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
项目总结
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.
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会议论文
Molecular -Scale Mapping of Rhodopsin Trafficking in Mammalian Rod Photoreceptors
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批准号:10593138
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项目类别:
-
资助金额:$29.05万
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财政年份:2022
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负责人:Michael Robichaux
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依托单位:
Molecular -Scale Mapping of Rhodopsin Trafficking in Mammalian Rod Photoreceptors
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批准号:10334877
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项目类别:
-
资助金额:$29.49万
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财政年份:2022
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负责人:Michael Robichaux
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依托单位:
海外基金