Simultaneous Knockout ofCXCR4andCCR5Genes in CD4+ T Cells via CRISPR/Cas9 Confers Resistance to Both X4- and R5-Tropic Human Immunodeficiency Virus Type 1 Infection

Abstract

Previous research has proven that disruption of either the CCR5 or the CXCR4 gene confers resistance to R5-tropic or X4-tropic human immunodeficiency virus type 1 (HIV-1) infection, respectively. However, the urgent need to ablate both of the co-receptors in individual post-thymic CD4+ T cells for dual protection remains. This study ablated the CCR5 and CXCR4 genes in human CD4+ cell lines and primary CD4+ T cells simultaneously using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, a well-developed, highly efficient genetic engineering tool. The efficiency of gene modification is as high as 55% for CCR5 and 36% for CXCR4 in CD4+ cell lines through infection of a single lentiviral vector (LV-X4R5), which were markedly protected from both HIV-1 (X4-using strain) and HIV-1 (R5-using strain) infection. Importantly, approximately 9% of the modified GHOST (3) CXCR4+CCR5+ cells harbor four bi-allelic gene disruptions in both the CXCR4 and CCR5 loci. Moreover, co-delivery of two single-guide RNAs loaded with Cas9: ribonucleoprotein (sgX4&R5 Cas9RNP) disrupted >12% of CCR5 and 10% of CXCR4 in primary human CD4+ T cells, which were rendered resistant to HIV-1 and HIV-1 in vitro. Further, the modified cells do not show discernible mutagenesis in top-ranked off-target genes by the Surveyor assay and Sanger sequencing analysis. The results demonstrate the safety and efficacy of CRISPR/Cas9 in multiplex gene modification on peripherally circulating CD4+ T cells, which may promote a functional cure for HIV-1 infection.

Altered adipose tissue and adipocyte function in the pathogenesis of metabolic syndrome

“…This may be part of a more complex regulatory loop in which TGF-β induces adipocyte secretion of miR-130b, which is then transferred to muscle, where it acts to reduce the expression of PGC-1α, reducing muscle oxidative metabolism (202). Skeletal muscle is also responsive to miR-27a, which is present in adipose-derived exosomes and induces insulin resistance via PPARγ repression (203). Serum levels of miR-27a are positively associated with obesity and insulin resistance in children and in mice with obesity, indicating that miR-27a may be another modulator of obesity-associated insulin resistance (203).…”

“…Skeletal muscle is also responsive to miR-27a, which is present in adipose-derived exosomes and induces insulin resistance via PPARγ repression (203). Serum levels of miR-27a are positively associated with obesity and insulin resistance in children and in mice with obesity, indicating that miR-27a may be another modulator of obesity-associated insulin resistance (203).…”

Genome editing of the HIV co-receptors CCR5 and CXCR4 by CRISPR-Cas9 protects CD4+ T cells from HIV-1 infection

“…Our group also reported that disruption of the CXCR4 co-receptor by CRISPR-Cas9 resulted in protection of primary CD4 + T cells from HIV-1 infection [ 37 ]. However, to date, only one study has investigated simultaneous CXCR4 and CCR5 modification using CRISPR-Cas9, which was reported to inhibit HIV-1 infection in cells [ 38 ]. In this study only one combination of CXCR4 and CCR5 sgRNA was assessed.…”

Reduce and Control: A Combinatorial Strategy for Achieving Sustained HIV Remissions in the Absence of Antiretroviral Therapy

“…As expected, CRISPR-Cas9-mediated knockout of the CCR5 gene inhibited HIV-1 infection in induced pluripotent stem cell-derived monocyte/macrophages [237], primary CD4+ T cells [238], and in vivo [239,240]. Similarly, CXCR4 knockout was shown to confer resistance to X4-tropic virus infection [241][242][243]. However, in view of the importance of CXCR4 in the development of hematopoietic cells [244], the clinical feasibility of this latter knockout approach is more problematic.…”