Work

284. High-Efficiency Targeted Introduction of an Anti-CD19 CAR Into the CCR5 Locus in Primary Human T Cells

Year: 2015

Type: article

Abstract: The safety and efficacy of anti-CD19 chimeric antigen receptor (CD19-CAR) therapeutic cell transfer for treatment of CD19+ B cell malignancies is currently being evaluated in multiple clinical trials. Current protocols employ randomly integrating lentiviral or gamma-retroviral vectors to introduce the CAR into patient- or donor-derived T cells. This strategy carries with it an inherent risk of insertional mutagenesis as well as variable CAR expression. Here we have developed a method of targeting a CD19-CAR construct directly into the CCR5 locus by homology directed recombination (HDR). Using a CCR5 megaTAL nuclease (an engineered homing endonuclease and TALEN chimera) and an AAV donor template (comprised of an MND promoter-CD19CAR-T2A-BFP expression cassette between CCR5 homology arms), we obtained efficient targeting rates in primary human T cells. CAR expression was confirmed by flow cytometry and homologous recombination within the CCR5 locus was verified by PCR analysis and by direct sequencing. Rates of biallelic HDR were assessed using single cell PCR. T cells with the CD19-CAR at the CCR5 locus demonstrated activation, cytokine production and specific killing in the presence of CD19+ cells. Activity levels were indistinguishable from T cells transduced with LV expressing an identical CD19-CAR construct. Seamless targeting to CCR5 has important therapeutic advantages over traditional viral delivery, including a reduced potential for oncogenic insertions and uniform levels of gene expression, in parallel with effective disruption of the co-receptor for CCR5-tropic HIV-1. Further, the targeting methods used are directly amenable to clinical application. As HIV patients develop aggressive B cell lymphomas at increased rates, we believe this technique has substantial potential for clinical implementation including the protection of autologous CD19-CAR effector T cell products from HIV infection in vivo.

Source: Molecular Therapy

DOI: 10.1016/s1525-0016(16)33893-x

Authors: Malika Hale, Guillermo S. Romano Ibarra, Iram F. Khan, Jaya Sahni, Andrew M. Scharenberg, David J. Rawlings

Institutions: Seattle Children's Research Institute, University of Washington

Language: English

Topic: CAR-T cell therapy research

Subfield: Oncology

Field: Medicine

Domain: Health Sciences

SDG: Good health and well-being

Open Access status: hybrid

Estimated APC paid: $4,500