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

Domain: chemistry
Published: 2026-02-26
Editorial outcome: major_revision

Abstract
mRNA cancer vaccines demonstrate potential in clinical trials, but existing platforms struggle to boost antitumor efficacy without added cost or complexity. Here, we present a streamlined linear cap-independent mRNA (LciRNA) cancer vaccine platform, achieved by fusing a UPA protective sequence, composed of a viral exoribonuclease-resistant RNA (xrRNA) and a poly(A) binding protein (PABP) motif, to an optimized Enterovirus A internal ribosome entry site. UPA impedes exonuclease-mediated decay and recruits RNA-binding proteins to stabilize LciRNA, enabling stable in vivo expression without 5’ capping or modifications. Moreover, LciRNA innately stimulates immune responses by engaging pattern-recognition receptors, promoting dendritic cell maturation, and upregulating proinflammatory signals. In murine melanoma and HPV-associated tumor models, this vaccine platform elicits strong systemic and intra-tumoral T cell responses, achieving superior tumor control, demonstrating how immune stimulation-translation synergy underpins its efficacy. Thus, we present a cost-effective platform with enhanced efficacy, and highlight coupled immune stimulation and translation as a paradigm for future mRNA cancer vaccines.

Introduction
Messenger RNA (mRNA) has emerged as a transformative vaccine platform owing to its rapid development, robust immunogenicity, and favorable safety profile. The successful application of mRNA vaccines during the COVID-19 pandemic demonstrated their exceptional safety and superior protective efficacy compared with traditional modalities1. Accordingly, mRNA technology is being actively explored for oncological applications, where it is anticipated to enable the development of a new generation of transformative cancer therapeutics2,3.

Recent clinical results underscore this promise. In a Phase IIb trial, the personalized mRNA-4157 vaccine plus pembrolizumab reduced the risk of recurrence or death by 44% in advanced melanoma patients4. Similarly, the personalized BNT-122 vaccine induced markedly prolonged median recurrence-free survival (RFS) in responders in a Phase I adjuvant trial for pancreatic ductal adenocarcinoma, and elicited long-lived effector T cell clones with an estimated lifespan of 7.7 years5,6.

Currently, mRNA platforms for cancer vaccines include conventional linear mRNA, circular RNA (circRNA), and self-amplifying mRNA7. Conventional linear mRNA has already been advanced to clinical application and represents the most mature mRNA vaccine technology to date. Wesselhoeft et al. described the circRNA expression platform in 2018, which enables efficient and stable protein expression via RNA circularization and internal ribosome entry site(IRES)-mediated translation8. We previously engineered an optimized circRNA vaccine platform specifically for cancer vaccine applications, which demonstrated superior antigen expression and immunogenicity compared to conventional linear mRNA9. However, circRNA production requires complex circularization and purification procedures, substantially complicating production workflows and increasing costs10,11. The self-amplifying mRNA (saRNA) vaccines, encode additional viral RNA replication proteins to achieve in vivo RNA amplification and robust efficacy at low doses12. Nevertheless, because these vaccines produce a large amount of double-stranded RNA (dsRNA) and express multiple heterologous viral proteins, they can induce systemic reactogenicity, potentially compromising tolerability and therapeutic efficacy13,14. Furthermore, chemically modified poly(A) mRNA15 and multi-capped mRNA16 strategies reported by Chen et al. substantially enhance stability and translation but introduce additional manufacturing complexity and tolerability concerns. To date, no existing platform achieves high potency without adding design or production complexity.

By contrast, conventional linear mRNA remains the most readily manufactured vaccine platform, with minimal intrinsic adverse effects17. However, linear mRNA’s optimal translation efficiency and stability requires two essential modifications: 5' capping18,19 and modified ribonucleotides20,21. These modifications also increase both production complexity and costs. Circumventing the reliance of linear mRNA on 5’ capping and ribonucleotide modifications could enable the development of a streamlined vaccine platform that balances affordability, manufacturability, and therapeutic efficacy. While uncapped linear mRNA designs have been explored previously22–25, existing evidence has not established their equivalence to capped mRNA in therapeutic efficacy. Furthermore, the potential of uncapped mRNA vaccines in cancer immunotherapy remains uncharacterized.

The mRNA 5’ cap not only recruits eIF4E to facilitate translation initiation but also shields the transcript’s 5’ terminus from XRN-1-mediated exonucleolytic decay26,27, whereas the incorporation of modified ribonucleotides enhances mRNA stability20,21. A survey of the literature on viral and eukaryotic mRNA metabolism reveals that these functions can, in principle, be conferred entirely by RNA sequences and structural elements. For example, Flavivirus genomes harbor structured elements that block XRN-1 to generate abundant subgenomic flaviviral RNAs (sfRNA) during infection28,29, which could be repurposed to shield synthetic transcripts from exonucleolytic digestion. Likewise, certain cellular RNA-binding proteins (RBP), such as poly(A) binding proteins (PABP) and IGF2BP1, stabilize mRNAs. Specifically, PABPs bind and stabilize the poly(A) tail of most mRNAs30, whereas IGF2BP1 associates with MYC and several other mRNAs to enhance their half-lives31. Integrating RBP-binding motifs is a viable strategy to further bolster mRNA stability. Finally, IRES, such as our previously characterized EV-A IRES, can efficiently drive cap-independent translation, effectively replacing the 5’ cap’s translational role8,9.

In this study, we introduce a fully sequence-encoded linear cap-independent mRNA (LciRNA) platform that achieves potent protein expression and immune stimulation without requiring 5' capping or ribonucleotide modifications. An optimal XRN1-resistant element, derived from flaviviral sfRNA, is identified through RNA structure modeling and luciferase reporter screening. This element is fused to a PABP-binding sequence and an optimized Enterovirus A (EV-A) IRES variant to generate LciRNA constructs that drive strong in vivo expression. Mechanistic analyses demonstrate that protective elements both block XRN-1 decay and recruit endogenous RBPs, including PABP, IGF2BP1, and YBX1, to stabilize LciRNA. Compared with conventional capped mRNA, LciRNA exhibits enhanced intrinsic adjuvant activity, activates multiple pattern-recognition receptors (PRR), and promotes maturation of dendritic cells. The streamlined LciRNA platform demonstrates superior efficacy in eliciting tumor-specific T-cell responses and controlling tumors in murine models, highlighting its potential for developing cost-effective cancer immunotherapies with global accessibility.

