Laboratory-scale lentiviral vector production and purification for enhanced ex vivo and in vivo genetic engineering

Abstract Lentiviral vectors (LVs) are increasingly employed in gene and cell therapy. Standard laboratory production of LV is not easily scalable and research-grade LVs often contain contaminants that can interfere with downstream applications. Moreover, purified LV production pipelines have been developed mainly for costly, large-scale clinical-grade settings. Therefore, a standardized and cost-effective process is still needed to obtain efficient, reproducible and properly executed experimental studies and pre-clinical development of ex vivo and in vivo gene therapies, as high infectivity and limited adverse reactions are important factors potentially influencing experimental outcomes also in pre-clinical settings. We describe here an optimized laboratory-scale workflow whereby LV-containing supernatant is purified and concentrated by sequential chromatographic steps, obtaining biologically active LVs with infectious titer and specific activity in the order of 109 Transducing Unit (TU)/ml and 5x104 TU/ng of HIV Gag p24, respectively. The purification workflow removes >99% of the starting plasmid, DNA and protein impurities resulting in higher gene transfer and editing efficiency in SCID-repopulating hematopoietic stem and progenitor cells (HSPCs) ex vivo as well as reduced activation of inflammatory responses ex vivo and in vivo as compared to TU-matched laboratory-grade vectors. Our results highlight the value of accessible purified LV production for experimental studies and pre-clinical testing.