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• W3021862015 abstract "There is a strong rationale to consider future cell therapeutic approaches for cystic fibrosis (CF) in which autologous proximal airway basal stem cells, corrected for CFTR mutations, are transplanted into the patient’s lungs. We assessed the possibility of editing the CFTR locus in these cells using zinc-finger nucleases and have pursued two approaches. The first, mutation-specific correction, is a footprint-free method replacing the CFTR mutation with corrected sequences. We have applied this approach for correction of ΔF508, demonstrating restoration of mature CFTR protein and function in air-liquid interface cultures established from bulk edited basal cells. The second is targeting integration of a partial CFTR cDNA within an intron of the endogenous CFTR gene, providing correction for all CFTR mutations downstream of the integration and exploiting the native CFTR promoter and chromatin architecture for physiologically relevant expression. Without selection, we observed highly efficient, site-specific targeted integration in basal cells carrying various CFTR mutations and demonstrated restored CFTR function at therapeutically relevant levels. Significantly, Omni-ATAC-seq analysis revealed minimal impact on the positions of open chromatin within the native CFTR locus. These results demonstrate efficient functional correction of CFTR and provide a platform for further ex vivo and in vivo editing. There is a strong rationale to consider future cell therapeutic approaches for cystic fibrosis (CF) in which autologous proximal airway basal stem cells, corrected for CFTR mutations, are transplanted into the patient’s lungs. We assessed the possibility of editing the CFTR locus in these cells using zinc-finger nucleases and have pursued two approaches. The first, mutation-specific correction, is a footprint-free method replacing the CFTR mutation with corrected sequences. We have applied this approach for correction of ΔF508, demonstrating restoration of mature CFTR protein and function in air-liquid interface cultures established from bulk edited basal cells. The second is targeting integration of a partial CFTR cDNA within an intron of the endogenous CFTR gene, providing correction for all CFTR mutations downstream of the integration and exploiting the native CFTR promoter and chromatin architecture for physiologically relevant expression. Without selection, we observed highly efficient, site-specific targeted integration in basal cells carrying various CFTR mutations and demonstrated restored CFTR function at therapeutically relevant levels. Significantly, Omni-ATAC-seq analysis revealed minimal impact on the positions of open chromatin within the native CFTR locus. These results demonstrate efficient functional correction of CFTR and provide a platform for further ex vivo and in vivo editing. There is still much to be learned about turnover of cells in the human airway, as well as the identity of potential stem/progenitor cells responsible for overall pulmonary architecture and maintenance. There is an emerging consensus, however, that pseudo-stratified epithelial tissue of the human proximal airway contains basal cells capable both of self-renewing cell division, as well as differentiation to other specialized cells, including ciliated and secretory cells. As such, proximal airway basal cells serve as a major class of stem/progenitor cells within the proximal airway.1Hogan B.L. Barkauskas C.E. Chapman H.A. Epstein J.A. Jain R. Hsia C.C. Niklason L. Calle E. Le A. Randell S.H. et al.Repair and regeneration of the respiratory system: complexity, plasticity, and mechanisms of lung stem cell function.Cell Stem Cell. 2014; 15: 123-138Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar, 2Rock J.R. Onaitis M.W. Rawlins E.L. Lu Y. Clark C.P. Xue Y. Randell S.H. Hogan B.L. Basal cells as stem cells of the mouse trachea and human airway epithelium.Proc. Natl. Acad. Sci. USA. 2009; 106: 12771-12775Crossref PubMed Scopus (1012) Google Scholar, 3Rock J.R. Randell S.H. Hogan B.L. Airway basal stem cells: a perspective on their roles in epithelial homeostasis and remodeling.Dis. Model. Mech. 2010; 3: 545-556Crossref PubMed Scopus (492) Google Scholar, 4Hong K.U. Reynolds S.D. Watkins S. Fuchs E. Stripp B.R. Basal cells are a multipotent progenitor capable of renewing the bronchial epithelium.Am. J. Pathol. 