Safe and Efficacious Permanent Removal of Large COL7A1 Exons for Gene Reframing as a Reliable Therapeutic Strategy for Recessive Dystrophic Epidermolysis Bullosa.

Mutations leading to premature termination codons in COL7A1 are commonly associated with severe generalized recessive dystrophic epidermolysis bullosa (RDEB). Previous research, including our own, has indicated that removing mutated COL7A1 exons along with the consequent reframing of COL7A1 may not pose noticeable impact on protein function, offering a potential therapeutic strategy. However, investigations into the long-term in vivo effects of genome editing-mediated removal of mutant exons have only focused on the small exon 80 thus far. Hence, this study focuses on exons 73 and 105 of COL7A1 to explore whether targeted exon removal, through a CRISPR/Cas9-assisted, Non-homologous end joining (NHEJ)-mediated approach, could be extended to other larger exons. Introducing ribonucleoprotein complexes carrying Cas9 and optimized sgRNA guide pairs for each exon (73 and 105) through electroporation efficiently led to their removal, consequently restoring type VII collagen (C7) synthesis in RDEB primary patient cells carrying frameshift mutations in these exons. In vitro tests indicated the normal stability of the resulting C7 variants expressed at physiological levels, while in vivo analyses of regenerated skin grafted onto immunodeficient mice using E73 or E105 RDEB edited cells demonstrated the proper deposition of C7 at the basement membrane zone, thereby restoring normal dermo-epidermal adherence. This study enhances the broader potential of the exon deletion approach in the treatment of RDEB.