Allogeneic hematopoietic stem and progenitor cell transplant (HSCT) of CCR5 null (CCR5Δ32) cells can be curative for HIV-1-infected patients. However, because allogeneic HSCT poses significant risk, CCR5Δ32 matched bone marrow donors are rare, and CCR5Δ32 transplant does not confer resistance to the CXCR4-tropic virus, it is not a viable option for most patients. We describe a targeted Cas9/AAV6-based genome editing strategy for autologous HSCT resulting in both CCR5- and CXCR4-tropic HIV-1 resistance. Edited human hematopoietic stem and progenitor cells (HSPCs) maintain multi-lineage repopulation capacity in vivo, and edited primary human T cells potently inhibit infection by both CCR5-tropic and CXCR4-tropic HIV-1. Modification rates facilitated complete loss of CCR5-tropic replication and up to a 2,000-fold decrease in CXCR4-tropic replication without CXCR4 locus disruption. This multi-factor editing strategy in HSPCs could provide a broad approach for autologous HSCT as a functional cure for both CCR5-tropic and CXCR4-tropic HIV-1 infections.

CCR5缺失 ( CCR5Δ32 ) 细胞的同种异体造血干细胞和祖细胞移植 (HSCT)可治愈 HIV-1 感染患者。然而，由于同种异体 HSCT 存在显着风险，CCR5Δ32匹配的骨髓供体很少见，而且CCR5Δ32移植不能赋予对 CXCR4 嗜性病毒的抵抗力，因此对于大多数患者来说不是一个可行的选择。我们描述了一种基于 Cas9/AAV6 的靶向基因组编辑策略，用于自体 HSCT，从而产生 CCR5 和 CXCR4 倾向的 HIV-1 耐药性。编辑的人类造血干细胞和祖细胞 (HSPC)在体内维持多谱系增殖能力，编辑的原代人类 T 细胞可有效抑制 CCR5 嗜性和 CXCR4 嗜性 HIV-1 的感染。修饰率促进了 CCR5 向性复制的完全丧失，并使 CXCR4 向性复制减少高达 2,000 倍，而无需破坏CXCR4位点。这种 HSPC 中的多因子编辑策略可以为自体 HSCT 提供一种广泛的方法，作为 CCR5 向性和 CXCR4 向性 HIV-1 感染的功能性治愈。