PKC agonists as small molecule inducing agents for enhancing lentiviral vector production
C Moore-Kelly(1) T M Evans(1) J Wright(1) D C Farley(1) K A Mitrophanous(1) N G Clarkson(1) R A Raposo(1)
1:Oxford Biomedica (UK) Ltd.
Achieving high upstream lentivector (LV) titres is fundamental to the development and manufacture of a commercially viable gene therapy product. Product titres can vary considerably with different therapeutic transgene sequences despite being produced in the same mammalian cell system. Implementing new technologies able to recover low titres or improve high titres further is invaluable.
Induction is a key stage of the upstream LV production process that typically involves increasing the expression of LV genes in production cells with the histone deacetylase inhibitor, sodium butyrate. A series of recent screening experiments revealed to us that titres of LV products can be further increased by using an additional class of molecules known as PKC agonists alongside the induction step.
Here, we describe how, through optimisation of dosing concentration and timing, LV titres can be enhanced 2- to 9-fold with the non-tumour promoting PKC agonists prostratin and ingenol 3-angelate in a product-specific manner. Importantly, LV produced using PKC agonists have comparable or superior particle-to-infectivity ratios, and residual PKC agonists are removed from the vector product following downstream processing. Furthermore, we demonstrate that PKC agonists act synergistically with an in-house technology based on LV RNA-targeted U1 snRNA to achieve log-fold-increases in titre without detriment to product quality attributes.
Having established the benefits of introducing PKC agonists to our platform process and in our packaging/producer cell lines, we provide insight into the mechanisms by which these agonists are acting on production cells and now intend to transfer this technology to GMP manufacturing for commercial products.