704. Enhanced Lentiviral Production Through Rational Design of Mammalian Host Cells

Abstract

The development of lentiviral-based therapeutics is hindered by the high costs of cGMP manufacturing, which is a particular concern for applications focused on in vivo delivery where both high titres and volumes are typically required. The key cost component of viral vector manufacturing is the production titre, which is typically several log-orders lower for lentiviral vectors than for non-enveloped vectors such as rAd or rAAV. Some progress has been made towards the generation of producer cell lines to reduce costs. However, the majority of effort to increase lentiviral production has focused on optimisation and scale-up of transient transfection-based processes, including: the selection of efficient gene transfer reagents, the ratio of plasmid DNA components, the composition of cell culture media, supplementation with growth substrates and bioreactor growth conditions (e.g. pH, O2, lactate, etc). Intriguingly, little attention has been paid to the optimisation of the mammalian host cell, with nearly all reported processes relying on commonly available and, readily transfectable, HEK 293T derivatives. We hypothesised that HEK 293T cells were not necessarily optimal for lentiviral production and have embarked on a rational design process to establish cell lines with enhanced manufacturing properties. General reviews of the HIV infectious life cycle typically describe only four host factors that limit lentiviral replication (APOBEC3G, SAMHD1, Tetherin and TRIM5α). We conducted an extensive literature review and identified a further 130 putative restriction factors active in the late phase of the lentiviral life cycle (from transcription to maturation) that are possibly relevant to lentiviral vector production. We have evaluated the effect of siRNA knock-down of these genes (3 days production, 2e5 HEK 293 cells/well, 100 nM siRNA/well in quadruplicate) on lentiviral vector production titre (3 days transduction, 5e4 HEK 293 cells/well, eGFP detection via Perkin Elmer Operetta) in a high-throughput 96-well format. Control studies with a non-target siRNA revealed that the assay variability was less than 0.2-fold (n=40). We showed that siRNA knockdown of the four canonical host restriction factors had no significant effect on lentiviral production (1.06 to 0.68-fold, n=4-23, p > 0.05). We identified 9 host factors, the knockdown of which significantly increased lentiviral production by 1.4 to 2.1-fold (threshold > 2 SD over control; p 80%). To extend the utility of these findings we are employing CRISPR/Cas9-mediated genetic disruption of these host factors. We anticipate that permanent disruption, either singly or synergistically in combination, will yield novel mammalian cell lines with a greater ability to support lentiviral production, which will be of considerable value to ongoing efforts to reduce lentiviral production costs and increase the speed of clinical development.