Departments of Ophthalmology and Neurobiology, Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, CA 90095, USA.
Gene Ther. 2013 Aug;20(8):824-33. doi: 10.1038/gt.2013.3. Epub 2013 Jan 24.
Usher 1 patients are born profoundly deaf and then develop retinal degeneration. Thus they are readily identified before the onset of retinal degeneration, making gene therapy a viable strategy to prevent their blindness. Here, we have investigated the use of adeno-associated viruses (AAVs) for the delivery of the Usher 1B gene, MYO7A, to retinal cells in cell culture and in Myo7a-null mice. MYO7A cDNA, under control of a smCBA promoter, was packaged in single AAV2 and AAV5 vectors and as two overlapping halves in dual AAV2 vectors. The 7.9-kb smCBA-MYO7A exceeds the capacity of an AAV vector; packaging of such oversized constructs into single AAV vectors may involve fragmentation of the gene. Nevertheless, the AAV2 and AAV5 single vector preparations successfully transduced photoreceptor and retinal pigment epithelium cells, resulting in functional, full-length MYO7A protein and correction of mutant phenotypes, suggesting successful homologous recombination of gene fragments. With discrete, conventional-sized dual AAV2 vectors, full-length MYO7A was detected, but the level of protein expression was variable, and only a minority of cells showed phenotype correction. Our results show that MYO7A therapy with AAV2 or AAV5 single vectors is efficacious; however, the dual AAV2 approach proved to be less effective.
乌谢尔 1 型患者生来就严重失聪，随后会出现视网膜变性。因此，在视网膜变性发生之前，他们很容易被发现，这使得基因治疗成为预防失明的可行策略。在这里，我们研究了腺相关病毒（AAV）在细胞培养和 Myo7a 基因敲除小鼠中向视网膜细胞传递乌谢尔 1B 基因（MYO7A）的用途。在 smCBA 启动子的控制下，MYO7A cDNA 被包装在单 AAV2 和 AAV5 载体中，并在双 AAV2 载体中作为两个重叠的半部分进行包装。7.9kb 的 smCBA-MYO7A 超过了 AAV 载体的容量；将如此超大的构建体包装到单个 AAV 载体中可能涉及基因片段的断裂。尽管如此，AAV2 和 AAV5 单载体制剂成功地转导了光感受器和视网膜色素上皮细胞，产生了功能性的全长 MYO7A 蛋白，并纠正了突变表型，表明基因片段的同源重组成功。使用离散的常规大小的双 AAV2 载体，检测到全长 MYO7A，但蛋白表达水平存在差异，只有少数细胞表现出表型纠正。我们的结果表明，AAV2 或 AAV5 单载体的 MYO7A 治疗是有效的；然而，双 AAV2 方法的效果较差。
