APPROACHES TO LON TERM GENE THERAPY BY TRANSIENT IMMUNOSUPPRESSION IN MACACA

通过短暂免疫抑制对猕猴进行长期基因治疗的方法

• 批准号: 6576605
• 负责人: STEVE E RAPER
• 金额: $ 28.24万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-15 至 2003-05-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6576605
• 关键词: Adenoviridae; CD4 molecule; FK506; Macaca mulatta; cell mediated lymphocytolysis test; cellular immunity; corticosteroids; cyclophosphamide; cyclosporines; disease /disorder model; flow cytometry; gene expression; gene therapy; genetic markers; histology; humoral immunity; immunosuppression; model design /development; mycophenolic acid; neutralizing antibody; transfection; transfection /expression vector; western blottings

We propose to study novel approaches to the prolongation of recombinant adenovirus-mediated gene expression and the ability to readminister recombinant adenoviruses in rhesus monkey liver. These experiments are based on results in analogous studies in the mouse, as well as preliminary experiences in the rhesus. Interspecies variability seen in immune responses during gene transfer experiments suggests that non-human primates are a necessary study cohort prior to consideration of human clinical trials. Although modification of the adenoviral backbone to further limit late gene expression may prove fruitful, modulation of the host immune response is more likely to prove fruitful in the short term. The purpose of this grant is to describe experiments designed to explore the feasibility of transient, selective immunosuppression as a strategy to improve the usefulness of recombinant adenoviruses as gene therapy vectors by either prolonging transgene expression, or by prevention of neutralizing antibody which will allow readministration of the vector. The approaches will include transient immunosuppression of rhesus macaques with a series of immunosuppressive agents all of which have been selective specificity for their utility in preventing rejection in organ transplants, and to act at different points in the developing immune response. These regimens include: Single agent tacrolimus (FK506); Single agent OKT4A; Combination cyclophosphamide and corticosteroids; Combination CyA, 15-deoxyspergualin, and anti-thymocyte globulin (ATGam); and Combination cyclosporin A, corticosteroid, and mycophenolate mofetil. All of the proposed regimens, in addition to having been proved useful in primate transplantation, have been utilized clinically for human organ transplantation, and as such could be rapidly translated into human clinical trials if proven effective. Each regimen has been chosen for a different mechanism of action to selectively alter aspects of the host immune system. We will perform extensive characterization of the immune host response to recombinant adenoviruses including cytotoxic T lymphocyte assays, proliferation assays, cytokine release, neutralizing antibody assays, and Western blots. We will also study the duration of transgene expression and the ability to readminister recombinant adenoviruses. Lastly, toxicity will be analyzed by routine H and E histology. Preliminary studies with high-dose cyclophosphamide suggest profound lymphocyte depletion, and that a gnotobiotic environment-administered with the help of the proposed gnotobiology core- will be required to optimize safety for the monkeys.

我们建议研究重组延长的新方法 腺病毒介导的基因表达和回教徒的能力 恒河猴的重组腺病毒。这些实验是 基于小鼠类似研究的结果以及初步 恒河所的经历。免疫中看到的种间变异性 基因转移实验期间的反应表明非人类 灵长类动物是在考虑人类之前的必要研究队列 临床试验。虽然修饰腺病毒主链 进一步限制晚期基因表达可能会证明富有成果，调节 宿主免疫反应在短期内更有可能证明富有成效。 这笔赠款的目的是描述旨在探索的实验 瞬态，选择性免疫抑制的可行性作为一种策略 提高重组腺病毒作为基因疗法载体的实用性 通过延长转基因表达或预防 中和抗体将允许对载体进行再入疗。这 方法将包括恒河猕猴的短暂免疫抑制 与一系列免疫抑制剂有关，所有这些都有选择性 他们在防止器官拒绝的效用方面的特异性 移植，并在发育中的免疫中的不同点起作用 回复。这些方案包括：单身克莫司（FK506）；单身的 代理Okt4a;联合环磷酰胺和皮质类固醇；组合 CYA，15-脱氧杂质蛋白和抗心理细胞球蛋白（ATGAM）；和 组合蛋白A，皮质类固醇和霉酚酸莫菲蒂组合。全部 在拟议的方案中，除了被证明有用 灵长类动物移植已在临床上用于人体器官 移植，因此可以迅速转化为人类 临床试验如果被证明有效。每个方案都被选为 选择性改变主机方面的不同作用机制 免疫系统。我们将对免疫进行广泛的特征 宿主对重组腺病毒（包括细胞毒性T淋巴细胞）的反应 测定，增殖测定，细胞因子释放，中和抗体 测定和蛋白质印迹。我们还将研究转基因的持续时间 表达和重组腺病毒的能力。 最后，将通过常规H和E组织学分析毒性。 高剂量环磷酰胺的初步研究表明 淋巴细胞的耗竭，并与gnotobiotic环境相关 拟议的gnotobiology核心的帮助将需要优化 猴子的安全。