Development of BEST1 reporter constructs for use in iPSC based high throughput drug screens and disease modeling

Abstract

Purpose: Best disease, a rare inherited form of dominant juvenile onset macular dystrophy, is characterized by electrophysiological dysfunction of the retinal pigment epithelium (RPE), accumulation of vitelliform material, death of the overlying photoreceptor cells and a subsequent irreversible loss of central vision. Although it is clear that mutations in the RPE gene BEST1 are responsible for this disease, the molecular pathophysiology of mutations in this gene is poorly understood. Induced pluripotent stem cell (iPSC) derived RPE cells are morphologically and biochemical similar to RPE cells in vivo, and iPSC-derived RPE cells generated from patients with Best disease can be used to elucidate the biological function of BEST1 mutations. Likewise, iPSC derived RPE cells make an attractive cell type for testing of potential therapeutics in a high throughput fashion. The purpose of this study was to develop BEST1 reporter constructs to be used in RPE cells generated from patients with Best disease for both elucidation of disease mechanism and as surrogate markers of therapeutic efficacy.

Methods: Keratinocyte-derived iPSCs were generated from patients with molecularly confirmed Best disease. Human iPSCs were differentiated into RPE cells using our previously developed step-wise differentiation protocol. Human BEST1 reporter constructs were synthetically generated and directionally cloned into an AAV vector. RT-PCR analysis was used to validated sequence and insert orientation.

Results: Human constructs containing a fluorescent reporter (GFP or tdTomato) flanked by 3’ and 5’ BEST1 UTRs, driven under control of the BEST1 promoter were generated, directionally cloned into AAV vectors, and packaged into AAV2 particles (Best1R-AAV2). Construct function was confirmed via both subretinal injection of Best1R-AAV2 into wildtype mice and in vitro transduction of human iPSC-RPE derived from patients with Best disease.

Conclusions: We have successfully developed a BEST1 reporter construct that can be utilized in high throughput drug screens and studies of disease pathophysiology.

Keywords: 688 retina • 721 stem cells • 462 clinical (human) or epidemiologic studies: outcomes/complications