title: Efficient Modification of the CCR5 Locus in Primary Human T Cells With megaTAL Nuclease Establishes HIV-1 Resistance.
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.
authors.affiliation: Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, Washington, USA.
authors.forename: Guillermo S
authors.initials: GS
authors.lastname: Romano Ibarra
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
authors.forename: Biswajit
authors.initials: B
authors.lastname: Paul
authors.affiliation: Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, Washington, USA.
authors.forename: Blythe D
authors.initials: BD
authors.lastname: Sather
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Younan
authors.affiliation: Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, Washington, USA.
authors.forename: Karen
authors.initials: K
authors.lastname: Sommer
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
authors.forename: John P
authors.initials: JP
authors.lastname: Kowalski
authors.affiliation: Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, Washington, USA.
authors.forename: Malika
authors.initials: M
authors.lastname: Hale
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
authors.forename: Barry
authors.initials: B
authors.lastname: Stoddard
authors.affiliation: bluebird bio, Inc., Seattle, Washington, USA.
authors.forename: Jordan
authors.initials: J
authors.lastname: Jarjour
authors.affiliation: bluebird bio, Inc., Seattle, Washington, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Astrakhan
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.|Department of Medicine, University of Washington, Seattle, Washington, USA.
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Kiem
authors.affiliation: Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, Washington, USA.|Department of Pediatrics, University of Washington, Seattle, Washington, USA.|Department of Immunology, University of Washington, Seattle, Washington, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Rawlings