Highly efficient correction of three recurrent pathogenic variants in COL7A1 using base editing to treat recessive dystrophic epidermolysis bullosa

Summary: We explored the therapeutic potential of Base editing mediated correction of three recurrent pathogenic <i>COL7A1 </i>variants in patient-derived cells with Recessive Dystrophic Epidermolysis Bullosa (RDEB), a life-threatening genetic disease of the skin and mucosa. We report highly efficient correction of c.425A>G (p.Lys142Arg); c.6187C>T (p.Arg2063Trp) and c.6508C>T (p.Gln2170*) in primary keratinocytes (KC) and fibroblasts (FC) from RDEB subjects. Fourteen candidate gRNAs were designed and tested. mRNAs encoding Cytosine Base Editors (CBE) and Adenine Base Editors (ABE) were synthesized by <i>in vitro</i> transcription and were delivered by nucleofection to RDEB KCs and FCs. For the c.425A>G variant, gRNA-N1 and gRNA-N2 achieved 73% and 91% editing, respectively, with minimal bystander effects and without apparently affecting protein sequence, as evaluated by high-throughput sequencing. mRNA analysis confirmed correct transcript expression. C7 protein levels were restored using gRNA-N2 together with TAD-CBED_V106W. Similarly, gRNA-N2 and gRNA-N3 in combination with ABE8eSprY achieved up to 79% of correction in patient’ cells homozygous for the c.6187C>T variant. For the c.6508C>T variant, six gRNAs were tested together with different ABEs (ABE8e, ABE-Max and ABE8e-NG) and results showed up to 91% of correction when using gRNA-N5 or gRNA-N6 in combination with ABE8e-NG. Other conditions showed up to 86% of correction, but were associated with up to four bystanders ranged from 95% at its highest to 13% at its lowest. Currently, genetically corrected 3D skin equivalents are being generated to validate functionality of this approach for future <i>ex vivo</i> clinical applications. At least 16.5% of patients (harboring one of these mutations) could benefit from this approach. Overall, by evaluating the feasibility and efficacy of base-editing of selected <i>COL7A1</i> recurrent variants, this project has the potential to accelerate clinical translation of gene-correcting strategies to treat RDEB<b>.</b> Marie Hautbois¹, Louise Penez¹, Aline Peynet¹, Cecile Masson¹, Christine Bole¹, Matthias Titeux¹, Alain Hovnanian¹, Araksya Izmiryan¹ 1. Imagine Institute for Genetic Diseases, Paris, Ile-de-France, France. Genetic Disease, Gene Regulation, Gene Therapy & Epigenetics