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Research Article

Engineering HIV-Resistant Human CD4+ T Cells with CXCR4-Specific Zinc-Finger Nucleases

  • Craig B. Wilen,

    Affiliation: Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America

    X
  • Jianbin Wang,

    Affiliation: Sangamo BioSciences, Richmond, California, United States of America

    X
  • John C. Tilton,

    Affiliation: Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America

    Current address: Center for Proteomics and Bioinformatics, Case Western Reserve University, Cleveland, Ohio, United States of America.

    X
  • Jeffrey C. Miller,

    Affiliation: Sangamo BioSciences, Richmond, California, United States of America

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  • Kenneth A. Kim,

    Affiliation: Sangamo BioSciences, Richmond, California, United States of America

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  • Edward J. Rebar,

    Affiliation: Sangamo BioSciences, Richmond, California, United States of America

    X
  • Scott A. Sherrill-Mix,

    Affiliation: Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America

    X
  • Sean C. Patro,

    Affiliation: Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America

    X
  • Anthony J. Secreto,

    Affiliation: Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America

    X
  • Andrea P. O. Jordan,

    Affiliation: Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America

    X
  • Gary Lee,

    Affiliation: Sangamo BioSciences, Richmond, California, United States of America

    X
  • Joshua Kahn,

    Affiliation: Sangamo BioSciences, Richmond, California, United States of America

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  • Pyone P. Aye,

    Affiliation: Divisions of Regenerative Medicine and Comparative Pathology, Tulane National Primate Research Center, Tulane University School of Medicine, Covington, Louisiana, United States of America

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  • Bruce A. Bunnell,

    Affiliation: Divisions of Regenerative Medicine and Comparative Pathology, Tulane National Primate Research Center, Tulane University School of Medicine, Covington, Louisiana, United States of America

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  • Andrew A. Lackner,

    Affiliation: Divisions of Regenerative Medicine and Comparative Pathology, Tulane National Primate Research Center, Tulane University School of Medicine, Covington, Louisiana, United States of America

    X
  • James A. Hoxie,

    Affiliation: Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America

    X
  • Gwenn A. Danet-Desnoyers,

    Affiliation: Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America

    X
  • Frederic D. Bushman,

    Affiliation: Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America

    X
  • James L. Riley,

    Affiliation: Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America

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  • Philip D. Gregory,

    Affiliation: Sangamo BioSciences, Richmond, California, United States of America

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  • Carl H. June,

    Affiliation: Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America

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  • Michael C. Holmes,

    Affiliation: Sangamo BioSciences, Richmond, California, United States of America

    X
  • Robert W. Doms mail

    doms@mail.med.upenn.edu

    Affiliation: Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America

    X
  • Published: April 14, 2011
  • DOI: 10.1371/journal.ppat.1002020

Reader Comments (1)

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Clarification sought

Posted by lukesnell on 18 Jan 2012 at 22:30 GMT

Next, we characterized CXCR4 cell surface expression over time by FACS. In the R5-ZFN control group, with intact cxcr4 genes, 88% of CD4+ T cells expressed CXCR4 protein at day 27 post engraftment, compared to 84% of cells in the X4-ZFN mice (∼24% cxcr4 gene disruption) as determined by a fluorescence minus-one (FMO) control
http://plospathogens.org/article/info:doi/10.1371/journal.ppat.1002020#article1.body1.sec3.sec7.p2

I'm sure I'm probably misreading this. Why is the surface expression of CXCR4 so high (84%) in CD4 cells from X4-ZFN mice, which have been treated to disrupt CXCR4 expression? How does this correspond to a '~24% CXCR4 gene disruption'? This seems comparable with the control CD4 cells which have not had CXCR disrupted.

No competing interests declared.

RE: Clarification sought

cwilen replied to lukesnell on 24 Jan 2012 at 02:39 GMT

It is likely necessary to disrupt both CXCR4 genes in a given cell to prevent expression of detectable CXCR4 protein. Assuming a Hardy-Weinberg distribution, 24% total gene disruption would translate to about 5% of cells that have both CXCR4 genes mutated. This is relatively consistent with the reduction of CXCR4 protein positive cells seen (88% in the controls vs 84% in the X4-ZFN treated cells).

No competing interests declared.