376 Tsc2 disruption in mesenchymal progenitors regulates hair follicles and TGF beta signaling

Abstract

Genome editing represents a promising strategy to correct COL7A1 gene mutations that cause recessive dystrophic epidermolysis bullosa (RDEB). Previously, we used programmable nucleases that create double-stranded DNA breaks (DSBs) to repair COL7A1 mutations through homology-directed repair (HDR) with an exogenous repair template. Delivery of this template can be cytotoxic and DSBs induce undesired insertions and deletions (indels) that compete with desired HDR. To overcome these limitations, we used base editors (BE), a CRISPR/Cas9-based system that uses naturally occurring or laboratory-evolved deaminating enzymes to directly convert A>G, C>T, T>C, or G>A. BE does not lead to significant DSBs, obviates the need for a repair template, and typically offers higher editing efficiencies for point mutations than HDR. We used an optimized A>G base editor (ABEmax) for the RDEB causative R185X and R525X nonsense mutations in the COL7A1gene. We delivered ABEmax mRNA with minimal toxicity into primary fibroblasts from two patients with RDEB, and observed mutation correction rates of up to 50% along with concomitant restoration of COL7A1 protein production. Indel occurrence was minimal, with an observed frequency of w2%, consistent with previous studies. Twenty predicted off-target loci were analyzed by high-throughput sequencing. Treatment with the R185X reagent showed no off-target effects, while the R525X candidate showed A>G editing at one exonic off-target site at a frequency of w6% with <0.5% indels. Base-edited human fibroblasts were injected into an immune-deficient mouse model of RDEB, and human COL7A1expressing cells were observed in vivo. 3D bioprinting was used to deposit base-edited fibroblasts in a biopolymer complex that allowed for significant fibroblast expansion in support of a scalable, ex vivo approach for skin graft generation. These findings suggest that an optimized base editing approach may provide an efficient and precise genome editing method for individualized autologous cell therapy for RDEB.