Laboratory-Scale Lentiviral Vector Production and Purification for Enhanced Ex Vivo and In Vivo Genetic Engineering

• Published in: Molecular therapy. Methods & clinical development Vol. 19; pp. 411 - 425
• Main Authors: Soldi, Monica, Sergi Sergi, Lucia, Unali, Giulia, Kerzel, Thomas, Cuccovillo, Ivan, Capasso, Paola, Annoni, Andrea, Biffi, Mauro, Rancoita, Paola Maria Vittoria, Cantore, Alessio, Lombardo, Angelo, Naldini, Luigi, Squadrito, Mario Leonardo, Kajaste-Rudnitski, Anna
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 11.12.2020; American Society of Gene & Cell Therapy; Elsevier
• Subjects: ex vivo; gene therapy; hematopoietic stem cells; in vivo; innate immunity; Lentiviral vectors; manufacturing; Original; purification process; innate immunity; purification process; Lentiviral vectors; manufacturing; gene therapy; in vivo; ex vivo; hematopoietic stem cells
• ISSN: 2329-0501; 2329-0501
• DOI: 10.1016/j.omtm.2020.10.009

Lentiviral vectors (LVs) are increasingly employed in gene and cell therapy. Standard laboratory production of LVs 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 preclinical 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 preclinical settings. We describe here an optimized laboratory-scale workflow whereby an LV-containing supernatant is purified and concentrated by sequential chromatographic steps, obtaining biologically active LVs with an infectious titer and specific activity in the order of 109 transducing unit (TU)/mL and 5 × 104 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 severe combined immunodeficiency (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 preclinical testing. [Display omitted] Lentiviral vectors (LVs) are powerful gene-transfer tools routinely exploited for distinct research and clinical applications. LVs produced in most research laboratories contain contaminants that can generate confounding effects in experimental studies. Soldi et al. describe a laboratory-scale workflow for purified LV production, highlighting enhanced gene-editing efficiency and diminished inflammatory responses.