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，并且需要潜在的应用