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.
Highlights
Lentiviral vectors (LVs) are used in some of the most successful gene therapies including correction of inherited blood, immune, and metabolic disorders, as well as in immunotherapy using chimeric antigen receptor redirected T cells (CAR-T cells)
HEK293T cells were co-transfected with LV producing plasmids and a panel of luciferase-encoding reporter constructs sensitive to activation by a variety of innate immune responses including type 1 IFN and transcription factors nuclear factor kB (NF-kB) and IRF3
We found that production of LV encoding GFP weakly induced synthetic NF-kB-sensitive reporter constructs bearing either NF-kB p50/p65 binding sites (5ÂNF-kB) or NF-kB binding sites from the immunoglobulin kappa light chain (Igk) promoter (LV-EV, Figure 1A)
Summary
Lentiviral vectors (LVs) are used in some of the most successful gene therapies including correction of inherited blood, immune, and metabolic disorders, as well as in immunotherapy using chimeric antigen receptor redirected T cells (CAR-T cells). Clinical LVs are typically produced by transient plasmidmediated co-expression of viral components in the HEK293T cell line.[1,2,3,4,5] Lentivirus production is a key limiting step in developing gene therapy as an efficient and cost-effective treatment. Innate immune activation caused by sensing of viral components could be an important factor in limiting maximal efficiency of LV production. Understanding whether vector components activate cell autonomous innate immune responses and whether these responses limit LV production is key to developing the most effective production strategies
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