Super resolution analysis of the subcellular effects of retinal gene therapy

项目摘要 Bardet-Biedl综合征（BBS）是一种以初级纤毛功能障碍为特征的人类纤毛病 以及通过受影响的视网膜中的感光细胞降解的视网膜变性。BBS相关基因 与纤毛病变相关的8个BBS蛋白均与初级纤毛相关，8个BBS蛋白组成一个BBSome蛋白 复杂. 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， 运输初级纤毛货物蛋白，但除了这些初步的发现，很少有人知道BBSome 在初级纤毛中起作用，包括视网膜中视杆光感受器的初级纤毛。值得注意的是， 通过AAV视网膜下注射的替代在BBS突变体的初步研究中产生了有希望的结果。 moue模型。在这个建议中，我概述了一系列的实验，利用STORM和PALM超分辨率 成像沿着冷冻电子断层扫描（cryo-ET）超微结构分析，以解决（1）精确的 BBSome在视杆细胞纤毛中的定位和（2）视网膜基因的亚细胞产物 替代疗法进入视杆细胞。(1)STORM超分辨率定位（分辨率为 ~ 20 nm）将用于定位野生型视杆细胞中的BBSome复合物，以及BBS 4- /-和BBS 1 M390 R突变小鼠，通过用特异性抗体的一系列STORM免疫染色实验。（二） 将与PALM蛋白标签融合的BBS 1和BBS 4克隆到优化的AAV载体中进行验证， 随后视网膜下注射到BBS 4-/-和BBS 1 M390 R突变小鼠视网膜中。治疗部位 视杆细胞将用STORM和PALM成像的组合进行评估，除了 冷冻电镜超微结构形态学分析。随着这些新的强大的成像技术的应用 技术，我将评估，第一次，视网膜基因治疗的亚细胞效应，并准确地跟踪 BBSome相对于杆状纤毛标记物的定位和相关的形态学缺陷 BBS突变体模型总之，这些研究目标的结果将有助于确定 野生型BBSome的功能，这是至关重要的视杆细胞活力，也将证明的可行性 对于BBS基因替代疗法进入突变的感光细胞作为BBS和其他疾病的可能治疗， 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.