474 Ratio-dependent adenine base editing engenders highly efficient correction of RDEB mutations with minimal bystander mutations

Recessive dystrophic epidermolysis bullosa (RDEB) is a currently incurable blistering genodermatosis caused by mutations in COL7A1 encoding type VII collagen (C7), the major component of anchoring fibrils. Adenine base editors (ABEs) are class gene editor which can install A-T to G-C pair changes and therefore hold promise for reversing pathogenic variants. Patient-derived fibroblasts harboring c.5047 C>T (p. Arg1683*) exon 54 were electroporated with 2ug mRNA ‘ABE8e’ ABE variant 5ug single guide (sg)RNA. Post-editing analysis uncovered 100% correction mutation edited cells, however 98% alleles contained an aberrant bystander nucleotide at position c.5052. To reduce this we tested range ratios concentrations sgRNA ABE8e found 1:2, 0.5 ug sgRNA: 1 was best, maintaining 64% efficiency diminishing just 2%. Western blot 35% normal C7 expression post-correction, likely be enough functional correction. Furthermore, top 10 predicted off-target sites higher concentration confirmed no detectable off-targeting activity screening entire other post editing, demonstrating promising safety profile. Ongoing work will use 3D skin equivalents evaluate restoration architecture also focus on vivo delivery strategies editing tools directly patient skin. Optimized appear both safe effective may clinical utility RDEB.