Results
Engineering LciRNA with Flaviviral xrRNA and PABP Motif for Enhanced Stability and Translation

Previously, we characterized an optimized EV-A IRES (hereafter EV-A-S1) that drives efficient cap-independent translation of target proteins9. Because a canonical 5’ cap both recruits eIF4E and protects the 5’ end from XRN-1 mediated degradation27, we sought RNA elements that could similarly impede XRN-1 decay for cap-independent LciRNA. Flavivirus sfRNAs contain structured exoribonuclease-resistant RNA (xrRNA) and dumbbell elements to stall XRN-1 progression28,32,33. Moreover, the mRNA 3’ poly(A) tail recruits PABPs, which shield transcripts from the 3’ terminus30, suggesting that integrating a PABP-binding motif may also enhance 5’-end protection. We therefore designed LciRNA constructs that incorporate, at the 5’ end, an xrRNA/dumbbell element and a PABP-binding motif upstream of the EV-A-S1 IRES, and, at the 3’ end, a 3’ untranslated region (UTR) followed by a poly(A) tail (Figure 1A). To identify the most effective XRN1-resistant RNA structure, we retrieved sfRNA sequences from five flaviviruses—Cellular fusion agent virus (CFAV), dengue virus (DENV), Usutu virus (USUV), yellow fever virus (YFV), and Zika virus (ZIKV)—from the NCBI Virus database. Leveraging both published sfRNA structural annotations28 and in silico secondary structures predicted by RNAfold34 (Supplementary Figure 1), we identified 18 candidate xrRNA and dumbbell motifs (Figure 1B).

We inserted each candidate motif at the 5’ end of a Gaussia luciferase (Gluc) reporter LciRNA carrying EV-A-S1 to enable rapid transcript stability screening via luminescence. All constructs were in vitro transcribed without cap analogs or modified nucleotides and transfected into HEK293T cells. After 48 h, luciferase assays revealed that UX1, DX1, ZX1, DD1, and DX2 significantly outperformed the unprotected control (Figure 1C). Notably, UX1, derived from USUV, yielded the highest expression and was therefore selected for further experiments.

Given that untranslated regions modulate translation in circRNAs and capped mRNAs, we next optimized all other untranslated regions including conventional 5’ and 3’ UTRs in LciRNA (Figure 1D). Incorporating a PABP motif between UX1 and EV-A-S1 yielded higher Gluc expression than polyC motif, polyU motif, or no insert (Empty) (Figure 1E). Screening various 5’ and 3’ UTRs revealed that human β-globin sequences at both sites yielded maximal translation (Figure 1F, 1G). We further evaluated these UTRs in LciRNA constructs containing an alternative xrRNA (DX1) to determine whether their effects were generalizable. Consistently, the PABP motif and human β-globin UTRs supported the strongest expression (Supplementary Figure 1B–D). Accordingly, all subsequent LciRNA designs incorporate β-globin 5’ and 3’ UTRs.

Ribonucleotide Modification and UX1’s Structural Alteration Impair LciRNA Performance

Ribonucleotide modifications often enhance mRNA stability and expression, so we evaluated their impact on LciRNA. Prior studies have shown that N1-methyl-pseudouridine (m1Ψ) improves mRNA stability and translational efficiency21,35, m6A enhances circRNA translation36, and N4-acetylcytidine (N4-Ac-C) boosts IRES-mediated translation37. We thus incorporated 5–100% m6A, m1Ψ, or N4-Ac-C into Gluc LciRNAs. When transfected into HEK293T cells, these modified RNAs exhibited dose-dependent reductions in luciferase activity at 48 h (Figure 2A). This decline likely reflects disruption of critical UX1 and IRES structures by noncanonical bases38, underscoring that LciRNA’s function relies on precise folding and is incompatible with extensive ribonucleotide modifications.

We subsequently examined whether UX1 structural modification could further enhance LciRNA protection. Previous studies demonstrated that xrRNA blocking of XRN-1 is regulated by pseudoknot length39. We therefore modeled UX1’s structure using SHAPE-MaP (selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling) and IPknot analysis, revealing two pseudoknots, PK1 and PK2 (Figure 2B, Supplementary Figure 2A). Then, we generated UX1 mutants with longer pseudoknots (Long-PK1, Long-PK2) or increased GC content in PK2 (mPK2) and assessed their performance in Gluc LciRNA. Unexpectedly, all three mutants showed significantly diminished expression levels compared with wild-type UX1 (Figure 2C), indicating that the native 88-nt UX1 sequence already achieves optimal XRN-1 resistance.

Engineered LciRNA Outperforms m1Ψ-modified Capped mRNA in in vivo Persistence

Building on our initial observation that coupling UX1 with a PABP-binding motif maximizes LciRNA expression, we designated their combination the UPA protective sequence. We hypothesized that tandem repeats of UPA could further enhance transcript stability. Accordingly, we designed Gluc-encoding LciRNAs featuring one (UPA), two (2UPA), or three (3UPA) tandem UPA units at the 5’ terminus. These constructs were evaluated alongside unmodified (CAP) and m1Ψ-modified (mCAP) Cap1-capped Gluc mRNAs, as well as uncapped RNAs containing only a 5’ PABP motif sequence (P-IRES) or the IRES alone. UPA-containing transcripts exhibited significantly increased luciferase activity, although their expression at 48 h post-transfection remained slightly below that of CleanCap-capped Cap1 mRNAs (Figure 2D). Notably, the 2UPA-LciRNA achieved the highest expression, 10.5-fold higher than IRES-only control, demonstrating that dual UPA repeats confer optimal protection.

To evaluate transcript expression dynamics, we transfected HeLa cells and sampled supernatants daily for five days. The 2UPA and 3UPA LciRNAs sustained stable expression at levels equivalent to capped mRNAs (Figure 2E). In contrast, single UPA LciRNA exhibited a modest decline, whereas the IRES-only control dropped by over 10-fold each day; the PABP-IRES LciRNA showed only slight improvement over the unprotected control (Figure 2E). To rule out the possibility that the enhanced stability of 2UPA LciRNA resulted simply from increased transcript length, we rendered the second UPA nonfunctional (UPA+mUPA) and compared its stability to that of intact 2UPA. The UPA+mUPA transcript remained markedly less stable (Supplementary Figure 2B). We further compared the stability of UPA-based LciRNAs with previously reported hairpin-protected capless RNAs (2HP/double-hairpin and 3HP/triple-hairpin). Both UPA and 2UPA LciRNA exhibited substantially greater stability than the hairpin-protected constructs (Supplementary Figure 2B). These data confirm that the UPA motif confers robust protection and that tandem UPA repeats restore cellular stability to match that of Cap1-capped transcripts.

To evaluate in vivo expression, BALB/c mice were injected intramuscularly with SM102 lipid nanoparticles (LNP) -encapsulated UPA and 2UPA LciRNA encoding firefly luciferase (Fluc) as well as CAP and mCAP Fluc mRNAs. Bioluminescence imaging over 120 h post-injection revealed that although 2UPA LciRNA Fluc yielded lower luminescence within the first 24 h, its signal surpassed both mCAP Fluc and CAP Fluc from 48 h to 120 h (Figure 2F, 2G, 2H). These findings demonstrate that 2UPA LciRNA confers greater in vivo transcript stability than m1Ψ-modified capped mRNA. This in vivo profile contrasts with our cell-culture results and underscores differences in mRNA translation and turnover between immortalized cell lines and mammalian tissues. These differences are likely driven by continuous proliferation and homogeneity in immortalized cells. Given that LciRNA vaccines are intended for clinical application, in vivo expression kinetics may more accurately predict therapeutic efficacy. In summary, our engineered LciRNA demonstrates superior in vivo expression stability compared to m1Ψ-modified Cap1-capped mRNA, suggesting advantages for cancer vaccine applications.

