O13 Preliminary results from correcting COL7A1 (c.G>A) mutation with adenine base editor 8e (ABE8e) in primary fibroblasts from dominant dystrophic epidermolysis bullosa (DDEB) patients

Abstract Introduction and aims Dominant dystrophic epidermolysis bullosa (DDEB) is a congenital blistering skin disease caused by mutant collagen VII (C7) chains interfering with the folding of wildtype (WT) C7, which weakens dermoepidermal junctions. Base editors (BEs) offer unique advantage in precisely correcting the mutant COL7A1 allele while preserving the WT allele. While our group has previously demonstrated the use of adenine base editor 8e (ABE8e) to correct recessive dystrophic epidermolysis bullosa (RDEB), this is the first report of ABE8e being applied to DDEB. Methods Primary fibroblasts from patients with DDEB carrying the COL7A1 exon 73 c.6127G>A mutation were electroporated with ABE8e mRNA and single guide (sg)RNA. Base editing efficiency was evaluated by semiquantitative analysis of Sanger sequencing chromatographs in EditR. Protein-level correction was assessed using a C7 Western blot. In preparation for future in vivo base editing, we validated a DDEB mouse model carrying the Col7a1 exon 73 c.6085C>G mutation using Sanger sequencing. Results Sanger sequencing analysis revealed that ABE8e achieved 19–41% editing efficiency in correcting the c.6127G>A mutation, with a 2 : 1 sgRNA-to-ABE8e ratio yielding higher efficiency than a 1 : 2 ratio. However, Western blot analysis did not show consistent protein-level correction. Sanger sequencing confirmed that our DDEB mouse model is heterozygous for the Col7a1 c.6085G>C mutation. Conclusions This study demonstrated partial DNA-level correction of the COL7A1 c.6127 G>A allele in ABE8e-electroporated primary DDEB fibroblasts ex vivo. However, this editing did not result in increased C7 protein levels. Notably, Western blots assess differences in C7 quantity, but not stability – a key factor in DDEB pathology. Further replication of DNA-level correction and evaluation of C7 stability using a trypsin digestion assay will be essential. These efforts will lay the foundation for transitioning from ex vivo experiments to in vivo correction of the Col7a1 c.6085C>G mutation in our DDEB mouse model.