157 Comparing adenine base editing and CRISPR-Cas9 gene editing strategies to correct recurrent mutations in RDEB primary fibroblasts

Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited blistering skin disorder which has no cure and only limited treatments. RDEB is caused by mutations in COL7A1 encoding type VII collagen (C7), the major component of skin anchoring fibrils. Most pathogenic COL7A1 mutations in RDEB are point mutations involving single nucleotides, often resulting in premature termination codons (PTC), leading to nonsense-mediated mRNA decay and no C7 protein expression. Adenine base editing (ABE) mediates A-T to G-C base pair changes and so is particularly suited for correcting PTC mutations which are caused by C>T mutations. Patient-derived fibroblasts carrying the RDEB PTC mutation c.5047 C>T (p.Arg1683*) in exon 54 of COL7A1 were electroporated with a single guide RNA and either ABE 7.10 mRNA or Cas9 ribonucleoprotein with a oligodeoxynucleotide donor template. We demonstrated permanent correction of the PTC causative mutation using both ABE and Homology Directed Repair-mediated CRISPR-Cas9 approaches and compared editing outcomes. Sanger sequencing, TOPO cloning and next-generation sequencing showed a correction efficiency of 23% using either ABE or CRISPR-Cas9. Notably, however, there were no detectable insertion or deletion (indel) mutations using ABE, but TIDE analysis uncovered indel mutations as a by-product of non-homologous end joining processes in CRISPR-Cas9 edited fibroblasts. Ongoing work will evaluate full-length COL7A1 mRNA expression post-correction, functional protein restoration and any off-target editing. ABE appears to be as efficient as Homology Directed Repair-mediated CRISPR-Cas9 gene editing in COL7A1 with a potentially better safety profile and may have clinical utility in RDEB.