UPA Sequence Stabilizes LciRNA Through XRN-1 Inhibition and RBP Binding

The above results showed that incorporation of the UPA sequence endows uncapped LciRNA with stability comparable to that of Cap1-capped mRNA. To elucidate the mechanism underlying UPA-mediated stabilization, we assessed its capacity to inhibit XRN-1 mediated degradation and to recruit intracellular RBPs. In an in vitro XRN-1 degradation assay (Figure 3A), the uncapped and unprotected RNA (IRES) was completely degraded after 2 h of XRN-1 treatment, whereas UPA and 2UPA LciRNAs as well as capped mRNA (CAP) remained largely intact (Figure 3B). Consistent with Gluc expression results, 2UPA conferred stronger protection than a single UPA, demonstrating that UPA impedes XRN-1 digestion of LciRNA and that tandem duplication further potentiates this effect.

We next sought UPA-binding proteins by fusing the UPA sequence to a streptavidin aptamer (SA) 40 and performing RNA pull-down with streptavidin-coated beads(Figure 3C). HeLa cell lysates were incubated with beads bearing either the UPA-SA aptamer fusion or SA aptamer alone, and bound proteins were resolved by SDS-PAGE (Figure 3D). Mass spectrometry, Western blotting, and RNA immunoprecipitation analyses identified YBX1, PABPC1, ILF2, ILF3, IGF2BP1, and PKR as UPA-specific interactors in the cytoplasm (Figure 3E, 3F, Supplementary Figure 2C). Notably, ILF2, ILF3, and PKR are immune regulatory proteins, suggesting UPA’s role in immune signaling 41–45. PABPC1, which binds poly(A) tails to stabilize transcripts and enhance translation, likely reinforces LciRNA stability and translational efficiency via the PABP motif in UPA. Similarly, YBX1 and IGF2BP1 are well-established RBPs that promote mRNA stability 31,46,47, and their association with UPA may further elevate LciRNA stability.

To assess each RBP’s role in LciRNA expression, we performed siRNA-mediated knockdown (excluding PABPC1 to preserve 3’ tail integrity) in HeLa cells (Figure 3G). We then transfected UPA-Gluc LciRNA, IRES-Gluc RNA, and capped Gluc mRNA and measured Gluc activity. PKR depletion significantly increased expression of all three RNA transcripts (Figure 3H), likely due to relief of PKR mediated eIF2α phosphorylation and global translational repression42,44,48. In contrast, knockdown of ILF2 or ILF3 reduced expression of all three RNAs (Figure 3I), consistent with their reported roles in mitosis and effects on cell viability49,50. Notably, silencing IGF2BP1 or YBX1 preferentially decreased UPA-LciRNA expression without affecting the other RNAs (Figure 3J). Given the established RNA-stabilizing functions of IGF2BP1 and YBX131,46,47, these findings indicate that the UPA motif enhances LciRNA stability not only by blocking XRN-1 mediated decay but also via recruitment of IGF2BP1, YBX1, and PABPC1.

Self-Adjuvanting LciRNA Drives PRR-Mediated DC Maturation with Minimal Toxicity

The ultimate goal of a cancer vaccine is to elicit potent, tumor-specific immune responses, which require both efficient antigen expression and the vaccine’s intrinsic or adjuvant mediated immunostimulatory capacity51,52. Unlike conventional capped mRNA, LciRNA possesses a 5’-triphosphate moiety that is recognized by pattern recognition receptors such as RIG-I, thereby potently triggering inflammatory signaling 53. Its viral-derived IRES contains extended stem-loop structures that may be sensed by protein kinase R (PKR) and toll-like receptors (TLR), activating downstream innate immune cascades54–56. Furthermore, the UPA’s interactions with PKR, ILF2 and ILF3 potentially enhance cellular recognition of LciRNA41–45,57. Collectively, these features suggest that LciRNA engages multiple PRRs to initiate innate immune pathways, conferring a self-adjuvant effect that can amplify adaptive antitumor immunity.

To assess the potential immunostimulatory ability of LciRNA vaccines, mouse bone marrow derived dendritic cells (BMDC) were transfected with Fluc-encoding LciRNAs (UPA-Fluc, 2UPA-Fluc) or capped Fluc mRNAs (CAP-Fluc, mCAP-Fluc). Total RNA was collected 24 h post-transfection for quantitative RT-PCR (qRT-PCR) and RNA sequencing. qRT-PCR analysis revealed that BMDCs transfected with either LciRNA construct upregulated MHC-I, the co-stimulatory molecules CD40, CD80, and CD86, and the proinflammatory cytokines IFN-α, IFN-β, and IL-6, to a significantly greater extent than cells receiving capped mRNA; only unmodified CAP-Fluc induced modest increases in CD40, IL-6, and IFN-β (Figure 4A). Transcriptomic analysis demonstrated that LciRNA strongly activated gene sets associated with antigen processing and presentation, RIG-I and TLR signaling, type I interferon response, and inflammatory cytokine production (Figure 4B, 4C). Further examination of PRR-induced dendritic cell (DC) maturation gene signatures58 revealed near-complete concordance between genes upregulated by LciRNA and canonical PRR-upregulated genes (Supplementary Figure 3A), indicating that LciRNA drives DC maturation via PRR engagement. Moreover, LciRNA markedly increased the expression of T cell co-stimulatory molecules, proinflammatory chemokines (Cxcl9, Cxcl10), and cytokines (Il6, Il12, Il15, Ifna1, Ifnb1) (Supplementary Figure 3B).

To evaluate whether these effects generalize across coding sequences and are maintained after LNP encapsulation, we stimulated BMDCs with SM102 LNP–encapsulated LciRNAs or capped mRNAs encoding either ovalbumin (OVA) or Fluc. In both cases, LciRNAs elicited substantially stronger BMDC activation than capped mRNAs or empty LNPs (Supplementary Figure 4A–H). Importantly, agarose gel electrophoresis and dsRNA ELISA confirmed no differences in RNA purity or dsRNA content among the tested RNAs (Supplementary Figure 4I, 4J). Collectively, these data confirm that LciRNA potently activates PRR signaling, promotes DC maturation, and induces proinflammatory chemokine and cytokine expression, supporting its strong self-adjuvant capacity to enhance antitumor immune responses.

