Investigating ectopic opsin-based gene therapies to restore pattern forming vision in blind mice

晚期年龄相关性黄斑变性（AMD）和遗传性视网膜疾病（IRD）通常导致不可逆的感光细胞损失。AMD是老年人法律的失明的主要原因，即使每个特定的IRD是罕见的，它们一起影响全世界3000人中的1人。目前视网膜退行性疾病的治疗方法仅旨在减缓或阻止疾病进展，而不是逆转它。此外，它们都依赖于退行性过程的生物学基础，因此大多数疾病特异性。大多数这些退行性视网膜疾病的共同特征是内层视网膜不受影响。已经采取了实验方法，通过视蛋白蛋白质的异位表达诱导视网膜内层细胞的光敏感性来恢复视觉功能。这种方法是有吸引力的，因为它可以独立于潜在的疾病原因发挥作用，并且它可以实现比替代视网膜植入技术高得多的空间分辨率和更大的视野。然而，它仍然不清楚到何种程度的复杂的光刺激的编码可以通过异位表达的视蛋白。缺乏这方面的知识主要是由于缺乏以足够分辨率监测对复杂光刺激的反应的方法。该计划旨在研究基于视蛋白的基因治疗是否能够通过应用新型电生理、活细胞成像和行为方法来恢复感光细胞缺陷小鼠的复杂图像形成视力。我们将建立多电极阵列和遗传编码的钙传感器作为记录电反应的工具。一系列模式刺激范式从外植视网膜引发。此外，我们将优化现有的行为测试，以评估小鼠的模式识别。主要的挑战将是达到最大的时空分辨率，从而能够分析最复杂的光刺激的响应。我们将首先应用这些方法学方法，通过选择性地向任一细胞群体递送快速光敏阳离子通道来鉴定基于视蛋白的基因治疗的最佳靶细胞群体。由于非生理刺激反应和高免疫原性，这种阳离子通道不适合基因治疗用途。因此，我们将测试光敏蛋白黑视蛋白和目前开发的快速转换黑视蛋白变体，并分析它们恢复失明小鼠模式形成视力的能力。黑视素是一种视网膜驻留蛋白，其激活生理上参与光反应转导的途径。因此，一个接近自然的反应，光在细胞水平上应获得和免疫反应的风险可以最小化。这项研究将确定视蛋白为基础的基因治疗，以恢复视觉功能的未来潜力，并将提供必要的知识，港口这种方法到临床。

Late-stage age related Macular Degeneration (AMD) and inherited retinal diseases (IRD) commonly lead to irreversible photoreceptor loss. AMD is the leading cause of legal blindness in the elderly and even if each particular IRD is rare, all together they affect 1 in 3000 humans worldwide. Current therapeutic approaches for degenerative retinal diseases only aim to slow down or halt disease progression, not to reverse it. In addition they all depend on the biology underlying the degenerative process and are therefore mostly disease specific.A common hallmark of most of these degenerative retinal disorders is that the inner retina remains unaffected. Experimental approaches have been taken to restore visual function by inducing light sensitivity to cells of the inner retina through ectopic expression of opsin proteins. This approach is attractive as it could function independently of the underlying disease cause and it may achieve much higher spatial resolution and a greater visual field than the alternative retinal implant technology. However, it remains unclear to what extend encoding of complex light stimuli can be obtained by ectopic expression of opsins. The lack of knowledge in this field is mainly caused by the fact that methods monitoring responses to complex light stimuli with sufficient resolution are missing. This program aims to investigate if opsin based gene therapy is capable of restoring complex image forming vision in photoreceptor deficient mice by applying novel electrophysiological, live-cell imaging and behavioral approaches.We will establish multi-electrode arrays and genetically encoded calcium sensors as tools to record electrical responses elicited by a range of pattern stimulus paradigms from explanted retinae. In addition we will optimize existing behavioral tests to assess pattern recognition in mice. The main challenge will be to reach maximal spatiotemporal resolution that enables analyzing the response also of the most complex light stimuli. We will first apply these methodological approaches to identify the optimal target cell population for opsin based gene therapy by selectively delivering a fast, light sensitive cation channel to either cell population. Due to non-physiologic stimuli responses and high immunogenicity such a cation channel would not be suitable for gene therapeutic use. Therefore we will test the light-sensitive protein melanopsin and currently developed fast-switching melanopsin variants and analyze their capacity to restore pattern forming vision in blind mice. Melanopsin is a retina-resident protein activating pathways physiologically involved in the transduction of light responses. Thereby a close-to-native response to light on cellular level should be obtained and the risk of immunoreactions can be minimized.This study will determine the future potential of opsin based gene therapy to restore visual function and will provide essential knowledge necessary to port this approach into clinics.