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• W2736022013 abstract "Designer nucleases allow specific and precise genomic modifications and represent versatile molecular tools for the correction of disease-associated mutations. In this study, we have exploited an ex vivo CRISPR/Cas9-mediated homology-directed repair approach for the correction of a frequent inherited mutation in exon 80 of COL7A1, which impairs type VII collagen expression, causing the severe blistering skin disease recessive dystrophic epidermolysis bullosa. Upon CRISPR/Cas9 treatment of patient-derived keratinocytes, using either the wild-type Cas9 or D10A nickase, corrected single-cell clones expressed and secreted similar levels of type VII collagen as control keratinocytes. Transplantation of skin equivalents grown from corrected keratinocytes onto immunodeficient mice showed phenotypic reversion with normal localization of type VII collagen at the basement membrane zone, compared with uncorrected keratinocytes, as well as fully stratified and differentiated skin layers without indication of blister development. Next-generation sequencing revealed on-target efficiency of up to 30%, whereas nuclease-mediated off-target site modifications at predicted genomic loci were not detected. These data demonstrate the potential of the CRISPR/Cas9 technology as a possible ex vivo treatment option for genetic skin diseases in the future. Designer nucleases allow specific and precise genomic modifications and represent versatile molecular tools for the correction of disease-associated mutations. In this study, we have exploited an ex vivo CRISPR/Cas9-mediated homology-directed repair approach for the correction of a frequent inherited mutation in exon 80 of COL7A1, which impairs type VII collagen expression, causing the severe blistering skin disease recessive dystrophic epidermolysis bullosa. Upon CRISPR/Cas9 treatment of patient-derived keratinocytes, using either the wild-type Cas9 or D10A nickase, corrected single-cell clones expressed and secreted similar levels of type VII collagen as control keratinocytes. Transplantation of skin equivalents grown from corrected keratinocytes onto immunodeficient mice showed phenotypic reversion with normal localization of type VII collagen at the basement membrane zone, compared with uncorrected keratinocytes, as well as fully stratified and differentiated skin layers without indication of blister development. Next-generation sequencing revealed on-target efficiency of up to 30%, whereas nuclease-mediated off-target site modifications at predicted genomic loci were not detected. These data demonstrate the potential of the CRISPR/Cas9 technology as a possible ex vivo treatment option for genetic skin diseases in the future. Programmable, tailored nucleases can be used to repair disease-causing mutations through specific gene editing in order to restore genetic functions and therefore have high therapeutic potential for patients with genetic diseases.1Rio P. Baños R. Lombardo A. Quintana-Bustamante O. Alvarez L. Garate Z. Genovese P. Almarza E. Valeri A. Díez B. et al.Targeted gene therapy and cell reprogramming in Fanconi anemia.EMBO Mol. Med. 2014; 6: 835-848Crossref PubMed Scopus (63) Google Scholar, 2Shinkuma S. Guo Z. Christiano A.M. Site-specific genome editing for correction of induced pluripotent stem cells derived from dominant dystrophic epidermolysis bullosa.Proc. Natl. Acad. Sci. U S A. 2016; 113: 5676-5681Crossref PubMed Scopus (75) Google Scholar, 3Biffi A. Clinical translation of TALENS: treating SCID-X1 by gene editing in iPSCs.Cell Stem Cell. 2015; 16: 348-349Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar, 4Tebas P. Stein D. Tang W.W. Frank I. Wang S.Q. Lee G. Spratt S.K. Surosky R.T. Giedlin M.A. Nichol G. et al.Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV.N. Engl. J. Med. 2014; 370: 901-910Crossref PubMed Scopus (1008) Google Scholar, 5Sebastiano V. Zhen H.H. Haddad B. Bashkirova E. Melo S.P. Wang P. Leung T.L. Siprashvili Z. Tichy A. Li J. et al.Human COL7A1-corrected induced pluripotent stem cells for the treatment of recessive dystrophic epidermolysis bullosa.Sci. Transl. Med. 2014; 6: 264ra163Crossref PubMed Scopus (145) Google Scholar, 6Crane 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, 7Ousterout 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 The versatility of CRISPR/Cas9, including simple design and the possibility of targeting multiple genes simultaneously, has aided the rapid evolution of this groundbreaking technology in recent years.8Guitart Jr., J.R. Johnson J.L. Chien W.W. Research techniques made simple: the application of CRISPR-Cas9 and genome editing in investigative dermatology.J. Invest. Dermatol. 