Generation of a packaging cell line for the production of gamma retroviral vectors

Abstract

The usage of chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of hematological malignancies, but its application still faces logistical challenges. These challenges, involve the complex coordination required between hospitals and pharmaceutical companies, resulting in longer production times and high costs. Academic hospitals would therefore benefit from the production of in-house CAR-T cell therapies. Gamma retroviral vectors are well-studied vectors for the transduction of T cells. The production of gamma retroviral vectors is preferred in a packaging cell line due to scalability and simplified production processes. In this study it was aimed to generate a packaging cell line for the production of gamma retroviral vectors at academic hospital sites. We transfected HEK293T cells with helper expression plasmids pRD114.EGFP.PuroR and pGagPol.mCherry.PuroR, followed by selection using Puromycin and sorting of eGFP and mCherry positive cells through fluorescent activated cell sorting (FACS). This process lead to the production of our HEK293T-PACK cell line. Our HEK293T-PACK cell line generated gamma retroviral vector particles that effectively transduced Jurkat cells and outperformed those produced by the commercially available 293Vec-RD114 packaging cell line. This indicates that our packaging cell line holds the potential for large-scale production of gamma retroviral vector for CAR-T cell therapy.