Investigating dynamics of lentiviral vector secretion from HEK293T producer cells using a fractionated perfusion system.

Mammalian cell culture is quickly becoming the go to engineering vehicle to mass produce viral vectors in a manner that is safe, convenient, reproducible, and cost and scale effective. Human embryonic kidney (HEK293) cells, in particular, have been utilized and customized (via differentiated transgene expression, modified culture parameters, addition of cytostatic culture agents) to increase vector yields. However, less attention has been made to understanding innate processes within the cells (such as, immune response, cell cycle, metabolism) themselves to better control or increase viral vector product yield. Accordingly, herein, we study the variation in viral production from HEK cells over time using a one-way perfusion system and bioreactor to study the impact of external factors on secretion dynamics without retrotransduction. Specifically, we study the impact of cell density on viral titer, transduction efficiency, and LDH. Next, we look at the impact of using an inflammatory reporter cell line on viral output, and the secretion dynamics from HEK cells when we use sodium butyrate (cell cycle arrest agent). Lastly, we assess how downregulation of the PDK pathway increases viral titer. Altogether, we investigated the impact of various interventions to increase transient protein expression and viral output from HEK cells in a controlled and measurable environment to ultimately increase the efficiency of HEK cells for downstream clinical applications. This article is protected by copyright. All rights reserved.