An in vitro-transcribed circular RNA targets the mitochondrial inner membrane cardiolipin to ablate EIF4G2⁺/PTBP1⁺ pan-adenocarcinoma

In vitro-transcribed (IVT) mRNA has arisen as a rapid method for the production of nucleic acid drugs. Here, we have constructed an oncolytic IVT mRNA that utilizes human rhinovirus type 2 (HRV2) internal ribosomal entry sites (IRESs) to selectively trigger translation in cancer cells with high expression of EIF4G2 and PTBP1. The oncolytic effect was provided by a long hGSDMD ^{c.825 T>A/c.884 A>G} -F1L^CT mutant mRNA sequence with mitochondrial inner membrane cardiolipin targeting toxicity that triggers mitophagy. Utilizing the permuted intron-exon (PIE) splicing circularization strategy and lipid nanoparticle (LNP) encapsulation reduced immunogenicity of the mRNA and enabled delivery to eukaryotic cells in vivo. Engineered HRV2 IRESs-GSDMD^{p.D275E/E295G}-F1L^CT circRNA-LNPs (GSDMD^ENG circRNA) successfully inhibited EIF4G2⁺/PTBP1⁺ pan-adenocarcinoma xenografts growth. Importantly, in a spontaneous tumor model with abnormal EIF4G2 and PTBP1 caused by KRAS G12D mutation, GSDMD^ENG circRNA significantly prevented the occurrence of pancreatic, lung and colon adenocarcinoma, improved the survival rate and induced persistent KRAS G12D tumor antigen-specific cytotoxic T lymphocyte responses.