REPLICATING VECTORS DELIVERED BY ADENOVIRUS AMPLICONS

复制腺病毒扩增子提供的载体

• 批准号: 2770668
• 负责人: VALERI A KROUGLIAK
• 金额: $ 16.85万
• 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: 1997-09-30 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2770668
• 关键词: Adenoviridae; SCID mouse; bioengineering /biomedical engineering; biotechnology; cell line; gene expression; gene therapy; genetic manipulation; genetic transduction; histopathology; immunity; immunocytochemistry; molecular cloning; natural gene amplification; plasmids; polymerase chain reaction; recombinant virus; transfection /expression vector; virus replication

DESCRIPTION (Abstract of the application) Recombinant adenovirus 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 is limited by the response of the host immune system to viral gene products and by the inability of these vectors to replicate in the absence of expression of these viral proteins. We propose that safe, efficient and persistent adenovirus-mediated gene transfer may be achieved through the systemic delivery of self-excising, self-replicating episomes by recombinant adenovirus amplicon vectors in which most or all viral genes have been deleted. The design of the self-excising, self-replicating transgene expression cassette will first be optimized in in vitro experiments. The optimized unit will then be inserted into a recombinant adenovirus vector for in vivo confirmation of the ability of this unit to perform its intended excision and replication functions. As the size of this DNA species exceeds the capacity of E1-deleted adenovirus vectors, in vivo delivery in these initial proof-of-concept studies will be mediated by an E1- and E4-deleted adenovirus vector. To negate any possible interference of the host immune system on the outcome of these studies, they will be performed in SCID) mice. The conversion of the virus to the replicating episome will be monitored by PCR. The structure of the episome will be confirmed by restriction analysis of the plasmid rescued from various organs. Once self-excision and self-replication has been validated in immunodeficient animals, this element will be incorporated into an adenovirus amplicon vector for further testing in immunocompetent animals. The use of an adenovirus amplicon to deliver a replicating vector should minimize any immune response against the adenovirally-transduced cells, allowing the long-term persistence of the replicating vector. High titers of the amplicon vector will be produced using a true adenovirus packaging cell line that does not rely on the use of helper virus. In addition to the tests mentioned above, histopatho-logical and immunohistochemical studies will be performed to determine the efficiency of episome excision and the safety of the gene transfer procedure. If successful, the vector developed in these studies will prove useful in the delivery of potentially therapeutic transgenes to the liver and other target organs for the treatment of a variety of human diseases, genetic and otherwise.

说明（申请摘要） 重组腺病毒载体具有许多吸引人的功能， 治疗应用，包括体内高转导效率， 对非分裂细胞的抑制能力。然而， 这些载体受到宿主免疫系统对病毒的应答的限制 基因产物，以及这些载体不能在细胞中复制， 缺乏这些病毒蛋白的表达。我们建议安全， 可以实现有效和持久的腺病毒介导的基因转移 通过系统递送自我切除、自我复制的附加体， 重组腺病毒扩增子载体，其中大多数或所有病毒基因 已被删除。自我切除自我复制 转基因表达盒将首先在体外优化 实验然后将优化的单元插入重组体中， 腺病毒载体，用于在体内确认该单位的能力， 执行其预期的切除和复制功能。的大小 这种DNA种类超过了E1缺失腺病毒载体的能力， 在这些最初的概念验证研究中，体内递送将由 E1和E4缺失的腺病毒载体。为了否定任何可能的 宿主免疫系统对这些研究结果的干扰， 将在SCID小鼠中进行。病毒转化为 通过PCR监测复制的附加体。附加体的结构 将通过从质粒中拯救的质粒的限制性分析来证实。 各种器官。一旦自我切除和自我复制得到验证， 在免疫缺陷的动物中，该元件将被掺入到免疫缺陷的动物中。 腺病毒扩增子载体用于在免疫活性动物中进一步测试。 使用腺病毒扩增子递送复制载体应 使针对腺病毒转导的细胞的任何免疫应答最小化， 允许复制载体的长期存在。高滴度 将使用真正的腺病毒包装来产生扩增子载体 不依赖于使用辅助病毒的细胞系。除了有 上述检查、组织病理学和免疫组织化学研究 将进行测定附加体切除的效率， 基因转移过程的安全性。如果成功的话， 在这些研究中，将证明在提供潜在的 将治疗性转基因转移到肝脏和其他靶器官， 治疗各种人类疾病，包括遗传疾病和其他疾病。