Generating photoreceptors by reprogramming RPE cells

通过重新编程 RPE 细胞生成光感受器

• 批准号: 8597424
• 负责人: SHU-ZHEN WANG
• 金额: $ 14.55万
• 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/8597424
• 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。 光感受器替代在治疗由以下原因引起的视觉障碍方面具有很大的希望： 光感受器变性与此同时，它提出了需要提供差异化的 光感受器，因为人类神经视网膜缺乏再生能力。为了解决这个 在发展光受体替代疗法的关键障碍，我们采取了相当 非传统的方法来产生分化的光感受器-重新编程RPE细胞 利用亲光感受器基因来引导RPE众所周知的增殖能力， 对感光细胞产生的可塑性。对鸡细胞的研究提出了令人兴奋的可能性， 从视网膜色素上皮细胞中通过基因导向的重编程获得新的感光细胞。有趣 目前，是时候验证哺乳动物RPE细胞可以重新编程的假设了， 产生感光细胞。假设的验证具有临床和社会意义 意义 为了验证这一假设，我们设计了两组互补的研究。第一集直接 检查培养的人RPE细胞在光刺激下产生光感受器细胞的能力。 前光感受器基因ngn 1的指导。人RPE细胞将被病毒转导， ngn 1启动感光细胞分化。然后对细胞培养物进行重新分析。 在基因表达水平，细胞形态， 和功能生理学，在体外和体内移植到眼睛后。的直接测试 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 研究新的感光细胞是否会从小鼠RPE异位产生 表达前光感受器基因ngn 1。将实现RPE中的异位ngn 1表达 使用病毒传递和转基因技术。ngn 1-RPE然后将经受条件，例如 光感受器退化的体内环境，这可能会释放实验性的 视网膜色素上皮细胞的潜力产生感光细胞。随后将进行从头分析 感光细胞在分子、细胞和生理水平上的生成。除了 为了验证我们的假设，小鼠中“RPE ->感光细胞”重编程的演示将 为将来研究RPE提供科学依据， 用于原位细胞替代而无需细胞移植的光受体。总之，这些研究 这些信息对于使用RPE来重建受感光细胞影响的视网膜至关重要 退化