2004; 164: 577-588Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar Thus, there is strong rationale to consider future cell therapeutic approaches for CF in which autologous proximal airway basal cells, corrected for CFTR gene mutations, are transplanted into the lungs of affected CF patients. To that end, our objective has been to prepare a population of CFTR corrected, patient-specific airway basal cells, which retain the ability to develop pseudo-stratified airway epithelium with restored CFTR function. Several groups, including our own, have utilized various methods to directly correct or compensate for CFTR mutations in relevant cell types via editing or gene transfer.5Harrison P.T. Hoppe N. Martin U. Gene editing & stem cells.J. Cyst. Fibros. 2018; 17: 10-16Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 6Firth A.L. Menon T. Parker G.S. Qualls S.J. Lewis B.M. Ke E. Dargitz C.T. Wright R. Khanna A. Gage F.H. Verma I.M. Functional gene correction for cystic fibrosis in lung epithelial cells generated from patient ipscs.Cell Rep. 2015; 12: 1385-1390Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 7Bednarski C. Tomczak K. Vom Hövel B. Weber W.M. Cathomen T. Targeted integration of a super-exon into the cftr locus leads to functional correction of a cystic fibrosis cell line model.PLoS ONE. 2016; 11: e0161072Crossref PubMed Scopus (36) Google Scholar, 8Merkert S. Bednarski C. Göhring G. Cathomen T. Martin U. Generation of a gene-corrected isogenic control iPSC line from cystic fibrosis patient-specific iPSCs homozygous for p.Phe508del mutation mediated by TALENs and ssODN.Stem Cell Res. (Amst.). 2017; 23: 95-97Crossref PubMed Scopus (25) Google Scholar, 9Suzuki S. Sargent R.G. Illek B. Fischer H. Esmaeili-Shandiz A. Yezzi M.J. Lee A. Yang Y. Kim S. Renz P. et al.Talens facilitate single-step seamless sdf correction of f508del cftr in airway epithelial submucosal gland cell-derived cf-ipscs.Mol. Ther. Nucleic Acids. 2016; 5: e273Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 10Maule G. Casini A. Montagna C. Ramalho A.S. De Boeck K. Debyser Z. Carlon M.S. Petris G. Cereseto A. Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing.Nat. Commun. 2019; 10: 3556Crossref PubMed Scopus (45) Google Scholar, 11Vaidyanathan S. Salahudeen A.A. Sellers Z.M. Bravo D.T. Choi S.S. Batish A. Le W. Baik R. de la O.S. Kaushik M.P. et al.High-efficiency, selection-free gene repair in airway stem cells from cystic fibrosis patients rescues cftr function in differentiated epithelia.Cell Stem Cell. 2020; 26: 161-171Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 12Jennings S. Ng H.P. Wang G. Establishment of a ΔF508-CF promyelocytic cell line for cystic fibrosis research and drug screening.J. Cyst. Fibros. 2019; 18: 44-53Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar, 13Ruan J. Hirai H. Yang D. Ma L. Hou X. Jiang H. Wei H. Rajagopalan C. Mou H. Wang G. et al.Efficient gene editing at major cftr mutation loci.Mol. Ther. Nucleic Acids. 2019; 16: 73-81Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 14Ramalingam S. London V. Kandavelou K. Cebotaru L. Guggino W. Civin C. Chandrasegaran S. Generation and genetic engineering of human induced pluripotent stem cells using designed zinc finger nucleases.Stem Cells Dev. 2013; 22: 595-610Crossref PubMed Scopus (42) Google Scholar, 15Valley H.C. Bukis K.M. Bell A. Cheng Y. Wong E. Jordan N.J. Allaire N.E. Sivachenko A. Liang F. Bihler H. et al.Isogenic cell models of cystic fibrosis-causing variants in natively expressing pulmonary epithelial cells.J. Cyst. Fibros. 2019; 18: 476-483Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 16Crane A.M. Kramer P. Bui J.H. Chung W.J. Li X.S. Gonzalez-Garay M.L. Hawkins F. Liao W. Mora D. Choi S. et al.Targeted correction and restored function of the CFTR gene in cystic fibrosis induced pluripotent stem cells.Stem Cell Reports. 