Nanoneedle-Mediated Gene Editing in Epidermolysis Bullosa Fibroblasts

Gene therapy can address the unmet clinical need for non-invasive treatments in dystrophic epidermolysis bullosa(DEB), a rare skin disease associated with trauma-induced erosions and blisters, which progress to chronic wounds and cancer. All forms of DEB, whether autosomal dominant(DDEB) or recessive(RDEB), result from mutations in the <i>COL7A1</i> gene, which encodes type VII collagen(C7). For RDEB, a small number of early phase gene therapy trials has been reported^1,2 while DDEB treatments are limited. In DDEB, glycine substitutions within the C7 triple helix compromises C7 assembly through dominant-negative interference.<br/><br/>Base editing is a highly effective approach to restore functionality of the mutated allele. The targeted nature of this approach is well suited for gene therapy for RDEB, if supported by an effective delivery strategy. Nanoneedles are a “bed of nail” substrate that interface tightly with cells and the superficial layers of tissues to directly transfer payloads with minimal perturbation of cell function³. Intracellular delivery using nanoneedles, known as nanoinjection, yields efficient transfection for a broad range of nucleic acids including RNAs in primary, hard to transfect cells⁴ and to the exposed layers of tissues⁵. Nanoinjection is highly biocompatible, with minimal cell toxicity and perturbation of cell function, making it an ideal candidate for efficient base editing in DEB cells⁶.<br/><br/>Here we develop nanoinjection for base editing of DEB fibroblasts. Nanoinjection to RDEB fibroblasts is highly efficienct without observable toxicity, providing significant improvement over cationic lipid transfection. We edited fibroblasts form an RDEB patient harbouring the compound heterozygous mutation for c.5047C&gt;T (p.Arg1683*) and c.7344+4G&gt;A (IVS95+4G&gt;A). Our nanoinjection approach of adenine base editing (ABE) targeted the c.5047C&gt;T mutation using conical nanoneedles (5μm height, 600nm base, 20nm tip, 2μm spacing).<br/>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&gt;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 expanded to assess C7 production DEJ assembly in organotypic skin models <i>in vitro</i>.