ESDR SkinChronicles, No. 4, January 2025, by Jos P.H. Smits

Paper:  Steinbeck BJ, Gao XD, McElroy AN, et. al. Twin Prime Editing mediated exon skipping/reinsertion for restored collagen VII expression in recessive dystrophic epidermolysis bullosa. J Invest Dermatol. 2024 in press;

Recessive dystrophic epidermolysis bullosa (RDEB) is a debilitating skin disease often caused by nucleotide variations in type VII collagen (C7, COL7A1). These variations lead to a loss of the dermal-epidermal anchoring, ultimately resulting in blister formation. Hundreds of pathogenic COL7A1 variations are residing in exons that can be skipped in order to restore C7 (Vermeer et al., 2021), for example using antisense oligonucleotides (Turczynski et al., 2012). Although promising, this treatment option is transient and repeated application remains necessary. Reports have shown that exon skipping can occur naturally (Saito et al., 2009), poising the idea to use genetic engineering to omit the exon of interest as a personalized therapeutic option. Genetic engineering is permanent and its efficacy has improved over the years to be broadly applicable and a feasible treatment option.

Steinbeck and colleagues anticipated that reframing of COL7A1 by deletion of exons harboring multiple pathogenic nucleotide variants would be a treatment approach useful for multiple patients (Steinbeck et al., 2024). The authors employed multiple prime editing strategies in RDEB fibroblasts to delete COL7A1 exon 5, maintaining the remaining 117 exons in-frame and effectively restoring C7 expression and functionality as displayed by 3D organotypic cultures and 2D adhesion assays. Similarly, prime editing of patient-derived RDEB keratinocytes restored C7 expression.

Ever since the first publications on CRISPR/Cas9, the promise for permanent treatment of debilitating diseases (like RDEB) has been highly anticipated. However, standard CRISPR/Cas9 has its drawbacks i.e., off-target, stochastic editing and issues delivering the CRISPR-machinery. With improved CRISPR-based and delivery methodologies, such as transient mRNA-based prime editing, off-target edits are much reduced while on-target, precise editing has improved markedly. Steinbeck et al. for the first time employed prime editing in human keratinocytes, positioning precise genome engineering as feasible tool for permanent treatment of RDEB. Additionally, the extended precise genome engineering toolbox will further propel in vitro basic research aimed to build disease models to understand (genetic) disease processes, not just limited to epidermolysis bullosa.

References:

Saito M, Masunaga T, Ishiko A. A novel de novo splice-site mutation in the COL7A1 gene in dominant dystrophic epidermolysis bullosa (DDEB): specific exon skipping could be a prognostic factor for DDEB pruriginosa. Clin Exp Dermatol 2009;34(8):e934-6.

Steinbeck BJ, Gao XD, McElroy AN, Pandey S, Doman JL, Riddle MJ, et al. Twin Prime Editing Mediated Exon Skipping/Reinsertion for Restored Collagen VII Expression in Recessive Dystrophic Epidermolysis Bullosa. J Invest Dermatol 2024.

Turczynski S, Titeux M, Pironon N, Hovnanian A. Antisense-mediated exon skipping to reframe transcripts. Methods Mol Biol 2012;867:221-38.

Vermeer FC, Bremer J, Sietsma RJ, Sandilands A, Hickerson RP, Bolling MC, et al. Therapeutic Prospects of Exon Skipping for Epidermolysis Bullosa. Int J Mol Sci 2021;22(22).