Enhancing titers of therapeutic lentiviral vectors using PKC agonists

Abstract

Lentiviral vector (LV)-based therapies employ the molecular machinery of HIV-1 to stably integrate therapeutic genes into patient cells for long-term disease correction. However, suboptimal expression of LV components in HEK293T-based production systems can limit titers and hinder clinical product development. Here, we identify protein kinase C (PKC) agonists as robust enhancers of LV production. PKC activation resulted in rapid transcription of LV genomic RNA and accelerated vector particle release in a manner that complemented the use of the histone deacetylase (HDAC) inhibitor, sodium butyrate. Stimulation of HEK293T cells strongly upregulated AP-1 transcription factor subunits independently of nuclear factor κB (NF-κB) pathway activation. Application of PKC agonists in LV production resulted in a ∼3-fold improvement in the titer of a chimeric antigen receptor (CAR)-LV. Furthermore, a ∼9-fold increase in titer was achieved when this induction method was combined with co-expression of an LV RNA-targeted U1 snRNA enhancer. Importantly, LV produced using PKC agonists had comparable particle-to-infectivity ratios and preserved T cell transduction efficiency. These findings suggest that incorporating PKC agonists into commercial LV manufacturing could considerably reduce the cost per patient dose of new LV-based gene therapies.

Keywords: LV manufacturing; PKC; PKC agonist; U1 snRNA-based lentiviral vector enhancer; bioprocessing; chimeric antigen receptor T cell therapy; gene therapy; lentiviral vector; protein kinase C.

Graphical abstract

Figure 1

PKC agonists increase LV titers in HEK293T-derived production cells (A) Fold change in functional titer of LV-CMV-GFP produced in suspension-adapted HEK293T using PKC agonists phorbol myristate 13-acetate (red: 250 pM–256 nM), ingenol-3-angelate (blue: 3.9 nM–4 μM), bryostatin-1 (green: 10 nM–2.56 μM), and prostratin (purple: 156 nM–40 μM). PKC agonist or 0.2% (v/v) DMSO was added to media immediately after 10 mM NaBu for all conditions. (B) Impact of residual PKC agonist on reported transduced cells. Percentage of GFP⁺ HEK293T was determined by flow cytometry three days post-transduction with vector reference control (LV-CMV-GFP) spiked with 16 nM I3A, 250 nM I3A, 0.2% (v/v) DMSO, or untreated (NT). (C) Effect of PKC inhibitor BIM-I on LV-CMV-GFP titer. Transfected cells were pre-treated with 2 μM BIM-I (red) or 0.1% (v/v) DMSO (blue) for 1 h prior to the addition of inducing agents. LV was harvested 24 h after the addition of 0.2% (v/v) DMSO, 10 mM NaBu + 0.2% (v/v) DMSO, 250 nM I3A, or 10 mM NaBu + 250 nM I3A. Functional titers were determined by flow cytometry of HEK293T three days post-transduction. Statistical significance was established using Welch’s t test: ∗∗p < 0.01 and ∗∗∗p < 0.001; ns, not significant. All data are mean average values ± SD of biological triplicates (n = 3).

Figure 2

Time course analysis of LV-CMV-GFP production in suspension-adapted HEK293T (A) Functional LV titer produced using the following induction conditions: 0.2% (v/v) DMSO (gray), 10 mM NaBu + 0.2% (v/v) DMSO (green), 250 nM I3A (blue), and 10 mM NaBu + 250 nM I3A (red). (B) HIV p24 capsid quantification measured by p24 ELISA. (C) Particle-to-infectivity ratio derived from functional titer and physical viral particles. (D) Cellular vRNA expression relative to RPPH1 mRNA determined by RT-qPCR and normalized to pre-induction values. (E) Representative western blot showing cellular expression of viral structural proteins using anti-p24 and anti-VSV-G antibodies. Gag-Pol cleavage products: p55 (Gag precursor), p39 (matrix-capsid), p24 (capsid), and envelope protein VSV-G are indicated. β-actin was used as loading control. (F and G) Changes in cellular expression of (F) p55 and (G) VSV-G relative to β-actin and normalized to pre-induction values, as determined by quantification of western blot in (E) and Figure S2. (H) Transgene protein expression level in production cells measured by flow cytometry. (I and J) Viability (I) and aggregation (J) of production cells. (K) Cell cycle analysis of end-of-production cells showing percentage of cells in G₁ (blue), S (gray), and G₂ (green). (L) Residual dsDNA in vector harvest measured using a PicoGreen assay showing pre-Benzonase- (solid lines) and post-Benzonase-treated (dashed lines) cell culture supernatant. (M) Functional titer of Benzonase-treated Mustang Q purified LV determined by flow cytometry of HEK293T cells three days post-transduction. (N) Transduction efficiency of T cells from two donors determined by flow cytometry 5 days post-transduction at MOIs 1, 2, and 5. All data are mean average values ± SD of biological triplicates (n = 3).

