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: 6888933
• 负责人: DONALD EUGENE REDMOND
• 金额: $ 103.98万
• 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/6888933
• 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（申请人提供）：本提案的目标是针对帕金森病进行有效的基因治疗，该提案将测试由两种改进的载体系统递送的人胶质细胞系源性神经营养因子（GDNF）的有效性和安全性，所述载体系统源自马传染性贫血病毒（EIAV）或腺相关病毒（腺相关病毒）。两种载体都将细胞标志物基因、核定位的lacZ（lacZnl）或GDNF有效且稳定地递送到黑质纹状体靶区域中，可以使用四环素启动子系统进行调节，并且提供额外的安全性，即相应的野生型病毒不会在人类中引起任何疾病。重组载体将在猴神经毒素MPTP产生的帕金森病模型中进行测试。GDNF已经显示出使用rAAV和rHIV预防或逆转啮齿动物和灵长类动物中帕金森病的其他模型中的形态、生化和功能缺陷的前景。但这些研究也显示了需要解决的重要问题，以确保GDNF基因治疗对患者安全有效。对炎症、细胞毒性、基因表达不足或过度、持久性、病毒重组或复制的担忧导致了具有可调控启动子的改进和更安全的载体的开发，这些载体将在本拟议项目中进行测试。初始研究将解决正常非洲绿色猴中的转基因表达（lacZnl或GDNF），确定转基因表达的有效滴度、转导效率、细胞向性、分布、水平和稳定性、神经病理学和通过rEIAV或rAAV递送后的宿主细胞应答。两种载体中的每一种都将用于将GDNF递送到MPTP帕金森病猴的黑质纹状体系统，以测试GDNF表达将改善中度和重度帕金森病猴的功能长达24个月的假设。将通过比较注射部位、可调控启动子对p53基因表达的影响以及所有程序（包括高注射滴度）的安全性来优化最有效的程序。疗效指标将包括行为参数、使用ELISA检测蛋白质、RT-PCR检测mRNA和PCR检测载体DNA的转基因表达的分子测定、DA及其代谢物的生化测定、神经解剖学和形态测定分析、神经病理学、临床化学、SPECT成像和放射自显影。这些研究旨在提供必要的数据，以便尽早在帕金森病患者中开展成功的临床试验。