CRISPR-Cas9-based non-viral gene editing therapy for topical treatment of recessive dystrophic epidermolysis bullosa

Xianqing Wang; Xi Wang; Yinghao Li; Sigen A; Bei Qiu; Albina Bushmalyova; Zhonglei He; Wenxin Wang; Irene Lara-Sáez

doi:10.1016/j.omtm.2023.101134

CRISPR-Cas9-based non-viral gene editing therapy for topical treatment of recessive dystrophic epidermolysis bullosa

Mol Ther Methods Clin Dev. 2023 Oct 13:31:101134. doi: 10.1016/j.omtm.2023.101134. eCollection 2023 Dec 14.

Authors

Xianqing Wang¹, Xi Wang², Yinghao Li¹, Sigen A², Bei Qiu¹, Albina Bushmalyova¹, Zhonglei He², Wenxin Wang^{1

2}, Irene Lara-Sáez¹

Affiliations

¹ Charles Institute of Dermatology, School of Medicine, University College Dublin, D04 V1W8 Dublin, Ireland.
² Research and Clinical Translation Center of Gene Medicine and Tissue Engineering, School of Public Health, Anhui University of Science and Technology, Huainan 232001, China.

Abstract

Recessive dystrophic epidermolysis bullosa (RDEB) is an autosomal monogenic skin disease caused by mutations in COL7A1 gene and lack of functional type VII collagen (C7). Currently, there is no cure for RDEB, and most of the gene therapies under development have been designed as ex vivo strategies because of the shortage of efficient and safe carriers for gene delivery. Herein, we designed, synthesized, and screened a new group of highly branched poly(β amino ester)s (HPAEs) as non-viral carriers for the delivery of plasmids encoding dual single-guide RNA (sgRNA)-guided CRISPR-Cas9 machinery to delete COL7A1 exon 80 containing the c.6527dupC mutation. The selected HPAEs (named PTTA-DATOD) showed robust transfection efficiency, comparable with or surpassing that of leading commercial gene transfection reagents such as Lipofectamine 3000, Xfect, and jetPEI, while maintaining negligible cytotoxicity. Furthermore, CRISPR-Cas9 plasmids delivered by PTTA-DATOD achieved efficient targeted deletion and restored bulk C7 production in RDEB patient keratinocyte polyclones. The non-viral CRISPR-Cas9-based COL7A1 exon deletion approach developed here has great potential to be used as a topical treatment for RDEB patients with mutations in COL7A1 exon 80. Besides, this therapeutic strategy can easily be adapted for mutations in other COL7A1 exons, other epidermolysis bullosa subtypes, and other genetic diseases.

Keywords: CRISPR-Cas9; gene delivery vectors; gene editing; hyperbranched polyβ amino esters; non-viral; polymers; recessive dystrophic epidermolysis bullosa.