Functional Validation of Known BEST1 Variants Using a Robust Halide-Sensitive YFP Quenching Assay

Abstract

Bestrophinopathies comprise a heterogeneous group of inherited retinal degenerative diseases caused by mutations in the Bestrophin-1 (BEST1) gene. Although more than 250 pathogenic BEST1 variants have been reported, only a small subset has undergone functional interrogation. OPGx-BEST1 is an investigational gene therapy designed to restore BEST1 function through augmented wild-type BEST1 expression in retinal pigment epithelium (RPE) cells. Previous studies in animal models and patient-derived cellular systems have demonstrated that loss-of-function (LOF) BEST1 variants can be complemented with wild-type protein expression and are therefore amenable to gene augmentation strategies. In contrast, known gain-of-function (GOF) mutations appear to retain pathogenic activity despite augmentation with wild-type BEST1. Therefore, given the therapeutic relevance of LOF/GOF disease mechanisms in bestrophinopathies, a comprehensive genotype-phenotype characterization across the BEST1 mutational landscape is critical to inform clinical trial enrollment. Conventional approaches to profile BEST1 variants have relied upon patient-derived RPE models and electrophysiologic assessment of channel function. While these systems provide valuable translational insight, they lack the scalability, speed, and experimental flexibility needed for a systematic evaluation of many variants for patient selection. Here, we describe a functional screening platform that leverages an engineered halide-sensitive reporter cell line to quantitatively assess the activity of BEST1 mutant proteins.