Re-engineering melanopsin as a tool for optogenetic vision restoration

背景：视网膜变性疾病是致盲的常见原因，并导致不可逆的感光细胞死亡。在光遗传基因治疗中，外源性光敏蛋白（视蛋白）在视网膜的存活细胞（例如双极细胞）中表达，以允许它们取代失去的光感受器的功能。虽然实验室研究正在进行中，但最近发表了第一位在临床试验中接受治疗的患者的功能数据。虽然数据是有希望的，但视力恢复的水平非常低。虽然观察到的有限功能结果的原因尚不清楚，但一个可能的原因是所使用的视蛋白不适合作为视力恢复的工具。在这个和所有正在进行的试验中，已经使用了微生物来源的视蛋白。然而，这些仅代表两种主要类型的光遗传学工具中的一种：另一种是哺乳动物起源的G蛋白偶联受体视蛋白。反对或赞成微生物视蛋白的争论自光遗传学基因治疗的早期以来一直在推进，并且各种哺乳动物视蛋白已被测试为视力恢复的工具。对于哪种脊椎动物视蛋白最适合于这一目的，目前还没有达成共识。可能每种视蛋白都有一定的优点和缺点，需要对可用的哺乳动物视蛋白进行系统的重新设计，以使其成为视力恢复的“完美”工具。人类天然的黑视素是一个理想的起点，因为它已经过广泛的测试，并且在没有外部视网膜供应的情况下也有功能。此外，它可以在ON-双极细胞内发挥作用，这是光遗传学视力恢复的优选靶点。 目的：在这个项目中，我们将系统地重新设计黑视蛋白，使其成为一种在ON双极细胞中表达时理想的视力恢复工具。方法：我们将建立尖端的荧光成像技术，专门监测黑视素信号在真实的时间。使用这些工具作为一个读数，我们将优化黑视素的兼容性的ON双极细胞转导级联G蛋白特异性和突触后靶向使用小鼠模型。此外，我们将通过系统地交换已知与G蛋白受体信号传导相关的候选残基和通过直系同源物筛选鉴定的新候选残基来动力学优化黑视素。由此产生的“优化”的黑视蛋白将被测试作为一种工具，用于视力恢复并排对微生物视蛋白在电生理体外和体内记录从视网膜和视觉cortic.Impact：该计划将，第一次，提供了一个系统优化的哺乳动物视蛋白用于视力恢复，并将提供潜在的克服在替代工具中观察到的局限性。在已建立的小鼠疾病模型中进行的测试将量化其附加值，并为转化为I期临床研究铺平道路。

Background: Degenerative retinal disorders are a common cause of blindness and lead to irreversible photoreceptor death. In optogenetic gene therapy exogenous light sensitive proteins (opsins) are expressed in surviving cells of the retina (e.g. in bipolar cells) to allow them to substitute the function of the lost photoreceptors. While laboratory research is ongoing, functional data from a first patient treated in a clinical trial have recently been published. While data are promising, the level of vision restored is extremely low.Though the reasons for the limited functional outcome observed are unknown, one possible reason is that the opsin used is not ideally suited as tools for vision restoration. In this and all ongoing trials opsins of microbial origin have been used. However, these represent only one of the two main classes of optogenetic tools: the other being G-protein coupled receptor opsins of mammalian origin.Arguments against or in favour of microbial opsins are being pushed forward since the early days of optogenetic gene therapy and various mammalian opsins have been tested as tools for vision restoration. There is no consensus on which vertebrate opsin could serve best for that purpose. Probably each of the opsins has certain advantages and disadvantages and systematic re-engineering of an available mammalian opsin is needed to make a “perfect” tool for vision restoration. The human-native melanopsin is an ideal starting point as it has been extensively tested and is functional without external retinal supply. Moreover, it can function inside ON-bipolar cells, the preferred target for optogenetic vision restoration. Purpose: In this project we will systematically re-engineer melanopsin to make an optogenetic tool ideal for the purpose of vision restoration when expressed in ON-bipolar cells. Methods: We will establish cutting-edge fluorescent imaging techniques to specifically monitor melanopsin signalling in real time. Using these tools as a readout, we will optimize the compatibility of melanopsin to the ON-bipolar cell transduction cascade in terms of G-protein specificity and postsynaptic targeting using a murine model. Moreover, we will kinetically optimize melanopsin by systematically exchanging candidate residues of known relevance for G-protein receptor signalling and novel candidate residues identified by ortholog screening. The resulting “optimized” melanopsin will be tested as a tool for vision restoration side-by-side against microbial opsins in electrophysiological ex-vivo and in-vivo recordings from retina and visual cortex.Impact: This program will, for the first time, provide a systematically optimized mammalian opsin for use in vision restoration and will offer the potential to overcome the limitations observed in alternative tools. Tests in an established murine disease model will quantify its added value and pave the way towards translation into Phase I clinical research.