612. Site-Specific Gene Editing of COL7A1 Restores Type VII Collagen in RDEB iPSCs
Authors: Christos Georgiadis, Anastasia Petrova, John A. McGrath, Adrian J. Thrasher, Wei-Li Di, Waseem Qasim
Journal: Molecular Therapy (2015)

Development of a treatment for the rare genetic skin disorder Recessive Dystrophic Epidermolysis Bullosa (RDEB) has included a number of approaches such as protein replacement, cell therapy and gene therapy. Over 600 mutations have been reported in the COL7A1 gene resulting in deposition of non-functional type VII collagen protein (C7) at the dermal-epidermal junction. Loss of the C7-encoded anchoring fibrils leads to severe blistering, tissue cleavage and a high incidence of mortality through progression to metastatic squamous cell carcinoma. All previous therapeutic advances have had notable limitations and alternative approaches based on gene editing of disease causing mutations have recently become feasible. Here we report the use of zinc finger nucleases (ZFN) for the targeted editing of human genomic COL7A1 and subsequent restoration of C7 expression in autologous RDEB patient cells. ZFNs were designed to target a mutation hot-spot clustered around exon 4 of COL7A1. The inefficiency of DNA transfection in keratinocytes (KC) led us to improve delivery of these DNA-binding nucleases by incorporating the ZFN pairs into a non-integrating lentiviral vector (NILV) platform, allowing for efficient yet transient expression. AKC cell line was initially used for the in-vitro modelling of C7 deficient RDEB demonstrating successful monoallelic and biallelic ZFN-mediated disruption of the gene in 26% of cells. Partial or complete knockdown of C7 expression was respectively seen, correlating with RDEB-like dysregulation of in-vitro cell migration when COL7A1 disrupted KC clones were functionally assessed. A dsDNA donor repair template encompassing a portion of endogenous sequence was designed and delivered via a third NILV. Introduction of silent mutations within the ZFN binding site of the template prevented ZFN-mediated cleavage, while incorporation of a restriction site within the intronic region allowed for PCR-based detection of integration. Sequencing across the ZFN binding sites confirmed donor template insertion by targeted homologous recombination (HR). Furthermore, to overcome the finite proliferative capacity of primary KCs we have previously established a 4-step differentiation protocol yielding a pure population of KCs from induced pluripotent stem cells (iPSCs) showing identical gene expression profiles to normal human KCs. The derived KCs are fully functional, forming fully-stratified 3D epidermal equivalents in-vitro. In this study, we reprogrammed RDEB patient fibroblasts (FB) carrying a missense mutation in a nearby exon 3 (p. K142R) into iPSCs which we subsequently transduced with a combination of NILVs delivering our ZFN pair and donor template. Direct sequencing revealed targeted template insertion by HR in 3/13 clones. We further demonstrated restoration of endogenous C7 expression in the template-bearing clones when protein level was assessed in contrast to null expressing patient cells. Efficient site specific targeting and correction of COL7A1 mutations in both KCs and iPSCs uncovers potential therapeutic applications of this approach in the otherwise incurable RDEB.