Generating photoreceptors by reprogramming RPE cells

该项目的长期目标是生产用于细胞替代的新的光感受器。 光感受器替代疗法在治疗由青光眼引起的视力障碍方面前景广阔 光感受器退化。与此同时，它提出了提供差异化供应的必要性 光感受器，因为人类的神经视网膜缺乏再生能力。要解决这个问题 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--重新编程RPE细胞 通过一个前光感受器基因来引导RPE众所周知的增殖和 光感受器产生的可塑性。对小鸡细胞的研究提出了令人兴奋的可能性 通过基因导向的重新编程从RPE获得新的光感受器。有意思的 目前，是时候测试哺乳动物RPE细胞可以重新编程以 产生感光细胞。这一假说的验证具有临床和社会意义 意义。 为了检验这一假设，我们设计了两组互补性研究。第一套直接 检测培养的人视网膜色素上皮细胞产生感光细胞的能力。 前光感受器基因Ngn1的引导。人RPE细胞将通过病毒转导 Ngn1启动光感受器分化。然后对细胞培养物进行从头分析。 光感受器样神经元在基因表达、细胞形态、 和功能生理学，体外和体内移植到眼睛内后。直接测试 与人类细胞有很高的相关性，具有潜在的治疗发展前景。第二盘 研究小鼠RPE是否会异位产生新的光感受器细胞 表达光感受器前基因Ngn1。将实现Ngn1在RPE中的异位表达 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 光感受器退化的体内环境，这可能会释放出实验 RPE产生感光细胞的潜力。在此之后，将对De nevo进行分析 在分子、细胞和生理水平上产生感光细胞。除了……之外 为了验证我们的假设，在小鼠身上演示了“RPE-&>光感受器”重新编程 为未来研究RPE作为方便的来源提供科学证据 无需细胞移植的原位细胞替代的光感受器。总之，这些研究 承诺信息对于使用RPE重新填充受光感受器困扰的视网膜至关重要 退化。

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.