CIS-REQUIREMENTS FOR RECOMBINANT AAV PRODUCTION

重组腺相关病毒可在体内有效地转导多种组织，使转基因产物持续表达。然而，它在遗传疾病治疗中的普遍应用受到实现足够高滴度的载体的困难的限制。在这项申请中，我们将从两个方面解决这一限制：我们收集了证据，证明包装可能需要病毒基因组中的额外顺式元件。我们建议对这些潜在信号进行定位和表征，并将它们整合到rAAV基因组中。使用rAAV生产的标准方法，我们将评估这些潜在的顺式作用信号对病毒滴度的影响。为了验证我们的假设，我们将产生一些pAV2(WtAAV)的缺失突变体。这些突变体将在无细胞系统中进行基因组复制测试，以及在存在辅助质粒的组织培养中进行包装测试。一个被广泛接受的假设是，重组AAV生产中的一个限速步骤是在生产细胞中表达衣壳(CAP)的水平。这一限制可以通过在一种能够在生产者细胞中复制但没有包装信号的结构中提供帽基因来解决。我们建议检验一种不同细小病毒的ITR可以用来实现这一目标的假设。这一目标的目的是在一种能够在生产细胞中复制但缺乏包装信号的构建中提供rep和capp基因。我们建议检验GPV的ITRS可以用来实现这一目标的假设。我们已经产生了一个嵌合的Rep蛋白，由GPV Rep1起源相互作用结构域和AAV Rep78的其余部分组成。使用这种方法，我们能够将Rep78的起源特异性从AAV起源重定向到GPV起源。我们建议测试该蛋白是否能够在组织培养中介导包含帽的结构的复制，该结构位于GPV ITRs两侧。此外，我们将测试GPV ITR侧翼的基因组是否缺乏AAV衣壳的包装信号。

Recombinant adeno-associated virus has been shown to efficiently transduce a variety of tissues in vivo, which results in persistent expression of transgene products. Its general application in the treatment of genetic disorders, however, is limited by difficulties in achieving sufficiently high titers of the vectors. In this application we will address this limitation twofold: We have collected evidence that packaging may require additional cis-elements within the viral genome. We propose to map and characterize these potential signals and to incorporate them into rAAV genomes. Using standard methods for rAAV production we will evaluate the effect of these potential cis- acting signals on viral titers. To test our hypothesis we will generate a number of deletion mutants of pAV2 (wtAAV). These mutants will be tested for genome replication in cell-free systems as well as for packaging in tissue culture in the presence of helper plasmids. A well-accepted hypothesis is that a rate-limiting step in recombinant AAV production is at the level of capsid (cap) expression in the producer cell. This limitation can be resolved by providing cap-genes in a construct which is capable of replicating in the producer cells but which is devoid of packaging signals. We propose to test the hypothesis that the ITRs of a different parvovirus can be used to achieve this goal. The goal of this aim is to provide rep and cap-genes in a construct, which is capable of replicating in the producer cells but which is devoid of packaging signals. We propose to test the hypothesis that the ITRS of a GPV can be used to achieve this goal. We have generated a chimeric Rep protein consisting of the GPV Rep1 origin interaction domain and the rest of AAV Rep78. Using this approach we were able to redirect the origin specificity of Rep78 from the AAV origin to the GPV origin. We propose to test if this protein is capable of mediating replication in tissue culture of a cap-containing construct, which is flanked by the GPV ITRs. Furthermore, we will test if genomes flanked by GPV ITRs lack the packaging signals for AAV capsids.