Article Title: An engineered linear cap-independent mRNA vaccine with intrinsic adjuvanticity induces potent anti-tumor immunity in mice

Journal: *Nature Communications*

Publication Date: Published online on February 26, 2026

Research Team: A collaboration between the research group of Prof. Huang Shenglin at the Institute of Biomedical Sciences, Fudan University, and Prof. Fang Zhuting at the Fujian Cancer Hospital.

Key Findings: The research team developed a streamlined, engineered linear cap-independent mRNA (LciRNA) platform. By fusing a virus-derived exonuclease-resistant RNA (xrRNA) with a PABP-binding motif (UPA protective sequence)—and incorporating an optimized Enterovirus A-type Internal Ribosome Entry Site (IRES)—this platform achieves highly efficient translation and *in vivo* stability without the need for 5’ capping or base modifications. Furthermore, LciRNA exhibits significant intrinsic adjuvant activity, capable of activating various pattern recognition receptors and promoting the maturation of dendritic cells. In melanoma and HPV-associated tumor models, this platform demonstrated superior tumor control capabilities compared to traditional modified mRNA vaccines.

Research Background

mRNA vaccines have garnered significant acclaim in the fight against the COVID-19 pandemic—thanks to their short development cycles, strong immunogenicity, and high safety profiles—and are rapidly emerging as a promising new frontier in cancer immunotherapy. However, existing mRNA platforms still face numerous limitations: traditional linear mRNA relies on expensive 5’ capping processes and complex nucleoside modifications to ensure stability and mitigate immunogenicity; while circular RNA (circRNA) boasts high stability, its production involves intricate *in vitro* circularization and purification steps, thereby increasing process complexity and cost; and although self-amplifying mRNA (saRNA) requires lower dosages, it frequently triggers systemic adverse reactions, compromising patient tolerability. Consequently, a central scientific challenge currently facing the field of cancer vaccines—one that urgently requires resolution—is how to construct an mRNA platform that is both cost-effective and capable of eliciting a potent immune response, without simultaneously increasing manufacturing complexity.

Detailed Explanation of Core Content

I. Design, Construction, and Element Screening of the LciRNA Platform

The researchers aimed to functionally replace the 5' cap structure through a purely sequence-encoded approach. The primary function of the cap is to recruit translation initiation factors and prevent XRN-1-mediated degradation. The research team first selected 18 exonuclease-resistant structures (xrRNAs) derived from flavivirus sfRNAs as candidate elements, inserting them upstream of a Gluc reporter gene.

Study Subjects and Methods: HEK293T cells were transfected with *in vitro* transcribed (IVT) products—synthesized without cap analogs or modified bases—and luciferase activity was measured 48 hours post-transfection.

Results: The experiments revealed that the UX1 element, derived from the Usutu virus (genus *Flavivirus*), demonstrated the most exceptional performance in terms of enhancing expression levels. Subsequently, the team inserted various motifs between the UX1 element and an EV-A-S1 IRES; they discovered that the inclusion of a PABP-binding motif significantly boosted expression, outperforming poly-C, poly-T, or empty-vector controls by a wide margin.

Experimental Summary: Through systematic screening, the UPA protective sequence—comprising the UX1 sequence and the PABP motif—was identified as the core functional element. Additionally, the study confirmed that utilizing the 5' and 3' untranslated regions (UTRs) of human β-globin yielded the strongest translational output.

II. Negative Impacts of Base Modifications and Structural Mutations on Function

Traditional mRNA vaccines rely on modifications—such as N1-methylpseudouridine (m1Ψ)—to enhance their performance; however, LciRNA exhibits fundamentally different characteristics.

**Study Subjects and Methods:** Varying proportions of m6A, m1Ψ, or N4-Ac-C modifications were incorporated into Gluc LciRNA, and structural pseudoknot mutants of UX1 were designed.

**Results:** The data demonstrated that as the proportion of modified bases increased, the luciferase activity of the LciRNA declined in a dose-dependent manner. Furthermore, structural analysis of UX1 using SHAPE-MaP technology revealed that it comprises two critical pseudoknots: PK1 and PK2. Artificially altering the lengths of these pseudoknots or increasing their GC content resulted in a drastic reduction in expression levels.

**Experimental Summary:** The results indicate that the function of LciRNA is highly dependent on its precise secondary structure folding. Complex base modifications disrupt this native folding; consequently, this platform neither requires nor is compatible with extensive base modifications—a feature that significantly simplifies the manufacturing process.

III. 2UPA-LciRNA Demonstrates Exceptional In Vivo Expression Persistence

To further enhance protective effects, researchers designed a tandem repeat sequence of UPA.

Research Methodology: The study compared the expression kinetics of LciRNAs containing one (UPA), two (2UPA), or three (3UPA) UPA units against those of traditional capped mRNA (CAP) and m1Ψ-modified capped mRNA (mCAP) in both HeLa cells and BALB/c mice.

Results: At the cellular level, the expression levels of 2UPA-LciRNA remained stable on Day 5, whereas the expression levels of the unprotected control group declined by more than 10-fold daily. More notably—following intramuscular injection of LNP-encapsulated mRNA into mice—although 2UPA-LciRNA performed slightly less robustly during the initial 24 hours, its bioluminescence signal significantly surpassed those of the mCAP and CAP groups during the 48-to-120-hour period.

Experimental Summary: The 2UPA repeat unit endows uncapped mRNA with in vivo stability that rivals—and even surpasses—that of modified capped mRNA. This finding challenges the conventional wisdom that "uncapped mRNA is unstable," thereby laying a solid foundation for its application as a cancer vaccine platform.

IV. Elucidation of the Mechanisms Underlying UPA Sequence's

Resistance to Enzymatic Degradation and Recruitment of Protective Proteins

Through a series of molecular biochemical experiments, the research team conducted an in-depth investigation into how 2UPA confers protection to RNA.

