AAV VECTOR DEVELOPMENT

这个子项目的主要重点是分子生物学， 腺相关病毒，因为它涉及AAV用于基因治疗的用途。 AAV已经显示出作为用于治疗多种肿瘤的载体的显著前景。 最有希望的靶点之一是神经系统。 尽管如此，重要的基本问题和技术问题仍有待解决 在rAAV载体可以有信心地使用之前。其中之一就是 病毒编码的Rep蛋白在病毒DNA复制、整合和 控制基因表达。为了解决这个问题，第一个目标是 子项目将是分离AAV复制品中的条件致死突变， 基因这些突变体将用于更精确地定义 Rep蛋白参与DNA的复制、激活和阻遏 此外，这些突变体将用于回答 关于Rep在位点特异性整合入人类的作用的问题 19号染色体。该分项目的第二个目标是改善 目前的rAAV病毒生产方法。以前的尝试发展 包装线或腺病毒/AAV杂合病毒显然 由于当AAV rep基因被转染时观察到的毒性， 在人类细胞中以高拷贝数表达。重点抓好 在非可挽救的细胞中携带AAV基因的腺病毒载体的开发 form.将尝试几种方法来工程化稳定的腺病毒/AAV 可用于有效提供所有Ad和 rAAV繁殖所需的AAV基因功能。这些机制将包括 使用上面提出的条件致死性REP突变，使用 诱导型REP启动子，以及使用新的细胞系， 调节rep表达。这个子项目的最后一个目标是 确定rAAV转导在原代细胞中的效率和持久性， 神经组织，在体内，特别是脊髓，作为一个功能， 时间和用于驱动转基因表达的启动子。rAAV 携带改进的绿色荧光蛋白（gfp）基因的病毒将 注射到体内正常大鼠神经组织中。GFP标记基因将 在神经特异性、胶质特异性或管家的控制下 发起人。注射部位包括脊髓、海马， 皮质和小脑。注射部位的表达将是 通过几种方法监测作为耳过程中时间的函数。 病毒基因组的物理状态，也就是说，它是否作为一种 附加体或整合的原病毒。 这些 实验将补充类似的研究，在视网膜组织提出的 次级项目2，并提供资料， 子项目3，其中rAAV将用于表达特异性 脊髓中的治疗性转基因

The primary focus of this subproject will be the molecular biology of adeno-associated virus as it relates to the use of AAV for gene therapy. AAV has shown significant promise as a vector for therapy in a variety of tissues and one of the most promising targets is the nervous system. Nevertheless, significant basic and technical issues need to be resolved before rAAV vectors can be used with confidence. One of these is the role of the virus encoded Rep protein in viral DNA replication, integration and control of gene expression. To address this issue, the first goal of this subproject will be to isolate conditional lethal mutations in the AAV rep gene. These mutants will be used to define more precisely the domains of the Rep protein involved in the DNA replication, activation and repression functions of Rep. In addition, these mutants will be used to answer questions about the role of Rep in site specific integration into human chromosome 19. The second goal of this subproject will be to improve the current methods of rAAV virus production. Previous attempts to develop packaging lines or adenovirus/AAV hybrid viruses have apparently floundered because of the toxicity observed when the AAV rep genes are expressed in human cells at high copy number. We will focus on the development of adenovirus vectors which carry AAV genes in a non-rescuable form. Several methods will be tried to engineer stable adenovirus/AAV hybrid vectors which can be used to provide efficiently all of the Ad and AAV gene functions required for rAAV propagation. These will include the use of the conditional lethal rep mutations proposed above, the use of inducible rep promoters, and the use of novel cell lines that down regulate rep expression. The last goal of this subproject will be to determine the efficiency and persistence of rAAV transduction in primary neural tissues, in vivo, specifically the spinal cord, as a function of time and the promoter used to drive expression of the transgene. rAAV virus carrying an improved green fluorescent protein (gfp) gene will be injected into normal rat nervous tissue in vivo. The gfp marker gene will be under the control of neural specific, glial specific or housekeeping promoters. Sites of injection will include spinal cord, hippocampus, cortex an cerebellum. Expression at the sites of injection will be monitored by several methods as a function of time over the course of ear. The physical state of the viral genome, that is, whether it exists as an episome or as an integrated provirus, will also be determined. These experiments will complement similar studies in retinal tissues proposed in Subproject 2, and provide information for the studies proposed in Subproject 3, in which rAAV will be used for the expression of specific therapeutic transgenes in the spinal cord.