Tags

Type your tag names separated by a space and hit enter

Lentiviral Vector Production Titer Is Not Limited in HEK293T by Induced Intracellular Innate Immunity.
Mol Ther Methods Clin Dev 2020; 17:209-219MT

Abstract

Most gene therapy lentiviral vector (LV) production platforms employ HEK293T cells expressing the oncogenic SV40 large T-antigen (TAg) that is thought to promote plasmid-mediated gene expression. Studies on other viral oncogenes suggest that TAg may also inhibit the intracellular autonomous innate immune system that triggers defensive antiviral responses upon detection of viral components by cytosolic sensors. Here we show that an innate response can be generated after HIV-1-derived LV transfection in HEK293T cells, particularly by the transgene, yet, remarkably, this had no effect on LV titer. Further, overexpression of DNA sensing pathway components led to expression of inflammatory cytokine and interferon (IFN) stimulated genes but did not result in detectable IFN or CXCL10 and had no impact on LV titer. Exogenous IFN-β also did not affect LV production or transduction efficiency in primary T cells. Additionally, manipulation of TAg did not affect innate antiviral responses, but stable expression of TAg boosted vector production in HEK293 cells. Our findings demonstrate a measure of innate immune competence in HEK293T cells but, crucially, show that activation of inflammatory signaling is uncoupled from cytokine secretion in these cells. This provides new mechanistic insight into the unique suitability of HEK293T cells for LV manufacture.

Authors+Show Affiliations

Molecular and Cellular Immunology Unit, Great Ormond Street Institute of Child Health, University College London, London WC1N 1EH, UK.Division of Infection and Immunity, University College London, London WC1E 6BT, UK.Division of Infection and Immunity, University College London, London WC1E 6BT, UK.Molecular and Cellular Immunology Unit, Great Ormond Street Institute of Child Health, University College London, London WC1N 1EH, UK.Division of Infection and Immunity, University College London, London WC1E 6BT, UK.Molecular and Cellular Immunology Unit, Great Ormond Street Institute of Child Health, University College London, London WC1N 1EH, UK. Great Ormond Street Hospital for Children NHS Foundation Trust, London WC1N 1EH, UK.Molecular and Cellular Immunology Unit, Great Ormond Street Institute of Child Health, University College London, London WC1N 1EH, UK. Great Ormond Street Hospital for Children NHS Foundation Trust, London WC1N 1EH, UK.Division of Infection and Immunity, University College London, London WC1E 6BT, UK.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31970199

Citation

Ferreira, Carolina B., et al. "Lentiviral Vector Production Titer Is Not Limited in HEK293T By Induced Intracellular Innate Immunity." Molecular Therapy. Methods & Clinical Development, vol. 17, 2020, pp. 209-219.
Ferreira CB, Sumner RP, Rodriguez-Plata MT, et al. Lentiviral Vector Production Titer Is Not Limited in HEK293T by Induced Intracellular Innate Immunity. Mol Ther Methods Clin Dev. 2020;17:209-219.
Ferreira, C. B., Sumner, R. P., Rodriguez-Plata, M. T., Rasaiyaah, J., Milne, R. S., Thrasher, A. J., ... Towers, G. J. (2020). Lentiviral Vector Production Titer Is Not Limited in HEK293T by Induced Intracellular Innate Immunity. Molecular Therapy. Methods & Clinical Development, 17, pp. 209-219. doi:10.1016/j.omtm.2019.11.021.
Ferreira CB, et al. Lentiviral Vector Production Titer Is Not Limited in HEK293T By Induced Intracellular Innate Immunity. Mol Ther Methods Clin Dev. 2020 Jun 12;17:209-219. PubMed PMID: 31970199.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Lentiviral Vector Production Titer Is Not Limited in HEK293T by Induced Intracellular Innate Immunity. AU - Ferreira,Carolina B, AU - Sumner,Rebecca P, AU - Rodriguez-Plata,Maria T, AU - Rasaiyaah,Jane, AU - Milne,Richard S, AU - Thrasher,Adrian J, AU - Qasim,Waseem, AU - Towers,Greg J, Y1 - 2019/12/24/ PY - 2019/07/24/received PY - 2019/11/18/accepted PY - 2020/1/24/entrez PY - 2020/1/24/pubmed PY - 2020/1/24/medline KW - HEK293T KW - Lentiviral vectors KW - SV40 large T-antigen KW - innate immunity KW - vector titer SP - 209 EP - 219 JF - Molecular therapy. Methods & clinical development JO - Mol Ther Methods Clin Dev VL - 17 N2 - Most gene therapy lentiviral vector (LV) production platforms employ HEK293T cells expressing the oncogenic SV40 large T-antigen (TAg) that is thought to promote plasmid-mediated gene expression. Studies on other viral oncogenes suggest that TAg may also inhibit the intracellular autonomous innate immune system that triggers defensive antiviral responses upon detection of viral components by cytosolic sensors. Here we show that an innate response can be generated after HIV-1-derived LV transfection in HEK293T cells, particularly by the transgene, yet, remarkably, this had no effect on LV titer. Further, overexpression of DNA sensing pathway components led to expression of inflammatory cytokine and interferon (IFN) stimulated genes but did not result in detectable IFN or CXCL10 and had no impact on LV titer. Exogenous IFN-β also did not affect LV production or transduction efficiency in primary T cells. Additionally, manipulation of TAg did not affect innate antiviral responses, but stable expression of TAg boosted vector production in HEK293 cells. Our findings demonstrate a measure of innate immune competence in HEK293T cells but, crucially, show that activation of inflammatory signaling is uncoupled from cytokine secretion in these cells. This provides new mechanistic insight into the unique suitability of HEK293T cells for LV manufacture. SN - 2329-0501 UR - https://www.unboundmedicine.com/medline/citation/31970199/Lentiviral_Vector_Production_Titer_Is_Not_Limited_in_HEK293T_by_Induced_Intracellular_Innate_Immunity L2 - https://linkinghub.elsevier.com/retrieve/pii/S2329-0501(19)30151-2 DB - PRIME DP - Unbound Medicine ER -