Chimeric Antigen Receptor (CAR)-T cell therapy has emerged as one of the most prominent cancer immunotherapies. To date, all marketed CAR-T cell products use viral vectors for permanent genomic integration of the CAR gene. However, this approach faces significant limitations. For example, it poses the risk of insertional mutagenesis, can lead to long-term side effects by on-target-off-tumor cytotoxicity, and complex manufacturing requirements contribute substantially to high therapy costs.

We are addressing these shortcomings by using RNA instead of viral vectors for CAR-T cell generation. As demonstrated by the recent breakthrough of mRNA vaccines, mRNA therapeutics can be considered safe and feasible for large-scale production. Nevertheless, the labile nature of mRNA presents a challenge in CAR-T cell therapy, as it leads to a rapidly declining CAR expressing and therefore a short-lived CAR-T cell efficacy. To address this challenge, we systematically compared innovative RNA formats, namely self-amplifying, trans-amplifying, and circular RNA, with a standard optimised mRNA in terms of transgene expression in T cells. To this end, the different RNAs were produced in vitro, optimised, and transfected into Jurkat T cells and primary T cells. Protein expression and cell viability were analysed using flow cytometry. The best RNA candidates will be used for CAR-T cell generation, with a focus on further improving RNA stability, transfection, and CAR-T cell efficacy. Ultimately, we pursue to pave the way for a safer, more cost-effective, and therefore more widely accessible alternative to the current CAR-T cell products.