GDNF Delivery to MPTP Monkeys by EIAV lentivirus and AAV

描述(申请人提供)：这项提案的目标是一种有效的帕金森氏病基因疗法，它将测试由马传染性贫血病毒(EIAV)或腺相关病毒(AAV)两种改进的载体系统运送的人胶质细胞系衍生神经营养因子(GDNF)的有效性和安全性。这两个载体都可以将细胞标记基因、核定位的lacZ(LacZn1)或GDNF高效稳定地输送到黑质纹状体靶区，可以通过四环素启动子系统进行调节，并提供了额外的安全性，即各自的野生型病毒不会对人类造成任何疾病。重组载体将在由神经毒素MPTP在猴子身上产生的帕金森病模型中进行测试。GDNF已经显示出利用rAAV和rHIV预防或逆转其他啮齿动物和灵长类动物帕金森病模型中的形态、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带有可调节启动子的改进和更安全载体的开发，这将在这个拟议的项目中进行测试。初步研究将针对正常非洲绿猴的转基因表达(lacZn1或GDNF)，确定有效滴度、转导效率、细胞趋向性、分布、水平和转基因表达的稳定性，以及rEIAV或rAAV交付后的神经病理学和宿主细胞反应。然后，这两个载体中的每一个都将被用来将GDNF输送到MPTP帕金森病猴子的黑质纹状体系统，以测试GDNF表达将在长达24个月的时间内改善中度和重度帕金森猴子功能的假设。通过比较注射部位、用于灭活基因表达的可调节启动子，以及包括高滴度注射在内的所有程序的安全性，将优化最有效的程序。疗效的测量将包括行为参数、使用ELISA法进行的转基因表达的分子分析、载体DNA的RT-PCRmRNA和PCR法、DA及其代谢物的生化分析、神经解剖学和形态计量学分析、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，以便尽早在帕金森氏症患者中启动成功的临床试验。

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.