[Enhanced translational efficacy of internal ribosomal entry site-mediated mRNA in dendritic cells contributes to efficient induction of antitumor immunity]

Abstract

Objective: To develop a more economic and applicable substitute for modification of dendritic cells (DCs) by capped mRNA in induction of anti-tumor immunity.

Methods: Four DNA plasmids as templates of mRNA in vitro transcription were constructed: pmRNA luciferase (Luc), pmRNA internal ribosomal entry site (IRES)-Luc, pmRNA ovalbumin (OVA), and pmRNA IRES-OVA. The translational efficiency of uncapped-Luc, capped-Luc, or IRES-Luc mRNA in murine DCs was detected via a Luc reporter system. Nine C57BL/6 mice were divided into 3 equal groups to be injected intraperitoneally with DCs transfected with IRES-OVA or capped-OVA mRNA and untreated DCs respectively, 1 weeks later the mice were injected with splenocytes wrapped with the target peptide or control peptide labeled by CFSE, and 4 hours later the mice were killed and suspension of splenocytes was made to test the activity of cytotoxic lymphocytes (CTLs). Another 30 mice were divided into 3 equal groups to undergo immunization by DCs as mentioned above, 1 week later mice melanoma cells of the line MO5 stably expressing OVA were injected the caudal vein, and 3 weeks later the mice were killed and their lungs were taken out to observe the metastasis of tumor, using OVA as a target antigen.

Results: Insertion of IRES into upstream of gene of interest in mRNA transcriptional templates didn't affect the yield of mRNA in vitro transcription. The level of Luc activity expressed by IRES-Luc mRNA in the DCs was 20 times higher than that by expressed by Uncapped Luc mRNA, and one time higher than that by Capped-Luc mRNA 8 h after transfection of DC. Expression of Luc could be detected up to 96 h after the transfection with IRES-Luc and Capped-Luc mRNA. Four hours after the injection of peptides the CTL activity of the mice immunized with DCs pulsed with Capped-OVA mRNA was (28 +/- 3)%, not significantly different from that of the mice immunized with DCs pulsed with IRES-OVA mRNA [(32 +/- 4)%, P > 0.05]. Mata static melanoma nodules could be seen in all control mice, only one mouse of the Capped-OVA mRNA group, and none of the mice of the IRES-OVA mRNA group.

Conclusion: IRES-containing tumor-associated antigen (TAA) mRNA as a more economic and applicable substitute fully replaces Capped-TAA mRNA for mRNA-based DC vaccines and DCs pulsed with IRES-containing TAA mRNA induces the same effective antigen-specific cellular response as the DCs pulsed with Capped-TAA mRNA.