Super resolution analysis of the subcellular effects of retinal gene therapy
Super resolution analysis of the subcellular effects of retinal gene therapy
批准号:
9190775
负责人:
Michael Robichaux
金额:
$5.8万
依托单位国家:
美国
项目类别:
财政年份:
2016
资助国家:
美国
项目状态:
已结题
起止时间:
2016-08-18 至 2019-08-17
关键词:
AddressAffectAntibodiesApoptoticBardet-Biedl SyndromeBiological ProcessBlindnessBullaCaliberCell DeathCell SurvivalCellsCellular MorphologyCiliaComplete BlindnessComplexDataDefectDiseaseEtiologyFellowshipFunctional disorderGenesGoalsHealthHereditary DiseaseHumanHuman GeneticsImageImaging TechniquesLengthLifeLightLong-Term EffectsMediatingMembraneMicroscopyModelingMolecularMusMutant Strains MiceMutateMutationOutcomePatientsPatternPhotoreceptorsProteinsRecovery of FunctionResearchResolutionRetinaRetinalRetinal DegenerationSeriesSiteStagingStructureTechniquesTherapeuticTherapeutic EffectTimeValidationVertebrate PhotoreceptorsVesicleVisualVisual impairmentWild Type Mouseadeno-associated viral vectorbaseciliopathyearly childhoodelectron tomographygene productgene replacementgene replacement therapygene therapyinsightmutantmutant mouse modelnovelprotein complexprotein transportresearch studyretinal rodssubretinal injectiontraffickingtreatment planning
中文摘要
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。
英文摘要
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.
期刊论文(0)
专著(0)
科研奖励(0)
会议论文
Molecular -Scale Mapping of Rhodopsin Trafficking in Mammalian Rod Photoreceptors
-
批准号:10593138
-
项目类别:
-
资助金额:$29.05万
-
财政年份:2022
-
负责人:Michael Robichaux
-
依托单位:
Molecular -Scale Mapping of Rhodopsin Trafficking in Mammalian Rod Photoreceptors
-
批准号:10334877
-
项目类别:
-
资助金额:$29.49万
-
财政年份:2022
-
负责人:Michael Robichaux
-
依托单位:
海外基金