Generating photoreceptors by reprogramming RPE cells

通过重新编程 RPE 细胞生成光感受器

• 批准号: 8403031
• 负责人: SHU-ZHEN WANG
• 金额: $ 34.79万
• 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/8403031
• 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

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.

该项目的长期目标是生产新的光感受器以替代细胞。 感光器更换在治疗由 感光体变性。同时，它提出了供应分歧的需求 光感受器，因为人类神经视网膜缺乏再生能力。解决这个问题 开发感光受体替代疗法的关键障碍，我们采取了相当的 产生分化感光体的非常规方法 - 重新编程RPE细胞 具有促肌感受器的基因，以引导RPE众所周知的增殖能力和 对感光受体产生的可塑性。雏鸡细胞的研究提高了令人兴奋的可能性 通过基因定向重编程从RPE中得出新的感光体。有趣的 就目前而言，是时候测试可以将哺乳动物RPE细胞重新编程为 产生感光细胞。验证假设带有临床和社会 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，以产生感光细胞。 促肌感受器基因NGN1的引导。人类RPE细胞将通过 NGN1启动感光细胞分化。然后将分析细胞培养 在基因表达水平，细胞形态，细胞形态，细胞形态的水平上产生类似感受器的神经元 和功能生理，在移植到眼睛后体外和体内。直接测试 人类细胞与潜在治疗的发展具有很高的相关性。第二组 研究是否会从小鼠RPE中生成新的感光细胞 表达pro-photoreceptor基因NGN1。将实现RPE中的异位NGN1表达 使用病毒递送和转基因。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产生感光细胞的潜力。随后将进行从头开始的分析 在分子，细胞和生理水平上生成感光细胞。此外 测试我们的假设，在小鼠中的“ RPE->光感受器”重编程的演示将会 提供科学证据，以将来对RPE进行调查，作为方便的来源 未经细胞移植的原位细胞置换的光感受器。一起研究 保证信息对于使用RPE重新填充患有光感受器的视网膜至关重要 退化。