ANTI-HIV GENE THERAPY VECTORS

摘要 通过插入抗艾滋病毒基因或基因编辑技术来修饰干细胞，理论上可以导致艾滋病毒 证明干细胞移植后患者的免疫系统。“柏林病人”接收的成功 CCR 5阴性细胞移植已经证明了这种方法的可行性。此后， 以CCR 5为靶点的基因治疗试验已经尝试过，但结果不确定。最近， 数学建模数据表明，CCR 5干扰策略本身将无法抑制HIV载量，除非 结合自杀基因因此，迫切需要开发新的联合基因治疗载体 以及可用于在移植前富集载体修饰的干细胞的方法， 移植我们的建议通过两步矢量系统解决了这些问题，该系统包含多种安全性 以及选择特征。我们的载体1是一个条件载体，它只在大肠杆菌中表达TK-SR 39基因。 HIV达特蛋白的存在。TK-SR 39是TK突变体，其在存在小的细胞因子的情况下高度有效地杀死细胞。 量的更昔洛韦，一种常规用于治疗HIV感染者的疱疹病毒的药物。此外，委员会还认为， 我们的载体结合了一个附加的独特特征，它也瞬时表达一个达特诱导的修饰的CD 4 在一个实施方案中，所述抗体是CD 4 mod，其是缺乏胞质结构域以及HIV Env结合区的CD 4分子。这将 提供了一种使用免疫磁珠纯化修饰细胞的机制， 艾滋病毒进入。为了诱导CD 4 mod的瞬时表达，我们将使用表达HIV达特的载体2和一种表达CD 4 mod的载体。 CRISPR-CCR 5盒以敲除CCR 5。因此，通过这种两步载体系统修饰的细胞将具有CCR 5 TK-SR 39和CD 4 mod的敲除以及条件表达将允许容易地选择细胞。因此，在本发明中， 这些细胞包含多层抗HIV活性，因为1）由于CCR 5敲除，它们将抵抗HIV 感染2）在CXCR 4病毒出现的情况下，可以通过GCV利用TK-SR 39的细胞毒性潜力 3）这将导致受感染细胞的快速死亡，从而使病毒传播最小化并防止感染。 建立潜在油气藏。我们的方法的优越性在于在多条战线上针对病毒， 允许一种分选修饰细胞的方法，这是体内长期植入的关键。

ABSTRACT Modifying stem cells by insertion of anti-HIV gene or gene editing technologies can theoretically result in an HIV proof immune system in patients after stem cell transplantation. The success of the “Berlin patient” receiving CCR5 negative cells for transplantation has demonstrated the feasibility of this approach. Thereafter, a myriad gene therapy trials targeting CCR5 to prevent HIV entry have been tried with inconclusive results. Recent, mathematical modeling data suggests that CCR5 disrupting strategies alone will fail to suppress HIV load unless combined with a suicide gene. Thus, there is an urgent need to develop new combination gene therapy vectors and methodologies that can be used to enrich vector modified stem cells before transplantation to ensure robust engraftment. Our proposal addresses these issues via a two-step vector system that incorporates multiple safety as well as selection features. Our vector 1 is a conditional vector that expresses the TK-SR39 gene only in the presence of HIV Tat protein. TK-SR39 is a TK mutant that is highly potent at killing cells in the presence of small amounts of Ganciclovir, a drug routinely used for Herpes virus treatment in HIV infected individuals. Furthermore, our vector incorporates an added unique feature that it also transiently expresses a Tat inducible modified CD4 (CD4mod) that is a CD4 molecule lacking the cytoplasmic domain as well as HIV Env binding region. This will provide a mechanism to purify modified cells using immunomagnetic beads while eliminating its use as a receptor for HIV entry. To induce transient expression of CD4mod we will use vector 2 that expresses HIV Tat and a CRISPR-CCR5 cassette to knockout CCR5. Thus, cells modified by this two-step vector system will have CCR5 knockout as well as conditional expression of TK-SR39 and CD4mod that will allow easy selection of cells. Thus, these cells encompass multiple layers of anti-HIV activity as 1) because of CCR5 knock out they will resist HIV infection 2) in case of CXCR4 virus emergence, the cytotoxic potential of TK-SR39 can be utilized via GCV administration 3) this will lead to rapid death of infected cells thereby minimizing virus spread and preventing establishment of latent reservoirs. The superiority of our approach lies in targeting the virus at multiple fronts and allowing a method to sort modified cells that is the key to long-term engraftment in vivo.