title: Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template.
abstract: Genetic mutations or engineered nucleases that disrupt the HIV co-receptor CCR5 block HIV infection of CD4(+) T cells. These findings have motivated the engineering of CCR5-specific nucleases for application as HIV therapies. The efficacy of this approach relies on efficient biallelic disruption of CCR5, and the ability to efficiently target sequences that confer HIV resistance to the CCR5 locus has the potential to further improve clinical outcomes. We used RNA-based nuclease expression paired with adeno-associated virus (AAV)-mediated delivery of a CCR5-targeting donor template to achieve highly efficient targeted recombination in primary human T cells. This method consistently achieved 8 to 60% rates of homology-directed recombination into the CCR5 locus in T cells, with over 80% of cells modified with an MND-GFP expression cassette exhibiting biallelic modification. MND-GFP-modified T cells maintained a diverse repertoire and engrafted in immune-deficient mice as efficiently as unmodified cells. Using this method, we integrated sequences coding chimeric antigen receptors (CARs) into the CCR5 locus, and the resulting targeted CAR T cells exhibited antitumor or anti-HIV activity. Alternatively, we introduced the C46 HIV fusion inhibitor, generating T cell populations with high rates of biallelic CCR5 disruption paired with potential protection from HIV with CXCR4 co-receptor tropism. Finally, this protocol was applied to adult human mobilized CD34(+) cells, resulting in 15 to 20% homologous gene targeting. Our results demonstrate that high-efficiency targeted integration is feasible in primary human hematopoietic cells and highlight the potential of gene editing to engineer T cell products with myriad functional properties.
authors.affiliation: Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA.
authors.forename: Blythe D
authors.initials: BD
authors.lastname: Sather
authors.affiliation: Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA.
authors.forename: Guillermo S
authors.initials: GS
authors.lastname: Romano Ibarra
authors.affiliation: Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA.
authors.forename: Karen
authors.initials: K
authors.lastname: Sommer
authors.affiliation: Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA.
authors.forename: Gabrielle
authors.initials: G
authors.lastname: Curinga
authors.affiliation: Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA.
authors.forename: Malika
authors.initials: M
authors.lastname: Hale
authors.affiliation: Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA.
authors.forename: Iram F
authors.initials: IF
authors.lastname: Khan
authors.affiliation: Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA.
authors.forename: Swati
authors.initials: S
authors.lastname: Singh
authors.affiliation: Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA.
authors.forename: Yumei
authors.initials: Y
authors.lastname: Song
authors.affiliation: Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA.
authors.forename: Kamila
authors.initials: K
authors.lastname: Gwiazda
authors.affiliation: Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA.
authors.forename: Jaya
authors.initials: J
authors.lastname: Sahni
authors.affiliation: Bluebird Bio, Seattle, WA 98102, USA.
authors.forename: Jordan
authors.initials: J
authors.lastname: Jarjour
authors.affiliation: Bluebird Bio, Seattle, WA 98102, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Astrakhan
authors.affiliation: Center for Global Infectious Disease Research, Seattle Children's Research Institute, Seattle, WA 98101, USA. Department of Pediatrics, University of Washington, Seattle, WA 98101, USA.
authors.forename: Thor A
authors.initials: TA
authors.lastname: Wagner
authors.affiliation: Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA. Department of Pediatrics, University of Washington, Seattle, WA 98101, USA. Department of Immunology, University of Washington, Seattle, WA 98101, USA.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Scharenberg
authors.affiliation: Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA. Department of Pediatrics, University of Washington, Seattle, WA 98101, USA. Department of Immunology, University of Washington, Seattle, WA 98101, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Rawlings
mesh_terms: D000328:Adult; D018952:Antigens, CD34 / Q000378:metabolism; D017252:CD3 Complex / Q000378:metabolism; D002478:Cells, Cultured; D004260:DNA Repair; D003851:Deoxyribonucleases / Q000378:metabolism*; D000229:Dependovirus / Q000378:metabolism*; D056426:Genetic Loci; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D017393:RNA Editing / Q000235:genetics; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D019713:Receptors, CCR5 / Q000378:metabolism*; D013601:T-Lymphocytes / Q000378:metabolism
chemical_list: D018952:Antigens, CD34; D017252:CD3 Complex; D011948:Receptors, Antigen, T-Cell; D019713:Receptors, CCR5; D049452:Green Fluorescent Proteins; D003851:Deoxyribonucleases