CRISPR-Cas9-mediated gene disruption of HIV-1 co-receptors confers broad resistance to infection in human T cells and humanized mice

Abstract

In this preclinical study, we evaluated the efficacy and feasibility of creating broad human immunodeficiency virus (HIV) resistance by simultaneously disrupting the human <i>CCR5</i> and <i>CXCR4</i> genes, which encode cellular co-receptors required for HIV-1 infection. Using a clinically scalable system for transient <i>ex vivo</i> delivery of Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complexes, we demonstrated that CRISPR-mediated disruption of <i>CCR5</i> and <i>CXCR4</i> in T lymphocyte 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. Similarly, disruption of <i>CCR5</i> alleles in human CD34⁺ hematopoietic stem and progenitor cells (HSPCs) successfully led to the differentiation of HIV-resistant macrophages. In a humanized mouse model under HIV-1 challenge, <i>CXCR4</i>-disrupted CD4⁺ T cells were enriched in the peripheral blood and spleen, indicating survival advantage because of resistance to viral infection. However, in human CD4⁺ T cells with both <i>CCR5</i> and <i>CXCR4</i> 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 <i>CXCR4</i>, which may abate engraftment of CD4⁺ T cells in bone marrow.