Development And Optimization Of In-House Serum Free Suspension-Adapted 293T Cell Line For Lentiviral Vector Production To Improve Cell & Gene Therapy Manufacturing

By Joshua Gutierrez, MA, Chakrapani Tripathi, PhD, Sergey Zolov, MA, PhD, Frank Luh, MD, MHA, MS, and Venkatesh Natarajan, MS, PhD

Lentiviral (LV) vectors hold immense potential for cell and gene therapy due to their efficient gene delivery capabilities. Traditionally, adherent HEK293T cells are transiently transfected with lentiviral plasmids to generate LV particles. This approach suffices for preclinical research. However, large-scale production for clinical or commercial applications using adherent cells presents significant challenges, including scalability and cost-effectiveness.

To address these limitations, our team at Theragent Inc. successfully adapted adherent HEK293T cells to suspension culture in a serum-free lentiviral production medium (SFM). These adapted cells demonstrated comparable or superior LV titers compared to commercially available production cell lines. Furthermore, we optimized the production process, eliminating the need for expensive commercial transfection reagents, enhancers, and packaging plasmids. This streamlined protocol not only reduces production costs but also minimizes processing time. Notably, the adapted cell line was validated for the production of clinically relevant lentiviral vectors encoding chimeric antigen receptors (CAR) and fluorescent proteins. Theragent currently leverages this technology to offer high-titer LV production services, accelerating cell and gene therapy research for academic institutions and advanced therapy developers. Learn more about how this adapted cell line can reduce costs and improve scalability in the production of lentiviral vectors for cell and gene therapy and examine its potential for further optimization to produce GMP-grade lentiviral vectors for clinical applications.