2015; 4: 569-577Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 17Sinn P.L. Hwang B.Y. Li N. Ortiz J.L.S. Shirazi E. Parekh K.R. Cooney A.L. Schaffer D.V. McCray Jr., P.B. Novel GP64 envelope variants for improved delivery to human airway epithelial cells.Gene Ther. 2017; 24: 674-679Crossref PubMed Scopus (18) Google Scholar, 18Cooney A.L. Thornell I.M. Singh B.K. Shah V.S. Stoltz D.A. McCray Jr., P.B. Zabner J. Sinn P.L. Novel aav-mediated gene delivery system corrects cftr function in pigs.Am. J. Respir. Cell Mol. Biol. 2019; 61: 747-754Crossref PubMed Scopus (27) Google Scholar, 19Cmielewski P. Donnelley M. Parsons D.W. Long-term therapeutic and reporter gene expression in lentiviral vector treated cystic fibrosis mice.J. Gene Med. 2014; 16: 291-299Crossref PubMed Scopus (36) Google Scholar, 20Yan Z. McCray Jr., P.B. Engelhardt J.F. Advances in gene therapy for cystic fibrosis lung disease.Hum. Mol. Genet. 2019; 28: R88-R94Crossref PubMed Scopus (47) Google Scholar, 21Steines B. Dickey D.D. Bergen J. Excoffon K.J. Weinstein J.R. Li X. Yan Z. Abou Alaiwa M.H. Shah V.S. Bouzek D.C. et al.CFTR gene transfer with AAV improves early cystic fibrosis pig phenotypes.JCI Insight. 2016; 1: e88728Crossref PubMed Scopus (64) Google Scholar There is currently uncertainty about the relative contribution of different cell types to overall CFTR activity in the airway epithelium. Does the rare ionocyte population expressing high levels of CFTR dominate22Montoro D.T. Haber A.L. Biton M. Vinarsky V. Lin B. Birket S.E. Yuan F. Chen S. Leung H.M. Villoria J. et al.A revised airway epithelial hierarchy includes CFTR-expressing ionocytes.Nature. 2018; 560: 319-324Crossref PubMed Scopus (537) Google Scholar,23Plasschaert L.W. Žilionis R. Choo-Wing R. Savova V. Knehr J. Roma G. Klein A.M. Jaffe A.B. A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte.Nature. 2018; 560: 377-381Crossref PubMed Scopus (464) Google Scholar or do other more abundant luminal cell types (secretory or ciliated), which apparently express CFTR at lower levels per cell, also contribute?24Okuda K. Kobayashi Y. Dang H. Nakano S. Barbosa Cardenas S.M. On V.K. Kato T. Chen G. Gilmore R.C. Chua M. et al.Regional regulation of cftr and ionocyte expression in normal human airways. 2019 North American CF Conference, Nashville TN, Pediatric Pulmonology. 54. 2019Google Scholar Importantly, the aforementioned cell types are derived from airway basal stem cells, which justifies our editing focus.2Rock J.R. Onaitis M.W. Rawlins E.L. Lu Y. Clark C.P. Xue Y. Randell S.H. Hogan B.L. Basal cells as stem cells of the mouse trachea and human airway epithelium.Proc. Natl. Acad. Sci. USA. 2009; 106: 12771-12775Crossref PubMed Scopus (1012) Google Scholar,3Rock J.R. Randell S.H. Hogan B.L. Airway basal stem cells: a perspective on their roles in epithelial homeostasis and remodeling.Dis. Model. Mech. 2010; 3: 545-556Crossref PubMed Scopus (492) Google Scholar,22Montoro D.T. Haber A.L. Biton M. Vinarsky V. Lin B. Birket S.E. Yuan F. Chen S. Leung H.M. Villoria J. et al.A revised airway epithelial hierarchy includes CFTR-expressing ionocytes.Nature. 2018; 560: 319-324Crossref PubMed Scopus (537) Google Scholar It is important to consider frequencies at which cells in the CF airway would need to be corrected, either as a consequence of transplantation of edited airway basal cells or direct in vivo editing, for therapeutic benefit. Johnson et al.25Johnson L.G. Olsen J.C. Sarkadi B. Moore K.L. Swanstrom R. Boucher R.C. Efficiency of gene transfer for restoration of normal airway epithelial function in cystic fibrosis.Nat. Genet. 1992; 2: 21-25Crossref PubMed Scopus (380) Google Scholar reported that as few as 6%–10% corrected cells was sufficient to restore CF chloride ion transport levels to normal. A second study showed that 5% of pseudostratified epithelial cells expressing CFTR corrected the CF chloride transport defect.26Goldman M.J. Yang Y. Wilson J.M. Gene therapy in a xenograft model of cystic fibrosis lung corrects chloride transport more effectively than the sodium defect.Nat. Genet. 1995; 9: 126-131Crossref PubMed Scopus (78) Google Scholar However, these two studies used highly efficient viral vector promoters to express CFTR and therefore it cannot be assured that comparable results would be obtained in differentiated human airway epithelia where CFTR is under the control of its endogenous promoter. In mixing experiments of human airway cells in air-liquid interface (ALI) cultures, it was reported that 20% of non-CF cells mixed into a background of 80% ΔF508/ΔF508 cells yielded CFTR chloride current at levels 70% of wild-type levels.27Farmen S.L. Karp P.H. Ng P. Palmer D.J. Koehler D.R. Hu J. Beaudet A.L. Zabner J. Welsh M.J. Gene transfer of CFTR to airway epithelia: low levels of expression are sufficient to correct Cl- transport and overexpression can generate basolateral CFTR.Am. J. Physiol. Lung Cell. Mol. Physiol. 