Ann Macdonald, Senior Business Development Manager at Thermo Fisher discussed the importance of non-viral engineering and how scientists can use automated instruments to optimize non-viral engineering techniques for resting or naive non-proliferative T cells.
Macdonald and her team focused on these T cells due to their potential to improve CAR-T therapy, she added: “We identify the naive and stem cell memory as being the ones that are most capable of longevity and perhaps higher tumorigenicity or anti-tumorigenicity by the marker CD62L and CD45RA.”
However, since naïve T cells are unstimulated and non-dividing, they are difficult to transfect. The research focuses on leveraging Thermo Fisher’s CTS Xenon electroporation device to overcome this challenge. The CTS Xenon gives scientists a great design space to optimise their transfection conditions at scale, Macdonald commented: “The Xenon has a single shot cassette for one mil, but it also then has a multi-shot which will do up to 2.5 billion T cells in a 25-mil volume over the course of about 25 minutes.”
Furthermore, introducing low-conductivity buffers also enhanced energy transfection. Macdonald’s research showed that adding a buffer supported high-efficiency gene editing and transfection without compromising cell viability or phenotype. Experiments showed successful mRNA and plasmid delivery with efficiencies ranging from 40% to 80% and demonstrated the potential for gene knockouts and knock-ins, although further optimization is needed for therapeutic applications.
The results showed that non-viral approaches provide a feasible solution for engineering these difficult cell populations and facilitating a more streamlined, shorter vein-to-vein CAR-T manufacturing process.
Macdonald explained that future work will centre on refining post-editing purification and exploring methods to enhance edited cell expansion for clinical use. To sum up, this work represents significant progress in adopting non-viral techniques to overcome the limitations of traditional CAR-T workflows.
