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• W2921119983 abstract "Gene editing constitutes a novel approach for precisely correcting disease-causing gene mutations. Frameshift mutations in COL7A1 causing recessive dystrophic epidermolysis bullosa are amenable to open reading frame restoration by non-homologous end joining repair-based approaches. Efficient targeted deletion of faulty COL7A1 exons in polyclonal patient keratinocytes would enable the translation of this therapeutic strategy to the clinic. In this study, using a dual single-guide RNA (sgRNA)-guided Cas9 nuclease delivered as a ribonucleoprotein complex through electroporation, we have achieved very efficient targeted deletion of COL7A1 exon 80 in recessive dystrophic epidermolysis bullosa (RDEB) patient keratinocytes carrying a highly prevalent frameshift mutation. This ex vivo non-viral approach rendered a large proportion of corrected cells producing a functional collagen VII variant. The effective targeting of the epidermal stem cell population enabled long-term regeneration of a properly adhesive skin upon grafting onto immunodeficient mice. A safety assessment by next-generation sequencing (NGS) analysis of potential off-target sites did not reveal any unintended nuclease activity. Our strategy could potentially be extended to a large number of COL7A1 mutation-bearing exons within the long collagenous domain of this gene, opening the way to precision medicine for RDEB. Gene editing constitutes a novel approach for precisely correcting disease-causing gene mutations. Frameshift mutations in COL7A1 causing recessive dystrophic epidermolysis bullosa are amenable to open reading frame restoration by non-homologous end joining repair-based approaches. Efficient targeted deletion of faulty COL7A1 exons in polyclonal patient keratinocytes would enable the translation of this therapeutic strategy to the clinic. In this study, using a dual single-guide RNA (sgRNA)-guided Cas9 nuclease delivered as a ribonucleoprotein complex through electroporation, we have achieved very efficient targeted deletion of COL7A1 exon 80 in recessive dystrophic epidermolysis bullosa (RDEB) patient keratinocytes carrying a highly prevalent frameshift mutation. This ex vivo non-viral approach rendered a large proportion of corrected cells producing a functional collagen VII variant. The effective targeting of the epidermal stem cell population enabled long-term regeneration of a properly adhesive skin upon grafting onto immunodeficient mice. A safety assessment by next-generation sequencing (NGS) analysis of potential off-target sites did not reveal any unintended nuclease activity. Our strategy could potentially be extended to a large number of COL7A1 mutation-bearing exons within the long collagenous domain of this gene, opening the way to precision medicine for RDEB. Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin fragility genodermatosis caused by loss-of-function mutations in the COL7A1 gene, encoding type VII collagen (C7). C7 deficiency results in generalized blistering of the skin and other stratified epithelia, scarring, fibrosis, mitten-like deformities of hands and feet, and a high risk of developing metastatic squamous cell carcinoma.1Fine J.D. Bruckner-Tuderman L. Eady R.A. Bauer E.A. Bauer J.W. Has C. Heagerty A. Hintner H. Hovnanian A. Jonkman M.F. et al.Inherited epidermolysis bullosa: updated recommendations on diagnosis and classification.J. Am. Acad. Dermatol. 2014; 70: 1103-1126Abstract Full Text Full Text PDF PubMed Scopus (625) Google Scholar Ex vivo gene addition therapies based on transplantation of keratinocyte sheets modified by retroviral vectors are already at the clinical stage for forms of epidermolysis bullosa, including junctional epidermolysis bullosa (JEB) and RDEB, with encouraging results.2Bauer J.W. Koller J. Murauer E.M. De Rosa L. Enzo E. Carulli S. Bondanza S. Recchia A. Muss W. Diem A. et al.Closure of a Large Chronic Wound through Transplantation of Gene-Corrected Epidermal Stem Cells.J. Invest. Dermatol. 2017; 137: 778-781Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 3Hirsch T. Rothoeft T. Teig N. Bauer J.W. Pellegrini G. De Rosa L. Scaglione D. Reichelt J. Klausegger A. Kneisz D. et al.Regeneration of the entire human epidermis using transgenic stem cells.Nature. 