IMPROVED METHODS OF ADENOVIRUS MEDIATED GENE TRANSFER

腺病毒介导的基因转移的改进方法

• 批准号: 2713442
• 负责人: RANDY C EISENSMITH
• 金额: $ 24.79万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-06-01 至 2000-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2713442
• 关键词: Adenoviridae; biotechnology; cell line; disease /disorder model; gene therapy; histopathology; host organism interaction; immunocytochemistry; immunomodulators; laboratory mouse; method development; phenylalanine 4 monooxygenase; phenylketonurias; polymerase chain reaction; transfection; transfection /expression vector; virus DNA

DESCRIPTION: Recombinant adenoviral vectors have many attractive features for gene therapy applications, including high transduction efficiencies in vivo and the ability to transduce non-dividing cells. However, the persistence of these vectors and their ability to be readministered are limited by the response of the host immune system to viral gene products, either the proteins of the viral capsid necessary for infection or viral proteins expressed in transduced cells. Dr. Eisensmith and his colleagues propose that safe, efficient and persistent adenovirus-mediated gene transfer may be achieved through vector modification, host immunomodulation, or the combined application thereof. Modification of adenoviral vectors to reduce or abolish late viral gene expression may increase the persistence of adenovirus-mediated gene transfer. To test this hypothesis, the investigators will construct novel E1/E4-deleted adenoviral vectors and adenoviral amplicon vectors in which most or all viral genes have been deleted. High titers of these latter vectors will be produced using a true adenoviral packaging cell line that does not rely on the use of helper virus. Host immunomodulation may also increase the persistence of adenovirus-mediated gene transfer. This hypothesis will be tested by developing and testing reagents designed to reduce the phagocytic, cytokine-releasing and antigen-presenting functions of macrophages, to limit T cell-mediated host immune responses by clonal deletion or anergization of T cells specific for adenoviral antigens or by inhibiting Th1 differentiation, or to specifically block the interaction between T cells and B cells that is necessary for the generation of a strong humoral immune response. Each of these approaches will be tested in vivo, first alone and then in combination if necessary, and if synergism is likely based on immunological studies, by expressing the mouse phenylalanine hydroxylase (PAH) gene in PAH-deficient Pahenu2 mice, an animal model of phenylketonuria. Because of the complete absence of humoral or cellular immune responses in Pahenu2 mice to the mouse PAH protein, this combination of transgene and animal model greatly simplifies the interpretation of experimental results regarding the immunogenicity of viral vectors administered in the presence or absence of various immunomodulatory strategies. Following treatment, serum phenylalanine (PHE) levels and hepatic levels of PAH and viral DNA will be monitored over time to determine efficacy and persistence. Histopathological and immunohistochemical studies will be performed to determine the mechanism of action of these procedures and their safety. Those approaches showing the most promise will be further tested to fully characterize the degree and duration of their immunosuppressive effects so that these may be minimized for potential future applications in humans. Strategies that are successful in the treatment of the phenylketonuria present in the Pahenu2 mouse should also be applicable to the treatment of may other human metabolic diseases for which there are not representative animal models.

描述：重组腺病毒载体具有许多吸引人的特征 用于基因治疗应用，包括高转导效率 体内和转导非分裂细胞的能力。 然而， 这些载体的持久性及其重新管理的能力是 受到宿主免疫系统对病毒基因产物的反应的限制， 感染所需的病毒衣壳蛋白或病毒 转导细胞中表达的蛋白质。 艾森史密斯博士和他的同事 提出安全、高效、持久的腺病毒介导基因 转移可以通过载体修饰、宿主免疫调节、 或其组合应用。 将腺病毒载体修饰为 减少或消除晚期病毒基因表达可能会增加病毒的持久性 腺病毒介导的基因转移。 为了检验这个假设， 研究人员将构建新型 E1/E4 缺失腺病毒载体 腺病毒扩增子载体，其中大部分或所有病毒基因已被 已删除。 后面这些载体的高滴度将使用真正的 不依赖辅助细胞的腺病毒包装细胞系 病毒。 宿主免疫调节也可能增加 腺病毒介导的基因转移。 这个假设将被检验 开发和测试旨在减少吞噬细胞的试剂， 巨噬细胞的细胞因子释放和抗原呈递功能，以限制 T 细胞通过克隆缺失或失活介导宿主免疫反应 T 细胞对腺病毒抗原具有特异性或通过抑制 Th1 分化，或特异性阻断 T 细胞之间的相互作用 和 B 细胞，这是产生强大的体液免疫所必需的 回复。 这些方法中的每一种都将首先单独进行体内测试，然后 然后在必要时进行组合，并且如果协同作用可能基于 通过表达小鼠苯丙氨酸羟化酶进行免疫学研究 PAH 缺陷 Pahenu2 小鼠（一种动物模型）中的 (PAH) 基因 苯丙酮尿症。 由于完全没有体液或细胞 Pahenu2 小鼠对小鼠 PAH 蛋白的免疫反应，这种组合 转基因和动物模型的结合大大简化了解释 病毒载体免疫原性的实验结果 在存在或不存在各种免疫调节剂的情况下施用 策略。 治疗后，血清苯丙氨酸（PHE）水平和 将随着时间的推移监测 PAH 和病毒 DNA 的肝脏水平，以确定 功效和持久性。 组织病理学和免疫组织化学研究 将进行以确定这些程序的作用机制 以及他们的安全。 那些最有希望的方法将进一步 进行测试以充分表征其程度和持续时间 免疫抑制作用，以便最大限度地减少潜在的免疫抑制作用 未来在人类中的应用。 成功的策略 Pahenu2 小鼠中存在的苯丙酮尿​​症的治疗也应该 适用于治疗其他多种人类代谢性疾病 没有代表性的动物模型。