HIV-based vector with predictable safety

描述（申请人提供）：慢病毒载体，包括 来源于HIV-1，为基因治疗带来了巨大的希望。但 产生可复制逆转录病毒（RCR）的可能性， 基因重组引起了对安全性的严重关注。报告受限 现有的动物模型，以评估这些载体在体内，安全性 最终在人类宿主中决定。因此，当务之急是， 载体本身的设计确保可达到的最大安全水平。 使用我们开发的一种高灵敏度的检测方法， 基因重组，我们已经证明，重组发生在 第3代和SIN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，我们已经表明，这些 表达缺失的重组体可以表达功能性gag/gag-pol， 当提供外源包膜时，能够动员逆转录病毒DNA， 基于我们早期的发现，全功能逆转录酶 (RT)和整合酶（IN）可以反式掺入HIV- 1颗粒中， 独立于Gag-Pol，我们已经开发了一种载体（“反式慢病毒”）， 载体），其防止含有RI-IN的重组体的产生 （gag-pol）。由于功能性gag-pol是绝对需要的出现或 任何类型的RCR和逆转录病毒DNA动员，反式慢病毒 vector在两个重要方面提高了安全性：首先， 涉及RT-IN的重组事件是产生重组体所必需的 第二，含有功能性gag-pol结构的反式慢病毒， 载体设计本身使得有可能在体外监测载体库存， 功能性gag-pol结构的再生。我们的核心假设是 反式慢病毒载体设计将确保最大水平的 可预测的安全性，同时保留产生高滴度的能力 能够有效转导非分裂细胞的载体储备物。测试 我们的假设是：（1）构建gag-pro包装质粒 含有最少的RT和IN编码序列;（2）遗传修饰 trans-RT-IN表达构建体;（3）分析反式慢病毒包装和表达载体。 用于基因重组的trans-RT-IN构建体;（4）建立可诱导的 能够产生高滴度反式慢病毒的稳定包装细胞系 （5）评估用于转导的反式慢病毒载体， 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.