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Chimeric antigen receptor T cell (CAR-T) therapy is a significant and costly immunotherapy strategy that has provided benefits to many patients with cancer and autoimmune diseases. As of January 2025, only a few FDA- and NMPA-approved CAR-T therapies were available for clinical use, targeting CD19 or B cell maturation antigen (BCMA) on cancer cells. The manufacturing process for these approved CAR-T cells mainly relies on lentiviral vectors (LVVs) due to their high efficiency in transducing functional genes in both dividing and nondividing cells, as they have the unique ability to integrate into the genomes of those types of cells stably, and post-mitotic mammalian cells, a capability that ɣ-retroviruses do not possess to the same extent. This review summarizes the main CAR-T therapies derived from LVVs, the basic biological principles of HIV derived LVVs, the LV structure, capacities, and functions in biotechnology, a comparison between different vectors, the CAR T structure, and a summary of manufacturing processes, with an emphasis on the microbiological perspective for human and environmental biosafety. Additionally, we outline the latest developments in LVV technology, providing insights into the production of next-generation CAR-T therapies by using in vivo approaches.