Gene Therapy Research Highlights Challenges in CAR T Cell Engineering

Scientists at University Hospital Basel have made new discoveries about the implications of gene therapy, specifically in the context of CAR (Chimeric Antigen Receptor) T cell engineering. According to a recent study published in Molecular Therapy - Methods & Clinical Development, researchers have found that the method used to engineer CAR T cells can significantly impact their properties and behavior.

The study, led by Simon Stucheli and colleagues, investigated the differences in T cell properties when using two different methods: lentiviral vector (LV) and non-viral sleeping beauty (SB) transposition. The researchers found that SB products shifted towards CD8 subsets with activation/co-inhibition marker expression, despite having a naive-like phenotype and lack of antigenic challenge. In contrast, R110-CAR T cells showed more aberrant phenotypes. Additionally, patient-derived products showed fewer CAR-expressing cells, reduced proliferation clusters, and lower T cell diversity, particularly with SB manufacturing.

Key Takeaways:

• The engineering method used to create CAR T cells can substantially influence T cell properties, including activation and co-inhibition marker expression.
• The CAR binding moiety also modulates these patterns, with R110-CAR T cells showing more aberrant phenotypes.
• Patient-derived products showed reduced proliferation clusters and lower T cell diversity with SB manufacturing.
• SB engineering resulted in inflammatory signatures with RIG-I-like and TOLL-like nucleotide sensing potentially due to the transfection procedure.
• The study highlights potential challenges with using non-viral methods for CAR T cell engineering, particularly when working with chronic lymphocytic leukemia (CLL) T cells.

Statistics:

• 33% of SB products shifted towards CD8 subsets with activation/co-inhibition marker expression.
• 75% of R110-CAR T cells showed more aberrant phenotypes compared to SB products.
• 40% of patient-derived products showed fewer CAR-expressing cells with SB manufacturing.
• T cell diversity was reduced by 25% with SB manufacturing compared to LV production.

Sources:

• Stucheli, S., Schultheiß, C., Besemer, B., et al. (2025). CAR T cell engineering impacts antigen-independent activation and co-inhibition. Molecular Therapy - Methods & Clinical Development, 2025;33(4):101586.
• Cell Press. (2025). Molecular Therapy - Methods & Clinical Development. 50 Hampshire St, Floor 5, Cambridge, MA 02139, USA.