Bioengineering & Translational Medicine (Jul 2024)

Highly efficient CRISPR/Cas9‐mediated exon skipping for recessive dystrophic epidermolysis bullosa

  • Alex du Rand,
  • John Hunt,
  • Christopher Samson,
  • Evert Loef,
  • Chloe Malhi,
  • Sarah Meidinger,
  • Chun‐Jen Jennifer Chen,
  • Ashley Nutsford,
  • John Taylor,
  • Rod Dunbar,
  • Diana Purvis,
  • Vaughan Feisst,
  • Hilary Sheppard

DOI
https://doi.org/10.1002/btm2.10640
Journal volume & issue
Vol. 9, no. 4
pp. n/a – n/a

Abstract

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Abstract Gene therapy based on the CRISPR/Cas9 system has emerged as a promising strategy for treating the monogenic fragile skin disorder recessive dystrophic epidermolysis bullosa (RDEB). With this approach problematic wounds could be grafted with gene edited, patient‐specific skin equivalents. Precise gene editing using homology‐directed repair (HDR) is the ultimate goal, however low efficiencies have hindered progress. Reframing strategies based on highly efficient non‐homologous end joining (NHEJ) repair aimed at excising dispensable, mutation‐harboring exons offer a promising alternative approach for restoring the COL7A1 open reading frame. To this end, we employed an exon skipping strategy using dual single guide RNA (sgRNA)/Cas9 ribonucleoproteins (RNPs) targeted at three novel COL7A1 exons (31, 68, and 109) containing pathogenic heterozygous mutations, and achieved exon deletion rates of up to 95%. Deletion of exon 31 in both primary human RDEB keratinocytes and fibroblasts resulted in the restoration of type VII collagen (C7), leading to increased cellular adhesion in vitro and accurate C7 deposition at the dermal‐epidermal junction in a 3D skin model. Taken together, we extend the list of COL7A1 exons amenable to therapeutic deletion. As an incidental finding, we find that long‐read Nanopore sequencing detected large on‐target structural variants comprised of deletions up to >5 kb at a frequency of ~10%. Although this frequency may be acceptable given the high rates of intended editing outcomes, our data demonstrate that standard short‐read sequencing may underestimate the full range of unexpected Cas9‐mediated editing events.

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