2016; 136: e87-e93Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar Directed by a specifically designed single guide RNA (sgRNA) to the respective DNA target site, the Cas9 nuclease performs DNA double-strand breaks (DSBs), leading to the activation of the cellular repair machinery to correct the DNA damage either by non-homologous end joining (NHEJ) or, in the presence of a homologous DNA donor template, by homology-directed repair (HDR).9March O.P. Reichelt J. Koller U. Gene editing for skin diseases: designer nucleases as tools for gene therapy of skin fragility disorders.Exp. Physiol. 2017; (Published online March 7, 2017)https://doi.org/10.1113/EP086044Crossref PubMed Scopus (27) Google Scholar To date, many gene or RNA therapeutic approaches, focusing on the phenotypic improvement of genetic disorders, require sustained expression of the therapeutic transgene, maintained by the use of viral vectors for transgene delivery. Approaches depending on viral delivery bear the risk of insertional genotoxicity.10Hacein-Bey-Abina S. Garrigue A. Wang G.P. Soulier J. Lim A. Morillon E. Clappier E. Caccavelli L. Delabesse E. Beldjord K. et al.Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1.J. Clin. Invest. 2008; 118: 3132-3142Crossref PubMed Scopus (1395) Google Scholar CRISPR/Cas9 does not rely on viral vectors to achieve permanent DNA repair of monogenetic mutations, circumventing the risk for viral DNA integration.11Maeder M.L. Gersbach C.A. Genome-editing technologies for gene and cell therapy.Mol. Ther. 2016; 24: 430-446Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar Type VII collagen, secreted from keratinocytes and fibroblasts, is the main constituent of anchoring fibrils, which are essential structures within the basement membrane zone of the skin, crosslinking the epidermis with the dermis. Pathogenic mutations within the COL7A1 gene, leading to absence or malfunction of type VII collagen, are associated with the severe skin blistering disease dystrophic epidermolysis bullosa (DEB), which can be inherited in an autosomal recessive (RDEB) or dominant (DDEB) way. So far, various therapeutic approaches to restore COL7A1 expression have been described.12Murauer E.M. Gache Y. Gratz I.K. Klausegger A. Muss W. Gruber C. Meneguzzi G. Hintner H. Bauer J.W. Functional correction of type VII collagen expression in dystrophic epidermolysis bullosa.J. Invest. Dermatol. 2011; 131: 74-83Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 13Peking P. Koller U. Hainzl S. Kitzmueller S. Kocher T. Mayr E. Nyström A. Lener T. Reichelt J. Bauer J.W. Murauer E.M. A gene gun-mediated non-viral RNA trans-splicing strategy for Col7a1 repair.Mol. Ther. Nucleic Acids. 2016; 5: e287Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar, 14Rashidghamat E. McGrath J.A. Novel and emerging therapies in the treatment of recessive dystrophic epidermolysis bullosa.Intractable Rare Dis. Res. 2017; 6: 6-20Crossref PubMed Scopus (63) Google Scholar However, the extremely large size of both the gene and its transcript and the presence of highly repetitive sequences pose challenges in the development of convenient therapies.15Titeux M. Pendaries V. Zanta-Boussif M.A. Décha A. Pironon N. Tonasso L. Mejia J.E. Brice A. Danos O. Hovnanian A. SIN retroviral vectors expressing COL7A1 under human promoters for ex vivo gene therapy of recessive dystrophic epidermolysis bullosa.Mol. Ther. 2010; 18: 1509-1518Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar Genome-editing technologies have the potential to overcome size-associated issues and have already been used in various therapeutic approaches for DEB. TALEN-based gene correction of COL7A1 by HDR in RDEB fibroblasts16Osborn 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 and keratinocytes17Chamorro 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 resulted in high type VII collagen expression levels. The CRISPR/Cas9 technology was applied for editing of induced pluripotent stem cells2Shinkuma S. Guo Z. Christiano A.M. Site-specific genome editing for correction of induced pluripotent stem cells derived from dominant dystrophic epidermolysis bullosa.Proc. Natl. Acad. Sci. U S A. 2016; 113: 5676-5681Crossref PubMed Scopus (75) Google Scholar, 18Webber 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 and skipping of disease-associated Col7a1 exon 80 in a mouse model.19Wu 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. U S A. 2017; 114: 1660-1665Crossref