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

Nature Cancer volume 5, pages 30–46 (2024)Cite this article

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Abstract

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 hGSDMDc .825 T>A/c.884 A>G-F1LCT 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-GSDMDp.D275E/E295G-F1LCT circRNA-LNPs (GSDMDENG circRNA) successfully inhibited EIF4G2+/PTBP1+ pan-adenocarcinoma xenografts growth. Importantly, in a spontaneous tumor model with abnormal EIF4G2 and PTBP1 caused by KRASG12D mutation, GSDMDENG circRNA significantly prevented the occurrence of pancreatic, lung and colon adenocarcinoma, improved the survival rate and induced persistent KRASG12D tumor antigen-specific cytotoxic T lymphocyte responses.

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Fig. 1: Construction of selective HRV2-IRES-driven GSDMDNT IVT mRNA.
Fig. 2: LNPs encapsulate HRV2 IRES-GSDMDNT IVT mRNA for eukaryotic cell delivery.
Fig. 3: The C-terminal F1L peptide translocates GSDMDNT into mitochondria.
Fig. 4: Acquired mitochondrial enzymatic hydrolytic activity via GSDMD base modification.
Fig. 5: PIE splicing to form circRNA.
Fig. 6: Safety and efficacy of GSDMDENG circRNA-LNPs.
Fig. 7: GSDMDENG circRNA-LNPs for the preventive treatment of pan-adenocarcinoma caused by KRAS mutation.

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Data availability

Source data for Figs. 1–7 and Extended Data Figs. 1–9 have been provided as Source Data files. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

This study was supported by the National Natural Science Foundation of China (82072739, 81800082, 81172790 and 81671586), The Recruitment Program of Overseas High-Level Young Talents, ‘Innovative and Entrepreneurial Talent and Team’ (No. (2018) 2015) of Jiangsu Province; Beijing Xisike Clinical Oncology Research Foundation (YBMS2019-071 and Y-MSDZD2021-0169), the start-up funding of Southeast University (No. 4024002312), the First Affiliated Hospital; Excellent Scientific Research and Innovation Team in Natural Sciences of Anhui province (2022AH010073); the National University of Singapore (NUHSRO/2020/133/Startup/08, NUHSRO/2023/008/NUSMed/TCE/LOA, NUHSRO/2021/034/TRP/09/Nanomedicine), National Medical Research Council (CG21APR1005, MOH-001388-00), Singapore Ministry of Education (MOE-000387-00) and National Research Foundation (NRF-000352-00). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Authors and Affiliations

  1. The Translational Research Institute for Neurological Disorders & Interdisciplinary Research Center of Neuromedicine and Chemical Biology of Wannan Medical College, Department of Neurosurgery, Department of Intensive Care Medicine, the First Affiliated Hospital of Wannan Medical College (Yijishan Hospital of Wannan Medical College), Wuhu, China

    Zunyong Feng, Guangfu Di, Qun Chen & Xiaochun Jiang

  2. Zhongda Hospital, School of Medicine & School of Biological Sciences and Medical Engineering, Advanced Institute for Life and Health & Interdisciplinary Innovation Institute for Medicine and Engineering, Southeast University, Nanjing, China

    Zunyong Feng, Liuxi Chu & Hongping Xia

  3. Departments of Diagnostic Radiology, Surgery, Chemical and Biomolecular Engineering, and Biomedical Engineering, Yong Loo Lin School of Medicine and College of Design and Engineering, National University of Singapore, Singapore, Singapore

    Zunyong Feng, Xuanbo Zhang & Xiaoyuan Chen

  4. Clinical Imaging Research Centre, Centre for Translational Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

    Zunyong Feng, Xuanbo Zhang & Xiaoyuan Chen

  5. Nanomedicine Translational Research Program, NUS Center for Nanomedicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

    Zunyong Feng, Xuanbo Zhang & Xiaoyuan Chen

  6. Institute of Molecular and Cell Biology, Agency for Science, Technology, and Research (A*STAR), Proteos, Singapore, Singapore

