HIV-based vector with predictable safety

描述(申请人提供)：慢病毒载体，包括 源于HIV-1，为基因治疗带来了巨大的希望。然而， 通过以下途径产生复制能力强的逆转录病毒(RCR)的可能性 基因重组引起了人们对安全性的严重担忧。考虑到这些限制 在体内评估这些载体的可用动物模型，安全性将 最终由人类宿主决定。因此，当务之急是 载体本身的设计确保了可达到的最大安全水平。 使用我们开发的一种高度敏感的分析方法，专门为 基因重组体，我们已经证明了重组发生在 第三代慢病毒的包装和载体组分 转导细胞中的载体系统。重要的是，我们已经证明了这些 去包膜重组子可以表达功能性的Gag/Gag-Poll，即 在提供外源包膜的情况下能够动员逆转录病毒DNA 互换。根据我们早先的发现，全功能逆转录酶 (RT)和整合酶(IN)可以反式掺入HIV-1颗粒， 独立于Gag-Pol，我们已经开发出一种载体(“反式慢病毒” 载体)，以防止产生包含RI-IN的重组子 (搞笑-Poll)。由于功能性口交是绝对必要的出现或 任何类型的RCR和用于逆转录病毒DNA动员的反式慢病毒 载体在两个重要方面提高了安全性：第一，增加了一个 涉及RT-IN的重组事件是产生重组子所必需的 含有功能性Gag-Poll结构，第二，反式慢病毒 媒介设计本身使在体外监测媒介库存成为可能 一个有功能的搞笑结构的再生。我们的中心假设是 转基因慢病毒载体的设计将确保最大水平的 可预测的安全性，同时保持产生高滴度的能力 能够有效地转导未分裂细胞的载体。为了测试 我们的假设是：(1)构建Gag-Pro包装质粒 包含最小RT和IN编码序列；(2遗传修改 反式RT-IN表达载体的构建；(3)分析反式慢病毒包装和 用于基因重组的反式RT-IN构建；(4)建立可诱导的 能产生高滴度反式慢病毒的稳定包装细胞系 (5)评价转导慢病毒载体的转导效果。 造血干细胞。

description (provided by applicant): Lentiviral vectors, including those derived from HIV-1, hold great promise for gene therapy. However, the possibility of generating replication competent retrovirus (RCR) through genetic recombination raises serious concerns for safety. Given the limitations of available animal models to evaluate these vectors in vivo, safety will ultimately be determined in human hosts. Therefore, it is imperative that the design of the vector itself ensures the maximal level of safety attainable. Using a highly sensitive assay that we developed to specifically select for genetic recombinants, we have demonstrated that recombination occurs between the packaging and vector components of the 3rd generation and SIN lentiviral vector systems in transduced cells. Importantly, we hay shown that these envelope-minus recombinants can express functional gag/gag-pol, which is capable c mobilizing retroviral DNA when exogenous envelope is provided in trans. Based on our earlier findings that full functional reverse transcriptase (RT) and integrase (IN) can be incorporated into HIV- 1 particles in trans, independently of Gag-Pol, we have developed a vector ("trans-lentiviral" vector) that prevents the generation a recombinants that contain RI-IN (gag-pol). Since functional gag-pol is absolutely required for the emergence or any type of RCR and for retroviral DNA mobilization, the trans-lentiviral vector improves safety in two important ways: First, an additional recombination events involving RT-IN is necessary to generate recombinants containing a functional gag-pol structure, and second, the trans-lentiviral vector design itself makes it possible to monitor vector stocks in vitro for the regeneration of a functional gag-pol structure. Our central hypothesis is that the trans-lentiviral vector design will ensure the greatest level of safety that is predictable while retaining the ability to produce high titer vector stocks capable of efficient transduction of nondividing cells. To test our hypothesis, we propose to: (1) Construct gag-pro packaging plasmids containing minimal RT and IN coding sequences; (2 Genetically modify the trans-RT-IN expression construct; (3) Analyze the trans-lentiviral packaging an trans-RT-IN constructs for genetic recombination; (4) Establish an inducible stable packaging cell line capable c producing high titer trans lentiviral vector; (5) Evaluate the trans-lentiviral vector for transduction o hematopoietic stem cells.