2017; 551: 327-332Crossref PubMed Scopus (419) Google Scholar, 4Mavilio F. Pellegrini G. Ferrari S. Di Nunzio F. Di Iorio E. Recchia A. Maruggi G. Ferrari G. Provasi E. Bonini C. et al.Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells.Nat. Med. 2006; 12: 1397-1402Crossref PubMed Scopus (512) Google Scholar, 5Siprashvili Z. Nguyen N.T. Gorell E.S. Loutit K. Khuu P. Furukawa L.K. Lorenz H.P. Leung T.H. Keene D.R. Rieger K.E. et al.Safety and Wound Outcomes Following Genetically Corrected Autologous Epidermal Grafts in Patients With Recessive Dystrophic Epidermolysis Bullosa.JAMA. 2016; 316: 1808-1817Crossref PubMed Scopus (130) Google Scholar However, significant hurdles face the gene addition approach that include suboptimal viral gene delivery to the stem cell population, inaccurate spatial-temporal gene expression, and potential insertional mutagenesis-derived adverse events, which are particularly relevant for RDEB patients given their high proneness to carcinoma development. Therefore, the implementation of gene therapy approaches for RDEB based on highly precise gene-editing technologies is warranted and has been pursued by employing different types of nucleases, i.e., meganucleases, transcription activator-like effector nucleases (TALENs), and CRISPR/Cas9, and target cells.6Osborn M.J. Starker C.G. McElroy A.N. Webber B.R. Riddle M.J. Xia L. DeFeo A.P. Gabriel R. Schmidt M. von Kalle C. et al.TALEN-based gene correction for epidermolysis bullosa.Mol. Ther. 2013; 21: 1151-1159Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 7Webber B.R. Osborn M.J. McElroy A.N. Twaroski K. Lonetree C.L. DeFeo A.P. Xia L. Eide C. Lees C.J. McElmurry R.T. et al.CRISPR/Cas9-based genetic correction for recessive dystrophic epidermolysis bullosa.NPJ Regen. Med. 2016; 1: 16014Crossref PubMed Google Scholar, 8Izmiryan A. Danos O. Hovnanian A. Meganuclease-Mediated COL7A1 Gene Correction for Recessive Dystrophic Epidermolysis Bullosa.J. Invest. Dermatol. 2016; 136: 872-875Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 9Hainzl S. Peking P. Kocher T. Murauer E.M. Larcher F. Del Rio M. Duarte B. Steiner M. Klausegger A. Bauer J.W. et al.COL7A1 Editing via CRISPR/Cas9 in Recessive Dystrophic Epidermolysis Bullosa.Mol. Ther. 2017; 25: 2573-2584Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 10Chamorro C. Mencía A. Almarza D. Duarte B. Büning H. Sallach J. Hausser I. Del Río M. Larcher F. Murillas R. Gene Editing for the Efficient Correction of a Recurrent COL7A1 Mutation in Recessive Dystrophic Epidermolysis Bullosa Keratinocytes.Mol. Ther. Nucleic Acids. 2016; 5: e307Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar Since neither homology-directed repair (HDR)- nor non-homologous end joining (NHEJ)-mediated gene-editing strategies tested so far in patient cells have reached a sufficient level of efficacy to enable therapeutic C7 replacement by direct transplantation of cells treated in bulk, isolation of corrected cell clones has been necessary, either from patient-derived induced pluripotent stem cells (iPSCs) and subsequent target cell derivation6Osborn M.J. Starker C.G. McElroy A.N. Webber B.R. Riddle M.J. Xia L. DeFeo A.P. Gabriel R. Schmidt M. von Kalle C. et al.TALEN-based gene correction for epidermolysis bullosa.Mol. Ther. 2013; 21: 1151-1159Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 7Webber B.R. Osborn M.J. McElroy A.N. Twaroski K. Lonetree C.L. DeFeo A.P. Xia L. Eide C. Lees C.J. McElmurry R.T. et al.CRISPR/Cas9-based genetic correction for recessive dystrophic epidermolysis bullosa.NPJ Regen. Med. 2016; 1: 16014Crossref PubMed Google Scholar or from patient keratinocytes.9Hainzl S. Peking P. Kocher T. Murauer E.M. Larcher F. Del Rio M. Duarte B. Steiner M. Klausegger A. Bauer J.W. et al.COL7A1 Editing via CRISPR/Cas9 in Recessive Dystrophic Epidermolysis Bullosa.Mol. Ther. 2017; 25: 2573-2584Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 10Chamorro C. Mencía A. Almarza D. Duarte B. Büning H. Sallach J. Hausser I. Del Río M. Larcher F. Murillas R. Gene Editing for the Efficient Correction of a Recurrent COL7A1 Mutation in Recessive Dystrophic Epidermolysis Bullosa Keratinocytes.Mol. Ther. Nucleic Acids. 