citations

                    Clinical Scale Production of Circular mRNA for the Manufacture of Base-Edited CAR T-cell Therapies
                

Authors:

Rosie Woodruff1,2, James Sillibourne1, Martin Pule1,2, Claire Roddie2

In:

Source:
Publication Date: (2023)
Issue: 01: 01

Research Area:

Cancer Research/Cell Biology

Immunotherapy / Hematology

Gene Expression

Basic Research

Cells used in publication:

T cell, human stim.: Species: human; Tissue Origin: blood

Platform:

4D-Nucleofector® LV-Unit

Experiment

T-cells were selected prior to activation with TransAct and interleukins-7 and -15. 25x10^6 stimulated T-cells were co-transfected with the PD-1 guide RNA and 1.00 µg circular mRNA per 1x10^6 cells. 24 hours post-editing, T-cells were transduced with anti-CD19 CAR-encoding lentivirus at an MOI 5 by spinoculation in RetroNectin-coated bags. Base-edited CAR T-cells were expanded to >50x10^6 T-cells, prior to cryopreservation.

Abstract

Cytosine base editors can be employed to disrupt the expression of inhibitory receptors in T-cells, and when combined with the introduction of chimeric antigen receptor (CAR) genes, can generate an improved drug product with tolerance to tumour-associated immunosuppressive signals. The manufacture of base-edited cellular therapies is limited by the large-scale production of mRNA encoding the base editor enzyme. Here, we present a novel method to produce mRNA encoding BE4max, that is efficient, scalable and financially viable.