P07 Optimizing DNA specificity and applicability of base and prime editors on COL7A1 variants causing recessive dystrophic epidermolysis bullosa

Abstract

Abstract Base and prime editors are novel CRISPR variants that reduce the risk of off-target effects by avoiding double-stranded DNA cleavage, bypassing the need for homology-directed repair. These tools offer great therapeutic potential for inherited skin diseases such as recessive dystrophic epidermolysis bullosa (RDEB). Biallelic loss-of-function mutations in COL7A1 cause RDEB, resulting from loss of functional type VII collagen (C7) in the basement membrane zone. Current therapies for RDEB are limited, but ex vivo and in vivo COL7A1 base or prime editing therapies can potentially restore normal protein production with a single intervention treatment. This study compared DNA editing specificity, safety and efficiency on RDEB fibroblasts harbouring a homozygous c.4448 G>A (p.Gly1483Asp) mutation in COL7A1. Firstly, transfected adenine base editor (ABE8e) mRNA together with single guide RNA (sgRNA) were introduced into patient primary fibroblasts with Lipofectamine MessengerMax. By varying concentrations and ratios of guide RNA and ABE8e mRNA, we have achieved 100% biallelic correction without introducing any bystander mutations using 200 ng ABE8e and 28 ng sgRNA by transfecting 100 000 cells. Initial prime editing experiments are underway with PEmax plasmid and engineered prime editing guide RNAs (epegRNA) to optimize the guide design and to compare PE2max and PE4max editing systems. The functionality of the 100% biallelic correction using ABE8e is being evaluated with Western blot and in three-dimensional skin models. These early experiments demonstrate the potential value of base editing as a gene-editing tool in RDEB therapeutics; however, more functional work and off-target analyses are needed.