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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.

Author information

1
Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, Washington, USA.
2
Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
3
bluebird bio, Inc., Seattle, Washington, USA.
4
Department of Medicine, University of Washington, Seattle, Washington, USA.
5
Department of Pediatrics, University of Washington, Seattle, Washington, USA.
6
Department of Immunology, University of Washington, Seattle, Washington, USA.

Abstract

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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