To evaluate potential in vivo toxicity of LciRNA vaccines, we formulated SM102 LNPs encapsulating UPA-E6E7 LciRNA, 2UPA-E6E7 LciRNA, or m1Ψ-modified CleanCap-E6E7 mRNA, and administered 5 μg RNA intramuscularly to C57BL/6 mice, followed by a booster dose one week later. Body weight and serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured before immunization and seven days after each dose. No vaccinated group exhibited significant deviations in body weight compared with untreated controls (Figure 4D). Similarly, ALT and AST levels remained comparable to baseline and those of untreated controls (Figure 4E, 4F). Two weeks after the booster dose, histopathological examination of heart, liver, spleen, lung, and kidney via H&E staining revealed no vaccine-related lesions (Figure 4G). These findings demonstrate that LciRNA vaccines are well tolerated in mice at a dosing regimen equivalent to 1 mg per dose in humans.

LciRNA Vaccination Provokes Potent Tumor-Specific T Cell Responses and Controls B16F10-OVA Melanoma Growth

To evaluate therapeutic efficacy of LciRNA-based cancer vaccines, we established an OVA-expressing B16F10 (B16F10-OVA) melanoma model in C57BL/6 mice. On day 3 and 10 post tumor inoculation, mice received intramuscular doses of 3 μg UPA-OVA LciRNA, 2UPA-OVA LciRNA, unmodified or m1Ψ-modified capped mRNA (unmodified: CAP-OVA, m1Ψ-modified: mCAP-OVA), or m1Ψ-modified capped Fluc control mRNA (mCAP-Fluc) (Figure 5A). Tumor growth monitoring revealed that all OVA-targeting vaccines significantly inhibited tumor growth compared with mCAP-Fluc, with the 2UPA-OVA cohort exhibiting the most pronounced inhibition (Figure 5B). On day 21, excised tumors from UPA-OVA and 2UPA-OVA groups were markedly smaller, and complete tumor regression occurred in 3/6 mice in 2UPA-OVA group, versus 1/6 in the CAP-OVA and mCAP-OVA group (Figure 5C, Supplementary Figure 5B). Body weights remained stable across all groups, indicating favorable tolerability (Supplementary Figure 5A). These results demonstrate that LciRNA vaccines provide superior antitumor efficacy over unmodified and m1Ψ-modified capped mRNA in mouse melanoma model.

To evaluate antigen-specific T cell responses, peripheral blood mononuclear cells (PBMC) were collected one week after the booster and stained with OVA (SIINFEKL) Tetramer. FACS analysis revealed that UPA-OVA, 2UPA-OVA, CAP-OVA and mCAP-OVA vaccines induced 25.3%, 32.6%, 20.0% and 23.2% OVA-specific CD8+ T cells on average, respectively, versus 0.6% in the control group (Figure 5D, 5E), with 2UPA-OVA eliciting the strongest response. On day 21, spleens and tumors were harvested to further characterize systematic and intratumoral T cell responses. Consistently, splenocytes exhibited the highest frequency of OVA-specific CD8⁺ T cells in the 2UPA-OVA group compared to UPA-OVA and capped mRNAs (Figure 5F, Supplementary Figure 5C). Analysis of tumor-infiltrating lymphocytes showed that both UPA-OVA and 2UPA-OVA increased the proportion of OVA-specific CD8+ T cells (Figure 5G) and total CD4+ and CD8+ T cell infiltration (Figure 5H, 5I) relative to unmodified or m1Ψ-modified capped OVA mRNAs. These findings suggest that LciRNA vaccines convert immunologically “cold” tumors into “hot” tumors by enhancing T cell infiltration. Collectively, the 2UPA-OVA LciRNA vaccine demonstrated the most robust antitumor efficacy and T cell induction, while UPA-OVA matched mCAP-OVA in systematic T cell response but achieved greater tumor control, presumably owing to its self-adjuvant activity that enhanced T cell infiltration.

Effective Clearance of HPV- associated Tumors by LciRNA-Based Therapeutic Vaccination

Human papillomavirus (HPV) infection is etiologically linked to cervical and oropharyngeal cancers, in which tumor cells commonly express the viral oncoproteins E6 and E759,60. Therefore, the E6 and E7 proteins serve as ideal target antigens for HPV-associated malignancies. To assess the therapeutic potential of our HPV E6E7 LciRNA vaccines, we established an HPV-associated tumor model by subcutaneously implanting C57BL/6 mice with HPV-E6/E7–expressing TC-1 cells. When tumors reached approximately 50 mm³ on day 9, mice received intramuscular injections of 3 μg UPA-E6E7 LciRNA, 2UPA-E6E7 LciRNA, unmodified or m1Ψ-modified capped mRNA (unmodified: CAP- E6E7, m1Ψ-modified: mCAP- E6E7), or m1Ψ-modified capped Fluc control mRNA (mCAP-Fluc) (Figure 6A). Following two immunizations, all E6E7 vaccine groups exhibited continuous tumor regression, whereas tumors in control mice progressed rapidly (Figure 6B, Supplementary Figure 7C). On day 23, mice were photographed, showing significantly smaller tumors in the E6E7 mRNA vaccinated groups (Supplementary Figure 7B). Survival analysis showed that all mice treated with UPA-E6E7 or 2UPA-E6E7 LciRNA maintained recurrence-free survival throughout the 40-day observation period, whereas one mouse in each of the CAP-E6E7 and mCAP-E6E7 groups experienced tumor relapse (Figure 6C, Supplementary Figure 7C). Together, these results demonstrate that the LciRNA-based E6E7 vaccines achieve more complete and more durable tumor control than both unmodified and m1Ψ-modified capped mRNAs.

PBMCs were harvested one week after the booster dose and restimulated with E6/E7 peptides. FACS results showed high frequencies of IFN-γ⁺ T cells in all E6E7 vaccine groups (mean IFN-γ⁺ CD8⁺ T cells proportions: UPA-E6E7 9.5%; 2UPA-E6E7 17.0%; CAP-E6E7 7.8%; mCAP-E6E7 10.7%; mCAP-Fluc 0.2%) (Figure 6D, 6E), with 2UPA-E6E7 eliciting the strongest response. On day 40, splenocytes restimulated ex vivo with E6/E7 peptides again showed the highest frequency of IFN-γ⁺ CD8⁺ T cells in the 2UPA-E6E7 group, significantly exceeding that of the two capped mRNA cohort (Figure 6F, S7A). Collectively, these results demonstrate that 2UPA-E6E7 LciRNA elicits robust and durable antigen-specific CD8⁺ T-cell responses that correlate with their superior antitumor efficacy.

Methods
Molecular cloning
All the LciRNA template sequences were cloned into the pUC57 plasmid through seamless cloning. The Cloning Kit for mRNA Template (Takara Bio) was used to construct all the capped mRNA templates. DNA fragments were synthesized by Genewitz (Suzhou, China) and amplified by PCR. ClonExpress II One Step Cloning Kit from Vazyme (Nanjing, China) were used for seamless cloning. All the related sequences are provided in Supplementary Data 1 and Supplementary Data 2 .

