175 Generation of a keratinocyte derived cell line via the CRISPR/Cas9 system for the study of corneodesmosin deficiency

Abstract

Recessive dystrophic epidermolysis bullosa (RDEB) is a devastating orphan skin disease caused by loss-of-function mutations in COL7A1 encoding type VII collagen (C7). C7 forms anchoring fibrils which are essential structures for dermal-epidermal adherence. Patients suffer from skin and mucosal blistering and develop severe complications resulting in poor prognosis. Local and systemic delivery of bone marrow-derived mesenchymal stromal cells (BM-MSCs) has shown that BM-MSCs reduce skin inflammation, contribute to skin regeneration and accelerate wound healing. Possible mechanisms of action include anti-inflammatory properties of BM-MSCs, enhanced expression of endogenous C7 through a paracrine effect, and/or C7 expression by injected cells. Here, we aim at assessing the therapeutic potential of local injections of BM-MSCs in a murine model for RDEB. Human BM-MSCs were grown in culture and expressed the established BM-MSC surface phenotype (CD45-, CD105+, CD90+, and CD73+). C7 transcript and protein levels of BM-MSCs were compared with normal human dermal fibroblasts in vitro and showed comparable expression of human C7 in platelet lysate culture conditions. The feasibility and efficacy of intradermal injections of BM-MSCs were evaluated in vivo using a xenograft murine model in which human RDEB reconstructed skin is grafted onto immune tolerant mice. Intradermal injection of a single dose of BM-MSCs within RDEB reconstructed skin led to the production of human C7 by injected cells and C7 deposition at the dermal-epidermal junction at least until two months post-injection. These data indicate that intradermal injected BM-MSCs have the potential to express C7 and to improve wound healing in RDEB patients.