2016; 5: e307Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 11Mencía Á. Chamorro C. Bonafont J. Duarte B. Holguin A. Illera N. Llames S.G. Escámez M.J. Hausser I. Del Río M. et al.Deletion of a Pathogenic Mutation-Containing Exon of COL7A1 Allows Clonal Gene Editing Correction of RDEB Patient Epidermal Stem Cells.Mol. Ther. Nucleic Acids. 2018; 11: 68-78Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar Our laboratory previously demonstrated long-term skin regeneration from single, gene-edited epidermal stem cell clones of primary RDEB patient keratinocytes.11Mencía Á. Chamorro C. Bonafont J. Duarte B. Holguin A. Illera N. Llames S.G. Escámez M.J. Hausser I. Del Río M. et al.Deletion of a Pathogenic Mutation-Containing Exon of COL7A1 Allows Clonal Gene Editing Correction of RDEB Patient Epidermal Stem Cells.Mol. Ther. Nucleic Acids. 2018; 11: 68-78Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar Our original approach involved the use of TALENs delivered by adenoviral vectors to induce NHEJ-mediated insertions or deletions (indels) able to restore the reading frame of COL7A1 in patient cells carrying the frameshift mutation c.6527insC12Hovnanian A. Rochat A. Bodemer C. Petit E. Rivers C.A. Prost C. Fraitag S. Christiano A.M. Uitto J. Lathrop M. et al.Characterization of 18 new mutations in COL7A1 in recessive dystrophic epidermolysis bullosa provides evidence for distinct molecular mechanisms underlying defective anchoring fibril formation.Am. J. Hum. Genet. 1997; 61: 599-610Abstract Full Text PDF PubMed Scopus (139) Google Scholar located at exon 80, which is highly prevalent in the cohort of Spanish RDEB patients.13Cuadrado-Corrales N. Sánchez-Jimeno C. García M. Escámez M.J. Illera N. Hernández-Martín A. Trujillo-Tiebas M.J. Ayuso C. Del Rio M. A prevalent mutation with founder effect in Spanish Recessive Dystrophic Epidermolysis Bullosa families.BMC Med. Genet. 2010; 11: 139Crossref PubMed Scopus (16) Google Scholar, 14Escámez M.J. García M. Cuadrado-Corrales N. Llames S.G. Charlesworth A. De Luca N. Illera N. Sánchez-Jimeno C. Holguín A. Duarte B. et al.The first COL7A1 mutation survey in a large Spanish dystrophic epidermolysis bullosa cohort: c.6527insC disclosed as an unusually recurrent mutation.Br. J. Dermatol. 2010; 163: 155-161Crossref PubMed Scopus (48) Google Scholar One of the edited patient keratinocyte clones described in this study carried a long deletion encompassing the whole exon 80, and it showed restoration of C7 expression and persistent phenotypic correction in vivo upon transplantation onto immunocompromised mice.11Mencía Á. Chamorro C. Bonafont J. Duarte B. Holguin A. Illera N. Llames S.G. Escámez M.J. Hausser I. Del Río M. et al.Deletion of a Pathogenic Mutation-Containing Exon of COL7A1 Allows Clonal Gene Editing Correction of RDEB Patient Epidermal Stem Cells.Mol. Ther. Nucleic Acids. 2018; 11: 68-78Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar The functionality of C7 variants lacking the amino acids encoded by specific exons within the collagenous domain (i.e., exons 73, 80, and 105) has been previously demonstrated.15Bornert O. Kühl T. Bremer J. van den Akker P.C. Pasmooij A.M. Nyström A. Analysis of the functional consequences of targeted exon deletion in COL7A1 reveals prospects for dystrophic epidermolysis bullosa therapy.Mol. Ther. 2016; 24: 1302-1311Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 16Turczynski S. Titeux M. Tonasso L. Décha A. Ishida-Yamamoto A. Hovnanian A. Targeted Exon Skipping Restores Type VII Collagen Expression and Anchoring Fibril Formation in an In Vivo RDEB Model.J. Invest. Dermatol. 2016; 136: 2387-2395Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar Further, Wu et al.17Wu W. Lu Z. Li F. Wang W. Qian N. Duan J. Zhang Y. Wang F. Chen T. Efficient in vivo gene editing using ribonucleoproteins in skin stem cells of recessive dystrophic epidermolysis bullosa mouse model.Proc. Natl. Acad. Sci. USA. 2017; 114: 1660-1665Crossref PubMed Scopus (67) Google Scholar also showed that Col7a1 exon 80-skipped mice generated with the CRISPR/Cas9 system were indistinguishable from their wild-type littermates. Building on these results, we sought more efficient and safe methods to achieve targeted deletion of mutation-carrying