Wood Shaun R, McClements Michelle E, Martinez-Fernandez de la Camara Cristina, Patrício Maria I, Uggenti Carolina, Sekaran Sumathi, Barnard Alun R, Manson Forbes D, MacLaren Robert E
1 Nuffield Laboratory of Ophthalmology, Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom.
2 Division of Evolution and Genomic Sciences, The University of Manchester, Manchester, United Kingdom.
Hum Gene Ther Methods. 2019 Apr;30(2):44-52. doi: 10.1089/hgtb.2018.267.
Mutations in the human gene are responsible for a number of distinct retinal disorders known as bestrophinopathies, for which there are no current treatments. The protein product, bestrophin-1, is expressed in the retinal pigment epithelium (RPE) where it localizes to the basolateral membrane and acts as a Ca-activated chloride channel. Recent studies have shown successful BEST1-mediated gene transfer to the RPE, indicating human clinical trials of BEST1 gene therapy may be on the horizon. A critical aspect of such trials is the ability to assess the efficacy of vector prior to patient administration. Here, an assay is presented that enables the quantitative assessment of AAV-mediated BEST1 chloride conductance as a measure of vector efficacy. Expression of BEST1 following transduction of HEK293 cells with AAV.BEST1 vectors was confirmed by liquid chromatography, Western blot, and immunocytochemistry. Whole-cell patch-clamp showed increased chloride conductance in BEST1-transduced cells compared to sham-transduced and untransduced controls. Exogenous chloride current correlated to BEST1 expression level, with an enhanced AAV.BEST1.WPRE vector providing higher expression levels of BEST1 and increases in chloride conductance. This study presents o electrophysical quantification of bestrophin-1 following AAV-mediated gene transfer, providing vital functional data on an AAV gene therapy product that will support a future application for regulatory approval.
人类基因中的突变会导致多种被称为贝斯特氏病的独特视网膜疾病，目前尚无针对这些疾病的治疗方法。其蛋白质产物贝斯特素-1在视网膜色素上皮（RPE）中表达，定位于基底外侧膜，并作为一种钙激活氯离子通道发挥作用。最近的研究表明，BEST1介导的基因已成功转移至RPE，这表明BEST1基因治疗的人体临床试验可能即将开展。此类试验的一个关键方面是在对患者给药前评估载体疗效的能力。在此，我们介绍一种检测方法，该方法能够对AAV介导的BEST1氯电导进行定量评估，以此作为载体疗效的衡量标准。通过液相色谱、蛋白质免疫印迹和免疫细胞化学方法证实了用AAV.BEST1载体转导HEK293细胞后BEST1的表达。全细胞膜片钳显示，与假转导和未转导的对照相比，BEST1转导细胞中的氯电导增加。外源氯电流与BEST1表达水平相关，增强型AAV.BEST1.WPRE载体可提供更高水平的BEST1表达并增加氯电导。本研究展示了AAV介导的基因转移后贝斯特素-1的电生理量化，提供了关于一种AAV基因治疗产品的重要功能数据，这将为未来申请监管批准提供支持。