LciRNA and capped mRNA synthesis
For both types of RNAs, the DNA templates for IVT were first amplified by PCR using 2×AdvanceFast PCR Master Mix (Yeasen, Shanghai) and primers with T7 promoter and 120nt poly(T) (to add 120nt poly(A) tail during transcription; the last two thymidines carried 2’-O-methyl modifications to reduce dsRNA byproduct formation). The LciRNAs were synthesized using T7 High Yield RNA Synthesis Kit (Yeasen, Shanghai) without addition of any cap analog or modified ribonucleotides. The modified LciRNAs were synthesized using T7 High Yield RNA Synthesis Kit (Yeasen, Shanghai) with indicated proportion of modified ribonucleotides added (m6A, m1Ψ, or N4-Ac-C from Syngenebio), but without addition of any cap analog. The capped mRNAs were synthesized using T7 High Yield RNA Synthesis Kit (Yeasen, Shanghai) with CleanCap Reagent AG (TriLink) and m1Ψ (Syngenebio) added co-transcriptionally. Both types of RNAs were subsequently treated with DNase I at 37℃ for 20 mins, and purified using GeneJET RNA Purification Kit (Thermofisher). Final RNA products were analyzed by 2% agarose gel or capillary electrophoresis.

Luciferase reporter assay
For Gaussia luciferase assay, cells were cultured in 96 well plate at the density of 5,000 cells per well. The next day, mRNAs were transfected with CALNP mRNA in vitro reagent (D-nano, Beijing) at 50ng per well according to manufacturer’s instructions. 24 hours later, 30μL cell culture medium was taken from each well, and luminescence counts were measured using Gaussia Luciferase Reporter Gene Assay Kit (Beyotime) according to manufacturer’s instructions.

SHAPE-MaP analysis of UX1 structure.
The UX1 RNA structure was analyzed by SHAPE-MaP method reported by Smola et al.66 with some modifications. Briefly, RNA was modified in 100mM 1-methyl-7-nitroisatoic anhydride (1M7) for 5min under 37℃, then purified with GeneJET RNA Purification Kit (Thermo Scientific). Unmodified and denatured control were prepared the same as Smola’s protocol. Purified RNA was fragmented, then reverse transcribed with Hieff NGS Ultima Dual-mode mRNA Library Prep Kit (Yeasen) with some modifications including incubating under 42℃ for 3 hours and adding 15 mM MnCl2 (final concentration). cDNAs were then purified with GeneJET RNA Purification Kit (Thermo Scientific). Subsequent RNA library construction was carried out according to the manufacturer’s instructions. The sequencing data in FASTQ format was subsequently analyzed by ShapeMapper program according to Smola’s protocol. Secondary structure prediction constrained by SHAPE reactivity were generated using RNAstructure program.

LNP encapsulation of RNA
All RNAs used in mouse studies were encapsulated by the SM102 LNP mix using GenNano-E0021 cancer vaccine kit (Micro&Nano, Shanghai) according to manufacturer’s instructions. Qubit 2.0 (Invitrogen) and Qubit RNA HS (Invitrogen) were used to determine the RNA concentration in the RNA-LNP vaccine (2% Triton X-100 was added to disrupt the RNA-LNP sample).

In vivo bioluminescence imaging in mice
The Fluc RNA-LNP (5 μg RNA per mouse) was intramuscularly injected into the right thigh of the mice. At the indicated time points, 200 μL of D-luciferin potassium salt (Meilun Biology, Dalian) was administered per mouse. Bioluminescence imaging was performed and analyzed using the IVIS in vivo imaging system (PerkinElmer) within 10–30 min after substrate injection.

XRN-1 Digestion Assay
For the XRN-1 digestion assay, 2 μg of RNA was first denatured at 70 °C for 2 min and immediately placed on ice. The RNA was then refolded in a 20 μL reaction containing 1× NEBuffer 3 (NEB) and 1 U/μL RNase Inhibitor murine (Vazyme) at 37 °C for 10 min. Subsequently, 1 U of XRN-1 (NEB) and 10 U of RppH (NEB) were added, followed by incubation at 37 °C for 2 h. After digestion, 5 μL of the reaction mixture was mixed with 5 μL of 2× RNA loading buffer, heat-denatured at 95 °C for 1 min, and analyzed alongside an equal amount of undigested RNA using 2% agarose gel electrophoresis.

RNA pull-down assay
Streptavidin-coated magnetic beads (QuarAcces, Diying, Shanghai) were equilibrated at room temperature for 30 min, washed twice with Solution A (0.1 M NaOH, 0.05 M NaCl in DEPC-treated H₂O) and once with Solution B (0.05 M NaCl in DEPC-treated H₂O), then resuspended in Solution B. In parallel, 30 µg of streptavidin-aptamer–tagged RNA was denatured at 56 °C for 5 min in RNase-free water, refolded in annealing buffer (100 mM Tris-HCl pH 7.5, 300 mM NaCl, 3 mM MgCl₂, 2 U/µL murine RNase inhibitor) at 37 °C for 10 min followed by 5 min at room temperature, and incubated with the pretreated beads for 10 min at room temperature and then rotated for 2 h at 4 °C. HeLa cell lysates were cleared by centrifugation (15 000 × g, 15 min, 4 °C), pre-adsorbed with empty beads for 30 min at 4 °C, and subsequently incubated with the RNA-bound beads in the presence of 1.5 mM MgCl₂ for 4 h at 4 °C. Beads were washed six times with NT2 buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1.5 mM MgCl₂), and proteins were eluted in 1× SDS loading buffer by boiling at 100 °C for 10 min. Finally, eluates were resolved on a 10% SDS-PAGE gel (18 × 16 cm) at 40 V, silver-stained (Beyotime Biotechnology Fast Silver Stain Kit), and differentially visible bands excised for mass spectrometry analysis.

Western blotting
Pull-down protein samples and 5% Hela cell lysate input were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred on to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membrane was next blocked with 5% non-fat milk and incubated with antibodies targeting indicated proteins or β-Actin antibody. The protein bands were detected using a Tanon 5200 Chemiluminescent Imaging System (Tanon, Shanghai, China) with Omni-ECL reagents (Epizyme Biotech, China). Antibodies used in western blotting were YBX1 (84993-4-RR, Proteintech), PABPC1 (66809-1-Ig, Proteintech), ILF2 (67685-1-Ig, Proteintech), ILF3 (68213-1-Ig, Proteintech), IGF2BP1 (22803-1-AP, Proteintech), EIF2AK2 (PKR) (66646-1-Ig, Proteintech), DDX3X (67915-1-Ig, Proteintech), HNRNPR (29980-1-AP, Proteintech), β-Actin (66009-1-Ig, Proteintech). All information on the antibodies is summarized in Supplementary Table 1.

