Large-scale manufacturing of base-edited chimeric antigen receptor T cells
Rosie Woodruff, Farhaan Parekh, Katarina Lamb, Leila Mekkaoui, Christopher Allen, Katerina Smetanova, Jasmine Huang, Alex Williams, Gerardo Santiago Toledo, Koki Lilova, Claire Roddie, James Sillibourne, Martin Pulé
Abstract
Base editing is a revolutionary gene-editing technique enabling the introduction of point mutations into the genome without generating detrimental DNA double-stranded breaks. Base-editing enzymes are commonly delivered in the form of modified linear messenger RNA (mRNA) that is costly to produce. Here, we address this problem by developing a simple protocol for manufacturing base-edited cells using circular RNA (circRNA), which is less expensive to synthesize. Compared with linear mRNA, higher editing efficiencies were achieved with circRNA, enabling an 8-fold reduction in the amount of RNA required. We used this protocol to manufacture a clinical dose (1 × 10 8 cells) of base-edited chimeric antigen receptor (CAR) T cells lacking expression of the inhibitory receptor, PD-1. Editing efficiencies of up to 86% were obtained using 0.25 μg circRNA/1 × 10 6 cells. Increased editing efficiencies with circRNA were attributed to more efficient translation. These results suggest that circRNA, which is less expensive to produce than linear mRNA, is a viable option for reducing the cost of manufacturing base-edited cells at scale.