Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells

Mol Ther. 2017 Apr 5;25(4):949-961. doi: 10.1016/j.ymthe.2017.02.005. Epub 2017 Feb 23.


Adoptive cellular therapy using chimeric antigen receptor (CAR) T cell therapies have produced significant objective responses in patients with CD19+ hematological malignancies, including durable complete responses. Although the majority of clinical trials to date have used autologous patient cells as the starting material to generate CAR T cells, this strategy poses significant manufacturing challenges and, for some patients, may not be feasible because of their advanced disease state or difficulty with manufacturing suitable numbers of CAR T cells. Alternatively, T cells from a healthy donor can be used to produce an allogeneic CAR T therapy, provided the cells are rendered incapable of eliciting graft versus host disease (GvHD). One approach to the production of these cells is gene editing to eliminate expression of the endogenous T cell receptor (TCR). Here we report a streamlined strategy for generating allogeneic CAR T cells by targeting the insertion of a CAR transgene directly into the native TCR locus using an engineered homing endonuclease and an AAV donor template. We demonstrate that anti-CD19 CAR T cells produced in this manner do not express the endogenous TCR, exhibit potent effector functions in vitro, and mediate clearance of CD19+ tumors in an in vivo mouse model.

Keywords: chimeric antigen receptor; gene editing; homology-directed repair.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • Alleles
  • Animals
  • Antigens, CD19 / genetics*
  • Batch Cell Culture Techniques*
  • Cell Engineering*
  • Dependovirus / genetics
  • Disease Models, Animal
  • Gene Editing*
  • Gene Expression
  • Gene Knockout Techniques
  • Gene Order
  • Genetic Loci
  • Genetic Vectors / genetics
  • Humans
  • Immunotherapy, Adoptive
  • Lymphoma / genetics
  • Lymphoma / immunology
  • Lymphoma / therapy
  • Mice
  • Neoplasms
  • Receptors, Antigen, T-Cell / genetics*
  • Receptors, Antigen, T-Cell, alpha-beta / genetics*
  • T-Lymphocytes / immunology*
  • T-Lymphocytes / metabolism*
  • Transduction, Genetic


  • Antigens, CD19
  • CD19-specific chimeric antigen receptor
  • Receptors, Antigen, T-Cell
  • Receptors, Antigen, T-Cell, alpha-beta