Generating photoreceptors by reprogramming RPE cells

通过重新编程 RPE 细胞生成光感受器

• 批准号: 8039470
• 负责人: SHU-ZHEN WANG
• 金额: $ 36.63万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8039470
• 关键词: 9-cis-retinal; Address; Cell Culture Techniques; Cell Transplantation; Cells; Cellular Morphology; Chick Embryo; Clinical; Development; Environment; Exhibits; Eye; Future; Gene Expression; Generations; Genes; Goals; Human; In Situ; In Vitro; Infection; Investigation; Light; Methods; Molecular; Motion; Mus; Natural regeneration; Neural Retina; Neurons; Pathway interactions; Photoreceptors; Phototransduction; Physiological; Physiology; Production; Property; Regulator Genes; Replacement Therapy; Retina; Retinal; Scheme; Source; System; Testing; Therapeutic; Time; Transgenic Mice; Transgenic Organisms; Transplantation; Ursidae Family; Validation; Viral; Virus; Visual impairment; design; in vivo; induced pluripotent stem cell; interest; neurogenesis; novel strategies; photoreceptor degeneration; programs; promoter; research study; response; trait; transcription factor

DESCRIPTION (provided by applicant): The long-term goal of this project is to produce new photoreceptors for cell replacement. Photoreceptor replacement holds great promise in treating visual impairments caused by photoreceptor degeneration. At the same time, it presents the need for a supply of differentiating photoreceptors, because the human neural retina lacks regeneration capability. To address this critical barrier in developing photoreceptor-replacement therapies, we take a rather unconventional approach to generate differentiating photoreceptors - reprogramming RPE cells with a pro-photoreceptor gene to channel RPE's well-known capabilities of proliferation and plasticity towards photoreceptor production. Studies with chick cells raise the exciting possibility of deriving new photoreceptors from the RPE through gene-directed reprogramming. Interesting as it stands, it is time to test the hypothesis that mammalian RPE cells can be reprogrammed to give rise to photoreceptor cells. Validation of the hypothesis bears clinical and societal significance. To test the hypothesis, we designed two sets of complementary studies. The first set directly examines cultured human RPE cells for their capacity to produce photoreceptor cells under the guidance of a pro-photoreceptor gene ngn1. Human RPE cells will be virally transduced with ngn1 to initiate photoreceptor differentiation. The cell culture will then be analyzed for de novo production of photoreceptor-like neurons at the levels of gene expression, cellular morphology, and functional physiology, in vitro and in vivo after transplantation into the eyes. A direct test with human cells bears high relevance to the development of potential therapy. The second set investigates whether new photoreceptor cells will be generated from the mouse RPE ectopically expressing pro-photoreceptor gene ngn1. Ectopic ngn1 expression in the RPE will be achieved using viral delivery and transgenics. The ngn1-RPE will then be subjected to conditions, such as the in vivo environment of photoreceptor degeneration that may unleash the experimental RPE's potential to give rise to photoreceptor cells. This will be followed by analyses for de novo generation of photoreceptor cells at molecular, cellular, and physiological levels. In addition to testing our hypothesis, a demonstration of "RPE -> photoreceptor" reprogramming in mice will provide scientific evidence for future investigation into RPE as a convenient source of photoreceptors for in situ cell replacement without cell transplantation. Together, the studies promise information vital to using the RPE to repopulate the retina afflicted with photoreceptor degeneration. PUBLIC HEALTH RELEVANCE: A critical barrier to progress in developing photoreceptor replacement therapy is a lack of reliable source of new photoreceptors. This project investigates gene-directed reprogramming of RPE cells as a novel approach to produce new photoreceptors for future cell replacement studies.

描述（由申请人提供）：该项目的长期目标是生产新的光感受器以替换细胞。光感受器的替换在治疗受感光器变性引起的视觉障碍方面有很大的希望。同时，由于人类神经视网膜缺乏再生能力，因此需要提供分化感光体的供应。为了解决这种关键的障碍在开发感光受体替代疗法时，我们采用一种相当非常规的方法来产生分化的光感受器 - 用亲核受体基因重编程RPE细胞，以通向RPE的众所周知的著名的对光感受器生产的增殖和可塑性的能力。对雏鸡细胞的研究提出了通过基因定向重编程从RPE中衍生出新的光感受器的令人兴奋的可能性。有趣的是，是时候测试可以对哺乳动物RPE细胞进行重编程以产生感光细胞的假设了。对假设的验证具有临床和社会意义。为了检验假设，我们设计了两组互补研究。第一组直接检查了培养的人RPE细胞的能力，其能力在pro-photoreceptor基因NGN1的指导下产生感光细胞。人RPE细胞将用NGN1病毒转导，以启动感光细胞分化。然后，将分析细胞培养物，以从基因表达，细胞形态和功能生理学水平，在移植到眼睛后的体外和体内从头产生。与人类细胞的直接测试与潜在治疗的发展具有很高的相关性。第二组研究是否将从小鼠RPE异位表达pro-Propotoreceptor基因NGN1中产生新的感光细胞。将使用病毒递送和转基因来实现RPE中的异位NGN1表达。然后，NGN1-RPE将受到条件，例如光感受器变性的体内环境，可能会释放实验RPE产生光感受器细胞的潜力。随后将分析分子，细胞和生理水平的从头产生感光细胞。除了检验我们的假设外，小鼠中“ RPE->感光器”重编程的演示还将为未来对RPE进行研究的科学证据，以作为原位细胞置换的方便光能来源，无需细胞移植。研究共同为使用RPE重新填充患有光感受器变性的视网膜至关重要的信息。 公共卫生相关性：开发感光受体替代疗法进展的关键障碍是缺乏可靠的新光感受器来源。该项目研究了RPE细胞的基因定向重编程，作为一种新的方法，用于产生新的光感受器，以供未来的细胞置换研究。