PubMed Scopus (67) Google Scholar But the likelihood of off-target effects is still an important issue to be addressed, and especially when applying CRISPR/Cas9 in vivo, adverse events are difficult to control. Ex vivo CRISPR/Cas9-mediated gene therapy offers the possibility to monitor the safety by investigating the on-target specificity of epidermal stem cells prior to transplantation of gene-edited epidermal skin sheets. Recently, mutant versions of the originally identified Cas9 protein, Streptococcus pyogenes Cas9 (spCas9), have been devised, such as the Cas9 D10A nickase (Cas9n), improving HDR efficiency, specificity, and thus safety of CRISPR/Cas9, which is a prerequisite for potential clinical applications.20Cong L. Ran F.A. Cox D. Lin S. Barretto R. Habib N. Hsu P.D. Wu X. Jiang W. Marraffini L.A. Zhang F. Multiplex genome engineering using CRISPR/Cas systems.Science. 2013; 339: 819-823Crossref PubMed Scopus (9981) Google Scholar Cas9n induces single-strand DNA nicks instead of DSBs, because of mutations in the conserved nuclease domain RuvC. As DNA nicks are preferentially repaired by the high-fidelity base excision repair (BER) pathway,21Dianov G.L. Hübscher U. Mammalian base excision repair: the forgotten archangel.Nucleic Acids Res. 2013; 41: 3483-3490Crossref PubMed Scopus (246) Google Scholar the activation of the error-prone NHEJ pathway is uncommon, while HDR is stimulated when donor DNA is provided.22Ran F.A. Hsu P.D. Lin C.Y. Gootenberg J.S. Konermann S. Trevino A.E. Scott D.A. Inoue A. Matoba S. Zhang Y. Zhang F. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.Cell. 2013; 154: 1380-1389Abstract Full Text Full Text PDF PubMed Scopus (2335) Google Scholar In this study we developed an ex vivo gene therapy approach using the CRISPR technology to correct a highly recurrent homozygous mutation in COL7A1 exon 80 (6527insC). This insertion, which results in a premature termination codon, accounts for 46% of alleles in the Spanish RDEB patient population.23Cuadrado-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 Gene editing of RDEB keratinocytes using either spCas9 or Cas9n, in combination with a corresponding donor template for HDR induction, resulted in phenotypic correction as demonstrated in vitro and in vivo in a xenograft mouse model. Specificity analysis of our CRISPR/Cas9 approach via next-generation sequencing (NGS) showed up to 30% on-target cutting efficiency in the absence of off-target effects at predicted genomic regions with various degrees of homology to the designed sgRNA. Our findings mark an important step toward the development of a CRISPR/Cas9-mediated ex vivo gene therapy for DEB patients. The design of the 20 nt sgRNA is crucial for both specificity and efficiency of CRISPR/Cas9-mediated gene editing. HDR efficiency can be increased by reducing the distance of the nuclease cleavage site to the targeted mutation.24Findlay G.M. Boyle E.A. Hause R.J. Klein J.C. Shendure J. Saturation editing of genomic regions by multiplex homology-directed repair.Nature. 2014; 513: 120-123Crossref PubMed Scopus (214) Google Scholar Further, targeting intronic sequences is advantageous in order to avoid possible gene disruption and impaired protein expression. We predicted 13 sgRNAs in silico, targeting intron 80 of COL7A1, varying in their binding sites within the target region, GC content, and degree of homology to potential genomic off-target regions. From these sgRNAs, one with no bioinformatically predicted off-target activity was selected. The target site spans from nucleotide 52 to nucleotide 71 within intron 80 of COL7A1 localized adjacent to a protospacer-adjacent motif (PAM) in near proximity to the 6527insC mutation within exon 80 (Figure 1A).25Montague T.G. Cruz J.M. Gagnon J.A. Church G.M. Valen E. CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing.Nucleic Acids Res. 2014; 42: W401-W407Crossref PubMed Scopus (677) Google Scholar Initially, the efficiency of DSB induction of the spCas9 nuclease in combination with the selected sgRNA was determined in human embryonic kidney 293 (HEK293) and RDEB keratinocytes. In contrast to treated HEK293 cells, in which >90% were GFP positive after transfection (data not shown), RDEB keratinocytes showed lower transfection efficiencies (∼17%) and were therefore selected for GFP-positive cells via cell sorting to enrich for the cell population expressing the Cas9/sgRNA combination, thus facilitating the subsequent detection of indel formation via T7E1 assay. T7 endonuclease I digestion of the PCR-amplified COL7A1 target region (exons 78–84, 1,311 bp), recognizing mismatches arising from NHEJ events upon Cas9-mediated DSB induction, resulted in cleavage products with the expected sizes of 717 and 594 bp only in CRISPR/Cas9-treated cells (Figure 1B). On the basis of these results, we used the selected Cas9/sgRNA combination for the following gene editing studies in RDEB patient keratinocytes. For spCas9 nuclease, unspecific cutting events resulting in unintentional indels (off-target events) at genomic sequences with varying degrees of homology to the sgRNA have been described in the literature.26Wu Z. Feng G. Progress of application and off-target effects of CRISPR/Cas9.Yi Chuan. 