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

虽然在过去的几十年里，艾滋病的治疗方法有了很大的进步，但到目前为止，唯一真正的治疗方法是 一直通过造血干细胞移植(HSCT)的细胞天然缺乏HIV共受体CCR5。 由于HSCT前的预适应方案的毒性，以及移植物对宿主的显著发病率 对于大多数患者来说，异基因造血干细胞移植并不是一个可行的选择。随着CRISPR的到来， 修改患者自己的细胞以预防GVHD现在是可能的，但到目前为止，细胞的效率 在没有抗逆转录病毒治疗的情况下，已经被修改的病毒不足以防止病毒反弹。排队 根据联合抗逆转录病毒药物治疗的原理，我们在复制过程中针对多个步骤， 开发了一种编辑CD34+造血干/祖细胞的多因素敲除/敲入策略 细胞中允许超过90%的CCR5缺失，以及高达50%的等位基因敲入两种抑制作用 靶向融合(C46V2O)或去涂层(人-恒河猴嵌合TRIM5a)的多肽。在使用此功能时 策略编辑原代人类CD4+T细胞，敲除/敲入的效率足以完成 对CCR5嗜性病毒(BAL)的抑制，对CXCR4嗜性病毒的平均抑制率超过700倍 (NL4-3)在5个不同的人类初级T细胞捐赠者身上进行测试时。虽然这些数据非常 有希望的是，在抑制CXCR4嗜性复制的效率上存在很大的差异。 恒河猴TRIM5a，一些T细胞捐赠者在hRhTRIM5a敲打中显示出超过1000倍的抑制- 在条件下，一些表现出很少或没有明显的复制抑制。这一观察结果是尽管 在感染过程中持续的等位基因敲入和hRhTRIM5a RNA表达水平，以及 终点时在传染性病毒中没有观察到抗药性突变。为此，我们建议在这里研究 潜在的T细胞/TRIM/病毒相互作用可能是独一无二的供体特异性并影响病毒复制 包括T细胞细胞因子的表达，病毒的重新激活，以及内源性细胞因子的表达，可能 除了对其他TRIM相关基因的研究外，还对我们的抑制性hRhTRIM5a起显性否定作用 抑制因子如TRIMCyp。虽然最终目标是通过移植开发一种治疗方法 关于编辑的造血干细胞和祖细胞，这一建议完全基于编辑和调查 限制分化的原代人类靶细胞如CD4+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并最终允许 平台的改进。成功完成本提案将使您更好地了解 在原代细胞中的限制机制，这些机制通常主要在 永生的细胞系。此外，这项改善我们治疗平台的提案的完成将扩大 可能使用转基因自体造血干细胞移植治疗的患者池，如患者 在以后的感染中可能主要是CXCR4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.