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Zinc-finger nuclease editing of human cxcr4 promotes HIV-1 CD4(+) T cell resistance and enrichment.

ABSTRACT: HIV-1-infected individuals can harbor viral isolates that can use CCR5, as well as CXCR4, for viral entry. To genetically engineer HIV-1 resistance in CD4(+) T cells, we assessed whether transient, adenovirus delivered zinc-finger nuclease (ZFN) disruption of genomic cxcr4 or stable lentiviral expression of short hairpin RNAs (shRNAs) targeting CXCR4 mRNAs provides durable resistance to HIV-1 challenge. ZFN-modification of cxcr4 in CD4(+) T cells was found to be superior to cell integrated lentivirus-expressing CXCR4 targeting shRNAs when CD4(+) T cells were challenged with HIV-1s that utilizes CXCR4 for entry. Cxcr4 disruption in CD4(+) T cells was found to be stable, conferred resistance, and provided for continued cell enrichment during HIV-1 infection in tissue culture and, in vivo, in peripheral blood mononuclear cell transplanted NSG mice. Moreover, HIV-1-infected mice with engrafted cxcr4 ZFN-modified CD4(+) T cells demonstrated lower viral levels in contrast to mice engrafted with unmodified CD4(+) T cells. These findings provide evidence that ZFN-mediated disruption of cxcr4 provides a selective advantage to CD4(+) T cells during HIV-1 infection.

SUBMITTER: Yuan J

PROVIDER: S-EPMC3321595 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Zinc-finger nuclease editing of human cxcr4 promotes HIV-1 CD4(+) T cell resistance and enrichment.

Yuan Jinyun J Wang Jianbin J Crain Karen K Fearns Colleen C Kim Kenneth A KA Hua Kevin L KL Gregory Philip D PD Holmes Michael C MC Torbett Bruce E BE

Molecular therapy : the journal of the American Society of Gene Therapy 20120124 4

HIV-1-infected individuals can harbor viral isolates that can use CCR5, as well as CXCR4, for viral entry. To genetically engineer HIV-1 resistance in CD4(+) T cells, we assessed whether transient, adenovirus delivered zinc-finger nuclease (ZFN) disruption of genomic cxcr4 or stable lentiviral expression of short hairpin RNAs (shRNAs) targeting CXCR4 mRNAs provides durable resistance to HIV-1 challenge. ZFN-modification of cxcr4 in CD4(+) T cells was found to be superior to cell integrated lenti ...[more]

PMID: 22273578

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Project description:Zinc finger nucleases (ZFN) are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of human ADPGK, which encodes an ADP-dependent glucokinase (ADPGK), in tumour cell lines. The hypothesis tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two tumour cell lines (H460 and HCT116). All four lines had frameshift mutations in all alleles at the target site in exon 1 of ADPGK, and were ADPGK-null by immunoblotting. ADPGK knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p=0.002) and 4.3±0.8% (p=0.001) for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when ADPGK was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of ADPGK in HCT116 cells caused few changes in global gene expression, knockout of ADPGK in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no effect on glycolysis as measured by glucose consumption or lactate formation under oxic or anoxic conditions, or extracellular acidification rate (Seahorse XF analyser) under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the ADPGK knockouts, in some cases markedly so. Collectively, the results demonstrate that ADPGK can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of ADPGK is cell line dependent and appears to be unrelated to priming of glycolysis. Use Affymetrix microarrays to examine the effecs of knockout of the gene ADPGK using Zinc Finger nucleases in two cultured cell lines: (i) HCT116 cells (three wild type cultures compared to three ADPGK-knockout cultures), (ii) H460 cells (two wild type cultures compared to two ADPGK-knockout cultures). Ten microarrays in total.

Dataset Information

Zinc-finger nuclease editing of human cxcr4 promotes HIV-1 CD4(+) T cell resistance and enrichment.

Publications

Zinc-finger nuclease editing of human cxcr4 promotes HIV-1 CD4(+) T cell resistance and enrichment.

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