INTEGRATION OF AAV2 GENOME TO MUSCLE-SPECIFIC DNA REGION

描述（复制自申请人摘要）：2型腺相关病毒 (AAV)是独特的能力，整合其基因组位点特异性地进入 位于人类19号染色体q臂上的一个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， 需要介导靶向整合，可能与宿主细胞 因素AAV的这一特性使得一种新的概念成为可能， 治疗性基因递送：将转基因靶向整合到 人类基因组中的一个特定位点。这一有用性的先决条件 方法是表征人类目标区域以及 这是由Rep介导的AAV DNA整合引起的重排。 我们发现编码骨骼肌慢肌钙蛋白T的基因 （TNTI）位于Ca。14 kb端粒的AA VS 1此外，我们表明， TNNTI可由于位点特异性AAV DNA整合而被破坏。这些 数据让我们回答以下问题：1。是转录活性 在AAV整合所需的AAVSI区域中，和/或其是否增强AAV整合 效率？2.从这个区域，即从TNNTJ基因， 影响插入AA VSI的转基因的表达。3.针对 将AAV或rAAV DNA插入AAVS 1可能导致表型变化 在受感染的细胞中，由于邻近基因的表达被破坏， (now显示为TNNT 1）。 到目前为止，骨骼肌已经是AAV最成功的组织之一 介导的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部位特异性 将基因递送到AAVS 1中。

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.