2015; 37: 1003-1010PubMed Google Scholar To reduce the risk of potential off-target effects, we included the modified Cas9 version Cas9n in our study, which preferentially produces single-strand nicks in the DNA. Because low HDR efficiencies were expected, we inserted an mRuby/puromycin selection cassette flanked by loxP sites into the donor plasmid (DP). Selection with puromycin enriched for cells showing HDR and possible CRISPR/Cas9-mediated gene correction. The cassette was removed by Cre recombinase treatment, leaving a 116 nt trace of the donor vector sequence within intron 80, which is removed during the splicing process. The designed DP for HDR induction contained two homologous COL7A1 arms, spanning from exon 76 to intron 80 and intron 80 to intron 86, flanking an mRuby/puromycin selection cassette under the control of an EF1 promoter. Co-transfection of the DP and the respective Cas9/sgRNA combination (spCas9 or Cas9n) into RDEB keratinocytes led to the integration of the selection cassette and repair of the C insertion within exon 80 of COL7A1 after successful HDR (Figure 1C). After puromycin treatment, to obtain a homogeneous cell population expressing the HDR-mediated integrated mRuby/puromycin cassette (Figure S1), the correct integration of the selection cassette was determined by PCR analysis on genomic DNA using primers binding exon 76 of COL7A1 and the integrated vector sequence, showing a PCR product at the expected size of 1,190 bp (Figure 2A). The mRuby/puromycin-expressing cell population was treated with Cre recombinase twice, resulting in a cell population after a final cell sorting step, in which >99% of the cells the selection cassette was removed, leaving a 116 nt vector sequence within the target intron (Figure S1). After Cre recombinase treatment, HDR of the mutation was determined at the genomic level by PCR amplification of the COL7A1 target region (1,040 bp) spanning from exon 76 to intron 80 and subsequent BglI digestion. Upon repair of the 6527insC mutation, a new BglI restriction site is generated in exon 80.17Chamorro 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 The resulting digestion pattern indicated efficient repair in CRISPR/Cas9-treated RDEB keratinocytes using either of the two Cas9 variants (Figure 2B, left). Clones were expanded from the mRuby/puromycin-selected population following limiting dilution. BglI digestion of the PCR-amplified target region from genomic DNA indicated a heterozygous repair in 22 spCas9-treated single-cell clones (30 clones analyzed) and in 19 Cas9n-treated single-cell clones (76 clones analyzed). We used two single-cell clones, spCas9-C28 and Cas9n-C21, for further analysis (Figure 2B, right). Sequence analysis of the target sites confirmed the correction of the mutation in these clones (Figure 2C). The number of corrected alleles in the mixed CRISPR/Cas9-treated cell populations (following puromycin selection) was calculated with 17% for spCas9 (n = 94) and 24% (n = 94) for Cas9n, determined by cloning of the respective PCR products into subcloning vectors and BglI digestion analysis of the resulting bacterial single-cell clones (data not shown). In vitro protein expression and secretion analysis of the repaired single-cell clones spCas9-C21 and Cas9n-C28 showed type VII collagen expression determined by western blot analysis (Figure 2D) and immunofluorescence staining (Figure 2E). Relative quantification of detected type VII collagen via western blot analysis revealed ∼37% and ∼38% protein expression in spCas9-C21 and Cas9n-C28, respectively, compared with human wild-type keratinocytes (Figure 2D). For in vivo analysis, skin equivalents generated with corrected RDEB keratinocytes were grafted onto the back of immune-deficient nude mice. Eight weeks after transplantation, the grafts were removed for histological and immunofluorescence analysis. Grafts derived from CRISPR/Cas9-corrected clones showed epidermal differentiation and stratification comparable