Circular RNA-based neoantigen vaccine for hepatocellular carcinoma immunotherapy

Abstract

mRNA vaccines are regarded as a highly promising avenue for next-generation cancer therapy. Nevertheless, the intricacy of production, inherent instability, and low expression persistence of linear mRNA significantly restrict their extensive utilization. Circular RNAs (circRNAs) offer a novel solution to these limitations due to their efficient protein expression ability, which can be rapidly generated in vitro without the need for extra modifications. Here, we present a novel neoantigen vaccine based on circRNA that induces a potent anti-tumor immune response by expressing hepatocellular carcinoma-specific tumor neoantigens. By cyclizing linearRNA molecules, we were able to enhance the stability of RNA vaccines and form highly stable circRNA molecules with the capacity for sustained protein expression. We confirmed that neoantigen-encoded circRNA can promote dendritic cell (DC) activation and enhance DC-induced T-cell activation in vitro, thereby enhancing T-cell killing of tumor cells. Encapsulating neoantigen-encoded circRNA within lipid nanoparticles for in vivo expression has enabled the creation of a novel circRNA vaccine platform. This platform demonstrates superior tumor treatment and prevention in various murine tumor models, eliciting a robust T-cell immune response. Our circRNA neoantigen vaccine offers new options and application prospects for neoantigen immunotherapy in solid tumors.

Keywords: circRNA; hepatocellular carcinoma; immunotherapy; neoantigen; vaccine.

FIGURE 1

Protein expression initiated by circular RNA (circRNA). (A) Schematic diagram of circRNA circularization via the permuted intron‒exon (PIE) system. (B) Agarose gel confirmation of RNA circularization. (C) Precursor RNA subjected to circularization conditions. C + RNase R: digested with RNase R. (C) q‐PCR and (D) Sanger sequencing detect cyclic sites. (E) Expression of circRNA and linearRNA (linRNA) in HEK293T cell transfected with circRNA enhanced green fluorescent protein (EGFP) or linRNA EGFP. Scale bars: 400 µm. (F and G) Gaussian luciferase test showing the expression of PTPN2‐GLuc in HEK293T and DC2.4 cells. (H) The expression stability of circRNA compared to linear mRNA after RNase R treatment. (I and J) The stability of circRNA compared to linear mRNA were by observing the differences in the expression of GFP and GLuc after placed at room temperature for different times, respectively. Scale bars: 400 µm. (K) Translation efficacy of circRNA compared to linear mRNA after transfected different times. ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001.

FIGURE 2

Immunogenicity validation of circular RNA (circRNA) vaccine in vitro. (A) Overview of in vitro immunogenicity validation. (B and C) Flow cytometry analysis of the percentage of CD80, CD86, major histocompatibility complex (MHC) I in dendritic cells (DCs) after treated with PBS, circRNAGluc, Neo‐Polypeptide, and circRNA3×PTPN2, the right panel shows the statistical analysis of the data. (D and E) Detection of interferon‐gamma (IFN‐γ) and tumor necrosis factor‐alpha (TNF‐α) secretion in culture medium by ELISA. (F and G) Flow cytometry analysis of the percentage of active T cell by tests CD25 and CD69, the right panel shows the statistical analysis of the data. (H) IFN‐γ spot‐forming cells and statistical data from stimulated T cell of PBS, circRNAGluc, Neo‐Polypeptide, and circRNA3×PTPN2 via ELISPOT assay. (I) LDH test the cytotoxicity of Hepa1‐6 cells by T cells. (J) Flow cytometry analysis the apoptosis rate of Hepa1‐6 cell after treated with activated T cells, the right panel shows the statistical analysis of the data. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, and ^**** p < 0.0001. LDH, lactate dehydrogenase; PBS, phosphate buffered saline.

