Enhancing titers of therapeutic lentiviral vectors using PKC agonists

Enhancing titers of therapeutic lentiviral vectors using PKC agonists

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 d...

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Main Authors: Charles Moore-Kelly, Rajesh Reddem, Ben M. Alberts, Jordan Wright, Thomas Evans, Anurag Kulkarni, Nicholas G. Clarkson, Daniel C. Farley, Kyriacos A. Mitrophanous, Rui André Saraiva Raposo
Format: Article
Language:English
Published: Elsevier 2025-06-01
Series:Molecular Therapy: Methods & Clinical Development
Subjects:
lentiviral vector
LV manufacturing
protein kinase C
PKC
PKC agonist
bioprocessing
Online Access:http://www.sciencedirect.com/science/article/pii/S2329050125000798
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Summary: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.
ISSN:2329-0501

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