with grafts derived from normal keratinocytes. Immunofluorescence analysis of the grafts revealed a deposition of type VII collagen exclusively in the basement membrane zone (BMZ), ensuring the epidermal-dermal adhesion. Skin sheets derived from uncorrected RDEB keratinocytes showed traces of mutated type VII collagen in the BMZ. The human origin of the transplanted skin equivalents was confirmed by staining with a human keratin 16 (K16)-specific antibody, which exclusively labeled the grafts. Neither were any specific signals for human K16 nor for human type VII collagen detectable in murine tissue (Figure 3). Minicircle plasmids (MCs) are known to increase transfection efficiencies because of their small size.13Peking P. Koller U. Hainzl S. Kitzmueller S. Kocher T. Mayr E. Nyström A. Lener T. Reichelt J. Bauer J.W. Murauer E.M. A gene gun-mediated non-viral RNA trans-splicing strategy for Col7a1 repair.Mol. Ther. Nucleic Acids. 2016; 5: e287Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar Therefore, we used a minicircle DP (MC-DP) for subsequent experiments, containing only the homologous arms, flanking the restriction sites for EcoRI and ClaI and a GFP-blasticidin selection cassette under the control of an EF1 promoter (Figure 4A). The production of the MC-DP led to a size reduction from ∼8,000 bp of the parental plasmid MC-DP to ∼3,500 bp, leading to a ∼1.5-fold increase of the transfection efficiency into RDEB keratinocytes (Figures S2A and S2B). The restriction sites and an additional silent mutation within the 5′ homology arm (HA) facilitated detection of accurate HDR events (Figure 4A). Compared with the DP, the MC-DP contained a reduced number of nucleotides (15 nt compared with 3,093 nt) between the homologous arms, which is expected to increase recombination efficiency. The transfection efficiency of the spCas9/sgRNA and the MC-DP into RDEB keratinocytes was >50%, and transfected, GFP-expressing cells were further enriched via fluorescence-activated cell sorting (FACS) for subsequent detection of successful repair of the 6527insC mutation at genomic and protein level (Figure S2C). Upon treatment of RDEB keratinocytes with CRISPR/Cas9 and pMC-DP, HDR-mediated integration of the restriction sites provided by the MC-DP into intron 80 of COL7A1 was confirmed by PCR analysis, using a forward primer specifically binding exon 76 of COL7A1 and a reverse primer binding to the integrated recognition sequences of EcoRI and ClaI (Figure 4B). BglI digestion revealed partial correction of the mutation in spCas9/MC-DP-treated RDEB Kc. Upon single-cell dilutions, 169 clones were analyzed, among which two clones showed a heterozygous digestion pattern and one clone (spCas9-C44) showed a digestion pattern comparable with that of wild-type keratinocytes, indicating a homozygous correction of the mutation (Figure 4C). Sequence analysis confirmed that spCas9-C44 was a homozygously corrected single-cell clone (Figure 4D). In vitro protein expression and secretion analysis of the repaired single-cell clone spCas9-C44 showed increased, full-length type VII collagen expression, determined by western blot analysis (Figure 5A) and immunofluorescence staining (Figure 5B). We evaluated the specificity of our selected Cas9/sgRNA combination regarding potential off-target effects at predicted genomic loci harboring a high homology to the sgRNA targeting site within intron 80 of COL7A1 (Figures 6A and S3A). RDEB keratinocytes treated with Cas9/sgRNA (spCas9 or rather Cas9n) were analyzed for indel formation within the selected genomic regions via NGS, showing no obvious off-target activity for either nuclease (Table S1). In contrast, the NHEJ efficiency at the COL7A1 locus was ∼30%, confirming the high on-target efficiency of the wild-type spCas9 nuclease (Figures 6B and 6C). Additionally, we analyzed potential off-target events at the predicted genomic loci of the single-cell clones spCas9-C21, Cas9n-C28 and spCas9-C44 via T7E1 assays, resulting in no detectable genomic modifications (Figure S3B). Several COL7A1 gene therapy clinical trials for RDEB are ongoing, some of which focus on intradermal injections of gene-modified fibroblasts and others on transplantation of epidermal equivalents.27Lwin S.M. McGrath J.A. Gene Therapy for Inherited Skin Disorders. John Wiley, 2017Crossref Google Scholar Siprashvili et al.28Siprashvili 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 recently published the outcome of the first clinical trial for RDEB, in which epidermal sheets, expanded from keratinocytes, genetically modified via COL7A1 cDNA replacement, were grafted onto severe wounds. The approach initially resulted in phenotypic correction, but type VII collagen expression declined over a period of 12 months. The deficit of long-lasting and effective treatment options still requires more scientific input for the development of permanent therapies. Currently, more than 810 monogenetic mutations are listed in the COL7A1 database,29Wertheim-Tysarowska K. Sobczyńska-Tomaszewska A. Kowalewski C. Skroński M. Swięćkowski G. Kutkowska-Kaźmierczak A. Woźniak K. Bal J. The COL7A1 mutation database.Hum. Mutat. 