Novel hematopoietic stem cell engineering and transplantation approaches for HIV cure

基因编辑有望提高基于工程造血干细胞和祖细胞（HSPC）的抗艾滋病毒基因疗法的准确性和安全性，从而重现柏林病人治疗的要素。靶向核酸酶技术已经被用于利用nhej介导的DNA断裂修复，从而破坏CCR5基因，这模仿了他的治疗方法的一个方面。然而，将这种治疗扩展到非癌症患者，接受工程化的自体HSPC，可能需要包括其他方法。为此，我们正在开发基于同源定向修复（homology-directed repair， HDR）的DNA断裂修复替代途径。这可用于将功能获得突变引入细胞限制因子，或指导可溶性抗病毒因子的受控分泌，包括广泛进入抑制剂eCD4-Ig。此外，在CCR5位点的eCD4-Ig上的位点特异性插入将hiv抗性与系统保护结合起来，从而提供了一种组合抗hiv方法。在目前的提案中，我们的目标是继续提高HSPC中基因编辑的安全性和有效性，并应用该技术提供局部和全身HIV抗性。我们将把我们已建立的抗hiv方法与一个新的方向结合起来，以设计生产广泛中和的抗体，从而提供一种新的合成免疫能力。将在适当的小鼠模型中评估个体策略和组合方法，以评估它们对HIV感染和潜伏库的影响。

Gene editing holds the promise of enhancing the precision and safety of anti-HIV gene therapies based on engineering hematopoietic stem and progenitor cells (HSPC), in order to recreate elements of the Berlin Patient cure. Targeted nuclease technology is already being used to exploit NHEJ-mediated repair of DNA breaks and thereby disrupt the CCR5 gene, which mimics one aspect of his cure. However, extending this treatment to noncancer patients, receiving engineered autologous HSPC, will likely need to include other approaches. To do this, we are exploiting the alternate pathway of DNA break repair based on homology-directed repair (HDR). This can be used to introduce gain-of-function mutations into cellular restriction factors, or to direct the controlled secretion of soluble anti-viral factors, including the broad entry inhibitor eCD4-Ig. Moreover, the site-specific insertion at eCD4-Ig at the CCR5 locus would combine HIV-resistance with systemic protection and thereby provide a combinatorial anti-HIV approach. In the current proposal we aim to continue to improve the safety and efficacy of gene editing in HSPC, and to apply the technology to provide both local and systemic HIV resistance. We will combine our established anti-HIV approaches with a new direction to engineer production of broadly neutralizing antibodies, and thereby provide a novel synthetic immune capability. Individual strategies and combination approaches will be evaluated in appropriate mouse models, to evaluate their impact on HIV infection and the latent reservoir.