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用于细胞替代的新光感受器。 光感受器替代疗法在治疗由以下原因引起的视力障碍方面具有巨大前景 光感受器变性。同时，它也提出了对提供差异化​​产品的需求。 光感受器，因为人类的神经视网膜缺乏再生能力。为了解决这个问题 开发光感受器替代疗法的关键障碍，我们采取了相当 产生分化光感受器的非常规方法 - 重编程 RPE 细胞 具有前光感受器基因，可引导 RPE 众所周知的增殖和 光感受器生产的可塑性。鸡细胞研究提出了令人兴奋的可能性 通过基因定向重编程从 RPE 衍生出新的光感受器。有趣的 就目前而言，是时候检验哺乳动物 RPE 细胞可以被重新编程为 产生感光细胞。该假设的验证具有临床和社会意义 意义。 为了检验这个假设，我们设计了两组互补的研究。直接第一套 检查培养的人类 RPE 细胞在以下条件下产生感光细胞的能力 前光感受器基因 ngn1 的指导。人类 RPE 细胞将被病毒转导 ngn1 启动光感受器分化。然后对细胞培养物进行从头分析 在基因表达水平、细胞形态、 以及移植到眼睛后的体外和体内功能生理学。直接测试 与人类细胞的结合与潜在疗法的开发具有高度相关性。第二组 研究小鼠 RPE 是否异位产生新的感光细胞 表达前光感受器基因ngn1。 RPE 中的异位 ngn1 表达将实现 使用病毒传递和转基因技术。然后ngn1-RPE将受到以下条件的影响： 光感受器变性的体内环境，可能会释放实验 RPE 具有产生感光细胞的潜力。随后将进行从头分析 在分子、细胞和生理水平上产生感光细胞。此外 验证我们的假设，在小鼠中演示“RPE -> 光感受器”重编程将 为未来研究 RPE 作为便捷来源提供科学证据 用于原位细胞替换的光感受器，无需细胞移植。一起，研究 提供对于使用 RPE 重新填充受光感受器影响的视网膜至关重要的信息 退化。