Mechanisms that Enhance and Suppress HIV-1 Resistance in Gene Edited Primary Human Cells

虽然在过去的几十年里，艾滋病毒疗法取得了巨大的进步，但迄今为止唯一真正的治愈方法是 已经通过造血干细胞移植（HSCT）的细胞自然缺乏HIV共受体CCR 5。 由于HSCT前预处理方案的毒性和移植物抗宿主引起的显著发病率， 然而，由于移植物抗宿主病（GVHD），同种异体HSCT对于大多数患者来说不是可行的选择。随着CRISPR的出现， 修改患者自己的细胞以防止GVHD现在是可能的，但到目前为止， 在没有抗逆转录病毒治疗的情况下，已经被修改的药物不足以防止病毒反弹。一致 基于联合抗逆转录病毒药物治疗靶向复制过程中多个步骤的基本原理， 开发了一种用于编辑CD 34+造血干细胞和祖细胞的多因子敲除/敲入策略 允许大于90%的CCR 5缺失，以及高达50%的两个抑制因子的等位基因敲入的细胞。 靶向融合（C46 V20）或未包被（人-恒河猴嵌合TRIM 5a）的肽。您在访问此 在编辑原代人CD 4 + T细胞的策略中，敲除/敲入的效率足以完成免疫应答。 CCR 5嗜性病毒（BaL）的抑制，以及CXCR 4嗜性病毒的平均超过700倍的抑制 （NL 4 -3）。虽然这些数据非常 有希望的是，在抑制CXCR 4嗜性复制的效率方面存在很大的差异， 恒河猴TRIM 5a，一些T细胞供体在hRhTRIM 5a敲除中显示出大于1,000倍的抑制， 在条件下，和一些显示很少或没有显着的复制抑制。这一观察结果尽管 在整个感染过程中持续的等位基因敲入和hRhTRIM 5a RNA表达水平，和 在终点时未观察到感染性病毒的耐药突变。因此，我们建议在此研究 潜在的T细胞/TRIM/病毒相互作用，可能具有独特的供体特异性并影响病毒复制 包括T细胞细胞因子表达、病毒再活化和内源性细胞因子的表达， 作为一个显性阴性，我们的抑制hRhTRIM 5a除了调查其他TRIM相关 抑制因子如TRIMCyp。虽然最终的目标是通过移植开发一种治疗方法， 对于编辑的造血干细胞和祖细胞，该建议完全基于编辑和调查 在分化的原代人靶细胞如CD 4 + T细胞，单核细胞， 和巨噬细胞，以便更好地了解我们编辑平台内的生物学， 平台的改进。成功完成本建议书将有助于更好地了解 原代细胞中的限制机制，通常这些机制主要在 永生化细胞系。此外，完成这项改善我们治疗平台的建议将扩大 可能能够使用基因修饰的自体HSCT治疗的患者群， 在感染后期，可能主要是CXCR 4嗜性病毒。

While there have been great advances in HIV therapies over the past decades, the only true cure so far has been through hematopoietic stem cell transplant (HSCT) of cells naturally lacking the HIV co-receptor CCR5. Due to the toxicity of conditioning regimens prior to HSCT and significant morbidity due to graft-verses-host disease (GVHD), allogeneic HSCT is not a viable option for most patients. With the advent of CRISPR, modification of a patient’s own cells to prevent GVHD is now possible, but thus far the efficiency at which cells have been modified was not enough to prevent viral rebound in the absence of anti-retroviral therapy. In line with the rationale for combination anti-retroviral drug therapy to target multiple steps in replication, we have developed a multi-factor knock-out/knock-in strategy for editing CD34+ hematopoietic stem and progenitor cells which allows greater than 90% deletion of CCR5, as well as up to 50% allelic knock-in of two inhibitory peptides targeting either fusion (C46V2o) or uncoating (a human-rhesus chimeric TRIM5a). When using this strategy to edit primary human CD4+ T cells, the efficiency of knock-out/knock-in is sufficient for complete inhibition of CCR5-tropic virus (BaL), and an average of more than 700-fold inhibition of CXCR4-tropic virus (NL4-3) when tested across 5 different primary human T cell donors. While these data are extremely promising, there was a large disparity in the efficiency of inhibition of CXCR4-tropic replication by the human- rhesus TRIM5a, with some T cell donors showing greater than 1,000-fold inhibition in the hRhTRIM5a knock- in condition, and some showing little or no significant inhibition of replication. This observation was in spite of sustained levels of allelic knock-in and hRhTRIM5a RNA expression throughout the course of the infection, and no resistance mutations observed in the infectious virus at endpoint. We therefor propose here to study the underlying T-cell/TRIM/virus interactions which may be uniquely donor specific and influence viral replication including T cell cytokine expression, viral reactivation, and expression of endogenous cellular factors that may act as a dominant negative to our inhibitory hRhTRIM5a in addition to investigation of other TRIM-related factors for inhibition such as TRIMCyp. Although the final goal is to develop a therapy through transplantation of edited hematopoietic stem and progenitor cells, this proposal is entirely based on editing and investigating the mechanisms of restriction in differentiated primary human target cells such as CD4+ T cells, monocytes, and macrophages in order to better understand the biology within our editing platform and allow eventual improvement of the platform. Successful completion of this proposal will allow a better understanding of mechanisms of restriction in primary cells where often these mechanisms have been predominantly studied in immortalized cell lines. In addition, completion of this proposal to improve our therapeutic platform will widen the pool of patients potentially able to be treated using genetically modified autologous HSCT such as patients later in infection that may have predominantly CXCR4-tropic virus.