COL7A1 exon 80 using the CRISPR/Cas9 system in RDEB patient keratinocytes. In this study, we show the remarkable efficacy and safety of a non-viral strategy employing a dual single-guide RNA (sgRNA)-guided Cas9 nuclease delivered as a ribonucleoprotein (RNP) complex by electroporation to precisely excise COL7A1 exon 80 carrying the c.6527insC mutation in RDEB patient keratinocytes. Moreover, we demonstrate the long-term skin regeneration ability of the corrected cells upon grafting of polyclonal and monoclonal populations of edited cells to immunodeficient mice, which is indicative of epidermal stem cell correction. This highly efficacious and one-step strategy would enable quick translation to clinical application. The dual sgRNA CRISPR/Cas9 deletion strategy of COL7A1 exon 80 (E80) is shown in the scheme (Figure 1A). Four different sgRNA pairs targeted to DNA sequences within introns 79 and 80 were designed (Figure 1B), with the aim of generating deletions of different sizes covering E80 (Figure 1C). RDEB keratinocytes from a homozygous carrier of the c.6527insC mutation were nucleofected with the CRISPR/Cas9 RNP complexes at two different electroporation pulse conditions (designated as A and B, Figure 1D). Once treated, keratinocytes reached confluency, genomic DNA was extracted, and E80 deletion (ΔE80) was assessed by PCR amplification of a fragment spanning the sgRNA target sites (Figure 1D). All sgRNA pairs led to E80 deletion, as shown by the presence of smaller molecular weight bands with sizes consistent with the distances between Cas9 cutting sites. Electroporation condition B performed better for all sgRNA pairs. Differences in deletion efficacy were found among the different sgRNA pairs (i.e., sg2 + sg3 > sg1 + sg3 > sg2 + sg4 > sg1 + sg4) (Figure 1D). In keratinocytes treated with sg2 + sg3, the most efficacious pair of guides, E80 deletions accounted for 66.5% of alleles, as determined by densitometric quantitation of the PCR products (Figure 1D). The presence of ΔE80 alleles in a ratio higher than 50% indicated that homozygous deletion of E80 was a frequent event. A variety of intron 79-intron 80 joining events after Cas9-mediated cleavage at sequences flanking E80 and subsequent NHEJ repair were expected to occur in a polyclonal cell population. The characterization of the DNA repair outcomes in RDEB keratinocytes from patient P1 treated with the most effective sgRNA pair (sg2 + sg3) was initially performed by thymidine-adenine (TA) cloning of PCR products spanning both Cas9 cutting sites and Sanger sequencing of individual colonies (n = 82). This analysis detected a spectrum of end joining repair variants, the majority of which (41 of 82) corresponded to the fusion of the predicted sgRNA-targeted Cas9 cleavage sites plus the insertion of a T (Figure 2). Other fusion events included small indels (both deletions and insertions) not affecting intron-splicing signals and, therefore, not likely to disrupt the splicing of the resulting chimerical intron. The analysis also showed that, in addition to dual Cas9 cuts leading to the intended E80 deletion, which accounted for 84% of alleles, indels corresponding to DNA repair after single cuts (i.e., at either intron 79 or 80) were also present. Taking these into account, 95% of alleles had been edited (Figure 2). For in-depth characterization of repair events at the on-target region and to analyze potential off-target cleavage activity at in silico-predicted sites, we performed next-generation sequencing (NGS) of PCR amplicons spanning the corresponding sites. Genomic DNA samples from keratinocytes from two homozygous c.6527insC carrier patients, P1 and P2, treated in duplicate with RNPs (biological replicates BR1 and BR2) as well as control DNA samples from both patients were subjected to this analysis. On-target NGS analysis confirmed highly efficient deletion of E80 in Cas9 RNP-treated patient cells. Sequence variations within a 60-bp window centered on the midpoint between both cut sites were considered. The two most frequent repair variants, i.e., fusion of the Cas9 cleavage sites with (62.2%) or without (9.7%) the insertion of a T (Figure S1), found in P1 (BR1), were also among the most frequently represented in the Sanger sequencing characterization (50.0% and 6.1%, respectively) for this sample (Figure 2). For less frequent repair variants, higher diversity was detected with NGS than with Sanger sequencing. Taking into account all variants resulting in E80 deletion, similar frequencies of deletion were found with either technique (84% versus 87% for Sanger and NGS, respectively) in P1 (BR1) patient keratinocytes. Patient P2 cells, analyzed by NGS only, showed 95% E80 deletion with a similar spectrum of allelic variants (Figure S1). The lower frequency of E80 deletion estimated by PCR genotyping in patient P1 (66%), as compared with Sanger or NGS sequencing estimates, might be explained by heteroduplex DNA formation between deleted and undeleted alleles that results in decreased intensity of the lower molecular weight PCR band.18Ousterout D.G. Kabadi A.M. Thakore P.I. Majoros W.H. Reddy T.E. Gersbach C.A. Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy.Nat. Commun. 2015; 6: 6244Crossref PubMed Scopus (314) Google Scholar To assess for potential off-target Cas9 cleavage activity, NGS was used to analyze PCR amplicons covering 278 in silico-predicted sites, which included all off-target sites up to 3 mismatches, all sites with 1- or 2-bp bulges, and the top 53 coding region sites with 4 mismatches for each guide. After establishing a 1,000-read cutoff for sequencing depth, 244 amplicons were studied. The only sequence variations considered were indels within a 6-bp window centered on the target site for each sgRNA. The threshold percentage for off-target activity was set at 0.52%, since this was the highest percentage of indel-containing reads (within the 6-bp window considered for the off-target evaluation) in unedited control samples. Thus, for each site, indel-containing reads below this percentage were considered as noise. For every off-target site analyzed, indel-containing reads represented less than 0.52% of the total (Figure 3), except for five predicted sites. Although these sites showed indel-containing reads at a slightly higher frequency (0.52%–0.6%) above the 0.52% threshold (Figure S2), the sequence variations found were also present in both controls and edited samples, suggesting that these were not bona fide Cas9 off-targets. We therefore concluded that our NGS analysis of 244 predicted off-target sites did not reveal any off-target events above the threshold of detection. Our NHEJ correction strategy was designed to generate, upon E80 deletion, new functional introns with donor and acceptor splicing sequences corresponding to those from exons 79 and 81, respectively. Still, the potential generation of cryptic splicing sites could result in inappropriately spliced transcripts. To confirm proper reading frame restoration derived from the precise in-frame exon 79-exon 81 junction, we studied COL7A1 transcription by performing RT-PCR analysis. For all three sgRNA pairs suitable for E80 deletion, a smaller band consistent with amplification of transcripts lacking E80 was found (Figure 4A). The intensity of the smaller band (Figure 4A) was proportional to the efficiency of the deletion, as detected by PCR analysis of genomic DNA (Figure 1D). The RT-PCR products corresponding to cells treated with the best-performing sgRNA pair (sg2 + sg3) were TA cloned (n = 49) and Sanger sequenced. This analysis revealed only two types of transcripts: 47 colonies contained the proper exon 79-exon 81 junction (96% of transcripts), and 2 colonies (4%) contained the E80 sequence originating from the unedited (c.6527insC) allele (Figure 4B). To further quantify the presence of E80-deleted and E80-containing transcripts in gene-edited cells, qRT-PCR was performed using Taqman probes specific for exon 80- and exon 64-encoded sequences. The exon 64-specific probe detects transcripts originating from both edited and unedited alleles, and the exon 80-specific probe detects only transcription from unedited E80 sequence-containing alleles. While in healthy donor control keratinocytes both probes detected similar expression levels, in the different RNP-treated pools of cells we observed higher expression with the exon 64 probe than with the exon 80 probe. The increased COL7A1 transcription detected with the exon 64 probe as compared to the exon 80 probe