Investigation of 2A Peptides to be used in Bicistronic Vector for CAR T-Cell

Abstract

The development of multi-directional promoter-driven chimeric antigen receptors (CARs) has great potential to enhance the effectiveness of antigen-targeting CAR-T therapies. In order to express two proteins or genes simultaneously, a 2A peptide site is added between the target proteins. However, based on research, there are many different types of these cleaving sites. Therefore, to determine the best 2A peptide to be utilized for the vector construction, a proper and detailed systematic review was conducted. Recent studies involving vector design were extracted, where the frequency in which different 2A peptides were used relative to one another, as well as the cleavage efficiencies of these peptides were observed. Based on these factors, it was found that P2A, with its high usage and cleavage efficiency, was the ideal 2A peptide. Thereafter, a bicistronic vector for CAR T cells involving a P2A cleaving site was constructed through PCR and ligation methods. The process outlined will provide further opportunities for the expansion of the development of bicistronic CARs.