Establishment of an ex vivo retinitis pigmentosa model by efficient delivery of PRPF31 siRNA to human organotypic retinal culture

Abstract

Purpose : The splicing factor PRPF31 is the most commonly mutated general splicing factor in the autosomal dominant retinitis pigmentosa. There is a high demand for safe and efficient delivery of siRNA into Human Organotypic Retinal Culture (HORC). We used a rapid, convenient and safe transfection method with an efficient PRPF31 knockdown in HORC in order to study the PRPF31 silencing effect on retinal gene expressions in this ex vivo model.

Methods : Donor human eyes were obtained within the 24 hours post mortem from Iranian eye bank in compliance with the Declaration of Helsinki. The retinas were isolated and cultured in DMEM serum free medium. HORC transfection using PRPF31 siRNA and scramble siRNA as negative control were done with modified calcium phosphate method. PRPF31 down regulation was assessed by quantitative real time PCR (qPCR) within 63 hours. The tissue viability of HORC was measured using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The PRPF31 gene down regulation effects on retinal gene expression such as RP1, GNAT and ROM were analyzed by qPCR.

Results : After 63 hours of calcium phosphate transfection method in HORC, the qPCR results showed that the levels of PRPF31 mRNA was reduced significantly in the PRPF31 siRNA treated samples in comparison to controls (over 90%). The PRPF31 mRNA silencing with calcium phosphate had no effect on cell viability in the time period of experiment. PRPF31 reduced expression in the siRNA treated samples led to reduction of retinal specific gene expression such as RP1 (over 100 times), GNAT (10 times) and ROM (10 times) compared to controls, based on qPCR results.

Conclusions : We represent an efficient and safe delivery method of PRPF31 siRNA to HORC that could be a useful tool for ex vivo modeling of RP disease. Other than significant effect of PRPF31 down regulation on reduction of RP1, GNAT and ROM gene expression detected by qPCR in this study, further examinations for understanding of PRPF31 down regulation effects in HORC are in progress.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.