INTEGRATION OF AAV2 GENOME TO MUSCLE-SPECIFIC DNA REGION

描述(复制自申请者摘要)：腺相关病毒2型 (AAV)的独特之处在于能够将其基因组位点整合到 人类19号染色体Q臂上指定的区域中的人类基因组 AAVSI。最有趣的是，只有一种病毒基因产物，即Rep蛋白，是 介导靶向整合所必需的，可能与宿主细胞协同作用 各种因素。AAV的这一特性使一种新的概念成为可能 治疗性基因传递：有针对性地将转基因整合到 人类基因组中已定义的基因座。这是有用的前提条件 方法是描述人类目标区域以及 Rep介导的AAV DNA整合引起的重排。 我们发现编码慢速骨骼肌肌钙蛋白T的基因 (TNNTI)位于加州。此外，我们还证明了AAVS1的端粒 由于特定部位的AAV DNA整合，TNNTI可能被打乱。这些 数据使我们能够回答以下问题：1.转录活动 在AAVSI所需的区域和/或是否增强AAV集成 效率？2.这个区域的转录，即来自TNNTJ基因的转录， 影响插入AA VSI的转基因的表达。3.是否有针对性 将AAV或rAAV DNA插入AAVS1中可能会导致表型变化 在受感染的细胞中由于近端基因的表达中断 (现在显示为TNNT1)。 到目前为止，骨骼肌一直是AAV最成功的组织之一。 介导性基因转移。我们建议回答这些问题，以便 确定骨骼肌是否可以作为特定部位的合适组织 将基因导入AAVS1。

DESCRIPTION (Copied from Applicant Abstract): Adeno-associated virus type 2 (AAV) is unique in the ability to integrate its genome site-specifically into the human genome in a region on the q-arm of human chromosome 19 designated AAVSI. Most intriguingly, only one viral gene product, the Rep protein, is required to mediate targeted integration, presumably in concert with host cell factors. This characteristic of AAV has made possible a novel concept for therapeutic gene delivery: the targeted integration of a transgene into a defined locus within the human genome. Prerequisite for the usefulness of this approach is the characterization of the human target region as well as of the rearrangements resulting from Rep-mediated AAV DNA integration. We have found that the gene encoding the slow skeletal muscle troponin T (TNNTI) is located Ca. 14 kb telomeric to AA VS1 Furthermore, we show that TNNTI can be disrupted as a result of site-specific AAV DNA integration. These data allow us to answer the following questions: 1. Is transcriptional activity in the region of AAVSI required for and/or does it enhance the AAV integration efficiency? 2. Does transcription from this region, i.e. from the TNNTJ gene, affect expression of a transgene inserted into AA VSI. 3. Does targeted insertion of AAV or rAAV DNA into AAVS1 potentially lead to phenotypic changes in the infected cell due to disruption of the expression of a proximal gene (now shown to be TNNT1). To date, skeletal muscle has been one of the most successful tissues for AAV mediated gene transfer. We propose to answer these questions in order to establish if skeletal muscle might serve as a suitable tissue for site-specific gene delivery into AAVS1.