FIGURE 3

Characterization of the circular RNA (circRNA)‒lipid nanoparticle (LNP) complex. (A) Zeta potential of the circRNA3×PTPN2 complex (pH 7.4). (B) Encapsulation of circRNA3×PTPN2 and size distributions of the circRNA3×PTPN2 complex. (C) Transmission electron microscopy (TEM) image of the circRNA3×PTPN2‒LNP complex. Scale bar: 100 nm. (D) Bioluminescence images of mouse after injected the circRNALuc, circRNALuc were given tail vein to C57/B6 mice (10 µg RNA per mouse), luciferase expression was measured 12 h post‐injection. (E) Intracellular localization of the Cy5‐labeled circRNA characterized by Confocal Laser Scanning Microscope. Scale bar: 20 µm. (F) The stability of circRNAs was analyzed by biofluorescence imaging of Gaussian luciferase expression in peripheral blood serum of mice at different times after injection of circRNAGluc. circRNAGluc and linRNAGluc were injected into the tail vein of C57/B6 mice (10 µg of RNA per mouse). ^*** p < 0.001.

FIGURE 4

Anti‐tumor efficacy of circular RNA (circRNA)‒lipid nanoparticle (LNP) vaccine in subcutaneous hepatocellular carcinoma (HCC) model. (A) Treatment timeline of the experiment to evaluate the anti‐tumor efficacy. (B) Body weight curves of mice during treatment. (C) Tumor growth curves of each group. (D) Average tumor growth curve of mice after treat with PBS, linRNA3×PTPN2, circRNA3×PTPN2 (n = 5). (E) Images of tumor size. (F) The representative immunofluorescence image of CD4⁺ and CD8⁺ T‐cell infiltration in tumor tissues. Scale bars: 20 and 100 µm. (G) The representative immunofluorescence image of terminal‐deoxynucleotidyl transferase‐mediated nick end labeling (TUNEL) in tumor tissues. Scale bars: 20 and 100 µm. (H) Hematoxylin and eosin (H&E) staining for vital organs in mice. Scale bars: 100 µm. ^** p < 0.01 ^**** p < 0.0001. PBS, phosphate buffered saline.

FIGURE 5

Anti‐tumor efficacy of circular RNA (circRNA)‒lipid nanoparticle (LNP) vaccine in orthotopic hepatocellular carcinoma (HCC) model. (A) Treatment timeline of the experiment to evaluate the anti‐tumor efficacy. (B) Tumor burden monitoring of PBS, linRNA3×PTPN2, and circRNA3×PTPN2‐treated mice by bioluminescence imaging. (C) Kaplan‒Meier survival curves of PBS, linRNA3×PTPN2, and circRNA3×PTPN2‐treated groups. (D) Serum cytokine interleukin‐6 (IL‐6) and tumor necrosis factor‐alpha (TNF‐α) release after circRNA3×PTPN2 administration via ELISA assays. (E) Flow cytometry analysis the percentage of matured dendritic cells (DCs) in lymph nodes after different treatment. (F and G) Flow cytometry analysis the percentage of CD25+ and CD69+ T cells in spleen after different treatment. (H) Interferon‐gamma (IFN‐γ) spot‐forming cells and statistical data from stimulated T cell of PBS, circRNAGluc, Neo‐Polypeptide, and circRNA3×PTPN2 via ELISPOT assay. (I) The representative immunofluorescence image of CD4+ and CD8+ T‐cell infiltration in tumor tissues. Scale bar: 20 µm. (J) Flow cytometry analysis the percentage of central memory T cells in spleen after different treatment and the statistical analysis. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, and ^**** p < 0.0001. PBS, phosphate buffered saline.

FIGURE 6

Anti‐tumor efficacy of circular RNA (circRNA)‒lipid nanoparticle (LNP) vaccine in prophylactic hepatocellular carcinoma (HCC) model. (A) Schematic diagram showing the timeline of establishing prophylactic HCC model. (B) Body weight curves of mice during treatment. (C) Tumor growth curves of each group and (D) average tumor growth curve of mice after treat with PBS, linRNA3×PTPN2, circRNA3×PTPN2 (n = 5). (E) Images of tumor size. (F and G) Flow cytometry analysis showed the percentage of PTPN2 tetramers and INF‐γ specific CD8+ T cells in infiltrating CD8+ T cells (n = 5). ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, and ^**** p < 0.0001. PBS, phosphate buffered saline.