[HTML][HTML] Homology-directed recombination for enhanced engineering of chimeric antigen receptor T cells

M Hale, B Lee, Y Honaker, WH Leung, AE Grier… - … Therapy-Methods & …, 2017 - cell.com
M Hale, B Lee, Y Honaker, WH Leung, AE Grier, HM Jacobs, K Sommer, J Sahni…
Molecular Therapy-Methods & Clinical Development, 2017cell.com
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 …
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 the CCR5 locus 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.
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