Base edited skin equivalents with clinical potential for ex vivo correction of recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe genetic disorder caused by bi-allelic null mutations in the COL7A1 gene. Among various therapeutic genome editing (GE) strategies, adenine base editors (ABEs) have demonstrated particular efficacy in nonsense mutations. Previous studies have predominantly focused on RDEB fibroblasts, which produce significantly less type VII collagen (C7) than keratinocytes. This study evaluates the efficacy and safety of ABE8e, via mRNA electroporation, to correct three different COL7A1 nonsense mutations (one homozygous and two heterozygous [patients 1, 2, and 3]), and a cryptic splice site mutation (patient 4) in primary cells from RDEB patients. Sanger and next-generation sequencing revealed high efficiency (>90%) at the intended mutation-causing nucleotides. Bystander effects were observed in two of the four loci analyzed. Deeper off-target study was performed in the first two patients. A safe off-target profile was observed in the homozygous patient (P1), while off-target effects were identified in the heterozygous patient (P2). The restoration of C7 expression in treated cells was consistent with genotypic correction. Additionally, functional assessments, including transplantation of skin equivalents formed by both base-edited keratinocytes and fibroblasts, demonstrated sustained in vivo skin regeneration and proper C7 deposition at the basement membrane. ABE8e-mediated base editing shows high efficacy and safety correcting COL7A1 mutations in the two cell types, broadening the scope of this GE therapeutic modality for RDEB treatment.