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Dec 13, 2019

Investigation and Restoration of BEST1 Activity in Patient-derived RPEs with Dominant Mutations

Changyi Ji, Yao Li, Alec Kittredge, Austin Hopiavuori, Nancy Ward, Peng Yao, Yohta Fukuda, Yu Zhang, Stephen H. Tsang, Tingting Yang | Scientific Reports | Vol 9 | 19026 | 13 Dec 2019 | https://doi.org/10.1038/s41598-019-54892-7


Abstract

BEST1 is a Ca2+-activated Cl− channel predominantly expressed in retinal pigment epithelium (RPE), and over 250 genetic mutations in the BEST1 gene have been identified to cause retinal degenerative disorders generally known as bestrophinopathies. As most BEST1 mutations are autosomal dominant, it is of great biomedical interest to determine their disease-causing mechanisms and the therapeutic potential of gene therapy. Here, we characterized six Best vitelliform macular dystrophy (BVMD)-associated BEST1 dominant mutations by documenting the patients’ phenotypes, examining the subcellular localization of endogenous BEST1 and surface Ca2+-dependent Cl− currents in patient-derived RPEs, and analyzing the functional influences of these mutations on BEST1 in HEK293 cells. We found that all six mutations are loss-of-function with different levels and types of deficiencies, and further demonstrated the restoration of Ca2+-dependent Cl− currents in patient-derived RPE cells by WT BEST1 gene supplementation. Importantly, BEST1 dominant and recessive mutations are both rescuable at a similar efficacy by gene augmentation via adeno-associated virus (AAV), providing a proof-of-concept for curing the vast majority of bestrophinopathies.


Introduction

Genetic mutation of the human BEST1 gene causes bestrophinopathies, which consist of a spectrum of retinal degeneration disorders including Best vitelliform macular dystrophy (BVMD)1,2, autosomal recessive bestrophinopathy (ARB)3, adult-onset vitelliform dystrophy (AVMD)4,5, autosomal dominant vitreoretinochoroidopathy (ADVIRC)6, and retinitis pigmentosa (RP)7. BVMD, featuring an early-onset and debilitating form of central macular degeneration, is the most common bestrophinopathy. Due to abnormalities in the fluid and/or electrolyte homeostasis between the RPE and photoreceptor outer segments8, the disease leads to the formation of serous retinal detachment and lesions that resemble egg yolk, or vitelliform, while rod and cone photoreceptor function remains unaffected. All types of bestrophinopathies, except for ARB, result from autosomal dominant mutation of BEST1. Patients are susceptible to untreatable, progressive vision loss, which significantly deteriorates life quality. Therefore, understanding the mechanisms of BEST1 disease-causing mutations and designing strategies to restore the damaged cellular function are critical for developing treatments for bestrophinopathies.


The protein encoded by the BEST1 gene is a Cl− channel named BESTROPHIN1 (BEST1), which is activated in response to intracellular Ca2+ and conducts Ca2+-dependent Cl− current on the cell membrane of retinal pigment epithelium (RPE)1,2,9,10. Consistently, Ca2+-dependent Cl− current has been suggested to generate a critical visual response upon light exposure, namely light peak (LP)11,12,13, which is defective in almost all BEST1-mutated patients as shown by electrooculography (EOG)14,15. This BEST1- Cl− current- LP correlation suggests gene supplementation as a promising approach for curing bestrophinopathies. Indeed, we reported that the impaired Cl− current in RPE derived from an ARB patient bearing a BEST1 recessive mutation was rescuable by baculovirus (BV) -mediated supplementation of the WT BEST1 gene9. Moreover, a recent study in canine models demonstrated that the retinal abnormalities caused by recessive mutation of BEST1 can be corrected by adeno-associated virus (AAV) -mediated subretinal BEST1 gene augmentation16. However, the rescue efficacy of gene augmentation for BEST1 dominant mutations is still unknown. This is a very important question because firstly, most of BEST1 mutations are dominant, and secondly, it will determine whether disruption/suppression of the dominant mutant allele is necessary in therapeutic interventions. In principle, the excess of WT BEST1 could overwhelm the mutant BEST1 despite the latter being dominant over the former at a 1:1 ratio. As canines do not have BEST1 dominant mutation genotypes while Best1 knockout mice do not show any retinal phenotype or Cl− current abnormality17,18, patient-derived RPEs offer a more relevant model for testing the rescue of BEST1 dominant mutations.


Here, we analyzed six BEST1 dominant mutations from BVMD patients, namely p.A10T, p.R218H, p.L234P, p.A243T, p.Q293K and p.D302A, using clinical examinations, patient-derived RPEs, electrophysiological recordings and structural models. Our results showed that these mutations are all loss-of-function with complete or partial deficiency of channel activity, while some of them affect the subcellular localization and/or Ca2+-sensitivity of BEST1. Remarkably, defective Ca2+-dependent Cl− currents in patient-derived RPE cells were restored by virus-mediated supplementation of the WT BEST1 gene in a time- and dose-dependent manner. Moreover, both dominant and recessive mutations of BEST1 are rescuable at a similar efficacy, and both BV and AAV can be used as the vector for gene delivery. Together, our findings underscore the great potential of gene augmentation therapy in treating bestrophinopathies, including those caused by BEST1 dominant mutations.


Results

Retinal phenotypes of six BVMD patients with different BEST1 mutations

We examined six BVMD patients from unrelated families. Generalized retinal dysfunction was found in all six patients. Fundus autofluorescence imaging and optical coherence tomography (OCT) revealed vitelliform lesions located in the subretinal space, as well as serous retinal detachments and cystic fluid in the maculae area (Fig. 1 and Supplementary Fig. S1). Unlike BEST1 recessive patients, whose electroretinography (ERG) and EOG results are significantly different from WT people9, BVMD patients display normal ERG but abnormal EOG results (Supplementary Fig. S2)19.


 
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