Department of Ophthalmology, College of Medicine, University of Florida, Gainesville, Florida.
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.
Hum Gene Ther. 2019 Nov;30(11):1361-1370. doi: 10.1089/hum.2019.132. Epub 2019 Sep 30.
Autosomal recessive Stargardt disease is the most common inherited macular degeneration in humans. It is caused by mutations in the retina-specific ATP binding cassette transporter A4 (ABCA4) that is essential for the clearance of all--retinal from photoreceptor cells. Loss of this function results in the accumulation of toxic bisretinoids in the outer segment disk membranes and their subsequent transfer into adjacent retinal pigment epithelium (RPE) cells. This ultimately leads to the Stargardt disease phenotype of increased retinal autofluorescence and progressive RPE and photoreceptor cell loss. Adeno-associated virus (AAV) vectors have been widely used in gene therapeutic applications, but their limited cDNA packaging capacity of ∼4.5 kb has impeded their use for transgenes exceeding this limit. AAV dual vectors were developed to overcome this size restriction. In this study, we have evaluated the expression of ABCA4 using three options: overlap, transplicing, and hybrid ABCA4 dual vector systems. The hybrid system was the most efficient of these dual vector alternatives and used to express the full-length ABCA4 in Abca4 mice. The full-length ABCA4 protein correctly localized to photoreceptor outer segments. Moreover, treatment of Abca4 mice with this ABCA4 hybrid dual vector system resulted in a reduced accumulation of the lipofuscin/N-retinylidene-N-retinylethanolamine (A2E) autofluorescence , and retinal A2E quantification supported these findings. These results show that the hybrid AAV dual vector option is both safe and therapeutic in mice, and the delivered transgene is functional and has a significant effect on reducing A2E accumulation in the Abca4 mouse model of Stargardt disease.
常染色体隐性遗传型斯塔加特病是人类最常见的遗传性黄斑变性。它是由视网膜特异性 ATP 结合盒转运蛋白 A4（ABCA4）的突变引起的，该蛋白对于从光感受器细胞清除全视网膜是必不可少的。该功能的丧失导致毒性双视黄醇在外节盘膜中的积累及其随后转移到相邻的视网膜色素上皮（RPE）细胞中。这最终导致斯塔加特病表型的视网膜自发荧光增加和 RPE 和光感受器细胞的进行性丧失。腺相关病毒（AAV）载体已广泛用于基因治疗应用，但它们有限的 cDNA 包装能力约为 4.5kb，限制了它们用于超过此限制的转基因。AAV 双载体已被开发用于克服这种大小限制。在这项研究中，我们使用三种选择来评估 ABCA4 的表达：重叠、剪接和杂交 ABCA4 双载体系统。杂交系统是这些双载体替代方案中最有效的，用于在 Abca4 小鼠中表达全长 ABCA4。全长 ABCA4 蛋白正确定位于光感受器外节。此外，用这种 ABCA4 杂交双载体系统治疗 Abca4 小鼠可减少脂褐素/N-视黄醛-N-视黄醇乙胺（A2E）自发荧光的积累，并且视网膜 A2E 定量支持了这些发现。这些结果表明，杂交 AAV 双载体选择在小鼠中既安全又具有治疗作用，并且递送的转基因是功能性的，并且对减少 Abca4 小鼠模型中 A2E 的积累具有显著影响。
