PILOT--DEELOPMENT OF A TRANS LENTIVRAL VECTOR

说明(改编自应用程序)： 慢病毒载体，特别是那些基于HIV-1的载体正在引起人们的兴趣 由于其稳定的诱人特性而在基因治疗领域 整合成非分裂细胞类型。它们在人类治疗中的应用 最终取决于基于慢病毒的安全载体的开发。我们 利用HIV病毒粒子相关辅助蛋白(VPR和VPX)作为 将病毒和非病毒来源的蛋白质输送到艾滋病毒的载体 粒子。最近，我们证明了反式RT和IN(源于 VPR-RT-IN融合蛋白)可以模拟顺式RT和IN(来源于Gag-Pol)。这个 反式RT和IN蛋白可有效挽救传染性和 来自RT-IN减去前病毒的病毒粒子通过完整的 生命周期。这些发现表明，这些关键的功能 酶可以反式形式提供，与Gag Pol无关。这些发现提供了 美国设计新一代安全慢病毒的独特方法 基因传递载体。我们的假设是VPR-RT-IN可以在 转至包装质粒和支持载体的GAG-Pro组分 传染性(转导)。这一假设的一个明显推论是 这一策略将减少重组事件的频率， 产生具有感染性/复制性的慢病毒(Ltr Gag-Poll-Ltr结构)。至 检验我们提出的这一假设：(1)构建“反式慢病毒”载体 并分析其转导原代呼吸道上皮细胞的能力； CFTR基因在原代呼吸道上皮细胞中的表达 使用转慢病毒载体。“转慢病毒”载体的核心 方法是从包装组件中分离RT和IN基因。 这与传统的慢病毒包装系统不同，在传统的包装系统中，RT和 In是Gag-Pol(Gag-PrRT-IN)顺式表达的一部分。通过表达 传输中的关键RT和IN功能将有可能减少 可能通过基因重组产生的致病病毒形式。 重要的是，我们公布的数据表明，反式RT-IN可以支持 减去HIV-1的RT-IN的传染性和复制力大约为 是野生型病毒的80%。我们相信这一代新一代 基于慢病毒的载体(反式慢病毒载体)将促进其 应用于人体临床试验。

Description (adapted from the application): Lentiviral vectors, specifically those based on HIV-1, are generating interest in the gene therapy community due to their attractive property of stable integration into non-dividing cell types. Their use for human therapy ultimately depends on the development of a safe lentiviral-based vector. We have exploited HIV virion associated accessory proteins (Vpr and Vpx) as vehicles to deliver protein of both viral and non-viral origin into HIV particles. Recently, we demonstrated that trans RT and IN (derived from Vpr-RT-IN fusion protein) can mimic cis- RT and IN (derived from Gag-Pol). The trans- RT and IN proteins can effectively rescue the infectivity and replication of virions derived from RT-IN minus provirus through the complete life cycle. These findings demonstrate that the functions of these critical enzymes can be provided in trans, independent of Gag Pol. These findings offer us a unique approach toward designing a new generation of safe lentiviral-based gene delivery vectors. It is our hypothesis that Vpr-RT-IN can be expressed in trans to the Gag-Pro component of the packaging plasmid and support vector infectivity (transduction). An obvious corollary to this hypothesis is that this strategy will reduce the frequency of recombination events that might generate an infectious/replicating lentivirus (LTR gag-pol-LTR structure). To test this hypothesis we propose to: (1) construct a "trans-lentivirus" vector and analyze its ability to transduce primary airway epithelial cells; and (2) analyze the expression of the CFTR gene in primary airway epithelial cells using the trans-lentivirus vector. Central to the "trans-lentiviral" vector approach is that the RT and IN genes are separated from packaging components. This is distinct from conventional lentiviral packaging systems in which RT and IN are expressed in cis a part of Gag-Pol (Gag-PRRT-IN). By expressing the critical RT and IN functions in trans it will be possible to decrease the generation of pathogenic viral forms that could arise by genetic recombination. Importantly, our published data show that trans-RT-IN can support the infectivity and replication of RT-IN minus HIV-1 at a level of approximately 80% that of wild type virus. We believe that this new generation of lentiviral-based vectors (trans-lentiviral vectors) will facilitate their application into human clinical trials.