Genotoxicity Associated with Retroviral CAR Transduction of ATM-Deficient T Cells

Abstract

Abstract Somatic variants in DNA damage response genes such as ATM are widespread in hematologic malignancies. ATM protein is essential for double-strand DNA break repair. Germline ATM deficiencies underlie ataxia–telangiectasia (A–T), a disease manifested by radiosensitivity, immunodeficiency, and predisposition to lymphoid malignancies. Patients with A–T diagnosed with malignancies have poor tolerance to chemotherapy or radiation. In this study, we investigated chimeric antigen receptor (CAR) T cells using primary T cells from patients with A–T (ATM−/−), heterozygote donors (ATM+/−), and healthy donors. ATM−/− T cells proliferate and can be successfully transduced with CARs, though functional impairment of ATM−/− CAR T-cells was observed. Retroviral transduction of the CAR in ATM−/− T cells resulted in high rates of chromosomal lesions at CAR insertion sites, as confirmed by next-generation long-read sequencing. This work suggests that ATM is essential to preserve genome integrity of CAR T-cells during retroviral manufacturing, and its lack poses a risk of chromosomal translocations and potential leukemogenicity. Significance: CAR T-cells are clinically approved genetically modified cells, but the control of genome integrity remains largely uncharacterized. This study demonstrates that ATM deficiency marginally impairs CAR T-cell function and results in high rates of chromosomal aberrations after retroviral transduction, which may be of concern in patients with DNA repair deficiencies.