2005; 289: L1123-L1130Crossref PubMed Scopus (101) Google Scholar These data supported the concept that even a small fraction of cells expressing CFTR from the endogenous CFTR locus would be sufficient to correct the chloride transport defect in CF cells.27Farmen S.L. Karp P.H. Ng P. Palmer D.J. Koehler D.R. Hu J. Beaudet A.L. Zabner J. Welsh M.J. Gene transfer of CFTR to airway epithelia: low levels of expression are sufficient to correct Cl- transport and overexpression can generate basolateral CFTR.Am. J. Physiol. Lung Cell. Mol. Physiol. 2005; 289: L1123-L1130Crossref PubMed Scopus (101) Google Scholar The two approaches for CFTR gene editing in CF airway basal cells pursued by this study are shown in Figure 1. Our rationale for performing gene editing specifically of the endogenous CFTR locus reflects an intent to achieve expression of the corrected CFTR gene at close to physiologic levels. The first approach is site-specific correction of CFTR mutations; namely, a footprint-free method replacing mutant CFTR sequences with the corrected sequences (Figure 1A). Because CF is a recessive disease, correction of only one CFTR allele per cell would be sufficient for rescue of CFTR function. Site-specific correction of CFTR mutations in the endogenous gene is expected to result in physiologically appropriate levels of CFTR protein and function. However, site-specific correction typically requires a distinct set of sequence-directed nuclease and oligonucleotide donor reagents for each CFTR mutation. Because approximately 2,000 different CF-associated mutations have been reported, the sequence-specific approach would only seem justified for the most frequently occurring variants such as ΔF508. The second approach is homologous recombination-mediated targeted integration (TI) of a codon optimized partial CFTR cDNA (also denoted as a “super-exon”)5Harrison P.T. Hoppe N. Martin U. Gene editing & stem cells.J. Cyst. Fibros. 2018; 17: 10-16Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar,7Bednarski C. Tomczak K. Vom Hövel B. Weber W.M. Cathomen T. Targeted integration of a super-exon into the cftr locus leads to functional correction of a cystic fibrosis cell line model.PLoS ONE. 2016; 11: e0161072Crossref PubMed Scopus (36) Google Scholar preceded by a splice acceptor (SA) and followed by a polyadenylation (pA) sequence, within an intron of the endogenous CFTR gene (Figure 1B). This approach is capable of providing correction for all CFTR mutations downstream of the targeted partial CFTR cDNA integration site. Furthermore, by targeting cleavage of the genomic DNA within intronic versus exonic sequences, the possibility that nuclease-induced indels will adversely impact uncorrected alleles is minimized. With the objective of achieving physiologically regulated levels of CFTR expression and function, this approach would also benefit, in principle, from endogenous CFTR promoter activity and native chromatin architecture, provided that TI of the partial CFTR cDNA does not disrupt chromatin architecture in edited cells. Starting with early passage primary human airway epithelial cells from explanted CF or non-CF lungs, we first compared feeder-free culture methodologies for their ability to expand airway basal cells for numerous passages while retaining functional capability. Both the previously reported dual SMAD inhibition medium28Mou H. Vinarsky V. Tata P.R. Brazauskas K. Choi S.H. Crooke A.K. Zhang B. Solomon G.M. Turner B. Bihler H. et al.Dual smad signaling inhibition enables long-term expansion of diverse epithelial basal cells.Cell Stem Cell. 2016; 19: 217-231Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar and Pneumacult Ex-Plus medium allowed for continuous proliferation to at least passage 12 (p12; corresponding to 35–37 population doublings [PDs]; Figure S1A) while retaining markers