**Research Methodology:** The study involved *in vitro* XRN-1 digestion assays and streptavidin-aptamer-based RNA pull-down experiments, complemented by mass spectrometry and siRNA interference techniques.

**Research Results:**

**Enzymatic Digestion Assays:** 2UPA-LciRNA remained intact even after two hours of treatment with XRN-1, whereas standard RNA lacking the UPA sequence was completely degraded.

**RBP Recruitment:** Mass spectrometry analysis and Western blot validation confirmed that the UPA sequence specifically recruits known RNA-stabilizing proteins, such as IGF2BP1, YBX1, and PABPC1.

**Functional Validation:** Knockdown of IGF2BP1 or YBX1 in HeLa cells resulted in a significant reduction in the expression levels of UPA-LciRNA, while having no discernible effect on other types of RNA.

**Experimental Summary:** The UPA sequence achieves RNA stabilization through a dual-mechanism strategy: On one hand, its precise, three-dimensional xrRNA structure physically blocks the progression of the XRN-1 exonuclease; on the other hand, it actively recruits endogenous RNA-binding proteins (RBPs) to form stable ribonucleoprotein complexes, thereby synergistically protecting the RNA from degradation.

V. Potent Natural Self-Adjuvant Effect and High Safety Profile

The success of a cancer vaccine depends not only on antigen expression but, more importantly, on its capacity to activate the immune system.

**Research Methods:** Mouse bone marrow-derived dendritic cells (BMDCs) were transfected with Fluc-LciRNA, followed by RT-qPCR analysis, transcriptome sequencing, and *in vivo* safety assessment in mice.

**Research Results:**

**Immune Activation:** Compared to capped mRNA, LciRNA significantly upregulated the expression of co-stimulatory molecules (CD40, CD80, CD86) on the surface of BMDCs, as well as pro-inflammatory cytokines (IL-6, IFN-α, IFN-β).

**Transcriptome Analysis:** Gene Set Enrichment Analysis (GSEA) revealed that LciRNA potently activated the RIG-I signaling pathway, TLR pathways, and antigen processing and presentation pathways.

**Safety:** Following the administration of two doses (5 μg per dose) to mice, no abnormalities were observed in body weight or liver function markers (ALT, AST); furthermore, histological sections of major organs (H&E staining) showed no signs of tissue damage.

**Experimental Summary:** LciRNA retains its 5'-triphosphate terminus and incorporates a complex virus-derived Internal Ribosome Entry Site (IRES) structure. This unique composition enables it to act as a natural ligand, activating various Pattern Recognition Receptors (PRRs)—such as RIG-I and PKR—thereby effectively driving DC maturation without the need for exogenous adjuvants. Concurrently, this platform demonstrates an excellent safety profile at equivalent dosages.

VI. Potent Tumor Control and T-Cell Activation in a Melanoma Model

**Study Subjects and Methods:** In a B16F10-OVA melanoma mouse model, 3 μg of 2UPA-OVA LciRNA (and corresponding controls) were administered via intramuscular injection on days 3 and 10; tumor volume was monitored, and immune infiltration was analyzed.

Results:：

**Tumor Control:** The 2UPA-OVA group demonstrated the most significant inhibition of tumor growth; by day 21, the average tumor volume in this group was substantially smaller than that in the modified mRNA group, and 3 out of 6 mice achieved complete tumor regression.

**Immune Response:** The proportion of OVA-specific CD8+ T cells in both peripheral blood and the spleen reached its peak in the 2UPA group (at 32.6% and an even higher frequency, respectively). Furthermore, the LciRNA significantly increased the infiltration of CD4+ and CD8+ T cells within the tumor microenvironment.

**Experimental Summary:** 2UPA-LciRNA successfully converted "cold tumors" into "hot tumors." Its high-level antigen expression synergized with an intrinsic adjuvant effect to induce a more robust systemic and tumor-specific T-cell response.

VII. Complete Eradication of HPV-Related Tumor Models and Long-Term Survival

**Study Subjects and Methods:** A therapeutic immune evaluation was conducted using the TC-1 tumor model (which expresses HPV E6/E7 proteins).

**Study Results:** Following the administration of two doses, all mice inoculated with LciRNA (UPA-E6E7 or 2UPA-E6E7) achieved relapse-free survival (RFS) throughout the 40-day observation period; in contrast, a subset of mice in the group treated with conventionally modified mRNA experienced tumor recurrence. Splenic analysis confirmed that the 2UPA group induced the highest frequency of IFN-γ+ CD8+ T cells.

**Experimental Summary:** The experiments demonstrated that the LciRNA platform exhibits superior therapeutic efficacy and induces more durable immune memory within a highly challenging HPV tumor model.

Summary

This study successfully established a linear, uncapped mRNA (LciRNA) vaccine platform driven entirely by RNA sequences. By ingeniously leveraging flavivirus-derived anti-exonuclease elements, this platform achieves exceptional *in vivo* stability and translational efficiency without relying on 5’ capping or nucleoside modifications. More importantly, the unique "immunity-translation coupling" mechanism inherent to LciRNA resolves the inherent conflict between innate immune activation and protein translation, thereby demonstrating superior anti-tumor immune responses and safety profiles. This work opens up a novel design paradigm for the development of next-generation mRNA cancer vaccines that are low-cost, highly efficient, and globally accessible.

References

[1] Yu H, Yang Y, Lin P, Liu C, Wen Y, Huang Z, Fang Z, Hu Z, Huang S. An engineered linear cap-independent mRNA vaccine with intrinsic adjuvanticity induces potent anti-tumor immunity in mice. Nat Commun. 2026 Feb 26.