    Zunyong Feng, Xuanbo Zhang & Xiaoyuan Chen

  7. Department of Anatomy, School of Basic Medicine & School of Medical Imageology, Anhui Province Key laboratory of Active Biological Macro-molecules Research, Wannan Medical College, Wuhu, China

    Jing Zhou, Qiang Li & Zhengyuan Xu

  8. Department of Neurosurgery, The Second Xiangya Hospital, Central South University, Changsha, China

    Ming Wang

  9. Department of Pathology, Nanjing Drum Tower Hospital Clinical College & Key Laboratory of Antibody Technique of National Health Commission & Jiangsu Antibody Drug Engineering Research Center, Nanjing Medical University, Nanjing, China

    Hongping Xia

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  1. Zunyong Feng
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Contributions

Z.F. and X.C. conceived and designed this study. Z.F., J.Z. and Q.L. performed the construction, breeding, reproduction and experiments of animal models. X.Z. assisted in the synthesis of LNPs and mRNA assembly, as well as electron microscope detection. L.C. and Z.X. performed data analysis, interpreted the results and maintained RNA quality control. Q.C. collected clinical samples. M.W. provided pathological support. Z.F., J.Z. and Q.L. performed the experiments and data collection. Z.F. wrote the original draft. X.J., H.X. and X.C. supervised the project and revised the manuscript, with all authors contributing to writing and providing feedback.

Corresponding authors

Correspondence to Xiaochun Jiang, Hongping Xia or Xiaoyuan Chen.

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Nature Cancer thanks Michael Mitchell, Dan Peer and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Expression of EIF4G2 and PTBP1 in tumors.

(a and b) Dot plot and (c and d) Bar plot of the EIF4G2 and PTBP1 mRNA expression profile across all tumor samples and paired normal tissues (www.gepia.cancer-pku.cn.). (e and f) Overall survival of eIF4G2 and PTBP1 in DLBC, PAAD, COAD, LUAD and GBM. (g) Detection of eIF4E2, eIF4G2 and PTBP1 mRNA in five types of tumor cell lines by qPCR. Data are shown as the mean ± SD, *p < 0.05 and **p < 0.01 by analysis of variance (ANOVA) followed by a post hoc test. All cell experiment data were derived from 3 independent experiments.

Source data

Extended Data Fig. 2 Expression of IRES-driven dicistronics in tumor cell lines.

The dicistronic IVT-mRNA constructs based on (a) EMCV-IRES, (b) PV_type1_Mahoney IRES and (c) PV_type3_Leon IRES were electrotransfected into tumor cell lines, and after 48 h, the fluorescence intensities of Renilla (480 nm) and Firefly (560 nm) luciferases were detected. Data are shown as the mean ± SD, *p < 0.05 and **p < 0.01 by analysis of variance (ANOVA) followed by a post hoc test. All cell experiment data were derived from 3 independent experiments.

Source data

Extended Data Fig. 3 Expression of HRV2 IRES-GSDMDNT IVT mRNA-LNPs in tumor cell lines.

(a) Representative single-cell fluorescence microscopy images of HRV2 IRES-GSDMDNT IVT mRNA-LNPs transfected with DLBC, COAD, LUAD and GBM and corresponding noncancer/nonmalignant cell lines. Scale bar: 5 μm. (b) After HRV2 IRES-GSDMDNT IVT mRNA-LNPs were transfected into DLBC, COAD, LUAD and GBM and corresponding noncancer/nonmalignant cell lines, the fluorescence intensity of eGFP on the cell surface was detected by flow cytometry. (c and d) The dose‒response relationship of transfected LNPs encapsulating HRV2 IRES-GSDMDNT IVT mRNA in WT or CHMP4A−/− DLBC, COAD, LUAD and GBM cell lines. All cell experiment data were derived from 3 independent experiments.

Source data

Extended Data Fig. 4 N/MTS-linked GSDMDNT IVT-mRNA targets mitochondria but is nonfunctional.

