Holguin, L. | bioRxiv Science

bioRxiv2021

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.

molecular biology↗

bioRxiv2021

Novel GVHD resistant humanized-PBMC mouse model for preclinical HIV research

Humanized mouse models are based on the engraftment of human cells in immunodeficient mouse strains, most notably the NSG strain. Most used models have a major limitation in common, the development of graft-versus-host disease (GVHD). GVHD not only introduces variabilities into the research data but also leads to animal welfare concerns. A new mouse strain, B6.129S-Rag2tm1Fwa CD47tm1Fpl Il2rgtm1Wjl/J which lacks Rag1, IL2rg, and CD47 (triple knockout or TKO), is resistant to GVHD development. We transplanted TKO mice with human peripheral blood mononuclear cells (PBMCs) to establish a new humanized PBMC (hu-PBMC) mouse model. A cohort of these mice was infected with HIV-1 and monitored for plasma HIV viremia and CD4+ T cell depletion. The onset and progression of GVHD were monitored by clinical signs. This study demonstrates that TKO mice transplanted with human PBMCs support engraftment of human immune cells in primary and secondary lymphoid tissues, rectum, and brain. Moreover, the TKO hu-PBMC model supports HIV-1 infection via intraperitoneal, rectal, or vaginal routes, as confirmed by robust plasma HIV viremia and CD4+ T cell depletion. Lastly, TKO mice showed a delayed onset of GVHD clinical signs ([~]21 days) and exhibited significant decreases in plasma levels of TNF{beta}. Based on these results, the TKO hu-PBMC mouse model not only supports humanization and HIV-1 infection but is also resistant to GVHD development, making this model a valuable tool in HIV research. ImportanceCurrently, there is no cure or vaccine for HIV infection, thus continued research is needed to end the HIV pandemic. While many animal models are used in HIV research, none is used more than the humanized mouse model. A major limitation with current humanized mouse models is the development of graft-versus-host disease (GVHD). Here, we show a novel humanized mouse model that is resistant to GVHD development and supports and models HIV infection comparable to well-established humanized mouse models.

molecular biology↗