was proportional to the deletion efficacy of the guide combinations. This analysis was consistent with the RT-PCR product quantification, and it clearly demonstrated the prevalence of E80-lacking transcripts in edited cells, confirming sg2 + sg3 guides as the best performing pair for E80 deletion (Figure 4C). COL7A1 reading frame restoration by targeted deletion of mutant E80 and splicing of the resulting chimerical intron should result in C7 expression, as we previously observed in a clone of RDEB keratinocytes containing a deletion that encompassed COL7A1 E80 entirely.11Mencía Á. Chamorro C. Bonafont J. Duarte B. Holguin A. Illera N. Llames S.G. Escámez M.J. Hausser I. Del Río M. et al.Deletion of a Pathogenic Mutation-Containing Exon of COL7A1 Allows Clonal Gene Editing Correction of RDEB Patient Epidermal Stem Cells.Mol. Ther. Nucleic Acids. 2018; 11: 68-78Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar Previous studies from others have also shown that C7-null cells expressing a retrovirally transferred ΔE80 COL7A1 cDNA construct were able to produce a functional C7 variant.16Turczynski S. Titeux M. Tonasso L. Décha A. Ishida-Yamamoto A. Hovnanian A. Targeted Exon Skipping Restores Type VII Collagen Expression and Anchoring Fibril Formation in an In Vivo RDEB Model.J. Invest. Dermatol. 2016; 136: 2387-2395Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar We therefore assessed C7 expression by immunofluorescence and western blot in RDEB keratinocytes nucleofected with the three most efficient E80-deleting RNP combinations (sg2 + sg4, sg1 + sg3, and sg2 + sg3). The number of C7-expressing cells detected by immunofluorescence analysis (Figures 5A and S3A) matched the E80 deletion efficacy found by PCR analysis (Figure 1D) and the COL7A1 mRNA transcription levels (Figures 4A and 4C). In fact, quantification of C7-positive cells by flow cytometry using a specific anti-C7 antibody showed that 81% of patient keratinocytes treated with the sg2 + sg3 RNPs expressed C7 (Figure S3B). Accordingly, western blot analysis performed on cellular extracts further demonstrated the highest expression of C7 in patient keratinocyte samples treated with sg2 + sg3 RNPs. The mobility of the C7 band detected in these samples was indistinguishable from that of the wild-type (WT) protein, as expected since the size difference between WT C7 and the variant lacking the 12 amino acids encoded by E80 cannot be resolved at the molecular weight range of C7 (290 kDa) (Figure 5B). In addition, to study C7 secretion, we performed western blot analysis of proteins precipitated from keratinocyte culture supernatants. As shown, C7 was present in the media of sg2 + sg3 RNP-treated keratinocytes (Figure 5C), confirming that removal of E80 does not impair C7 secretion. The high proportion of C7-expressing keratinocytes obtained with the non-viral CRISPR/Cas9 approach indicated that a C7 amount sufficient to restore epidermal-dermal adhesion would be attainable. To test this, polyclonal populations of edited RDEB cells, obtained with the different combinations of sgRNAs, and control unedited cells were used to produce bioengineered skin constructs that were subsequently grafted onto immunodeficient mice. Animals were monitored for engraftment, and biopsies were obtained at different time points for histopathological analysis of regenerated skin and assessment of C7 expression. Macroscopic examination clearly showed human skin engraftment for the most efficient guide combination (sg2 + sg3) (Figures 6A and 6B ). Routine histological analysis (H&E staining) of 12-week-old grafts showed normal skin architecture and uninterrupted dermal-epidermal attachment in grafts from these RDEB-edited cells (Figure 6C). On the contrary, epidermal-dermal separation was evident in grafts from non-edited cells (Figure 6D). Both types of grafts showed correct suprabasal human involucrin expression indicating normal epidermal differentiation (Figures 6E and 6F). Immunoperoxidase staining clearly exhibited C7 expression with appropriate localization at the basement membrane zone (BMZ) exclusively in grafts from edited patient cells (Figures 6G and 6H). Ultrastructural analysis by electron microscopy accordingly showed the presence of abundant anchoring