Abstract
Purpose :
As the most common pathology of the group of macular dystrophies known as bestrophinopathies, Best Vitelliforme Macular Dystrophy or Best disease (BD) is a severe disease of the central retina caused by dominant-negative mutations in the bestrophin 1 (BEST1) gene. The transcript encodes an intrinsic membrane protein shown to act as a homo-pentameric Ca2+-activated and volume-regulated chloride channel. In a proof-of-concept study, we designed a treatment for BD by selectively eliminating BEST1 mutant transcripts via the CRISPR/Cas9 system in patient-derived human induced pluripotent stem cells (hiPSCs) prior to retinal pigment epithelium (RPE) differentiation.
Methods :
Fibroblasts were obtained from skin biopsies of BD patients and reprogrammed into hiPSCs. After transfection of hiPSCs with the Cas9/sgRNA RNP complex the percentage of indel formation was determined by Sanger sequencing. Cas9-treated stem cell populations were differentiated into hiPSC-derived RPE cells. BEST1 localization and protein half-life was determined by immunocytochemistry and Western blot analysis, respectively. To analyze chloride transport function, we developed a halide assay using the yellow fluorescent protein (YFP)-based halide sensor YFPH148Q/I152L.
Results :
Based on computational prediction we identified sgRNAs with high on-target efficacy and reduced off-target effects targeting the most frequent mutations found in European BD patients. We show that truncated 17-nt sgRNA coupled with SpCas9 are highly efficient at specifically suppressing expression of mutant BEST1 transcripts in hiPSC-derived RPE cells from two heterozygous BD patients while leaving wildtype alleles intact. Consequently, elimination of mutant transcripts resulted in an increase of functional BEST1 channel activity and protein stability. In addition, we generated a self-limiting “all-in-one” AAV vector to minimize prolonged expression of SpCas9 applicable for future in vivo use. We show that such an AAV vector solution is preferable over a dual AAV system.
Conclusions :
So far, there is no treatment for BEST1-associated retinopathy. We show that haploinsufficiency of normal BEST1 protein is sufficient to restore cellular function in cells of primary BD pathology, namely the RPE. These results support further studies to corroborate the therapeutic potential of CRISPR/Cas9-based strategies to treat diseases with dominant-negative modes of action.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.