Generating photoreceptors by reprogramming RPE cells

描述(申请人提供)：该项目的长期目标是生产用于细胞替换的新的光感受器。光感受器替代技术在治疗由光感受器变性引起的视力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，因此需要提供差异化的光感受器。为了解决光感受器替代疗法开发中的这一关键障碍，我们采取了一种相当非传统的方法来产生分化的光感受器-用前光感受器基因重新编程RPE细胞，以引导RPE众所周知的增殖和可塑性能力产生光感受器。对小鸡细胞的研究提出了通过基因指导的重新编程从RPE获得新的光感受器的令人兴奋的可能性。有趣的是，现在是时候测试哺乳动物RPE细胞可以重新编程以产生光感受器细胞的假设了。这一假说的验证具有临床和社会意义。为了检验这一假设，我们设计了两组互补性研究。第一组直接检测培养的人类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RPE细胞将通过病毒转导Ngn1来启动光感受器分化。然后，细胞培养将在基因表达、细胞形态和功能生理学水平上分析光感受器样神经元的从头产生，在体外和体内移植到眼睛中。对人类细胞的直接测试与潜在疗法的开发具有很高的相关性。第二组研究是否会从异位表达前光受体基因Ngn1的小鼠RPE中产生新的光感受器细胞。异位Ngn1在RPE中的表达将通过病毒传递和转基因实现。然后，Ngn1-RPE将受到一些条件的影响，例如体内光感受器退化的环境，这可能会释放实验RPE产生光感受器细胞的潜力。随后将在分子、细胞和生理水平上对光感受器细胞的从头产生进行分析。除了验证我们的假设外，在小鼠身上进行的“RPE-&>光感受器”重新编程的演示将为未来的研究提供科学证据，RPE是一种方便的光感受器来源，用于在不进行细胞移植的情况下进行原位细胞替代。总而言之，这些研究提供了使用RPE重新填充患有光感受器退化的视网膜至关重要的信息。 公共卫生相关性：发展光感受器替代疗法的一个关键障碍是缺乏可靠的新光感受器来源。这个项目研究了RPE细胞的基因导向重编程，作为一种新的方法来产生新的光感受器，用于未来的细胞替代研究。

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.