characteristic of basal cells (e.g., CD49f, NGFR; Figure S1B). In order to assay functionality, expanded cells at various passage numbers were plated onto porous membranes and subject to ALI culture to assess development of well-differentiated airway epithelium. With increasing passage number, particularly for the dual SMAD inhibition culture, we noted progressive loss of ciliated cells, as measured by acetylated tubulin (Figure S1C). In order to measure CFTR ion transport function, ALI cultures were subjected to Ussing chamber assays. Airway basal cells expanded under Pneumacult Ex-Plus culture conditions exhibited robust levels of CFTR function through at least p8, while basal cells expanded in dual SMAD cultures exhibited lower levels of activity (Figures S1D and S1E). Under either culture condition, further passaging beyond p8 resulted in decreased levels of CFTR-dependent transepithelial transport (Figure S1E). Based on the above, we chose to perform our gene-editing manipulations on airway basal cells maintained maximally until p6 to p8 (∼18–25 PDs) in the Pneumacult Ex-Plus medium. Zinc-finger nucleases (ZFNs), targeted to recognize and cleave CFTR ΔF508 sequences in exon 11 (ZFN11; Figure S2A), were delivered as mRNA to ΔF508/ΔF508 airway basal cells via electroporation. For correction of the ΔF508 mutation, we evaluated two types of donor sequences encoding wild-type CFTR sequence. First, we co-delivered single-stranded (ss) 200-mer oligo DNAs together with the ZFN mRNAs via electroporation (Figure 2A). The ss oligo, centered on the ΔF508 mutation, included the wild-type restoring “CTT” bases (Figure S2A) spanned by approximately 100 bases of homology sequence on either side. In order to achieve maximal levels of correction while retaining cell viability, optimization was performed with respect to amount of ZFN mRNA (data not shown) and ssDNA donor oligo (e.g., Figures S2B and S2C). Next generation sequencing (NGS) was utilized to quantify the frequency of ΔF508 CFTR alleles exhibiting either no modification, indels, or correction (Figures 2A and 2B, Table S1). Delivery of ZFN mRNA alone resulted in 44.6% ± 2.4% of CFTR alleles exhibiting indels. When ssDNA donor was co-delivered with ZFN mRNA, the frequency of ΔF508 correction was 10.6% ± 2.6% (all editing frequencies are expressed on a per CFTR allele basis) (Figure 2B; Table S1). We sought to modify our methodology in order to achieve significantly higher efficiencies of correction. To this end, we utilized a longer donor (∼2.0 kb, again centered on the correcting “CTT” bases), but in this case delivered via adeno-associated virus type 6 (AAV-6) (Figure 2A); AAV-6 was selected due to its favorable tropism for the lung.29Limberis M.P. Vandenberghe L.H. Zhang L. Pickles R.J. Wilson J.M. Transduction efficiencies of novel AAV vectors in mouse airway epithelium in vivo and human ciliated airway epithelium in vitro.Mol. Ther. 2009; 17: 294-301Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar,30Yan Z. Lei-Butters D.C. Keiser N.W. Engelhardt J.F. Distinct transduction difference between adeno-associated virus type 1 and type 6 vectors in human polarized airway epithelia.Gene Ther. 2013; 20: 328-337Crossref PubMed Scopus (21) Google Scholar Optimization was again performed, in this case focused on the amount of ZFN and the AAV-6 dose (Figure S2D). Delivery of ZFN mRNA via electroporation followed immediately thereafter by AAV-6 donor transduction resulted in a correction efficiency of 31.0% ± 4.0% (Figure 2B; Table S1). In order to assess the functional consequences of ΔF508 correction, cells edited with the 2 kb AAV-6 donor were transferred onto porous supports and subjected to ALI culture conditions to generate well-differentiated airway epithelium containing basal cells (p63, keratin 5), secretory cells (mucin 5AC), ciliated cells (acetylated tubulin, FOXJ1), and ionocytes (FOXI1; Figures 2C and S3). Importantly, the manipulations required for editing did not alter the development of the pseudostratified epithelium. For example, cellular composition of the derived epithelium (i.e., % basal cells, % secretory cells, % ciliated cells, % ionocytes) was not affected by the manipulations (Figure S3). As expected, ALI cultures derived from unmanipulated ΔF508/ΔF508 