Аннотация
Viral vectors are indispensable for advanced therapeutics such as chimeric T cell antigen receptor (CAR-T) therapy. CAR-T is a revolutionary treatment for blood cancers in patients who have relapsed or refractory disease after standard therapy. The therapeutic success is driving CAR-T's wider adoption, but the prospects for wider use are currently limited by high price, including the price of a packaged CAR vector, and the limited supply of such a vector on the market. The production of large quantities of packaged lentiviral vector using transfection with multiple plasmids in academic settings is associated with technical difficulties. Here we describe a technology for packaging a lentiviral vector using autonomously replicating RNAs that are delivered to production cultures by infection with replicon particles. Chimeric vectors capable of cytoplasmic replication were assembled using a replicative backbone from the RNA-virus Venezuelan equine encephalitis virus, into which the HIV-1 sequences for genomic integration and the CAR transgene were cloned. These vectors are packaged into high-titered infectious alphavirus replicon particles, allowing subsequent infection of large culture volumes to produce lentivirus particles. Using this method, enough vector was generated to produce therapeutic amounts of CAR+ cells in an academic setting.