296 Nanoneedles for gene editing in Epidermolysis Bullosa

Recessive Dystrophic Epidermolysis Bullosa is an autosomal recessive disorder caused by loss of function mutations in COL7A1 causing mucocutaneous blistering and scarring, with considerable morbidity mortality. A few early phase gene therapy trials have been reported as potential therapeutic approaches. The targeted nature base editing well suited to restore functionality RDEB, if supported effective delivery strategy. Nanoneedles are a “bed nail” substrate that interface tightly cells for transfer primary, hard transfect exposed tissue layers. Nanoneedle-based transfection (nanoinjection) has minimal toxicity perturbation cell function, making it ideal candidate efficient RDEB. Here we show RED fibroblasts nanoinjection highly without observable toxicity, providing significant improvement over lipofection. We edited form RDEB patient harbouring the compound heterozygous mutation c.5047C>T (p.Arg1683*) c.7344+4G>A (IVS95+4G>A). Our approach adenine (ABE) using conical nanoneedles (5μm height, 600nm base, 20nm tip, 2μm spacing). fibroblast cultured on showed retained morphology, high viability proliferative capacity indicative good cytocompatibility. compared ABE7.10 ABE8e editors (mRNA + gRNA) targeting loaded at different ratios concentrations mRNAs gRNA nanoneedles, lipofection control. interfaced delivered ABEs directly intracellular space. At 48h, optimal efficiency was 17%, 6% When nanoinjection, increased 46%, resulting 82% total wild-type G allele 65% were assessed C7 production DEJ assembly.