scAAV.7m8 and VMD2 Promoter as Novel Gene Delivery Tools Targeting Chicken RPE

Abstract

Purpose : This study investigated the feasibility and specificity of novel gene delivery tools targeting chicken RPE (retinal pigment epithelium) using a new AAV serotype 7m8 and an RPE-specific promoter VMD2.

Methods : Cultured RPE cells and scleral fibroblasts were obtained from 3-day-old chickens and cultured up to 6 weeks. RPE cells were infected with either scAAV.7m8-VMD2-eGFP or scAAV.7m8-CAG-eGFP at concentration of 1 x 10¹⁰ vg/ml after they were confluent. eGFP fluorescence in cultured RPE cells was monitored with a Zeiss LSM 710 Confocal Laser Scanning Microscope every week, for up to 4 weeks. Scleral fibroblasts were also infected with these two viral vectors at the same pattern and observed for up to 2 weeks after administration of viral vectors.

Results : Cultured chicken RPE cells, but not scleral fibroblasts, were successfully transduced with scAAV.7m8-VMD2-eGFP, as indicated by eGFP fluorescence. Specifically, eGFP fluorescence was observed in RPE cells one week after transduction. Its intensity increased thereafter and was sustained throughout the 4-weeks observation period, indicating AAV serotype 7m8 and VMD2 promoter combination specifically transduced chicken RPE cells. On the other hand, both RPE and scleral fibroblasts were successfully transduced with scAAV.7m8-CAG-eGFP, as recorded by strong eGFP fluorescence three days after transduction, suggesting that AAV serotype 7m8 nonselectively infects chicken cells.

Conclusions : The results for scAAV.7m8-VMD2 showing selective transduction of the chicken RPE in vitro suggest that it may serve as a tool for gene delivery targeting RPE. They also open up the possibility of exploring gene therapy in an animal model for myopia as a novel approach to myopia control.

This is a 2020 ARVO Annual Meeting abstract.