Woodruff, Rosie;
Parekh, Farhaan;
Lamb, Katarina;
Mekkaoui, Leila;
Allen, Christopher;
Smetanova, Katerina;
Huang, Jasmine;
... Pule, Martin; + view all
(2023)
Large-scale manufacturing of base-edited chimeric antigen receptor T cells.
Molecular Therapy: Methods & Clinical Development
, 31
, Article 101123. 10.1016/j.omtm.2023.101123.
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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.
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