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Recessive dystrophic epidermolysis bullosa is a debilitating blistering skin disorder caused by loss-of-function mutations in COL7A1, which encodes type VII collagen, the main component of anchoring fibrils at the dermal−epidermal junction. Although conventional gene therapy approaches through viral vectors have been tested in preclinical and clinical trials, they are limited by transgene size constraints and only support unregulated gene expression. Genome editing could potentially overcome some of these limitations, and CRISPR/Cas9 has already been applied in research studies to restore COL7A1 expression. The delivery of suitable repair templates for the repair of DNA cleaved by Cas9 is still a major challenge, and alternative base editing strategies may offer corrective solutions for certain mutations. We show highly targeted and efficient cytidine deamination and molecular correction of a defined recessive dystrophic epidermolysis bullosa mutation (c.425A>G), leading to restoration of full-length type VII collagen protein expression in primary human fibroblasts and induced pluripotent stem cells. Type VII collagen basement membrane expression and skin architecture were restored with de novo anchoring fibrils identified by electron microscopy in base-edited human recessive dystrophic epidermolysis bullosa grafts recovered from immunodeficient mice. The results show the potential and promise of emerging base editing technologies in tackling inherited disorders with well-defined single nucleotide mutations.

Emerging base editing technology exploits CRISPR RNA-guided DNA modification effects for highly specific C > T conversion, which has been used to efficiently disrupt gene expression. These tools can enhance synthetic T cell immunity by restricting specificity, addressing histocompatibility leukocyte antigen (HLA) barriers, and promoting persistence. We report lentiviral delivery of a hepatitis B-virus (HBV)-specific recombinant T cell receptor (rTCR) and a linked CRISPR single-guide RNA for simultaneous disruption of endogenous TCRs (eTCRs) when combined with transient cytosine deamination. Discriminatory depletion of eTCR and coupled expression of rTCR resulted in enrichment of HBV-specific populations from 55% (SEM, ±2.4%) to 95% (SEM, ±0.5%). Intensity of rTCR expression increased 1.8- to 2.9-fold compared to that in cells retaining their competing eTCR, and increased cytokine production and killing of HBV antigen-expressing hepatoma cells in a 3D microfluidic model were exhibited. Molecular signatures confirmed that seamless conversion of C > T (G > A) had created a premature stop codon in TCR beta constant 1/2 loci, with no notable activity at predicted off-target sites. Thus, targeted disruption of eTCR by cytosine deamination and discriminatory enrichment of antigen-specific T cells offers the prospect of enhanced, more specific T cell therapies against HBV-associated hepatocellular carcinoma (HCC) as well as other viral and tumor antigens.