(a) Schematic diagram of the ligation of MTS peptide mRNA to the N terminus of GSDMDNT mRNA. (b) After encapsulating LNPs with N/MTS-GSDMDNT IVT-mRNA, they were transfected into AsPC-1 and hTRET-hPNE cells, and the expression of GSDMDNT in the isolated cytoplasm and mitochondria was detected. (c) Rb.sN/MTS-GSDMDNT was incubated with lipid strips, followed by detection of specific lipid binding with anti-GSDMDNT antibody. (d) Cleavage of PI(4,5)P2 and cardiolipin liposomes by Rb.sN/MTS-GSDMDNT was monitored by measuring released Tb3+. (e) Schematic diagram of the construction of His-tagged GSDMDNT truncated polypeptides of different lengths. (f) Coimmunoprecipitation of truncated His-tagged GSDMDNT with Flag-N-MTS polypeptide. (g) Coimmunoprecipitation of truncated His-tagged GSDMDNT with Flag-FL1CT polypeptide. All cell experiment data were derived from 3 independent experiments.

Source data

Extended Data Fig. 5 GSDMDNT-F1LCT-LNPs target mitochondria without plasma membrane toxicity.

(a) Validation of polystyrene bead size standard resolution at the 1, 2 and 4 μm levels by FACS. Forward scatter (FSC-A) x-axis and side scatter (SSC-A) y-axis. (b) The effects of GSDMDNT-F1LCT-LNPs on the expression of p-DRP1S616 and p-DRP1S637 in AsPC-1 and hTRET-hPNE cells. (c) Representative fluorescence microscopy images of the expression distribution of CHMP4A and Tsg101 in hTRET-hPNE and AsPC-1 cells 24 h after transfection with GSDMDNT-F1LCT-LNPs. Scale bar: 10 μm. (d)The AsPC-1 cells were pretreated with the pRIP inhibitor Nec-1, MLKL siRNA, and mtROS scavenger Necrox-5 before induction of cell death by GSDMDNT-F1LCT-LNPs for 48 h. All cell experiment data were derived from 3 independent experiments.

Source data

Extended Data Fig. 6 GSDMDp. E295G-F1LCT mRNA-LNP targets mitochondria without subcellular organelle membrane toxicity.

(a, b) Representative histograms of cell lysosomal and endoplasmic reticulum volumes detected by flow cytometry after GSDMDp. E295G-F1LCT mRNA-LNP-transfected hTRET-hPNE cells; the right panel shows the quantitative statistics. (c) Representative scatter plots of mitochondrial membrane potential measured by GSDMDp. E295G-F1LCT mRNA-LNPs after transfection of hTRET-hPNE and AsPC-1 cells by flow cytometry. The right panel shows the percentage quantification of cells with low mitochondrial membrane potential. (d) The ratio of MitoSox (cytoplasmic ROS) and MitoTracker Green (MTG, mitochondrial ROS) was measured to assess the cytoplasmic leakage of mtROS. (e) The cytosolic fraction of mtDNA (ND1) and control nuclear DNA (β-actin) were assayed by qPCR to assess mtDNA leakage. (f) Representative fluorescence microscopy images of the expression distribution of CHMP4A and Tsg101 in hTRET-hPNE and AsPC-1 cells. Scale bar: 10 μm. (g) The dose‒response relationship of transfected LNPs encapsulating HRV2 IRES-GSDMDp. E295G-F1LCT mRNA in WT or DLBC, COAD, LUAD and GBM cell lines. Data are shown as the mean ± SD, *p < 0.05 and **p < 0.01 by analysis of variance (ANOVA) followed by a post hoc test. All cell experiment data were derived from 3 independent experiments.

Source data

Extended Data Fig. 7 m6A-modified GSDMDENGcircRNA-LNPs attenuates non-antiviral immunogenic in vitro.

(a–f) Detection of rigi, mda5, oas1 and ifnb1 mRNA in five types of human normal cell lines and Primary Dermal Fibroblast (HDFa) by qPCR. Data are shown as the mean ± SD, *p < 0.05 and **p < 0.01 by analysis of variance (ANOVA) followed by a post hoc test. All cell experiment data were derived from 3 independent experiments.

Source data

Extended Data Fig. 8 GSDMDEngcircRNA-LNPs shrink tumors in xenogeneic tumor-bearing mice.

