof 1,2-Distearoyl-sn-glycero-3-phosphorylcholine (DSPC),Dioleoylphosphatidylethanolamine (DOPE), Polyethylene glycol lipid (PEG-lipid), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), or Dilinoleyl-methyl-4-dimethylaminobutyrate, MC3 (D-Lin-MC3).

28. The method of any one of claims 17-27, wherein the gene editing system is deliveredby a viral vector selected from an adeno-associated virus (AAV) vector, a bocavirus vector,or a herpesvirus vector.29.The method of claim 28, wherein the AAV vector is selected from AAV1, AAV2,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVPHP.B, AAV rh74, AAV 2.5T, or a variant thereof.

30. The method of any one of claims 17-29, wherein the subject is a human patient.31.A composition comprising a gene editing construct comprising a nucleic acidencoding a modified cystic fibrosis transmembrane conductance regulator (CFTR)polypeptide or a functional fragment thereof, wherein the modified CFTR polypeptide or thefunctional fragment thereof differs from a wild-type human CFTR protein of SEQ ID NO: 1by the presence of at least one mutation selected from:a deletion of amino acids at positions 404-435, a 1539T mutation, a G550E mutation, a S495Pmutation, or a R553K mutation,wherein the gene editing construct is configured to insert the nucleic acid encoding themodified CFTR polypeptide or functional fragment thereof into the native CFTR locus of acell.

32. The composition of claim 31, wherein the modified CFTR polypeptide or thefunctional fragment thereof comprises any one of SEQ ID NO: 2-5.

33. The composition of claim 31 or claim 32, wherein the gene editing construct iscontained within a nanoparticle.

34. The composition of claim 33, wherein the nanoparticle is a lipid nanoparticle or apolymer nanoparticle.

35. The composition of claim 33 or claim 34, wherein the nanoparticle comprises one ormore of 1,2-Distearoyl-sn-glycero-3-phosphorylcholine (DSPC),Dioleoylphosphatidylethanolamine (DOPE), Polyethylene glycol lipid (PEG-lipid), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), or Dilinoleyl-methyl-4-dimethylaminobutyrate, MC3 (D-Lin-MC3).36.The composition of any one of claims 31-35, wherein the gene editing constructfurther comprises a viral vector selected from an adeno-associated virus (AAV) vector, abocavirus vector, or a herpesvirus vector.

37. The composition of claim 36, wherein the AAV vector is selected from AAV1,AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13,AAV PHP.B, AAV rh74, AAV 2.5T, or a variant thereof.38.The composition of any one of claims 31-37, wherein the gene editing constructcomprises a CRISPR-Cas system, a base editing system, a prime editing system, a ZincFinger Nuclease system, or a TALEN system.

39. The composition of claim 38, wherein the CRISPR-Cas system comprises a Cas9ribonucleoprotein (RNP) and a single guide RNA (sgRNA).

40. The composition of any one of claims 31-39, further comprising a pharmaceuticallyacceptable carrier.41.A method for treating cystic fibrosis in a subject having a base-editable mutation or aprime-editable mutation in the CFTR gene, the method comprising:administering to the subject a gene editing system selected from a base editing system or aprime editing system, the gene editing system comprising:a base editor or a prime editor; anda first single guide RNA (sgRNA) that targets the base-editable mutation or the prime-editable mutation; anda second sgRNA to install a gain-of-function mutation in the native CFTR locus, wherein thesecond sgRNA targets one of (i) codon 1539, (ii) codon G550, (iii) codon S495, or (iv) codonR553 with respect to SEQ ID NO: 1, wherein the base editing system or the prime editingsystem is configured to correct the base-editable mutation or the prime-editable mutation andintroduce the gain-of-function mutation in the native CFTR locus of a cell of the subject.

42. The method of claim 41, wherein the gene editing system is administeredintravenously.

43. The method of claim 41 or claim 42, wherein the gene editing system is administeredto airway epithelial cells of the subject.44.The method of any one of claims 41-43, wherein the base editing system isadministered by a nebulizer.

45. The method of any one of claims 42-44, wherein gene editing system is administeredas mRNA.

46. The method of any one of claims 41-45, wherein the gene editing system isadministered within a nanoparticle.

47. The method of claim 46, wherein the nanoparticle is a lipid nanoparticle or a polymernanoparticle.

48. The method of claim 46 or claim 47, wherein the nanoparticle comprises one or moreof 1,2-Distearoyl-sn-glycero-3-phosphorylcholine (DSPC),Dioleoylphosphatidylethanolamine (DOPE), Polyethylene glycol lipid (PEG-lipid), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), or Dilinoleyl-methyl-4-dimethylaminobutyrate, MC3 (D-Lin-MC3).

