Generating photoreceptors by reprogramming RPE cells

描述（由申请人提供）：该项目的长期目标是生产用于细胞替代的新型光感受器。光感受器替代在治疗由光感受器变性引起的视觉障碍方面具有很大的前景。与此同时，由于人类神经视网膜缺乏再生能力，它提出了对分化光感受器供应的需求。为了解决开发光感受器替代疗法中的这一关键障碍，我们采用了一种相当非常规的方法来产生分化的光感受器——用前光感受器基因对RPE细胞进行重编程，以引导RPE众所周知的增殖和可塑性能力来产生光感受器。对小鸡细胞的研究提出了通过基因定向重编程从RPE中获得新的光感受器的令人兴奋的可能性。有趣的是，现在是时候验证哺乳动物RPE细胞可以被重新编程以产生光感受器细胞的假设了。该假设的验证具有临床和社会意义。为了验证这一假设，我们设计了两组互补研究。第一组直接检测培养的人RPE细胞在前光感受器基因ngn1的指导下产生光感受器细胞的能力。人类RPE细胞将通过病毒转染ngn1来启动光感受器分化。细胞培养将在基因表达、细胞形态和功能生理学水平上进行分析，在体外和体内移植到眼睛后重新产生光感受器样神经元。人类细胞的直接试验与潜在疗法的发展高度相关。第二组研究小鼠RPE是否会产生新的光感受器细胞，表达前光感受器基因ngn1。ngn1在RPE中的异位表达将通过病毒传递和转基因技术实现。然后，ngn1-RPE将受到诸如光感受器退化的体内环境等条件的影响，这些条件可能会释放实验RPE产生光感受器细胞的潜力。接下来将分析光感受器细胞在分子、细胞和生理水平上的新生生成。除了验证我们的假设外，“RPE ->光受体”在小鼠中的重编程演示将为未来研究RPE作为不需要细胞移植的原位细胞替代的光受体的便利来源提供科学证据。总之，这些研究为使用RPE重新填充受光感受器退化困扰的视网膜提供了至关重要的信息。

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.