title: Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy.
abstract: Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period of pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells.
authors.affiliation: Laboratory of Biotechnology and Therapeutics, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Hotaka
authors.initials: H
authors.lastname: Kusabuka
authors.affiliation: Laboratory of Biotechnology and Therapeutics, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Kento
authors.initials: K
authors.lastname: Fujiwara
authors.affiliation: Laboratory of Biotechnology and Therapeutics, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Tokunaga
authors.affiliation: Laboratory of Biotechnology and Therapeutics, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Sachiko
authors.initials: S
authors.lastname: Hirobe
authors.affiliation: Laboratory of Biotechnology and Therapeutics, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address: nakagawa@phs.osaka-u.ac.jp.
authors.forename: Shinsaku
authors.initials: S
authors.lastname: Nakagawa
authors.affiliation: Laboratory of Biotechnology and Therapeutics, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address: okada@phs.osaka-u.ac.jp.
authors.forename: Naoki
authors.initials: N
authors.lastname: Okada
mesh_terms: D000818:Animals; D000941:Antigens / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology; D016207:Cytokines / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D005818:Genetic Engineering; D005822:Genetic Vectors; D019337:Hematologic Neoplasms / Q000628:therapy; D016219:Immunotherapy, Adoptive / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D041681:NIH 3T3 Cells; D011691:Puromycin / Q000737:chemistry; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D013601:T-Lymphocytes / Q000166:cytology*
chemical_list: D000941:Antigens; D016207:Cytokines; D011948:Receptors, Antigen, T-Cell; D011691:Puromycin
keywords: Chimeric antigen receptor; Gene transduction; Retroviral vector; T cell