1. Large-scale manufacturing of base-edited chimeric antigen receptor T cells
- Author
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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, and Martin Pule
- Subjects
base editing ,BE4max ,chimeric antigen receptor ,circular RNA ,PD-1 ,TIM3 ,Genetics ,QH426-470 ,Cytology ,QH573-671 - 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 × 108 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 × 106 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.
- Published
- 2023
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