RNA immunoprecipitation assay
2×5μg UPA-SA aptamer RNA was transfected into two 10cm plates of Hela cells 24 hours before harvesting the cells. Cells were harvested by scraping in 1 mL PBS, and pelleted by centrifugation at 800 ×g for 5 min at 4°C. The cell pellet was lysed in 800 μL RIP lysis buffer supplemented with RNase inhibitor (Vazyme) and protease inhibitor (MedChemExpress), and incubated on ice for 30 min before storage at −80°C. For immunoprecipitation, 50 μL protein G magnetic beads per sample were washed twice with NT2 buffer, incubated with 5 μg target antibody or control IgG in 200 μL NT2 buffer for 1 h at room temperature with rotation, washed again, and resuspended in 500 μL ice-cold NT2 buffer. Cell lysates were cleared by centrifugation (15,000 ×g, 20 min, 4°C), and 100 μL supernatant was incubated overnight at 4°C with antibody-bound beads in 900 μL precipitation buffer (NT2 buffer with 35 μL 0.5 M EDTA and 5 μL RNase inhibitor). Beads were washed six times with NT2 buffer, and 50 μL was reserved for Western blot validation. RNA was extracted using TRIzol (Invitrogen), reverse transcribed, and analyzed by qPCR to assess enrichment. Antibodies used in RIP assay were Rabbit IgG (30000-0-AP, Proteintech), Mouse IgG (B900620, Proteintech), YBX1 (84993-4-RR, Proteintech), PABPC1 (66809-1-Ig, Proteintech), ILF2 (67685-1-Ig, Proteintech), ILF3 (68213-1-Ig, Proteintech), IGF2BP1 (22803-1-AP, Proteintech), EIF2AK2 (PKR) (66646-1-Ig, Proteintech), DDX3X (67915-1-Ig, Proteintech), HNRNPR (29980-1-AP, Proteintech). All information on the antibodies is summarized in Supplementary Table 1.

RNA extraction and RT-qPCR
Total RNA was extracted using TRIzol (Invitrogen) following the manufacturer’s instructions. cDNA was synthesized using Evo M-MLV Reverse Transcription Kit (Accurate Biotechnology, Huan) following the manufacturer’s instructions. mRNA levels were quantified using SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology, Huan). Primer sequences are provided in Supplementary Table 2 . All qPCR reactions were performed on QuantStudio 5 Real-Time PCR System (Applied Biosystems).

siRNA knockdown
Hela cells were cultured in 24 well plates at 100,000 cells per well. siRNAs targeting corresponding proteins were transfected at 30pmol per well with Hieff Trans siRNA/miRNA reagent (Yeasen). 48 hours later, total RNAs of three of the replicate wells were extracted and assayed by RT-qPCR to analysis knockdown efficiency. mRNAs were transfected into the other three replicates, at 200ng per well using CALNP mRNA in vitro reagent (D-nano, Beijing). 24 hours later, 30μL cell culture medium was taken from each well to measure Gaussia luciferase signal. siRNA sequences were synthesized by Hipobio (Huzhou, Zhejiang) and provided in Supplementary Table 3 .

BMDC stimulation assay
BMDCs were generated using the method reported by Lutz et al. 67 For the stimulation assays, BMDCs were seeded in 24-well plates at 250,000 cells per well and cultured in medium supplemented with mGM-CSF (Peprotech) at a final concentration of 4 ng/mL. After 6 hours, 800 ng of the indicated RNAs or the transfection control RNA were transfected into each well using CALNP mRNA in vitro reagent (D-nano, Beijing). After 24 hours, total RNA was extracted for RT-qPCR and RNA-seq analyses. For RNA-LNP assays, LNPs encapsulating 600 ng mRNA were added directly to the culture medium. For the empty-LNP control, an equivalent volume of LNPs formulated without mRNA using the same preparation procedure was added. After 24 hours, total RNA was extracted for RT-qPCR analysis.

dsRNA ELISA
DsRNA contamination in all mRNA samples was quantified using the NovoFast dsRNA ELISA Kit (Novoprotein, China) according to the manufacturer’s instructions. Briefly, 600 ng of each mRNA sample was diluted to 300 μL with sample dilution buffer, and 100 μL of the diluted sample was added into each well of the assay plate. The detection antibody was then added, gently mixed, and the plate was sealed and incubated at 37°C for 1 hour. After incubation, the wells were washed five times with wash buffer, followed by addition of the substrate solution. The reaction was terminated with stop reagent, and absorbance was measured at 450 nm.

RNA-Seq Analysis
Total RNA from BMDC samples was isolated using TRIzol (Invitrogen) following the manufacturer’s instructions. To eliminate DNA contamination, total RNAs were treated with DNase I (Yeason). The RNA-seq libraries were prepared by using SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian (Yeason). Quality control was performed using Qubit (Thermo Fisher Scientific, USA) and Qsep100 (BiOptic, China) before the libraries were sequenced on the BGI DNBSEQ-T7 Sequencing Platform using a 150 bp paired-end run. The RNA-seq data sequencing reads were aligned to the reference genome (Genome Reference Consortium GRCh38) using the spliced read aligner STAR, which was provided with the Ensembl human genome assembly. The gene expression matrix was obtained by featureCounts and be normalized by TPM for downstream analysis.
For data analysis, genes with |log₂FC| > 1 and adjusted p-value (FDR) < 0.05 were classified as differentially expressed (DEG). Gene Set Enrichment Analysis (GSEA) was performed using the clusterProfiler v4.2.2 R package. DEGs were ranked by log₂FC values and tested against the Molecular Signatures Database (MSigDB). Pattern recognition receptor (PRR)-related genes were curated from literature-defined sets encompassing Toll-like receptors (TLR), NOD-like receptors (NLR), RIG-I-like receptors (RLR), and C-type lectins (CLR). Heatmaps were generated using the pheatmap v1.0.12 R package: DEG expression values were transformed to z-scores by row, and visualized with diverging color gradients.