airway basal cells only expressed the core glycosylated CFTR protein (band B; Figure 2D), whereas non-CF ALI cultures primarily exhibited the mature, fully-glycosylated, membrane-bound form of CFTR (band C; Figure 2D). Importantly, the AAV-6 edited ΔF508/ΔF508 cultures demonstrated the emergence of band C, corresponding to the presence of corrected CFTR alleles in the treated cells (Figures 2D and S4). Electrophysiological measurement of CFTR channel function was performed via Ussing chamber analysis. In Figure 2E, tracings for a representative experiment are presented; results of several experiments are presented in Table S2A and summarized in Figure 2F and Table S2B. As expected, non-treated ΔF508/ΔF508 cultures exhibit negligible forskolin-activated CFTR current (Figure 2F). ALI cultures derived from ΔF508/ΔF508 cells treated with ZFNs plus AAV-6 donor exhibited CFTR-dependent current (20.2 ± 3.5 μA/cm2 (Figure 2F), robustly blocked by the CFTR channel inhibitor (CFTRinh-172; Figures 2E and 2F). This level of restored CFTR activity in edited cultures is significant in two respects. First, this level of rescue in bulk-treated ΔF508/ΔF508 cultures (with a mean correction efficiency of 31.0%; Figure 2B) is 40.2% of that seen in the non-CF culture (50.2 ± 4.7 μA/cm2; Figure 2F). Second, this activity is 152% of that resulting from exposure of non-edited ΔF508/ΔF508 cells to the clinically approved CFTR modulators VX-809 and VX-770 (13.3 ± 0.9 μA/cm2; Figure 2F). This particular combination of modulators has been shown to be of therapeutic benefit in ΔF508/ΔF508 CF patients31Wainwright C.E. Elborn J.S. Ramsey B.W. Marigowda G. Huang X. Cipolli M. Colombo C. Davies J.C. De Boeck K. Flume P.A. et al.TRAFFIC Study GroupTRANSPORT Study GroupLumacaftor-ivacaftor in patients with cystic fibrosis homozygous for phe508del cftr.N. Engl. J. Med. 2015; 373: 220-231Crossref PubMed Scopus (955) Google Scholar and thus provides a metric against which the CFTR activity in the edited ΔF508/ΔF508 cells can be compared. Thus, by both measures (% of non-CF, % of VX-809/VX-770 treated ΔF508/ΔF508), CFTR function resulting from this efficiency of ΔF508 correction is meaningful (see Discussion). Similar analysis of ALI cultures derived from the 200-mer ssDNA donor-corrected airway basal cells (mean correction frequency of 10.6%; Figure 2B; Table S1) also demonstrated restored expression of mature CFTR protein (Figure S4), albeit at lower levels. For ssDNA donor edited cells, the level of CFTR current was 13.2% of the non-CF control (Tables S2A and S2B). However, rescue in this case did not reach the level of the VX-809/VX-770 treated ΔF508/ΔF508 control (21.3% of the non-CF control; Tables S2A and S2B). For sequence-specific ΔF508 correction, we observed an approximately linear correlation (R2 = 0.92) between the frequency of correction and level of restored CFTR current (Figure 2G). Taken together, these data, comparing the ssDNA and AAV-6 donors, support the importance of achieving significant levels of editing. We selected CFTR intron 8 to demonstrate proof of principle for the homologous recombination-mediated TI approach. ZFNs targeting CFTR intron 8 were designed and verified in K562 cells (data not shown). In order to facilitate simultaneous quantification of both the TI and indel frequencies in the same population of edited cells, an intron 8 specific primer, followed by a random sequence and an EcoRI site (to permit a rapid assessment of TI), was incorporated into the donor immediately downstream of the SA-CFTR9-27-pA cassette (Figure 3A); PCR amplification would yield a PCR product of identical size as that from unmodified cells and NGS would permit an accurate determination of TI and indel rates. We note the downstream primer was positioned outside of donor sequences to only amplify from transgene sequences integrated at the targeted site (Figure 3A). Electroporation-mediated delivery of intron 8 ZFN mRNA to ΔF508/ΔF508 airway basal cells was followed almost immediately by AAV-6 transduction of SA-CFTR9-27-pA. Initial evidence for sequence-specific TI of SA-CFTR9-27-pA into intron 8 came from upstream inside-outside PCR amplification (Figure S5A) and from EcoRI digestion of downstream PCR amplicons (Figure S5B). These results were