GDNF Delivery to MPTP Monkeys by EIAV lentivirus and AAV

EIAV 慢病毒和 AAV 将 GDNF 传递给 MPTP 猴

• 批准号: 7059939
• 负责人: DONALD EUGENE REDMOND
• 金额: $ 104.58万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7059939
• 关键词: Cercopithecidae; Parkinson' s disease; adeno associated virus group; autoradiography; biotechnology; brain derived neurotrophic factor; enzyme linked immunosorbent assay; equine infectious anemia virus; gene therapy; genetic screening; genetic transcription; host organism interaction; lac operon; methylphenyltetrahydropyridine; neuropathology; polymerase chain reaction; single photon emission computed tomography; transfection /expression vector

DESCRIPTION (provided by applicant): An effective gene therapy for Parkinson's disease is the goal of this proposal, which will test the effectiveness and safety of human glial cell line derived neurotrophic factor (GDNF) delivered by two improved vector systems derived from equine infectious anemia virus (EIAV) or from adenoassociated virus (AAV). Both vectors deliver the cellular marker gene, nuclear localized lacZ (lacZnl) or GDNF efficiently and stably into nigrostriatal target regions, can be regulated using a tetracycline promoter system, and offer additional safety that the respective wild-type viruses do not cause any disease in humans. The recombinant vectors will be tested in the parkinsonian model produced by the neurotoxin MPTP in monkeys. GDNF has shown promise for preventing or reversing morphological, biochemical and functional deficits in other models of Parkinson's disease in rodents and primates, using rAAV, and rHIV. But these studies also showed important problems to be solved to ensure that a GDNF gene therapy will be safe and effective in patients. Concerns about inflammatory, cytotoxic, inadequate or excessive gene expression, persistence, viral recombination or replication have led to the development of improved and safer vectors with regulatable promoters, which will be tested in this proposed project. Initial studies will address transgene expression (lacZnl or GDNF) in normal African green monkeys, determining effective titers, transduction efficiency, cellular tropism, distribution, level, and stability of transgene expression, neuropathology and host cellular responses after delivery by rEIAV or rAAV. Each of the two vectors will then be used to deliver GDNF to the nigrostriatal system of MPTP parkinsonian monkeys to test hypotheses that GDNF expression will improve function in both moderate and severely parkinsonian monkeys for periods up to 24 months. The most effective procedures will be optimized by comparing injection sites, a regulatable promoter to inactivate gene expression, and safety of all procedures including high injection titers. Measures of efficacy will include behavioral parameters, molecular assays of transgene expression using ELISA for protein, RT-PCR for mRNA and PCR for vector DNA, biochemical assays of DA and its metabolites, neuroanatomical and morphometric analyses, neuropathology, clinical chemistry, SPECT imaging, and autoradiography. These studies aim to provide the necessary data to initiate successful clinical trials in Parkinson's patients at the earliest possible time.

描述（由申请人提供）：该提案的有效基因疗法是该提案的目标，该疗法将测试由两个改良的载体系统衍生出的人神经胶质细胞系的有效性和安全性，该疗法是由两种改进的载体系统衍生而来的，该载体系统源自马的感染性贫血病毒（EIAV）或adenosiped Virus（aav）。两个载体都可以使用四环素启动子系统来调节两个载体，将细胞标记基因，核局部LACZ（LACZNL）或GDNF有效且稳定地稳定地稳定地进入黑质纹状体靶区域，并提供相应的野生型病毒不会引起任何人类疾病的其他安全性。重组向量将在猴子中神经毒素MPTP产生的帕金森氏菌模型中进行测试。 GDNF显示了预防或逆转其他使用RAAV和RHIV的帕金森氏病中其他模型中帕金森氏病和灵长类动物中其他模型中的形态，生化和功能缺陷的希望。但是这些研究还显示了要解决的重要问题，以确保GDNF基因疗法对患者安全有效。人们对炎症，细胞毒性，不充分或过度基因表达，持久性，病毒重组或复制的关注导致与可调节启动子的改善和更安全的向量的发展，这将在该拟议项目中进行测试。最初的研究将解决非洲正常绿猴中的转基因表达（LACZNL或GDNF），确定有效的滴度，转导效率，细胞向上主义，分布，分布，水平和稳定性，通过REIAV或RAAV传递后，神经病理学和宿主细胞反应。然后，两个向量中的每一个都将用于将GDNF传递到MPTP Parkinsonian猴子的Nigrostriatal System，以测试GDNF表达的假设，即GDNF表达将在中度和严重的帕金森尼猴子中提高功能长达24个月。最有效的程序将通过比较注射位点，可调节的启动子与失活的基因表达以及包括高注射滴度在内的所有程序的安全性进行优化。疗效的度量将包括行为参数，使用ELISA用于蛋白质的转基因表达的分子测定，mRNA的RT-PCR和PCR用于载体DNA，DA及其代谢产物的生化测定，神经植物学和形态学分析，神经病理学，神经病理学，临床化学，临床化学，临床化学，图像Imageing，Specting Imagicagraphy。这些研究旨在提供必要的数据，以尽早在帕金森患者的患者中成功进行临床试验。