title: Homology-Directed Recombination for Enhanced Engineering of Chimeric Antigen Receptor T Cells.
abstract: Gene editing by homology-directed recombination (HDR) can be used to couple delivery of a therapeutic gene cassette with targeted genomic modifications to generate engineered human T cells with clinically useful profiles. Here, we explore the functionality of therapeutic cassettes delivered by these means and test the flexibility of this approach to clinically relevant alleles. Because CCR5-negative T cells are resistant to HIV-1 infection, CCR5-negative anti-CD19 chimeric antigen receptor (CAR) T cells could be used to treat patients with HIV-associated B cell malignancies. We show that targeted delivery of an anti-CD19 CAR cassette to theCCR5locus using a recombinant AAV homology template and an engineered megaTAL nuclease results in T cells that are functionally equivalent, in both in vitro and in vivo tumor models, to CAR T cells generated by random integration using lentiviral delivery. With the goal of developing off-the-shelf CAR T cell therapies, we next targeted CARs to the T cell receptor alpha constant (TRAC) locus by HDR, producing TCR-negative anti-CD19 CAR and anti-B cell maturation antigen (BCMA) CAR T cells. These novel cell products exhibited in vitro cytolytic activity against both tumor cell lines and primary cell targets. Our combined results indicate that high-efficiency HDR delivery of therapeutic genes may provide a flexible and robust method that can extend the clinical utility of cell therapeutics.
authors.affiliation: Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA 98101, USA; 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: bluebird bio, Seattle, WA 98102, USA.
authors.forename: Baeckseung
authors.initials: B
authors.lastname: Lee
authors.affiliation: Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA 98101, USA; Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA.
authors.forename: Yuchi
authors.initials: Y
authors.lastname: Honaker
authors.affiliation: bluebird bio, Seattle, WA 98102, USA.
authors.forename: Wai-Hang
authors.initials: WH
authors.lastname: Leung
authors.affiliation: Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA 98101, USA; Department of Immunology, University of Washington, Seattle, WA 98101, USA.
authors.forename: Alexandra E
authors.initials: AE
authors.lastname: Grier
authors.affiliation: Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA 98101, USA.
authors.forename: Holly M
authors.initials: HM
authors.lastname: Jacobs
authors.affiliation: Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA 98101, USA; 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, Seattle Children's Research Institute, Seattle, WA 98101, USA; 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: Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA 98101, USA; Department of Pediatrics, University of Washington, Seattle, WA 98101, USA.
authors.forename: Shaun W
authors.initials: SW
authors.lastname: Jackson
authors.affiliation: Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA 98101, USA; 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: bluebird bio, Seattle, WA 98102, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Astrakhan
authors.affiliation: Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA 98101, USA; 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
keywords: BCMA; CCR5; CD19; T cell; TCR; cell therapy; chimeric antigen receptor; gene editing