Animal model
Male C57BL/6 (113169, Biocytogen) and BALB/c (113170, Biocytogen) mice aged between 6-8 weeks were purchased from Biocytogen (Beijing, China). Sex was not considered in the study design and analysis. Sex of the animals does not affect the results of this study, and this study was not designed to detect sex differences. All experiments were conducted in a specific pathogen-free (SPF) barrier facility, and experimental and control animals were housed separately. Animals were euthanized using carbon dioxide inhalation at the end of the animal experiments. All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of Fudan University (approval number: FUSCC-IACUC-S2022-0205). According to the regulations of the Institutional Animal Care and Use Committee of Fudan University, the tumor volume in mice must not exceed 2000 mm3. Throughout the experiment, we measured the tumor size twice a week using a vernier caliper, and euthanasia was performed once the tumor reached the critical size.
For in vivo bioluminescence assay, BALB/c mice were injected with 100μL Fluc RNA-LNPs (5μg RNA per mouse) via intramuscular injection to the right thigh. At the indicated time points, 200 μL D‑luciferin potassium salt solution (15mg/mL) was injected to each mouse through intraperitoneal injection. In vivo luminescence signal was measured using an IVIS Spectrum system (PerkinElmer) 10 min later. Data were analyzed using Living image software.
For serum ALT and AST evaluation, mouse blood was collected at the indicated time points, and kept at room temperature for 60 min for coagulation. Serum samples were collected through centrifugation at 3000 rpm/min under 4℃. Then, serum samples were sent to Fudan University Laboratory Animal Center for analysis using ADVIA XPT (SIEMENS). For H&E staining of mouse tissue, after euthanizing the mice, organ samples were collected, fixed and paraffin-embedded, sectioned, and stained for H&E (Servicebio).
For the murine melanoma model, 1x105 B16F10-OVA cells in 200ul RPMI 1640 medium (without fetal bovine serum) were subcutaneously injected into the right flank of C57BL/6N mice. For the HPV-associated TC-1 model, 1x105 TC-1 cells in 200ul RPMI 1640 medium (without fetal bovine serum) were subcutaneously injected into the right flank of C57BL/6N mice. Mice body weight and tumors were measured twice every week. Tumor volumes were determined by using the formula volume = (length × width2)/2 (L: largest diameter of the tumor; W: smallest diameter of the tumor). For immunization, the mice were vaccinated with 100μL RNA-LNPs (3μg RNA per dose) via intramuscular injection to the right thigh.

Flow cytometry analysis
For OVA (SIINFEKL) Tetramer staining, PBMCs were separated by Mouse 1× Lymphocyte Separation Medium (DAKEWE, Shenzhen) according to the manufacturer’s instructions. Cells were then stained with Fixable Viability Dye eFluor 450 (65-0863-14, eBioscience), anti-CD3 (APC-65077, Proteintech), anti-CD8a (FITC-65069, Proteintech), H-2Kb&B2M&OVA (SIINFEKL) Tetramer (PE, H2A-MP2H7-25ug, ACROBiosystems), and analyzed by CytoFLEX Flow Cytometer (Beckman).
For intratumoral T cell staining, tumor tissues were dissected into 1–2 mm³ fragments in ice-cold RPMI 1640 medium (10% FBS). Samples were digested in a cocktail of 1 mg/mL collagenase IV (Yeasen) and 100 U/mL DNase I (Yeasen) for 30 min at 37°C with constant agitation (200 rpm). Cells were then stained with Fixable Viability Dye eFluor 450 (65-0863-14, eBioscience), anti-CD3 (APC-65077, Proteintech), anti-CD8a (FITC-65069, Proteintech), anti-mouse CD4 (PE-CY7, 116016，Biolegend), H-2Kb&B2M&OVA (SIINFEKL) Tetramer (PE, H2A-MP2H7-25ug, ACROBiosystems) , and analyzed by CytoFLEX Flow Cytometer (Beckman).
For intra-cellular IFNγ staining, PBMCs or splenocytes were separated by Mouse 1× Lymphocyte Separation Medium (DAKEWE, Shenzhen) according to the manufacturer’s instructions. Cells were then stained with Fixable Viability Dye eFluor 450 (65-0863-14, eBioscience), anti-CD3 (APC-65077, Proteintech), anti-CD8a at 4℃ for 30min. Subsequently, cells were fixed and permeabilized with Intracellular Fixation and Permeabilization Buffer Set (eBioscience) according to the manufacturer’s instructions, and stained with anti-IFNγ (PE, 163503, Biolegend) at room temperature for 20min, and analyzed by CytoFLEX Flow Cytometer (Beckman). All information on the antibodies is summarized in Supplementary Table 1.

