Real-time transfer of lentiviral particles by producer cells using an engineered coculture system
- 15 Downloads
Lentiviruses are quite effective gene delivery systems for stable production of genetically engineered human cells. However, prior to using lentivirus to deliver genetic materials to cells of interest, the normal course of production of these lentiviruses involves a lengthy collection, purification, preservation, and quantification process. In this report, we demonstrate the ability for producer HEK293T cells to simultaneously produce lentiviral particles and transduce (i.e., infect) target cells through a membrane-based coculture system in a continuous, real-time mode which negates the need for a separate viral collection and quantification process. The coculture system was evaluated for major design features such as variations in HEK293T seeding density, target cell type densities, as well as membrane porosities to identify key relationships between lentiviral particle production rate and infection kinetics for adherent and suspension cell types. As a proof-of-concept for the creation of an engineered cell immunotherapy, we describe the ability to engineer human T cells isolated from PBMCs under the control of this coculture system in under 6 days with a GFP construct. These studies suggest the capability to combine and more closely automate the transfection/transduction process in order to facilitate well-timed and cost-effective transduction of target cell types. These experiments provide novel insight into the forthcoming transition into improved manufacturing systems for viral production and subsequent cell engineering.
KeywordsLentivirus transduction Engineered cell therapy Scalable engineering systems
This work was supported in part by the National Institutes of Health Grants (R01GM127353 and R01EB012521).
LMT and BP formulated the research study design, LMT conducted experiments, acquired data, analyzed data, wrote manuscript. RP assisted in conducting experiments and acquiring data. MT assisted in conducting experiments.
- Ausubel L, Hall C, Sharma A et al (2012) Production of CGMP-grade lentiviral vectors. Bioprocess Int 10:32–43Google Scholar
- Germeraad WT, Asami N, Fujimoto S, Mazda O, Katsura Y (1994) Efficient retrovirus-mediated gene transduction into murine hematopoietic stem cells and long-lasting expression using a transwell coculture system. Blood 84:780–788Google Scholar
- Kumru O, Wang Y, Gombotz C, Wayne R, Kelley-Clarke B et al (2018) Physical characterization and stabilization of a lentiviral vector against adsorption and freeze–thaw. Pharm Biotechnol 107:2764–2774Google Scholar
- Rahman H, Taylor J, Clack B, Stewart RS, Canterberry SC (2013) Effects of storage conditions on the morphology and titer of lentiviral vectors. Faculty Publications. 94Google Scholar