Generating photoreceptors by reprogramming RPE cells

描述（由申请人提供）：本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希望。与此同时，由于人类神经视网膜缺乏再生能力，因此需要提供分化的光感受器。为了解决开发光感受器替代疗法中的这一关键障碍，我们采取了一种相当非常规的方法来产生分化的光感受器-用前光感受器基因重编程RPE细胞，以引导RPE众所周知的增殖和可塑性能力朝向光感受器产生。鸡细胞的研究提出了通过基因定向重编程从RPE衍生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细胞可以重新编程以产生感光细胞的假设了。该假说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的研究。第一组直接检查培养的人RPE细胞在前感光基因ngn 1的指导下产生感光细胞的能力。人RPE细胞将用ngn 1病毒转导以启动感光细胞分化。然后在移植到眼睛中后，在体外和体内分析细胞培养物在基因表达、细胞形态和功能生理学水平上的光感受器样神经元的从头产生。用人类细胞进行的直接测试与潜在疗法的开发具有高度相关性。第二组研究是否会产生新的感光细胞从小鼠视网膜色素上皮异位表达前感光基因ngn 1。RPE中的异位ngn 1表达将使用病毒递送和转基因实现。ngn 1-RPE然后将经受条件，例如体内光感受器变性的环境，其可以释放实验RPE产生光感受器细胞的潜力。随后将在分子、细胞和生理水平上分析感光细胞的从头生成。除了测试我们的假设之外，在小鼠中“RPE ->光感受器”重编程的证明将为将来研究RPE作为光感受器的方便来源用于原位细胞替代而无需细胞移植提供科学证据。总之，这些研究承诺的信息至关重要的使用视网膜色素上皮细胞重新填充视网膜感光细胞变性折磨。 公共卫生相关性：发展光感受器替代疗法的关键障碍是缺乏新光感受器的可靠来源。该项目研究了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.