Twin prime editing mediated exon skipping/reinsertion for restored collagen VII expression in recessive dystrophic epidermolysis bullosa

Benjamin J Steinbeck; Xin D Gao; Amber N McElroy; Smriti Pandey; Jordan L Doman; Megan J Riddle; Lily Xia; Weili Chen; Cindy R Eide; Andre H Lengert; Sang Won Han; Bruce R Blazar; Hans H Wandall; Sally Dabelsteen; David R Liu; Jakub Tolar; Mark J Osborn

doi:10.1016/j.jid.2024.04.013

Twin prime editing mediated exon skipping/reinsertion for restored collagen VII expression in recessive dystrophic epidermolysis bullosa

J Invest Dermatol. 2024 May 17:S0022-202X(24)00372-5. doi: 10.1016/j.jid.2024.04.013. Online ahead of print.

Authors

Benjamin J Steinbeck¹, Xin D Gao², Amber N McElroy¹, Smriti Pandey², Jordan L Doman², Megan J Riddle¹, Lily Xia¹, Weili Chen¹, Cindy R Eide¹, Andre H Lengert³, Sang Won Han³, Bruce R Blazar¹, Hans H Wandall⁴, Sally Dabelsteen⁴, David R Liu², Jakub Tolar¹, Mark J Osborn⁵

Affiliations

¹ Department of Pediatrics, Division of Blood and Marrow Transplant and Cellular Therapy, University of Minnesota, Minneapolis, MN, USA.
² Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.
³ Universidade Federal de Sao Paulo Escola Paulista de Medicina: Sao Paulo, SP, BR.
⁴ University of Copenhagen, Centre for Glycomics, Department of Cellular and Molecular Medicine, Copenhagen, Denmark.
⁵ Department of Pediatrics, Division of Blood and Marrow Transplant and Cellular Therapy, University of Minnesota, Minneapolis, MN, USA. Electronic address: osbor026@umn.edu.

PMID: 38763174
DOI: 10.1016/j.jid.2024.04.013

Abstract

Gene editing nucleases, base editors, and prime editors are potential locus specific genetic treatment strategies for recessive dystrophic epidermolysis bullosa (RDEB); however, many RDEB COL7A1 mutations are unique, making the development of personalized editing reagents challenging. 270 of the ∼320 COL7A1 EB mutations reside in exons that can be skipped, and antisense oligonucleotides (ASO) and gene editing nucleases have been used to create in-frame deletions. ASOs are transient and nucleases generate deleterious double stranded DNA breaks (DSB) and uncontrolled mixtures of allele products. We developed a twin prime editing (twinPE) strategy using the PEmax and recently evolved PE6 prime editors and dual prime editing guide RNAs flanking COL7A1 exon five. Prime editing-mediated deletion of exon 5 with a homozygous premature stop codon was achieved in RDEB fibroblasts, keratinocytes, and iPSC with minimal DSBs, and collagen type VII (C7) protein was restored. TwinPE can replace the target exon with recombinase attachment sequences, and we exploited this to re-insert a normal copy of exon 5 using the Bxb1 recombinase. These findings demonstrate that twinPE can facilitate locus-specific, predictable, in-frame deletions and sequence replacement with few DSBs as a strategy that may enable a single therapeutic agent to treat multiple RDEB patient cohorts.

Keywords: Prime editing; dystrophic epidermolysis bullosa.