CONVERTING BIPOLAR CELLS INTO RED-SHIFTED OPTOGENETIC SENSORS FOR RETINAL THERAPY

描述(申请人提供)：光遗传学在恢复晚期视网膜变性患者的视力方面具有巨大的潜力，特别是那些失去了大部分光感受器的患者。一种有希望的治疗策略是通过基因治疗在非光敏双极细胞中表达光敏蛋白。目前的方法受到低效的双极靶向和表达的限制，并且需要应用潜在的 光毒性水平的蓝绿光刺激光发生致动器。本提案的目的是通过利用定向进化和合成生物学来设计一种基于AAV的递送系统来克服这些挑战，以将红移的光遗传设备靶向开和关双极细胞，并使用该系统来治疗小鼠的失明。在目标1中，我们将使用定向进化来设计新的AAV血清型，在玻璃体液中注射后，能够高效地感染双相AAV。在目标2中，我们将利用一种名为Cre-seq的新技术来设计数千个紧凑的、位于双极特异启动子上的启动子，这些启动子表现出极好的特异性和广泛的表达强度。此外，我们将设计一种AAV可交付的合成基因电路，专门针对双极细胞的光遗传抑制物。在目标3中，我们将把在目标1和目标2中开发的工具与红移光遗传设备的使用相结合，以恢复Rd1突变小鼠的功能视力。我们最近在脊椎动物身上发现了负责“视紫红质-卟啉素”转换的酶，我们将使用这种酶来红移光遗传设备，使它们对远红光(&gt；650 nm)敏感。这种治疗方法有可能显著提高光敏感度。 在获救的小鼠身上，并将避免与高强度蓝光暴露相关的视网膜损伤，从而实现前所未有的功能恢复，并为未来在人类患者身上进行试验奠定基础。

DESCRIPTION (provided by applicant): Optogenetics holds tremendous potential for restoring vision to individuals with late-stage retinal degeneration, particularly those patients who have lost most of their photoreceptors. One promising therapeutic strategy is to express a light-sensitive protein in non-photosensitive bipolar cells by gene therapy. Current approaches are limited by inefficient bipolar targeting and expression, and require application of potentially phototoxic levels of blue-green light to stimulate the optogenetic actuator. The objective of the present proposal is to overcome these challenges by utilizing directed evolution and synthetic biology to engineer an AAV-based delivery system to target red-shifted optogenetic devices to both ON and OFF bipolar cells, and to employ this system to treat blindness in mice. In Aim 1, we will use directed evolution to engineer new AAV serotypes capable of highly efficient bipolar AAV infection after injection into the vitreous humor. In Aim 2, we will utilize a novel technolog called CRE-seq to engineer thousands of compact, ON bipolar-specific promoters that exhibit excellent specificity and a wide range of expression strengths. In addition, we will engineer an AAV-deliverable synthetic gene circuit to target an optogenetic inhibitor specifically to OFF bipolar cells. In Aim 3, we will combine the tools developed in Aims 1 and 2 with the use of red-shifted optogenetic devices to restore functional vision to rd1 mutant mice. We recently discovered the enzyme responsible for the 'rhodopsin- porphyropsin' switch in vertebrates, and we will use this enzyme to red-shift optogenetic devices, making them sensitive to far red light (> 650 nm). This therapeutic approach has the potential to dramatically improve light- sensitivity in the rescued mice and will avoid the retinal damage associated with high-intensity blue light exposure, thereby permitting unprecedented levels of functional restoration and setting the stage for future trials in human patients.