Molecular Therapy: Methods & Clinical Development – JOURNAL PRE-PROOF
Authors: Xianqing Wang, Xi Wang, Yinghao Li, Sigen A, Bei Qiu, Albina Bushmalyova,
Zhonglei He, Irene Lara-Sáez, Wenxin Wang
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 due to 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 18 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 c.6527dupC mutation. The selected HPAEs (named PTTA-DATOD) showed robust transfection efficiency, comparable to or surpassing that of leading commercial gene transfection reagents like LipofectamineTM 3000, XfectTM 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.