Development of an RNA Expression Platform Controlled by Viral Internal Ribosome Entry Sites

Since 1990, many nucleic acid expression platformsconsisting of DNA or RNA have been developed. However, although RNA expression platformshave been relatively neglected, several such platformscapped at the 5’ end of RNA by an anti-reverse cap analog have now been developed. At the same time, the capping reaction is a bottleneck in the production of such platforms, with high cost and low efficiency. Here, we investigated several viral and eukaryotic internal ribosome entry sites(IRESs) to develop an optimal RNA expression platform, because IRES-dependent translation does not require a capping step. RNA expression platformsconstructed with IRESs from the 5’ untranslated regions of the encephalomyocarditis virus (EMCV) and the intergenic regionof the cricket paralysis virus(CrPV) showed sufficient expression efficiency compared with cap-dependent RNA expression platforms. However, eukaryotic IRESs exhibited a lower viral IRES expression efficiency. Interestingly, the addition of a poly(A) sequence to the 5’ end of the coxsackievirusB3 (CVB3) IRES (pMA-CVB3) increased the expression level compared with the CVB3 IRES without poly(A) (pCVB3). Therefore, we developed two multiexpression platforms(termed pMA-CVB3-EMCV and pCrPV-EMCV) by combining the IRESs of CVB3, CrPV, and EMCV in a single-RNA backbone. The pMA-CVB3-EMCV-derived RNA platformshowed the highest expression level. Moreover, it clearly exhibited expression in mouse muscles in vivo. These RNA expression platformsprepared using viral IRESs will be useful in developing potential RNA-based prophylactic or therapeutic vaccines, because they have better expression efficiency and do not need a capping step.