Abstract
Purpose :
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 promising therapeutic strategy for treating retinal diseases, however conventional adeno-associated virus (AAV) vectors cannot accommodate cDNAs exceeding 4.7kb, such as the 15.6kb-long USH2A coding sequence. We investigated an alternative non-viral strategy using episomal DNA plasmid vectors containing a scaffold/matrix attachment region (S/MAR) and the human USH2A cDNA.
Methods :
USH2A-S/MAR vectors were generated by inserting the human USH2A coding sequence into the pS/MAR backbone in five cloning steps. HEK293 cells and USH2A-/- patient-derived dermal fibroblasts were transfected using the Neon transfection system. Wild-type and ush2au507 zebrafish were microinjected with USH2A-S/MAR vector at the single-cell stage of development. Expression of GFP and USH2A protein (usherin) was assessed in both human cell lines and zebrafish using qRT-PCR, immunostaining and Western blot analysis
Results :
USH2A-S/MAR vectors were generated, containing a GFP reporter gene and CAG (pS/MAR-CAG-USH2A) or CMV (pS/MAR-CMV-USH2A) promoters, reaching a size of 23kb. The vectors drove persistent transgene expression in patient fibroblasts and zebrafish with usherin detection, and up to 12 months of GFP expression detected in zebrafish retinal photoreceptor cells.
Conclusions :
To our knowledge, this is the first reported vector generating expression of full-length usherin in USH2A models. S/MAR DNA plasmid vectors have shown promise as a novel non-viral retinal gene therapy, warranting further translational development.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.