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IMPROVED METHODS OF ADENOVIRUS MEDIATED GENE TRANSFER

腺病毒介导的基因转移的改进方法

基本信息

批准号：
2905915
负责人：
RANDY C EISENSMITH
金额：
$ 25.38万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
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/2905915
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

项目摘要

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对非分裂细胞的增殖能力。但这些载体的持久性和它们被重新识别的能力是受宿主免疫系统对病毒基因产物反应的限制，感染所必需的病毒衣壳蛋白或病毒衣壳蛋白在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了一种安全、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。腺病毒载体的修饰，减少或消除晚期病毒基因表达可能会增加腺病毒介导的基因转移。为了验证这一假设，研究人员将构建新型E1/E4缺失的腺病毒载体，腺病毒扩增子载体，其中大多数或所有病毒基因已被的页面不存在或这些后者载体的高滴度将使用真不依赖于使用辅助物的腺病毒包装细胞系病毒宿主的免疫调节也可能会增加腺病毒介导的基因转移。这一假设将由以下人员进行检验：开发和测试旨在减少吞噬细胞的试剂，巨噬细胞的精氨酸释放和抗原呈递功能，以限制 T细胞介导的宿主免疫应答通过克隆性缺失或失能的T细胞介导的宿主免疫应答。腺病毒抗原特异性T细胞或通过抑制Th 1 分化，或特异性阻断T细胞之间的相互作用，和产生强体液免疫所必需的B细胞反应这些方法中的每一种都将在体内进行测试，首先单独测试，然后在必要时进行组合，并且如果协同作用可能基于免疫学研究，通过表达小鼠苯丙氨酸羟化酶 (PAH)PAH缺陷型Pahenu 2小鼠中的基因，苯丙酮尿症由于完全缺乏体液或细胞 Pahenu 2小鼠对小鼠PAH蛋白的免疫应答，这种组合转基因和动物模型极大地简化了对关于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。战略布局治疗后，血清苯丙氨酸（PHE）水平和将随时间监测PAH和病毒DNA的肝脏水平，以确定有效性和持久性。组织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和他们的安全那些最有希望的方法将进一步测试，以充分表征其程度和持续时间，免疫抑制作用，以便这些可能被最小化，未来应用于人类。成功的战略， Pahenu 2小鼠中存在的苯丙酮尿症的治疗也应该是适用于治疗许多其它人类代谢疾病，没有代表性的动物模型。