Super resolution analysis of the subcellular effects of retinal gene therapy

视网膜基因治疗的亚细胞效应的超分辨率分析

• 批准号: 9190775
• 负责人: Michael Robichaux
• 金额: $ 5.8万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-18 至 2019-08-17
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9190775
• 关键词: Address; Affect; Antibodies; Apoptotic; Bardet-Biedl Syndrome; Biological Process; Blindness; Bulla; Caliber; Cell Death; Cell Survival; Cells; Cellular Morphology; Cilia; Complete Blindness; Complex; Data; Defect; Disease; Etiology; Fellowship; Functional disorder; Genes; Goals; Health; Hereditary Disease; Human; Human Genetics; Image; Imaging Techniques; Length; Life; Light; Long-Term Effects; Mediating; Membrane; Microscopy; Modeling; Molecular; Mus; Mutant Strains Mice; Mutate; Mutation; Outcome; Patients; Pattern; Photoreceptors; Proteins; Recovery of Function; Research; Resolution; Retina; Retinal; Retinal Degeneration; Series; Site; Staging; Structure; Techniques; Therapeutic; Therapeutic Effect; Time; Validation; Vertebrate Photoreceptors; Vesicle; Visual; Visual impairment; Wild Type Mouse; adeno-associated viral vector; base; ciliopathy; early childhood; electron tomography; gene product; gene replacement; gene replacement therapy; gene therapy; insight; mutant; mutant mouse model; novel; protein complex; protein transport; research study; retinal rods; subretinal injection; trafficking; treatment planning

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.

项目概要 Bardet-Biedl 综合征 (BBS) 是一种人类纤毛病，其特征是初级纤毛功能障碍 以及通过受影响视网膜中的感光细胞降解而导致的视网膜变性。 BBS基因相关 与纤毛病相关的蛋白均与初级纤毛有关，8个BBS蛋白形成BBSome蛋白 复杂的。对 BBS 突变小鼠初级纤毛动力学的有限分析表明 BBSome 至关重要 用于运输初级纤毛货物蛋白，但除了这些初步发现之外，我们对 BBSome 知之甚少 初级纤毛的功能，包括视网膜中视杆光感受器的初级纤毛。值得注意的是，视网膜基因 通过 AAV 视网膜下注射进行替代在 BBS 突变体的初步研究中取得了有希望的结果 鼠标模型。在这个提案中，我概述了一系列利用 STORM 和 PALM 超分辨率的实验 成像与冷冻电子断层扫描（cryo-ET）超微结构分析相结合，以解决（1）精确 BBSome 在视杆光感受器纤毛中的定位和 (2) 视网膜基因的亚细胞结果 视杆细胞的替代疗法。 (1) STORM超分辨率定位（分辨率为 ~20nm) 将用于定位野生型视杆细胞中的 BBSome 复合物以及 BBS4- /- 和 BBS1M390R 突变小鼠，通过使用特定抗体进行的一系列 STORM 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 随后视网膜下注射到 BBS4-/- 和 BBS1M390R 突变小鼠视网膜中。治疗的定位 除 通过冷冻胚胎学进行超微结构形态分析。随着这些新的、强大的成像技术的应用 技术，我将首次评估视网膜基因治疗的亚细胞效应，并准确跟踪 BBSome 相对于杆状纤毛标记的定位以及相关的形态缺陷 与这些 BBS 突变模型。这些研究目标的结果将共同帮助确定 野生型 BBSome 的功能，这对于杆状细胞的活力至关重要，也将证明其可行性 将 BBS 基因替代疗法引入突变感光细胞作为 BBS 和其他疾病的可能治疗方法 视网膜退行性缺陷。