Objective:

To improve the efficiency of generating CAR T cells with large lentiviral transgenes exceeding 10 kilobases, addressing a significant manufacturing challenge.

Key Findings:

• Transduction efficiency for the largest construct (10.1 kb) increased by 10-12 fold.
• Functional viral titers improved by about an order of magnitude.
• T-cell transduction rates increased by up to 14.8-fold compared to conventional methods.
• Routine transduction rates of 60-70% for ~9 kb constructs and 15-20% for 10 kb vectors were achieved.

Interpretation:

The optimized workflow addresses a significant manufacturing bottleneck in CAR T cell therapy, enabling the use of larger genetic constructs without the need for cell sorting, potentially impacting clinical translation and reducing manufacturing costs.

Limitations:

• The study primarily focuses on CAR T cells, and further validation is needed for other immune cell types, such as natural killer cells or macrophages.

Conclusion:

This workflow could enhance the development of complex CAR T cell designs and potentially reduce manufacturing costs for lentivirus-modified cell therapies.