CRISPRCas9-mediated gene disruption of endogenous co-receptors confers broad resistance to HIV-1 in human primary cells and humanized mice

In this project, we investigated the CRISPR/Cas9 system for creating HIV resistance by targeting the human CCR5 and CXCR4 genes, which encode cellular co-receptors required for HIV-1 infection. Using a clinically scalable system for transient ex vivo delivery of Cas9/gRNA ribonucleoprotein (RNP) complexes, we demonstrated that CRISPR-mediated disruption of CCR5 and CXCR4 in T-lymphocytes cells significantly reduced surface expression of the co-receptors, thereby establishing resistance to HIV-1 infection by CCR5 (R5)-tropic, CXCR4 (X4)-tropic, and dual (R5/X4)-tropic strains. CRISPR-mediated disruption of the CCR5 alleles in human CD34+ hematopoietic stem and progenitor cells (HSPCs) led to the differentiation of HIV-resistant macrophages. In human CD4+ T cells transplanted into a humanized mouse model, disruption of CXCR4 inhibited replication of X4-tropic HIV-1, thus leading to the virus-mediated enrichment CXCR4-disrupted cells in the peripheral blood and spleen. However, in human CD4+ T cells with both CCR5 and CXCR4 disruption, we observed poor engraftment in bone marrow, although significant changes were not observed in the lung, spleen, or peripheral blood. This study establishes a clinically scalable strategy for the dual knockout of HIV-1 co-receptors as a therapeutic strategy, while also raising caution of disrupting CXCR4, which may abate engraftment of CD4+ T cells in bone marrow.