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Kinase Multitargeting for Glaucoma Neuroprotection

激酶多靶点治疗青光眼神经保护

基本信息

批准号：
10426103
负责人：
Derek Stuart Welsbie
金额：
$ 38.32万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10426103
关键词：
Ablation Affect Animals Axon Biological Assay Biology Blindness Breeding CRISPR screen CRISPR therapeutics Cell Death Cell Survival Cells Cessation of life Clustered Regularly Interspaced Short Palindromic Repeats Complement Cytoprotection DNA Polymerase II DNA Polymerase III Dependovirus Development Drug Targeting Electrophysiology (science)Electroretinography Enhancers Evaluation Gene Combinations Gene Targeting Genes Genetic Genetic Transcription Genome Glaucoma Glycogen Synthase Kinases Guide RNA Health Promotion Histological Techniques Knock-out Label Lead Leucine Zippers Mechanics Mediator of activation protein Methods Modeling Mus Muscle Cells N-terminal Nature Nerve Crush Neurites Neurodegenerative Disorders Neuroprotective Agents Ocular Hypertension Optic Nerve Optical Coherence Tomography Outcome Pathway interactions Patients Pattern Phosphorylation Phosphotransferases Physiologic Intraocular Pressure Play Proteins RNA Interference RNA interference screen Rattus Retina Retinal Ganglion Cells Rodent Model Role SOX11 gene Signal Transduction Specificity Streptococcus pyogenes Structure System Testing Therapeutic Venous Virus Visual Work adeno-associated viral vector axon injury axonal degeneration base design disability fluorescence imaging functional genomics glycogen synthase kinase 3 beta improved in vivo insight interest jun Oncogene knockout gene neuronal survival neuroprotection novel off-target site optic nerve disorder preservation pressure prevent promoter response restriction enzyme retinal ganglion cell degeneration screening synergism therapeutic gene vector

项目摘要

PROJECT SUMMARY – Glaucoma is a neurodegenerative disease in which there is specific loss of retinal ganglion cells (RGCs). Current therapies center around lowering intraocular pressure (IOP) although this can be challenging in some patients. In order to advance towards a neuroprotective strategy that could complement IOP-lowering, we have been identifying potential neuroprotective targets in primary RGCs using high- throughput functional genomic screening. The first iteration of this work, using RNA interference, identified dual leucine zipper kinase (DLK) and leucine zipper kinase (LZK) as key mediators of RGC cell death and validated the biology in rodent models of optic neuropathy, including glaucoma. Since then, we have completed a clustered regularly-interspaced short palindromic repeat (CRISPR)-based screen in order to identify genes whose knockout further potentiates the RGC protection conferred by DLK/LZK inhibition. The top new hit in this screen was glycogen synthase kinase three beta (GSK-3β). Highlighting the utility of our agnostic screening approach, multiple groups have previously found that while GSK-3β is indeed activated in RGCs after axonal injury, GSK- 3β loss alone does not increase RGC survival. We have shown however, in the setting of DLK/LZK pathway inhibition, GSK-3β loss does lead to a further increase in RGC survival. Moreover, we found an unexpected synergy in neurite degeneration with inhibition of DLK/LZK and GSK-3β leading to robust neurite protection. The central hypothesis of this proposal is that DLK/LZK and GSK-3β cooperate, potentially as a result of their ability to dually phosphorylate myocyte enhancer factor 2A (MEF2A), to cause somal and axonal degeneration and that simultaneous inhibition of DLK, LZK and GSK-3β is required for maximal neuroprotection. In order to test this hypothesis in vivo and to create a generalizable method for gene multitargeting in vivo, we have developed a novel adeno-associated virus (AAV)/CRISPR vector. This uses a novel insight about the compact H1 promoter which allows both guide RNA (gRNA) and S. pyogenes Cas9 (SpCas9) to be delivered in a single AAV virus, overcoming a major hurdle in the field of therapeutic gene editing. Specific aim 1 (SA1) will develop AAV/CRISPR vectors to multitarget DLK/LZK/GSK-3β, validate them in primary RGCs and then use the resulting cells to explore the role of MEF2A as a key convergence point of GSK-3β and DLK/LZK signaling. SA2 will use AAV/CRISPR vectors in vivo to test whether DLK/LZK/GSK-3β inhibition affects normal retinal structure/function and whether multitargeting leads to long-term preservation of electrophysiologically-active RGCs and decreased axon degeneration in the mouse optic nerve crush model. Finally, SA3 will use a more therapeutically-relevant design, in which the AAV/CRISPR virus delivers all of the CRISPR components, to test the hypothesis that kinase multitargeting in RGCs improves visual outcomes in a rat glaucoma model. Together, we anticipate this proposal will lead to a robust RGC neuroprotective strategy for combined axonal and somal preservation and the development of a novel AAV/CRISPR therapeutic.

青光眼是一种神经退行性疾病，伴有视网膜神经节的特异性丧失