Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a severe skin disease caused by mutations in COL7A1 gene, encoding a dysfunctional type VII collagen (C7). As a result, it leads to chronic blistering, progressive fibrosis, and high risk of developing cancer. Some of these mutations are located in exons 72, 80 and 94 of the gene. Premature termination codons due to a C>T substitution occur in exons 72 and 94 while in exon 80 there is a frameshift mutation (c.6527insC). Adenine base editors and non-viral delivered CRISPR based homology-directed repair (HDR) have been used to correct these mutations, respectively, and restore C7 in cell lines lacking this protein.

On the one hand, the base editing results indicate a highly efficient edit in the C>T mutation in exons 72 and 94 (76.2% and 92.3%, respectively). Removing the premature stop codon in the majority of the cases in both exons. Bystander edits are also generated in these cases but with no impact in the amino acid sequence due to silent mutations. Hence, the wildtype amino acid sequence is recovered.

On the other hand, the HDR delivered without viral vectors for correcting exon 80 mutation showed 90% of genome modification near to the Cas9 cleavage site (editing the PAM sequence), and around 60% 15 nucleotides apart (correcting the exon 80 mutation) and, therefore, restoring the C7 expression.

We now plan to evaluate these approaches on primary patient cells, allowing us to assess efficacy and functionality in bona fide pre-clinical settings.