Successful large gene augmentation of USH2A with non-viral episomal vectors

• Published in: Molecular therapy 2023-09, Vol.31 (9), p.2755-2766
• Main Authors: Toms, Maria, Toualbi, Lyes, Almeida, Patrick V., Harbottle, Richard, Moosajee, Mariya
• Format: Article
• Language: English
• Subjects: Animals; DNA; Extracellular Matrix Proteins - genetics; HEK293 Cells; Humans; Mutation; non-viral gene therapy; Original; Plasmids - genetics; retinitis pigmentosa; scaffold matrix attachment region; USH2A; Usher syndrome; Usher Syndromes - genetics; Usher Syndromes - therapy; Zebrafish - genetics
• ISSN: 1525-0016; 1525-0024; 1525-0024
• DOI: 10.1016/j.ymthe.2023.06.012

USH2A mutations are a common cause of autosomal recessive retinitis pigmentosa (RP) and Usher syndrome, for which there are currently no approved treatments. Gene augmentation is a valuable therapeutic strategy for treating many inherited retinal diseases; however, conventional adeno-associated virus (AAV) gene therapy cannot accommodate cDNAs exceeding 4.7 kb, such as the 15.6-kb-long USH2A coding sequence. In the present study, we adopted an alternative strategy to successfully generate scaffold/matrix attachment region (S/MAR) DNA plasmid vectors containing the full-length human USH2A coding sequence, a GFP reporter gene, and a ubiquitous promoter (CMV or CAG), reaching a size of approximately 23 kb. We assessed the vectors in transfected HEK293 cells and USH2A patient-derived dermal fibroblasts in addition to ush2au507 zebrafish microinjected with the vector at the one-cell stage. pS/MAR-USH2A vectors drove persistent transgene expression in patient fibroblasts with restoration of usherin. Twelve months of GFP expression was detected in the photoreceptor cells, with rescue of Usher 2 complex localization in the photoreceptors of ush2au507 zebrafish retinas injected with pS/MAR-USH2A. To our knowledge, this is the first reported vector that can be used to express full-length usherin with functional rescue. S/MAR DNA vectors have shown promise as a novel non-viral retinal gene therapy, warranting further translational development. [Display omitted] Moosajee and colleagues have generated episomal plasmid vectors containing the coding region of the very large USH2A gene, a common cause of the retinal disease retinitis pigmentosa. These vectors were assessed in cellular and zebrafish models of USH2A, showing promise as a novel non-viral retinal gene therapy.