fibrils in edited grafts (Figure 6I), but not in control unedited grafts where dermal-epidermal separation was evident (Figure 6J). Patient keratinocytes modified with the other sgRNA pairs able to induce alternative E80 deletions, albeit at lower efficiencies, were also tested for skin regeneration. Histological and immunohistochemical analysis 12 weeks after grafting showed full dermal-epidermal adhesion (Figure S4D) and continuous C7 expression (Figure S4F) at the BMZ in grafts of keratinocytes edited with sg1 + sg3. In contrast, microblisters (Figure S4A) and reduced and patchy C7 expression (Figure S4C) were observed in grafts generated from cells treated with the less efficient sg2 + sg4 RNP combination, suggesting that the C7 amount provided by these cells was not sufficient to sustain continuous dermal-epidermal adhesion. Human involucrin expression demonstrated normal epidermal differentiation of these grafts (Figures S4B and S4E). The very high efficiency of E80 deletion attained through our non-viral CRISPR/Cas9 RNP complex delivery and the long-term skin regeneration achieved with a polyclonal population of corrected cells suggested that targeting of the epidermal stem cell compartment had occurred. To confirm this, 11 clones from the bulk RDEB keratinocyte population edited with sg2 + sg3 RNPs were isolated by limiting dilution. Genotype analysis revealed that all of these clones had been edited (Figure S5A). Two clones, one monoallelic and the other biallelic for the E80 deletion (named as ΔE80/ΔE80 and ΔE80/mut), were selected for in vivo skin regeneration performance. These clones were accurately genotyped by sequencing (Figure S5C) and COL7A1 expression was precisely assessed (Figures S5B and S5D–S5F). Keratinocytes from these clones were labeled with GFP by lentiviral transduction to facilitate the monitoring of graft persistence over time. Macroscopic analysis under white and blue (GFP) light illumination of grafts 20 weeks post-grafting, when several epidermal turnover cycles had occurred, revealed clearly distinguishable human skin morphological characteristics (Figures 7A and 7B ). Grafts from both clones displayed normal histopathological features with continuous dermal-epidermal attachment (Figures 7C and 7D). As shown with the polyclonal ΔE80 RDEB keratinocyte population and consistent with the epidermal-dermal attachment observed histologically, both C7 expression (Figures 7E and 7F) and anchoring fibrils (Figures 7G and 7H) were also clearly detectable in the monoallelic and biallelic ΔE80 clonal grafts. Appropriate epidermal differentiation was confirmed by human involucrin expression immunodetection (Figures S6B and S6E). In addition to graft endurance over time, a robust p63 immunostaining also indicated that a pool of keratinocytes with persistent regenerative functionality was maintained19Pellegrini G. Dellambra E. Golisano O. Martinelli E. Fantozzi I. Bondanza S. Ponzin D. McKeon F. De Luca M. p63 identifies keratinocyte stem cells.Proc. Natl. Acad. Sci. USA. 2001; 98: 3156-3161Crossref PubMed Scopus (1181) Google Scholar (Figures S6C and S6F). Long-term clonal skin regeneration and persistence of the corrective effect, as determined by this stringent stemness in vivo test, indicated that the epidermal stem cell compartment had been effectively targeted. Mechanical strength of human skin grafts regenerated from patient P1 cells was assessed by using a suction device able to exert precisely controlled negative pressure on circular areas of a 3-mm diameter. A negative pressure of 10 ± 2 kPa20Petrof G. Lwin S.M. Martinez-Queipo M. Abdul-Wahab A. Tso S. Mellerio J.E. Slaper-Cortenbach I. Boelens J.J. Tolar J. Veys P. et al.Potential of Systemic Allogeneic Mesenchymal Stromal Cell Therapy for Children with Recessive Dystrophic Epidermolysis Bullosa.J. Invest. Dermatol. 2015; 135: 2319-2321Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar was applied for 5 min on two different points of each graft. No blist" @default.
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• W2921119983 title "Clinically Relevant Correction of Recessive Dystrophic Epidermolysis Bullosa by Dual sgRNA CRISPR/Cas9-Mediated Gene Editing" @default.
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