Using light-sensitive channels to restore vision for blinding retinal diseases

描述（由申请人提供）：视网膜退行性疾病中感光细胞的严重丧失常常导致完全失明。目前，还没有治疗或治愈盲人的方法。目前考虑的视杆和视锥变性后视力恢复的前瞻性策略包括正常感光细胞或祖细胞的移植和通过视网膜植入物对存活的视网膜神经元进行直接电刺激。我们正在探索另一种策略来恢复退化视网膜的光敏性：在存活的二级或三级视网膜神经元中表达光敏膜通道。这种方法是合理的。首先，直接光控的微生物型视紫红质，通道视紫红质，最近已经被克隆。我们的初步研究结果表明，在正常和视网膜营养不良动物的视网膜神经元中，通道视紫红素-2 （ChR2）具有功能性表达能力，并且能够通过光使表达ChR2的视网膜神经元的膜电位去极化。其次，对失明患者和动物模型的研究表明，第二和第三级视网膜神经元部分保留在病变视网膜中。此外，外源基因有可能通过基于病毒的基因治疗引入视网膜神经元。这种方法的一个潜在优点是它不涉及将组织或装置引入视网膜，因此可以避免免疫反应和生物相容性并发症。本提案的目的是通过使用啮齿动物模型来解决对该策略的可行性和成功至关重要的问题。本课题的具体目的是：1)在体内研究ChR2在视网膜神经元中表达的寿命和生物相容性，探索ChR2在视网膜内神经元某些功能亚群中的靶向表达；2)表征表达ChR2的视网膜内神经元的光诱发电流和电压响应；3)观察光感受器变性后存活的视网膜内神经元的生理特性；4)表征ChR2表达后退行性视网膜的光响应特性。这些拟议的研究可以为进一步的工作奠定必要的基础，从而导致致盲疾病的潜在治疗以及适用于视网膜研究的功率技术。

DESCRIPTION (provided by applicant): The severe loss of photoreceptor cells in retinal degenerative diseases often leads to total blindness. At present, there is no available treatment or cure to the blind. Prospective strategies currently being considered for restoration of vision following rod and cone degeneration include transplantation of normal photoreceptor or progenitor cells and direct electrical stimulation of the surviving retinal neurons via retinal implants. We are exploring another strategy to restore light sensitivity to the degenerate retinas: the expression of light-sensitive membrane channels in surviving second- or third-order retinal neurons. This approach is plausible. First, directly light-gated microbial-type rhodopsins, channelrhodopsins, have been recently cloned. Our preliminary results have shown the ability of functional expression of channerhodopsin-2 (ChR2) in retinal neurons in normal and retinal dystrophic animals in vivo and the capability to depolarize the membrane potential of ChR2-expressing retinal neurons by light. Second, studies in blind patients and in animal models suggest that the second- and third-order retinal neurons remain, in part, preserved in the diseased retinas. Furthermore, it is possible that foreign genes can be introduced into retinal neurons by viral-based gene therapy. A potential advantage of this approach is that it does not involve the introduction of tissues or devices into the retina and, thus, may avoid the immune reactions and biocompatibility complications. The objective of this proposal is to address questions that are fundamental to the feasibility and success of this strategy by using rodent models. The specific aims of this proposal are: 1) To examine the longevity and biocompatibility of the expression of ChR2 in retinal neurons in vivo and explore the targeting expression of ChR2 in certain functional sub-populations of inner retinal neurons; 2) To characterize the light-evoked current and voltage responses of ChR2 expressing inner retinal neurons; 3) To examine the physiological properties of the surviving inner retinal neurons after photoreceptor degeneration; 4) To characterize the light response properties in the degenerate retinas following the expression of ChR2. These proposed studies could establish the necessary foundation for further work leading to a potential treatment for blinding disorders as well as power techniques with applicability in retinal research.