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Brianna Bowman Young, Karina E Guziewicz, Alfred S Lewin, william W hauswirth, Cristhian Ildefonso; Development of an Allele-Independent Gene Replacement Therapy for Best Vitelliform Macular Dystrophy Using RNAi Technology. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2457.
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© ARVO (1962-2015); The Authors (2016-present)
Best Vitelliform Macular Dystrophy (BVMD) is an autosomal dominant form of macular degeneration caused by mutations in the human bestrophin (BEST1) gene. The onset of BVMD occurs at a young age and causes vision impairment due to retinal pigment epithelium atrophy. There is no approved therapy. The purpose of our research is to develop an allele-independent gene replacement therapy using RNA interference.
HEK293T cells expressing BEST1 were generated by lentiviral vector transduction and puromycin selection. Cells were transfected with polyethyleneimine (PEI) and siRNA oligos or plasmids containing a shRNA that targeted different sequences within the BEST1 gene. Expression was determined by western blot. Bestrophin knock-down was quantified by standardization to GAPDH or tubulin expression. Band intensities were measured using Image J software. Relative bestrophin values between groups were determined by standardization to ratios of cells transfected with either a non-targeting siRNA or a GFP containing plasmid.
HEK293T cells transduced with lentiviral vector delivering BEST1 and selected with puromycin expressed bestrophin as determined by western blot. Cells transfected with siRNA5 had a 28% expression level compared to control siRNA transfected cells. This sequence was converted into shRNA 5 gene by introducing an H1 promoter, a simple loop, and a six T termination signal. When transfected, this plasmid did not knock down BEST1 expression. In another experiment, six BEST1 shRNA containing plasmids were transfected into the HEK293T-BEST1 cells. The plasmid delivering the shRNA 744 knocked down bestrophin expression compared to the cells transfected with a GFP plasmid or no shRNA control.
Although siRNA 5 significantly knocked down the expression of bestrophin, when converted into a shRNA this sequence lost its efficacy. This could be due to a suboptimal promoter. shRNA 744 sufficiently knocked down bestrophin expression compared to the other shRNA’s and this could be due to it binding to the most open region of BEST1 mRNA. We will introduce silent mutations to the shRNA 744 target sequence within the BEST1 gene and this will create a de-targeted BEST1 gene. The constructed AAV vector containing shRNA 744 and the de-targeted BEST1, will knock down the endogenous mutant version of BEST1, and reintroduce a functioning copy of the gene.
This is a 2020 ARVO Annual Meeting abstract.
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