TARGETING OF AAV REP MEDIATED SPECIFIC DNA INTEGRATION

靶向 AAV REP 介导的特异性 DNA 整合

• 批准号: 2824606
• 负责人: RALPH MICHAEL LINDEN
• 金额: $ 16.66万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-15 至 2001-03-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2824606
• 关键词: DNA binding protein; adeno associated virus group; gene targeting; nucleic acid sequence; protein binding; recombinant proteins; tissue /cell culture

The primary focus of this project will be the targeting of DNA integration as mediated by the Rep proteins of adeno-associated virus (AAV). As shown previously, AAV integrates its genome site-specifically into human chromosome 19. Using an episome-based assay, we were able to demonstrate that a 33 nucleotide signal, present within the target sequence, is necessary and sufficient to mediate this highly specific integration/recombination event. The DNA targeting signal contains two sequence motifs which are also present in the viral origin of replication and which are bound by the viral Rep proteins. The hypothesis is that the viral Rep proteins determine site-specific integration through recognition and binding of these origin signals present on chromosome 19. This project will test the feasibility of retargeting Rep-mediated site-specific DNA integration by means of exchanging the DNA binding domain of the AAV Rep proteins. In order to establish a model system, we first propose to exchange the DNA binding domain of AAV Rep78 with its counterpart from goose parvovirus (GPV). Interestingly, the similarity between the two proteins exceeds 90 percent within the active domain of Rep. The DNA-binding domains share much lower similarity consistent with the observation that GPV Rep1 recognizes a different DNA motif. Following biochemical characterization of the hybrid protein a recombinant AAV will be generated containing a rep gene that encodes the hybrid protein. This virus will be used in an episome based integration assay to test for altered site-specific DNA integration into the sequence motif that is recognized by the GPV Rep1 protein. The second goal will be to redirect integration to sites within the human genome other than AAVS1. A genetic screen will be used to identify mutants of the Rep protein capable of binding defined cellular target sequences with high affinity. Once these mutant proteins are purified and biochemically characterized, the episomal integration assay will be used to test for integration into the new target sequences. The experiments proposed are designed to provide further insights into the mechanism of targeted DNA integration with regard to functions of AAV Rep as well as to provide us with a tool to target selected sites within the human genome.

该项目的主要重点是 DNA 的靶向 由腺相关病毒的 Rep 蛋白介导的整合 （AAV）。 如前所述，AAV 将其基因组位点特异性整合 进入人类染色体 19。使用基于附加体的测定，我们能够 证明目标内存在 33 个核苷酸信号 顺序，是必要且充分的来介导这种高度特异性的 整合/重组事件。 DNA靶向信号包含两个 序列基序也存在于病毒起源中 复制并与病毒 Rep 蛋白结合。 这 假设是病毒 Rep 蛋白决定位点特异性 通过识别和结合这些起源信号进行整合 存在于 19 号染色体上。该项目将测试 通过以下方式重新定位 Rep 介导的位点特异性 DNA 整合 交换 AAV Rep 蛋白的 DNA 结合域。 为了 建立模型系统，我们首先提出交换DNA结合 AAV Rep78 的结构域及其来自鹅细小病毒 (GPV) 的对应结构域。 有趣的是，两种蛋白质之间的相似度超过90 Rep 活性结构域内的百分比。DNA 结合结构域共享 相似度低得多，与 GPV Rep1 的观察结果一致 识别不同的 DNA 基序。 继生化 重组 AAV 杂合蛋白的表征 生成的包含编码杂合蛋白的rep基因。这 病毒将用于基于附加体的整合测定，以测试 改变位点特异性 DNA 整合到序列基序中 被 GPV Rep1 蛋白识别。 第二个目标是重定向 整合到人类基因组内除 AAVS1 之外的位点。 一个 基因筛选将用于鉴定 Rep 蛋白的突变体 能够以高亲和力结合确定的细胞靶序列。 一旦这些突变蛋白被纯化并进行生化表征， 游离整合测定将用于测试整合到 新的目标序列。 所提出的实验旨在 提供对靶向 DNA 整合机制的进一步见解 关于 AAV Rep 的功能以及为我们提供的工具 以人类基因组内的选定位点为目标。