quantified by NGS, with TI rates of 56.5% ± 7.4% observed 4 days post editing (Figure 3B; Table S3A). Efficient intron 8 TI was confirmed by Southern blotting of genomic DNA isolated from the edited ΔF508/ΔF508 cells (Figure 3C). Edited cells were plated in ALI cultures to develop well differentiated epithelium as described previously. In the one experiment for which we performed the analysis, we observed roughly comparable frequencies of intron 8 TI in basal cells prior to plating in the ALI cultures (50.1%) versus frequency of correction in mature, well differentiated airway epithelium in ALI cultures (43.9%) (Table S3B). The TI editing and minimal expansion required for early passage airway basal cell cultures also did not adversely affect the ability to establish well-differentiated airway epithelium comprising basal, secretory, and ciliated cells (Figure 3D). Successful TI-mediated correction of bulk cultures was confirmed by detection of CFTR transgene mRNA (Figure 3E) and by restoration of CFTR band C protein (Figure 3F). Ussing chamber assays from two independent experiments demonstrated restoration of forskolin-activated CFTR currents in intron 8 TI cultures (Figure 3G; sample trace in Figure S5C). CFTR current levels in edited cells from two independent experiments were 34.4% to 42.7% of the level seen in non-CF cultures (Figure 3G). Furthermore, the mean level of restored CFTR channel activity was 157% of the level seen for ΔF508/ΔF508 cultures exposed to VX-809/VX-770 (Figure 3G). Thus, with our current TI efficiency, we are in a therapeutically relevant range for a mixed population of corrected and uncorrected ΔF508/ΔF508 cells. We also evaluated, in a limited number of experiments, the TI strategy for intron 7 by delivery of intron 7 specific ZFNs followed shortly thereafter by AAV-6 delivery of a donor construct now including CFTR exons 8 to 27 (SA-CFTR8-27-pA). Evidence for intron 7 TI, first suggested via EcoRI digestion (Figure S6A), was confirmed by NGS (Figure S6B). Successful targeting was confirmed by expression of the transgenic mRNA (Figure S6C), restored CFTR band C protein (Figure S6D), and restored CFTR current (Figure S6E; sample trace in Figure S5C). This demonstrates that potential effectiveness of the TI approach is not limited to intron 8. The level of TI-8 or TI-7 restored CFTR function in ΔF508/ΔF508 cells, as a function of editing efficiency, is shown in Figure 3H. These results confirm significant CFTR functional restoration with the TI approach. Given the limited number of experimental data points for either the sequence-specific correction or TI approaches, it is premature to draw any firm conclusions, However, the tendency of the TI data points to perhaps lie slightly below the sequence-specific trendline suggests that further improvements may be possible (see Discussion). Our rationale for directly targeting integration of the partial CFTR cDNA at the endogenous CFTR locus was to exploit the native CFTR promoter and chromatin architecture, with the goal of achieving physiologically relevant levels of restored CFTR protein and function. The presence in ALI cultures of transgene-derived CFTR mRNA (Figure 3E), protein (Figure 3F), and function (Figure 3G) from bulk TI-8 edited cells provides support for this targeting strategy. We further wished to confirm that neither the integrated SA-CFTR9-27-pA transgene nor indels at the target site significantly disrupted the sites of open chromatin normally present in the native CFTR locus. To this end, we mapped, via Omni-ATAC-seq, the sites of open chromatin in ALI cultures established from TI-8 edited ΔF508/ΔF508 cells (Exp. 2; Table S3B; Figure 3G). At this time point, 43.9% and 47.3% of the CFTR alleles exhibited TI and indels, respectively (Table S3B). ALI cultures derived from unmanipulated ΔF508/ΔF508 cells or non-CF cells were used as controls. Significantly, Omni-ATAC-seq analysis of intron 8 targeted ALI cultures revealed minimal impact on the positions of open chromat" @default.
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• W3021862015 title "Highly Efficient Gene Editing of Cystic Fibrosis Patient-Derived Airway Basal Cells Results in Functional CFTR Correction" @default.
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