TY - JOUR
T1 - Laboratory-Scale Lentiviral Vector Production and Purification for Enhanced Ex Vivo and In Vivo Genetic Engineering
AU - Soldi, Monica
AU - Sergi Sergi, Lucia
AU - Unali, Giulia
AU - Kerzel, Thomas
AU - Cuccovillo, Ivan
AU - Capasso, Paola
AU - Annoni, Andrea
AU - Biffi, Mauro
AU - Rancoita, Paola Maria Vittoria
AU - Cantore, Alessio
AU - Lombardo, Angelo
AU - Naldini, Luigi
AU - Squadrito, Mario Leonardo
AU - Kajaste-Rudnitski, Anna
N1 - Funding Information:
We would like to thank Annapaola Andolfo and Cinzia Magagnotti from the ProMeFa facility (IRCCS San Raffaele Scientific Institute, Milan) for MS data acquisition; Luigi Gianolli for giving us the authorization to use the MCS instrument at the Department of Nuclear Medicine, San Raffaele Hospital; Maria Grazia Minotti and Cristina Monterisi for help with MCS usage; Tiziana Plati for help in setting the VSV.G ddPCR quantification; and Margherita Neri, Francesca Bellintani, and Simona La Seta Catamancio (MolMed S.p.A.) for helpful exchanges to set up the PDL workflow. This work was supported by the European Research Council (ERC-CoG 819815-ImmunoStem) and the Telethon Foundation (TELE20-C3) to AKR.
Funding Information:
We would like to thank Annapaola Andolfo and Cinzia Magagnotti from the ProMeFa facility (IRCCS San Raffaele Scientific Institute, Milan) for MS data acquisition; Luigi Gianolli for giving us the authorization to use the MCS instrument at the Department of Nuclear Medicine, San Raffaele Hospital; Maria Grazia Minotti and Cristina Monterisi for help with MCS usage; Tiziana Plati for help in setting the VSV.G ddPCR quantification; and Margherita Neri, Francesca Bellintani, and Simona La Seta Catamancio (MolMed S.p.A.) for helpful exchanges to set up the PDL workflow. This work was supported by the European Research Council (ERC-CoG 819815-ImmunoStem) and the Telethon Foundation (TELE20-C3) to AKR.
Publisher Copyright:
© 2020 The Author(s)
Copyright:
Copyright 2020 Elsevier B.V., All rights reserved.
PY - 2020/12/11
Y1 - 2020/12/11
N2 - 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. 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.
AB - 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. 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.
KW - ex vivo
KW - gene therapy
KW - hematopoietic stem cells
KW - in vivo
KW - innate immunity
KW - Lentiviral vectors
KW - manufacturing
KW - purification process
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U2 - 10.1016/j.omtm.2020.10.009
DO - 10.1016/j.omtm.2020.10.009
M3 - Article
AN - SCOPUS:85097176577
VL - 19
SP - 411
EP - 425
JO - Molecular Therapy - Methods and Clinical Development
JF - Molecular Therapy - Methods and Clinical Development
SN - 2329-0501
ER -