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）通常导致不可逆的光感受器丧失。AMD是老年人法定失明的主要原因，即使每种特定的IRD都很罕见，但全世界每3000人中就有1人受其影响。目前对退行性视网膜疾病的治疗方法仅旨在减缓或停止疾病进展，而不是逆转它。此外，它们都依赖于退化过程的生物学基础，因此大多是疾病特异性的。大多数这些退行性视网膜疾病的一个共同特征是内视网膜不受影响。通过异位表达视蛋白诱导视网膜内细胞的光敏性，已经采取了恢复视觉功能的实验方法。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，它可以实现更高的空间分辨率和更大的视野比替代视网膜植入技术。然而，目前尚不清楚视蛋白的异位表达能在多大程度上编码复杂的光刺激。这一领域缺乏知识的主要原因是缺乏足够分辨率的方法来监测对复杂光刺激的反应。本项目旨在研究基于视蛋白的基因疗法是否能够通过新的电生理、活细胞成像和行为方法恢复光感受器缺陷小鼠的复杂图像形成视觉。我们将建立多电极阵列和基因编码钙传感器作为工具，记录由外植视网膜的一系列模式刺激范式引起的电反应。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，通过选择性地向两个细胞群传递快速、光敏的阳离子通道，来确定基于视蛋白的基因治疗的最佳靶细胞群。由于非生理性刺激反应和高免疫原性，这种阳离子通道不适合用于基因治疗。因此，我们将测试光敏蛋白黑视素和目前开发的快速转换黑视素变体，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，激活生理上参与光反应转导的途径。因此，应该在细胞水平上获得接近天然的对光的反应，并且可以将免疫反应的风险降至最低。这项研究将确定视蛋白基因治疗恢复视觉功能的未来潜力，并将提供必要的知识，将这种方法移植到临床。

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.