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Mol Ther Nucleic Acids. 2016 Aug 23;5(8):e352. doi: 10.1038/mtna.2016.56.

Efficient Modification of the CCR5 Locus in Primary Human T Cells With megaTAL Nuclease Establishes HIV-1 Resistance.

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Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, Washington, USA.
Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
bluebird bio, Inc., Seattle, Washington, USA.
Department of Medicine, University of Washington, Seattle, Washington, USA.
Department of Pediatrics, University of Washington, Seattle, Washington, USA.
Department of Immunology, University of Washington, Seattle, Washington, USA.


A naturally occurring 32-base pair deletion of the HIV-1 co-receptor CCR5 has demonstrated protection against HIV infection of human CD4+ T cells. Recent genetic engineering approaches using engineered nucleases to disrupt the gene and mimic this mutation show promise for HIV therapy. We developed a megaTAL nuclease targeting the third extracellular loop of CCR5 that we delivered to primary human T cells by mRNA transfection. The CCR5 megaTAL nuclease established resistance to HIV in cell lines and disrupted the expression of CCR5 on primary human CD4+ T cells with a high efficiency, achieving up to 80% modification of the locus in primary cells as measured by molecular analysis. Gene-modified cells engrafted at levels equivalent to unmodified cells when transplanted into immunodeficient mice. Furthermore, genetically modified CD4+ cells were preferentially expanded during HIV-1 infection in vivo in an immunodeficient mouse model. Our results demonstrate the feasibility of targeting CCR5 in primary T cells using an engineered megaTAL nuclease, and the potential to use gene-modified cells to reconstitute a patient's immune system and provide protection from HIV infection.

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