INTEGRATION OF HUMAN PARVOVIRUS AAV 2 DNA

腺相关病毒（AAV）是一种缺陷型人细小病毒，是目前已知的最大的病毒学缺陷之一。 已知的最小DNA病毒。 虽然超过一半的 成人人群中有中和抗体的AAV，没有已知的 与AAV感染相关的病理学。 然而，AAV很容易 在体内和体外建立持续感染。 证据 这表明病毒的持久性是在AAV DNA的水平上，至少 在体外，可以整合到细胞DNA中。 AAV DNA的整合是 可能是AAV末端序列的位点特异性。 AAV DNA的末端 具有规则和反向重复序列，类似于末端 原核和真核转座因子的结构和长 逆转录病毒的末端重复序列。 这些元素已被证明 整合到宿主DNA中并有可能导致突变。 在 此外，感染性AAV可以在体外从潜伏感染的AAV中拯救出来， 用辅助腺病毒双重感染后的细胞。 这些发现表明，AAV可能是一种独特的非致病性病毒， 特别适用于真核生物研究的整合真核载体 基因调控，从文库中分离可选择的基因，以及 最终是基因治疗。 我们将开发载体， 由一个或两个AAV末端界定的特异性真核基因。 这些 将测试载体以确定它们的转化效率， 转化的稳定性，以及载体DNA是否整合或 受体细胞中的游离体。 将它们与类似的 缺乏AAV末端的载体。 如果如预期的那样，AAV载体不 促进克隆基因整合到受体细胞的基因组中， 用辅助腺病毒（Ad）的重复感染拯救 将测试来自这些转化细胞的载体序列。 类似地 将确定含有少于4,000个核苷酸的基因 可以在用辅助Ad进行双重感染后包装成AAV病毒粒子 和AAV。

Adeno-associated virus (AAV), a defective human parvovirus, is one of the smallest DNA containing viruses known. Although more than half of the adult population has neutralizing antibody to AAV, there is no known pathology associated with AAV infection. However, AAV does readily establish persistent infections both in vivo and in vitro. Evidence suggests that virus persistence is at the level of AAV DNA which, at least in vitro, can integrate into cellular DNA. Integration of AAV DNA is probably site-specific for the AAV terminal sequences. The ends of AAV DNA have both regular and inverted repeats and are analogous to the terminal structures of prokaryotic and eukaryotic transposable elements and the long terminal repeats of retroviruses. These elements have been shown to integrate into host DNA and have the potential for causing mutation. In addition infectious AAV can be rescued in vitro from latently infected cells following superinfection with a helper adenovirus. These findings suggest that AAV might be a unique non-pathogenic integrative eukaryotic vector especially useful for studies of eukaryotic gene regulation, isolation of selectable genes form libraries, and eventually gene therapy. We will develop vectors which will contain specific eukaryotic genes bounded by one or both of the AAV termini. These vectors will be tested to determine their efficiency of transformation, stability of transformation, and whether the vector DNA is integrated or episomal in the recipient cells. They will be compared with similar vectors lacking the AAV termini. If, as expected, the AAV vector does promote the integration of cloned genes into the genome of recipient cells, the ability of superinfection with helper Adenovirus (Ad) to rescue the vector sequences from these transformed cells will be tested. Similarly it will be determined whether genes containing fewer than 4,000 nucleotides can be packaged into AAV virions following superinfection with helper Ad and AAV.