Gene therapies differentially rescue disease phenotype in patient-specific hiPSC-RPE models of Best disease

Abstract

Purpose : Best disease (BD) is a common inherited macular dystrophy in which affected individuals gradually lose their central vision. Mutations in BESTROPHIN1 (BEST1) gene are known to be the underlying cause of BD, where the majority of missense mutations have a dominant inheritance pattern. Protein encoded by BEST1 has been postulated to form a homo-pentameric calcium activated chloride channel (CaCC) in the retinal pigment epithelium (RPE) within the retina. Currently, there is no treatment available for BD patients. Our objective was to test if gene therapy approaches (either gene augmentation or gene editing) could be used to treat BD.

Methods : Polarized monolayers of RPE derived from BD patient-specific induced pluripotent stem cells (iPSC-RPE) were cultured using protocols established by our lab. For gene augmentation, viral vectors were used to express wild-type hBEST1 under the hVMD2 promoter. For gene editing, iPSC-RPE cells were transduced with viral vectors expressing spCas9 and mutant-allele-specific guide RNA (gRNA). Single-cell patch clamp was used to assess changes in CaCC currents. Bovine photoreceptor outer segments (POS) were fed to iPSC-RPE to test phagocytic function of control and transduced iPSC-RPE. Post-phagocytosis, levels of Rhodopsin were assessed via Western blots using anti-Rhodopsin antibody.

Results : All BD iPSC-RPE cells had negligible baseline CaCC currents compared to wild-type and isogenic control iPSC-RPE. Efficient transduction of iPSC-RPE cells was confirmed through co-expression of GFP from viral vectors. Gene augmentation successfully restored CaCC currents and improved POS phagocytosis in iPSC-RPE with mutations in calcium clasp or in chloride ion binding region. However, a mutation predicted to be in a structural region of BEST1 channel was not rescued by gene augmentation. Gene editing was used as an alternative approach to specifically silence the mutant allele, which restored CaCC currents in all BD iPSC-RPE lines.

Conclusions : A subset of autosomal dominant BD missense mutations are amenable to gene augmentation. Gene augmentation is already in clinical trials for some inherited ocular disorders, thus it is likely to reach BD patients sooner compared to gene editing. However, patients with mutations that are unresponsive to gene augmentation are candidates for gene editing aimed at silencing the mutant allele.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.