Super resolution analysis of the subcellular effects of retinal gene therapy

项目摘要 Bardet-Biedl综合征(BBS)是一种以原发纤毛功能障碍为特征的人类纤毛疾病 以及受影响视网膜中的光感受器细胞降解导致的视网膜退化。与BBS相关的基因 与纤毛病变有关的都是初生纤毛，8个BBS蛋白形成一个BBSome蛋白 很复杂。对BBS突变小鼠初级纤毛动力学的有限分析表明，BBSome是必不可少的 对于初级纤毛货运蛋白的运输，但除了这些初步发现外，对BBSome知之甚少 初级纤毛的功能，包括视网膜中杆状光感受器的初级纤毛。值得注意的是，视网膜基因 AAV视网膜下注射置换在BBS突变体的初步研究中取得了令人满意的结果 老鼠模特。在这个提案中，我概述了利用Storm和Palm的超分辨率进行的一系列实验 成像与冷冻电子断层扫描(CRYO-ET)超结构分析一起解决(1)精确 BBSome在视杆状光感受器纤毛中的定位及(2)视网膜基因的亚细胞结局 视杆感光细胞的替代疗法。(1)风暴超分辨率局部化(分辨率为 ~20 nm)将用于定位野生型杆状感光细胞中的BBSome复合体以及BBS4- /-和BBS1M390R突变小鼠，通过一系列特定抗体的风暴免疫染色实验。(2) 与Palm蛋白标签融合的BBS1和BBS4将被克隆到优化的AAV载体中进行验证和 随后向BBS4-/-和BBS1M390R突变小鼠视网膜下注射。治疗的本地化 Rod感光细胞将用Storm和Palm成像相结合的方法进行评估，除了 冷冻电子显微镜超微结构形态分析。随着这些新的和强大的成像技术的应用 技术，我将第一次评估视网膜基因治疗的亚细胞效应，并准确跟踪 纤毛杆状标志BBSome的定位及其相关的形态缺陷 有了这些论坛突变模型。总而言之，这些研究目标的结果将有助于确定 野生型BBSome的功能，这对杆状细胞的活性至关重要，并将证明其可行性 对突变光感受器细胞进行BBS基因替代治疗，作为治疗BBS和其他疾病的可能方法 视网膜退行性缺陷。

Project Summary Bardet-Biedl syndrome (BBS) is a human ciliopathy characterized by the dysfunction of primary cilia and retinal degeneration via photoreceptor cell degradation in the affected retina. The BBS genes associated with the ciliopathy are all associated with the primary cilia, and 8 BBS proteins form a BBSome protein complex. Limited analysis of primary cilia dynamics in BBS mutant mice suggests that the BBSome is essential for transport of primary cilia cargo proteins, but beyond these preliminary findings, little is known of BBSome function in the primary cilia, including the primary cilia of rod photoreceptors in the retina. Notably, retinal gene replacement via AAV subretinal injection has yielded promising results in preliminary studies in BBS mutant moue models. In this proposal, I outline a series of experiments that utilize STORM and PALM super resolution imaging along with cryo-electron tomography (cryo-ET) ultra-structural analysis to address (1) the precise localization of the BBSome in rod photoreceptor cilia and (2) the subcellular outcome of retinal gene replacement therapy into rod photoreceptor cells. (1) STORM super resolution localization (to a resolution of ~20nm) will be used to localize the BBSome complex in rod photoreceptor cells of wild-type, as well as BBS4- /- and BBS1M390R mutant mice, via a series STORM immunostaining experiments with specific antibodies. (2) BBS1 and BBS4 fused to PALM protein tags will be cloned into an optimized AAV vector for validation and subsequent subretinal injection into the BBS4-/- and BBS1M390R mutant mouse retinas. Localization of treated rod photoreceptors cells will be assessed with a combination of STORM and PALM imaging, in addition to ultra-structural morphological analysis via cryo-Et. With the application of these new and powerful imaging techniques, I will assess, for the first time, the subcellular effects of retinal gene therapy, and accurately track the localization of the BBSome relative to markers of the rod cilium and the morphological defects associated with these BBS mutant models. Together, the results from these research aims will aid in determining the function of the wild-type BBSome, which is critical for rod cell viability, and will also demonstrate the feasibility for BBS gene replacement therapy into mutant photoreceptor cells as a possible treatment for BBS and other retinal degenerative defects.