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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

基本信息

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
项目类别：
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

项目摘要

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.

项目总结