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.

我们建议研究延长重组体的新方法 腺病毒介导的基因表达和重新管理的能力 恒河猴肝脏中的重组腺病毒。这些实验是 基于小鼠类似研究的结果以及初步 恒河猴的经历。免疫中观察到的种间变异 基因转移实验期间的反应表明非人类 在考虑人类之前，灵长类动物是一个必要的研究队列 临床试验。虽然腺病毒主干修饰 进一步限制晚期基因表达可能会被证明是富有成效的， 宿主免疫反应更有可能在短期内取得成果。 这笔赠款的目的是描述旨在探索的实验 短暂的、选择性的免疫抑制作为一种策略的可行性 提高重组腺病毒作为基因治疗载体的实用性 通过延长转基因表达，或通过预防 中和抗体将允许载体的重新施用。这 方法将包括恒河猴的短暂免疫抑制 一系列免疫抑制剂，所有这些都是选择性的 其在预防器官排斥方面的效用的特异性 移植，并在免疫发展的不同阶段发挥作用 回复。这些方案包括： 单药他克莫司 (FK506)；单身的 代理OKT4A；环磷酰胺和皮质类固醇的组合；组合 CyA、15-脱氧精胍菌素和抗胸腺细胞球蛋白 (ATGam)；和 环孢菌素 A、皮质类固醇和吗替麦考酚酯的组合。全部 所提议的方案，除了已被证明有用之外 灵长类移植，已应用于临床人体器官移植 移植，因此可以迅速转化为人类 临床试验如果证明有效。每个治疗方案均经过选择 不同的作用机制选择性地改变宿主的各个方面 免疫系统。我们将对免疫进行广泛的表征 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 表达和重新施用重组腺病毒的能力。 最后，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， 淋巴细胞耗竭，以及施用的限菌环境 拟议的侏儒生物学核心的帮助 - 将需要优化 猴子的安全。