2012; 33: 327-331Crossref PubMed Scopus (52) Google Scholar exhibiting COL7A1 as an ideal target for mutation-specific gene editing using the CRISPR/Cas9 technology. In this study, we have successfully developed an ex vivo gene therapy approach using CRISPR/Cas9, leading to correction of the disease phenotype in a xenograft mouse model. Although the CRISPR/Cas9 technology has emerged to a promising gene-editing tool applicable for various inherited or acquired diseases,30Valletta S. Dolatshad H. Bartenstein M. Yip B.H. Bello E. Gordon S. Yu Y. Shaw J. Roy S. Scifo L. et al.ASXL1 mutation correction by CRISPR/Cas9 restores gene function in leukemia cells and increases survival in mouse xenografts.Oncotarget. 2015; 6: 44061-44071Crossref PubMed Scopus (48) Google Scholar, 31Park C.Y. Kim D.H. Son J.S. Sung J.J. Lee J. Bae S. Kim J.H. Kim D.W. Kim J.S. Functional correction of large factor VIII gene chromosomal inversions in hemophilia A patient-derived iPSCs using CRISPR-Cas9.Cell Stem Cell. 2015; 17: 213-220Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 32Hung S.S. McCaughey T. Swann O. Pébay A. Hewitt A.W. Genome engineering in ophthalmology: application of CRISPR/Cas to the treatment of eye disease.Prog. Retin. Eye Res. 2016; 53: 1-20Crossref PubMed Scopus (34) Google Scholar low HDR efficiencies, including high risks for off-target effects, impeded its path to the clinic as a curative treatment for monogenetic disorders. Recently, Wu et al.19Wu 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. U S A. 2017; 114: 1660-1665Crossref PubMed Scopus (67) Google Scholar described a promising in vivo CRISPR/Cas9 approach used in an RDEB mouse model targeting epidermal stem cells. Although an increased type VII collagen expression level at the BMZ was detectable, only ∼2% of epidermal cells showed expression of a reporter molecule upon its HDR-mediated activation. We are focusing on a future ex vivo application, facilitating the challenge of low HDR efficiency by isolation and characterization of single-patient cell clones, homogeneously expressing type VII collagen due to CRISPR/Cas9-mediated genome editing prior to grafting onto the patient’s skin. A DEB mouse model expressing only 10% of the normal type VII collagen level is viable,33Fritsch A. Loeckermann S. Kern J.S. Braun A. Bösl M.R. Bley T.A. Schumann H. von Elverfeldt D. Paul D. Erlacher M. et al.A hypomorphic mouse model of dystrophic epidermolysis bullosa reveals mechanisms of disease and response to fibroblast therapy.J. Clin. Invest. 2008; 118: 1669-1679Crossref PubMed Scopus (169) Google Scholar and it has been reported that ∼35% of type VII collagen expression is required to maintain the mechanical stability of the skin in DEB.34Kern J.S. Loeckermann S. Fritsch A. Hausser I. Roth W. Magin T.M. Mack C. Müller M.L. Paul O. Ruther P. Bruckner-Tuderman L. Mechanisms of fibroblast cell therapy for dystrophic epidermolysis bullosa: high stability of collagen VII favors long-term skin integrity.Mol. Ther. 2009; 17: 1605-1615Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar A case study showed that even low expression levels of truncated procollagen VII polypeptide result in significant improvement of the phenotype in patients.35Schwieger-Briel A. Weibel L. Chmel N. Leppert J. Kernland-Lang K. Grüninger G. Has C. A COL7A1 variant leading to in-frame skipping of exon 15 attenuates disease severity in recessive dystrophic epidermolysis bullosa.Br. J. Dermatol. 2015; 173: 1308-1311Crossref PubMed Scopus (24) Google Scholar We have shown that the correction of only one COL7A1 allele, accounting for 50% of type VII collagen expression in the cell, leads to protein expression at levels comparable with normal, which is also known from heterozygous parents, who are carriers of a null mutation and do not display the RDEB phe" @default.
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• W2736022013 title "COL7A1 Editing via CRISPR/Cas9 in Recessive Dystrophic Epidermolysis Bullosa" @default.
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