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 and mortality. A few early phase gene therapy trials have been reported as potential therapeutic approaches. The targeted nature of base editing is well suited to restore COL7A1 functionality in RDEB, if supported by an effective delivery strategy. Nanoneedles are a “bed of nail” substrate that interface tightly with cells for gene transfer in primary, hard to transfect cells and exposed tissue layers. Nanoneedle-based transfection (nanoinjection) has minimal toxicity and perturbation of cell function, making it an ideal candidate for efficient base editing in RDEB. Here we show that base editing of RED fibroblasts by nanoinjection is highly efficient without observable toxicity, providing significant improvement over lipofection. We edited fibroblasts form an RDEB patient harbouring the compound heterozygous mutation for c.5047C>T (p.Arg1683*) and c.7344+1G>A (IVS95+1G>A). Our nanoinjection approach of adenine base editing (ABE) targeted the c.5047C>T mutation using conical nanoneedles (1.52μm height, 80nm tip). The fibroblast cultured on nanoneedles showed retained morphology, high viability and proliferative capacity indicative of good cytocompatibility. We compared the ABE7.10 and ABE8e editors (mRNA + gRNA) targeting the c.5047C>T mutation loaded at different ratios and concentrations of mRNAs and gRNA on nanoneedles, using lipofection as control. Nanoneedles interfaced with the cells delivered the ABEs directly to the intracellular space. At 48h, the optimal editing efficiency for ABE7.10 by nanoinjection was 17%, as compared to 6% by lipofection. When using ABE8e nanoinjection, editing efficiency increased to 46%, resulting in 82% total of the wild-type G allele as compared to 65% for lipofection. The edited cells were assessed for C7 production and DEJ assembly.

ESDR296 - Nanoneedles for gene editing in Epidermolysis Bullosa

The scientific meeting of the European Society for Dermatological Research (ESDR) is held yearly, usually in September. With an international attendance of more than 1000 skin scientists from around the globe, the four-day event includes short oral presentations from peer-reviewed abstracts, keynote lectures from international experts, sponsored symposia, satellite programs, educational events as well as an important focus on posters, networking and building collaborations amongst scientists. For more information about this year’s meeting visit our website.