(a) After 42 days of 400 μg/kg GSDMDEngcircRNA-LNPs i.v., the levels of AST, ALT, BUN, creatinine and platelets in the blood of mice were detected. (b, c) Human SU-DH-5, SW480, HCC-827 and U118-MG cells were subcutaneously inoculated into NGS mice with a humanized immune system, and 400 μg/kg GSDMDEngcircRNA-LNPs were injected peritumorally. Tumor size was continuously monitored for the following 42 days, and tumor weights were recorded on day 42. Data are shown as the mean ± SD, *p < 0.05 and **p < 0.01 by Student’s t test. All cell experiment data were derived from 3 independent experiments.

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Extended Data Fig. 9 Tumor-specific mitochondrial toxicity of GSDMDENGcircRNA-LNPs in vivo.

(a) Flow cytometry detection of FSC-A of isolated mitochondria from AsPC-1 tumor-bearing mouse tissues relative to a representative histogram of standard beads 42 days after peritumoral injection of 400 μg/kg GSDMDEngcircRNA-LNPs. (b) qPCR detection of mtDNA leakage in tissues of AsPC-1 tumor-bearing mice peritumorally injected with 400 μg/kg GSDMDENGcircRNA-LNPs. (c) Representative scatter plot of mitochondrial membrane potential measured by flow cytometry in tissues of AsPC-1 tumor-bearing mice peritumorally injected with 400 μg/kg GSDMDENGcircRNA-LNPs. (d) STING−/− AsPC-1 and MLKL−/− AsPC-1 cells were subcutaneously inoculated into NGS mice with a humanized immune system, and on day 3, 400 μg/kg GSDMDENG circRNA-LNPs were injected peritumorally, and the tumor size was continuously monitored for the following 42 days (bottom). (e) Mice were injected with AsPC-1-mtOVA cells encoding mtOVA fused with mCherry. 400 μg/kg GSDMDENG circRNA-LNPs were injected peritumorally, and on day 14 mice were sacrificed and cells from DLNs were stained and mCherry+ CD11b + CD11c+of APCs were identified, and the expression of CD86/CD80/CD40 in mCherry+ CD11c + CD103 + CD11b- or mCherry- CD11c + CD103 + CD11b-. (f) Adoptive transfer of CFSE-labeled OVA-specific transgenes CD8+ (OT-I) and CD4+ (OT-II) into WT, STING−/− and MLKL−/− AsPC-1-mtOVA tumor-bearing mice, followed by 400 μg/kg GSDMDENG circRNA-LNPs were injected peritumorally, and the proliferation activity of DLNs OT-I (left) and OT-II (right) cells was detected by flow cytometry. (g) Analysis of OVA OT-I (top)- or OVA OT-II (bottom)-specific IFN-γ T-cell responses in the DLNs of AsPC-1-mtOVA tumor-bearing mice by IFN-γ ELISPOT. The quantification plot on the right depicts the number of puncta from a single mouse DLNs.Data are shown as the mean ± SD, *p < 0.05 and **p < 0.01 by analysis of variance (ANOVA) followed by a post hoc test. All cell experiment data were derived from 3 independent experiments.

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Extended Data Fig. 10 Schematic diagram of GSDMDENG circRNA-LNPs therapy and prevention of adenocarcinoma.

GSDMDENG circRNA-LNPs exhibit dual properties of tumor-specific expression and conditional activation. The HRV2-IRES structure ensures translation initiation in KRAS mutation-driven EIF4G2/PTBP1-activated cancer cells, while engineered double mutations and C-terminal signal peptides enable their mitochondrial localization and activation. Cyclization strategies and lipid nanoparticle encapsulation enhance their expression and delivery efficiency in vivo. These comprehensive strategies hold great promise for using GSDMDENG circRNA-LNPs in the treatment and prevention of KRAS mutation-induced pan-adenocarcinoma.

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Feng, Z., Zhang, X., Zhou, J. et al. An in vitro-transcribed circular RNA targets the mitochondrial inner membrane cardiolipin to ablate EIF4G2+/PTBP1+ pan-adenocarcinoma. Nat Cancer 5, 30–46 (2024). https://doi.org/10.1038/s43018-023-00650-8

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