Small- to Large-Scale Production of Lentivirus Vectors

Lentiviral vectors have traditionally been produced in human embryonic kidney 293T cells (1 ), involving transient transfection procedures that were originally established for the production of retroviral vectors based on Moloney murine leukemia (MoMLV) virus (2 ). Simian virus 40 (SV40)-transformed African green monkey kidney (COS-7) cells (3 ) and human TE671 rhabdomyosarcoma cells (4 ) have also been used to generate human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors (5 –7 ), but titers obtained in 293T cells are generally higher than those observed in other cell lines. Page et al. (5 ) were the first to describe an HIV-1-based vector harboring a selectable transgene. The HIV-1 genome was rendered replication defective in this study by replacing the envelope (Env)-encoding gp160 sequence with a guanine-phosphoribosyl transferase (gpt ) gene driven by the SV40 early promoter. Transient cotransfection of COS-7 cells with this env -deleted vector and a gp160 expression vector resulted in packaging of the defective HIV-gpt genome into infectious virions. Upon infection of susceptible cells, the gpt drug resistance gene was transmitted and expressed, allowing transduced cells to be selected in mycophenolic acid (5 ). Landau et al. (8 ) subsequently demonstrated that expression of heterologous Env proteins, including the MoMLV Env and the human T-cell leukemia virus type I (HTLV-I) Env, in cells transfected with the HIV-gpt vector resulted in the production of vector pseudotypes capable of infecting human as well as murine cells with titers reaching 10⁵ colony-forming units (CFU) per mL.