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

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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 4.7kb, such as the 15.6kb-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 23kb. We assessed the vectors in transfected HEK-293 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 localisation in the photoreceptors of ush2au507 zebrafish retina injected with pS/MAR-USH2A. To our knowledge, this is the first reported vector which 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.

Journal details

Volume 31
Issue number 9
Pages 2755-2766
Available online
Publication date