49. The method of any one of claims 41-48, wherein the gene editing system furthercomprises a viral vector selected from an adeno-associated virus (AAV) vector, a bocavirusvector, or a herpesvirus vector.

50. The method of claim 49, wherein the AAV vector is selected from AAV1, AAV2,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVPHP.B, AAV rh74, AAV 2.5T, or a variant thereof.

51. The method of any one of claims 41-50, wherein the gene editing system isadministered to airway basal stem cells of the subject ex vivo, and wherein the methodfurther comprises transplanting the edited airway basal stem cells to the subject's airways.

52. The method of claim 51, wherein the airway basal stem cells are P63+, KRT5+, andNGFR+.

53. The method of claim 51 or claim 52, further comprising conditioning the subject'sairways to remove diseased airway epithelial cells prior to transplanting the edited airwaybasal stem cells.54.The method of claim 53, wherein the conditioning comprises introducing an acuteairway injury to the subject's airways.

55. The method of claim 54, wherein the acute airway injury is induced a chemical injuryinduced by polidocanol (PDOC) or a mechanical injury induced by an endoscopic device.

56. The method of any one of claims 53-55, wherein the conditioning comprisesremoving between at least 30% and 99% of diseased airway epithelial cells.

57. The method of any one of claims 41-56, wherein the subject is a human patient.58.A method for treating cystic fibrosis in a subject having an R553X mutation in theCFTR gene, the method comprising:(a) modifying airway basal stem cells from the subject ex vivo using a base editing systemcomprising:a base editing system; anda first single guide RNA (sgRNA) that targets the R553X mutation to revert a premature stopcodon to an arginine codon; anda second sgRNA to install a gain-of-function mutation in the native CFTR locus, wherein thesecond sgRNA targets one of (i) codon 1539, (ii) codon G550, or (iii) codon E1371 withrespect to SEQ ID NO: 1,wherein the base editing system is configured to correct the R553X mutation and introducethe gain-of-function mutation in the native CFTR locus of the airway basal stem cells;(b) conditioning the subject's airways to remove diseased airway epithelial cells; and(c) transplanting the airway basal stem cells to the subject's airways.59.The method of claim 58, wherein the airway basal stem cells are P63+, KRT5+, andNGFR+.

60. The method of claim 58 or 59, wherein the conditioning in (b) comprises introducingan acute airway injury to the subject's airways.

61. The method of claim 60, wherein the acute airway injury is a chemical injury inducedby polidocanol (PDOC) or a mechanical injury induced by an endoscopic device.

62. The method of any one of claims 58-61, wherein the conditioning comprisesremoving between at least 30% and 99% of diseased airway epithelial cells.

63. The method of any one of claims 58-62, wherein the first sgRNA and the secondsgRNA are delivered simultaneously with the adenosine base editor (ABE).

64. The method of any one of claims 58-63, wherein the adenosine base editor (ABE) isdelivered as mRNA.

65. The method of any one of claims 58-64, wherein the base editing system is deliveredby a nebulizer.

66. The method of any one of claims 58-65, wherein the base editing system is deliveredwithin a nanoparticle.

67. The method of claim 66, wherein the nanoparticle is a lipid nanoparticle or a polymernanoparticle.

68. The method of claim 66 or claim 67, wherein the nanoparticle comprises one or moreof 1,2-Distearoyl-sn-glycero-3-phosphorylcholine (DSPC),Dioleoylphosphatidylethanolamine (DOPE), Polyethylene glycol lipid (PEG-lipid), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), or Dilinoleyl-methyl-4-dimethylaminobutyrate, MC3 (D-Lin-MC3).

69. The method of any one of claims 58-68, wherein the base editing system is deliveredby a viral vector selected from an adeno-associated virus (AAV) vector, a bocavirus vector,or a herpesvirus vector.

70. The method of claim 69, wherein the AAV vector is selected from AAV1, AAV2,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVPHP.B, AAV rh74, AAV 2.5T, or a variant thereof.71.The method of any one of claims 58-70, wherein the subject is a human patient.

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