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

将双极细胞转化为红移光遗传学传感器用于视网膜治疗

• 批准号: 8989104
• 负责人: JOSEPH CORBO
• 金额: $ 44.25万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8989104
• 关键词: Adult; Automobile Driving; Behavioral Assay; Blindness; Cells; Devices; Directed Molecular Evolution; Electrophysiology (science); Engineering; Enzymes; Exhibits; Future; Gene Transfer; Genes; Goals; Health; Human; Individual; Infection; Injection of therapeutic agent; Interneurons; Light; Metabotropic Glutamate Receptors; Modeling; Molecular; Mus; Mutant Strains Mice; Patients; Photophobia; Photoreceptors; Proteins; Retina; Retinal; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Risk; Serotyping; Site; Specificity; Staging; Synthetic Genes; System; Therapeutic; Time; Tropism; Vertebrates; Vision; Vitreous humor; adeno-associated viral vector; base; blind; functional restoration; gene therapy; improved; in vivo; inhibitor/antagonist; innovation; new technology; novel; optogenetics; photoreceptor degeneration; promoter; recombinase; restoration; retinal damage; sensor; signal processing; subretinal injection; synthetic biology; tool

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.

描述（由申请人提供）：光遗传学对恢复视网膜变性的人的视力具有巨大的潜力，尤其是那些失去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感性双极细胞中表达光敏蛋白。 当前方法受到双极靶向和表达效率低下的限制，并且需要应用潜在的 光毒性水平的蓝绿色光刺激光遗传执行器。 本提案的目的是通过利用定向的进化和合成生物学来克服这些挑战，以设计基于AAV的输送系统，以将红移的光遗传学设备靶向开机和关闭双极细胞，并使用该系统来治疗小鼠的失明。 在AIM 1中，我们将使用定向进化来设计新的AAV血清型在注入玻璃体幽默后能够高效的双极AAV感染。 在AIM 2中，我们将利用一种名为Cre-Seq的新技术来设计成千上万的紧凑型，在表现出极好特异性和广泛表达强度的双极特异性启动子上。 此外，我们将设计一个可溶解的合成基因回路，以针对光遗传学抑制剂，专门针对双极细胞。 在AIM 3中，我们将将在AIM 1和2中开发的工具与使用红移的光遗传学设备将功能视觉恢复为RD1突变小鼠的功能视觉。 最近，我们发现了负责脊椎动物中“视紫红质”开关的酶，我们将使用该酶来进行红移光学遗传器件，使其对远红光（> 650 nm）敏感。 这种治疗方法有可能显着提高光敏性 在被救出的小鼠中，将避免与高强度蓝光暴露有关的视网膜损伤，从而允许空前的功能恢复水平，并为人类患者的未来试验奠定基础。