ANTI-HIV GENE THERAPY VECTORS

摘要 通过插入抗HIV基因或基因编辑技术来修饰干细胞理论上可以导致HIV 干细胞移植后患者的免疫系统验证。《柏林病人》的成功接诊 CCR5阴性细胞移植证明了该方法的可行性。此后，无数的 针对CCR5以防止艾滋病毒进入的基因治疗试验已经尝试，但结果不确定。最近， 数学模型数据表明，仅有CCR5干扰策略将无法抑制艾滋病毒负载，除非 与自杀基因相结合。因此，迫切需要开发新的组合基因治疗载体。 以及可用于在移植前浓缩载体修饰干细胞以确保健壮的方法学 嫁接。我们的方案通过包含多个安全性的两步载体系统来解决这些问题 以及选择功能。我们的载体1是一个条件载体，只在 存在HIV Tat蛋白。TK-SR39是一种TK突变体，在小分子病毒存在的情况下能高度有效地杀死细胞 更昔洛韦是一种常规用于治疗艾滋病毒感染者的疱疹病毒的药物。此外， 我们的载体还加入了一个额外的独特功能，即它还瞬时表达TAT可诱导的修饰CD4 (CD4mod)，这是一种缺乏细胞质结构域和HIV Env结合区的CD4分子。这将是 提供一种使用免疫磁珠纯化修饰细胞的机制，同时消除其作为受体的用途 为艾滋病病毒入境做准备。为了诱导CD4mod的瞬时表达，我们将使用表达HIV Tat和a的载体2 CRISPR-CCR5盒式磁带到击倒CCR5。因此，由该两步载体系统修饰的细胞将具有CCR5 基因敲除以及TK-SR39和CD4mod的条件表达，将允许轻松选择细胞。因此， 这些细胞包含多层抗艾滋病毒活性：1)由于CCR5基因被敲除，它们将抵抗艾滋病毒 感染2)在CXCR4病毒出现的情况下，TK-SR39的细胞毒潜力可以通过GCV被利用 管理3)这将导致受感染细胞的快速死亡，从而最大限度地减少病毒传播并防止 建立隐蔽油气藏。我们方法的优势在于从多个方面针对病毒和 允许一种方法来对修饰细胞进行分类，这是在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.