Efficient reframed gene therapy for recessive dystrophic epidermolysis bullosa using CRISPR/Cas9

The CRISPR/Cas9 system induces site-specific double-strand breaks, which stimulate cellular DNA repair through either the homologous recombination (HR) or non-homologous end-joining (NHEJ) pathways. The HR is an ideal gene treatment strategy; however, the frequency of HR is quite low. On the other hand, NHEJ pathway, which occurs more frequently than HR, is prone to introducing small insertions and/or deletions at the DSB site, leading to change in the reading frame. We hypothesize that the NHEJ pathway is applicable to genetic diseases caused by a frameshift mutation, namely reframed gene therapy. Recessive dystrophic epidermolysis bullosa (RDEB) is a hereditary skin disorder caused by mutations in the COL7A1 gene. In this study, we applied the reframed gene therapy to a recurrent frameshift mutation, c.5819delC, in COL7, which results in a premature termination codon. The mRNA and protein expression levels of COL7 were increased in the reframed RDEB fibroblasts. In addition, the reframed COL7 was functional as demonstrated by triple-helix formation assay in vitro and was correctly distributed in basement membrane zone in mice that were intradermally treated with reframed RDEB fibroblasts. Our data establish the feasibility that mutation site-specific NHEJ could be a highly efficient gene therapy for inherited disorders caused by frameshift mutations.