Figure 3

PKC treatment upregulates cellular immediate-early genes during LV production (A–F) Label-free quantitative peptide mass spectrometry of suspension-adapted HEK293T during production of LV-CMV-GFP. Volcano plots showing differentially expressed proteins in production cells collected 8 h (top) and 22 h (bottom) after treatment with 10 mM NaBu + 0.2% (v/v) DMSO (left), 250 nM I3A (middle), or 10 mM NaBu + 250 nM I3A (right). Changes are shown with respect to vehicle (0.2% [v/v] DMSO)-treated cells collected at the same respective time points. Highlighted proteins are shown with |log₂ (FC)| > 1 and −log₁₀ (FDR) > 1.3 (n = 3). (G and H) STRING network association graphs of up- (red) and downregulated (blue) human proteins in production cell pellets 8 h after induction with (G) I3A and (H) NaBu + I3A. Protein candidates were selected on basis of |log₂ (FC)| > 1 and −log₁₀ (FDR) > 1.3 (19 and 41 protein candidates, respectively). Node color illustrates fold change with respect to DMSO-treated cells, and line thickness illustrates confidence of supporting data (interaction score cut-off >0.4). (I) Western blot probing for cellular expression of AP-1 proteins JUN, JUNB, and FOS at 8 h post-induction with 0.2% (v/v) DMSO, 10 mM NaBu + 0.2% (v/v) DMSO, 250 nM I3A, or 10 mM NaBu + 250 nM I3A (n = 1).

Figure 4

PKC activation induces the production of therapeutic CAR LV and synergizes with a modified U1 snRNA-based LV enhancer to increase titers (A and B) Integrating titer of (A) LV-EF1α-GFP and (B) LV-EF1α-CAR.5T4 harvested 23 h post-induction. LV was produced in suspension-adapted HEK293T using 3^rd generation LV plasmids ± co-transfection with a plasmid coding for the retargeted U1 snRNA enhancer “256U1.” The following conditions were used for induction: 0.2% (v/v) DMSO (gray), 10 mM NaBu + 0.2% (v/v) DMSO (green), 250 nM I3A (blue), and 10 mM NaBu + 250 nM I3A (red). Integrating titers were determined by duplex qPCR assay probing for Ψ and RPPH1 10 days post-transduction of HEK293T cells. (C and D) HIV p24 capsid titer of (C) LV-EF1α-GFP and (D) LV-EF1α-CAR.5T4 measured by p24 ELISA. (E and F) Particle-to-infectivity ratio of (E) LV-EF1α-GFP and (F) LV-EF1α-CAR.5T4 derived from integrating titer and physical virus particles. Statistical significance was established using Welch’s t test: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; ns, not significant. All data are mean average values ± SD of biological triplicates (n = 3).

Figure 5

Phosphopeptide mass spectrometry of suspension-adapted HEK293T during production of LV-EF1α-CAR.5T4 (A) Functional titer of LV-EF1α-CAR.5T4 harvested 24 h after induction with 10 mM NaBu + 0.2% (v/v) DMSO (blue) or 10 mM NaBu + 10 μM prostratin (red). LV was produced in suspension-adapted HEK293T transfected with 3^rd generation LV plasmids and retargeted U1 snRNA enhancer “256U1.” Data are mean average values ± SD of biological triplicates (n = 3). Statistical significance was established using Welch’s t test: ∗∗p < 0.01. (B–D) Volcano plots showing differentially phosphorylated proteins (B) 10 min, (C) 60 min, and (D) 360 min after treatment with NaBu + prostratin. Changes are shown with respect to NaBu + DMSO treated cells collected at the same respective time points. Highlighted phosphorylation sites are shown with |log₂ (FC)| > 3 and log₁₀ (FDR) > 1.3 (n = 3). (E and F) (E) GO biological process and (F) GO molecular function enrichment analysis of phosphorylated protein candidates (|log₂ (FC)| > 3 and −log₁₀ (FDR) > 1.3) showing top 20 significantly enriched terms (left) and number of proteins detected for each term (right). (G) Kinase substrate enrichment analysis showing kinase Z score (left) and corresponding number of identified kinase substrates (right). Analysis was performed using PhosphoSitePlus and NetworKIN datasets with p value <0.05 and substrate count >5. Kinases with a |Z score| > 2 at one or more time points are shown. (H) Protein-protein interaction enrichment analysis of differentially phosphorylated protein (|log₂ (FC)| > 5 and −log₁₀ (FDR) > 1.3) collated from all collected time points (264 total protein candidates) using the Metascape Webb App with the following databases: STRING, BioGrid, OmniPath, and InWeb_IM. The molecular complex detection (MCODE) algorithm has been applied to identify densely connected network components. Pathway and process enrichment analysis has been applied to each MCODE component independently, and the three best-scoring terms by p value have been retained as the functional description of the corresponding components.

Figure 6

LV production induced by PKC agonist is independent of NF-κB activation (A) Fold change in NanoLuc NF-κB response element luciferase activity during production of LV-CMV-GFP in suspension-adapted HEK293T (blue, left axis). Luciferase activity was measured 5 h after stimulation of cells with 0.2% (v/v) DMSO, 10 mM NaBu + 0.2% (v/v) DMSO, 250 nM I3A, 10 mM NaBu + 250 nM I3A, PBS, or 20 ng/mL hTNF-α. Corresponding functional titers of LV harvested 22 h post-induction are shown (red, right axis). (B) Functional titers of LV-CMV-GFP harvested 22 h post-induction with 0.2% (v/v) DMSO, 10 mM NaBu + 0.2% (v/v) DMSO, 250 nM I3A, or 10 mM NaBu + 250 nM I3A. All conditions were treated with 20 ng/mL hTNF-α (red) or PBS (blue) immediately after induction. Functional LV titers were determined by flow cytometry of HEK293T three days post-transduction. Statistical significance was established using Welch’s t test: ∗p < 0.05 and ∗∗∗p < 0.001; ns, not significant. All data are mean average values ± SD of biological triplicates (n = 3).