Statistical analysis
All data in this study were analyzed using GraphPad Prism and presented as mean (SD) unless otherwise indicated. Two-tailed unpaired Student’s t-test was used to compare two groups. One-way ANOVA tests were used to compare more than two groups. For survival curves, the data was performed via Kaplan-Meier analysis. *P < 0.05 was considered statistically significant. **P < 0.01, ***P < 0.001 and ****P < 0.0001 were considered highly significant. ns, not significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Yu et al describes an mRNA platform in which the translation of mRNA is cap-dependent and such mRNa is capable of immune system activation through a combibation of PRRs.\nMajor comments:\n- in figure 2F and 2G, authors mention clearly more potent in vivo effiency of their platform compared to especially capped mRNA with modification (mCAP) which is not directly evident from the data shown. authors need to either show more quantitative data (like AUC) or change their title for this session\n- in Figure 4a and espeically in 4B, it is astonishing to see that unmodified but capped mRNA is not activating BMDCs which is contrary to existing literature. Can the authors comment on that?\n- In the tumor experiments, a proper control to their mRNA platform is indeed unmodified capped mRNA and not modified capped mRNA. do the authors have data in one of the tumor experiments for this comparison?\n- Can the authors provide data on the purity of the mRNAs used (e.g. double stranded mRNA content, etc)?\n- Can the authors comment on the immunostimulatory properties of the lipids used in their vaccine platform?\n- Have the authors investigated the antigen-specific immune responses that oculd be elicited against the viral motifs they introduced to the mRNa backbone?", "reviewer_2": "The authors have submitted a manuscript aiming to optimize an mRNA vaccination approach by developing a linear cap-independent mRNA with a goal to improve the stability and translatability of the mRNA construct. This is done with modifications of every aspect of the non-translated regions. The approach is comprehensive, the data clearly presented, and the results encouraging. Ultimately, the culmination of “proof of concept” in vivo testing of this approach is performed in three murine models, the first is a non-tumor model which shows that demonstrates superiority of driving immune responsiveness with UPA-FLUC and 2UPA-FLUC constructs over control, and then two tumor models, one B16-OVA melanoma and HPV positive tumor model, both of which demonstrate potent efficacy compared to mCAP-FLUC and mCAP-OVA. There are a few issues to be addressed.\nFirstly, the initial construct selected UX1 as sfRNA and then all modifications were built around this. Where other sfRNAs selected for optimization as well? For example, were modifications to a construct starting with sfRNA of ZX1 or DX1 made showing that similar approach with selection of PABP motif over polyC or polyT or optimization of 3’ and 5’ UTRs were similarly beneficial? Mainly, it would be helpful to know if these approaches generally improve the performance broadly or if this is just a function of UX1 as sfRNA.\nSecondly, please explain why you are using GLUC-mRNA constructs for in vitro and FLUC-constructs in vivo.\nThirdly, given the robust PPR activity of UPA-FLUC and 2UPA-FLUC, it would be helpful to understand the difference in outcomes between a non-translating mRNA and translating RNA against OVA or E6/E7 in context of UPA/2UPA constructs. For example, what is the influence of the PPR activity of UPA/2UPA-FLUC compared to UPA/2UPA-OVA or UPA/2UPA-E6E7?", "reviewer_3": "The manuscript ‘Engineering Linear Cap-independent mRNA vaccines with intrinsic adjuvanticity for potent cancer immunotherapy’ describes the construction of linear mRNA transcripts that can function without a 5’ cap or modified nucleotides. The construct initiates translation from an Enterovirus A internal ribosomal entry site (IRES). To protect the 5’ end from exonucleases, the authors tested several Flavivirus subgenomic RNAs that contain structured exoribonuclease-resistant RNA, settling on an 88nt sequence from the Usutu virus.\nThe manuscript has three main parts, the description of how this capless mRNA was constructed, characterized, and tested against canonical (capped) mRNA (Figures 1, 2, 3), its effect on gene expression when transfected into dendritic cells in vitro and mice in vivo (Figure 4), and testing its efficacy as a cancer vaccine in two mouse models (Figures 5,6).\nGeneral Comments:\nOverall, this is a well-written paper. The experiments are well designed and executed. The methodology is clear and easy to follow. The characterization of the mRNA construct is thorough and the experiments using siRNA to assess the relative importance of the different RNA binding proteins on the constructs in Figure 3 are very well done. The cancer vaccine studies in Figures 5 and 6 are also well done and demonstrate that mRNA vaccines can activate cell mediated immunity sufficiently to cause tumor regression.\nThe major concern is that the principal concept described in this manuscript, that it is possible to construct a functional linear capless mRNA comprised of unmodified nucleotides whose 5’ end is protected by secondary structures and whose translation is driven by an IRES, is not novel. This was originally described in 2023 (PMID: 36828937) in cells and its efficacy as a protective vaccine against a lethal dose of Influenza A confirmed in mice in 2025 (PMID: 39988620) by the same group. While the specific structure of the 5’ terminal protective sequences (hairpins) in those papers differ from the terminal protective sequences described in this manuscript (the 88 nucleotide UX1), they are similar in length and have been clearly shown to protect the 5’ terminus sufficiently to allow protein expression in vitro and in vivo.\nIn the introduction, the authors cite three published paper that describe linear capless mRNA transcripts. These constructs are dismissed as not likely to be very effective, however, because they generally fail to provide sufficient protection to the 5’ end (lines 83-85). The first cited manuscript (PMID: 39195827) describes a mRNA transcript that lacks a 5’ cap and initiates translation of the envelope protein E2 of the bovine viral diarrhea virus-1 using the coxsackievirus B3 IRES. This construct did not contain structured elements in the 5’ terminus but was able to generate essentially equivalent neutralizing antibodies as capped mRNA containing modified nucleotides in rodents and guinea pigs. It was not tested in a live viral challenge infection.\nThe second cited paper (PMID: 39648715) describes capless mRNA constructs lacking modified nucleotides that initiated translation through the EMCV IRES. These constructs also lacked structured sequences in the 5’ terminus but effectively expressed various proteins/antigens (eGFP, luciferase, SARS-CoV Delta spike, etc.) to generate neutralizing antibodies in vivo. This mRNA construct was not tested in a live animal challenge.\nThe third cited paper (also referenced above ((PMID: 39988620)) describes a linear capless mRNA without modified nucleotides containing the EMCV IRES to express the full-length Influenza A surface protein hemagglutinin. This construct provided essentially full protection against a lethal influenza A infection when used as a vaccine in mice. In contrast to the constructs in the other two papers, this construct did contain structured sequences (hairpins) in the 5’ terminus and thus is most similar to the construct described in this paper.\nWhile it is possible (although not demonstrated) that the mRNA transcript described in this manuscript is more stable and more effective at protein expression than those used in the above papers, there is no disputing that the capless mRNA constructs described in those papers are effective at generating neutralizing antibodies in vivo. Furthermore, there is no single universal \"Xrn1 stop sequence\"; instead, Xrn1 stalls at specific structural features within RNA molecules, such as G-rich regions forming G-quadruplexes and stem-loop (SL) and dumbbell (DB) structures. The structures must be stable and \"rigid\" enough to physically block Xrn1's progression. Technically, hairpins on the 5’ end are, in fact, a Xrn1 stop sequence. So, the novelty of this system is not clear. A difference in degree perhaps, but not a substantive difference.\nIf the authors have evidence that the constructs described in these three papers fail to provide sufficient protection to the mRNA 5’ end, it would be helpful to include it.\nSpecific comments/Questions:\nFigure 3I and 3J: Knocking down ILF2 and ILF3 decreases GLuc activity in all three constructs (UPA, IRES, mCAP). If ILF2 and ILF3 are specifically binding and stabilizing UPA and not the other constructs tested (IRES and mCAP), then why does siRNA knockdown decrease GLuc activity in all three constructs? (In contrast, knocking down IGF2BP1 and YBX1 seems to selectively decrease GLuc activity in the UPA construct).\nFigures 4A and 4B: Figure 4A indicates that UPA and 2UPA increase IL6, IFN-a, and IFN-b to a significantly greater degree than the capped mRNA without modified nucleotides (CAP-FLUC). The prevailing theory since the Kariko paper in 2005 is that it is the unmodified nucleotides that tend to activate the inflammatory/interferon pathways in dendritic cells. Therefore, gene activation should be similar in any construct that contains unmodified nucleotides. Is the difference in activation due to the number of unmodified nucleotides present in the UPA/2UPA constructs compared to the CAP-FLUC construct or is it due to something else?\nThis same issue also relates to Figure 4B. The heat map indicates that the capped mRNA construct without modified nucleotides (CAP-FLUC) does not upregulate inflammatory/type 1 interferon genes when transfected into mouse dendritic cells in contrast to the UPA/2UPA constructs that do. What specifically is activating the dendritic cells when they are transfected with UPA and 2UPA in Figure 4B? If it is the presence of unmodified nucleotides, why does the CAP-FLUC not activate the dendritic cells. Is it another component of UPA/2UPA?\nFigure 5B and 6B: ‘volumes’ is misspelled in the body of the graph.\nLine 128: ….we extracted sfRNA sequences from five flaviviruses:\nIs ‘extracted’ the best word here? Perhaps, ‘ we generated DNA templates that contained the sequences from five flaviviruses’\nLine 179: Notably, the 2UPA LciRNA achieved the highest expression:\nRNA expression and stability depend in part, on the size of the 5’UTR. If a long sequence upstream of IRES is present mRNA tends to degrade slower. A better comparison would be to compare UPA + a random sequence of the same length to 2UPA. This will confirm that stability of the 2UPA is specific to the sequence and not a side effect of increased length.\nLine 249…initiate innate immune pathways, conferring a self-adjuvant effect that can amplify adaptive antitumor immunity:\nDo the authors believe that inducing local intracellular inflammation by using unmodified nucleotides helps develop a better immune response even if this strategy (using unmodified RNA) can result in lower antigen expression? If so, then using capped mRNA with unmodified nucleotides should induce a better immune response than capped mRNA with modified nucleotides, but it does not.\nThe activation of the intracellular inflammatory/interferon 1 pathways by unmodified nucleotides is a two-edged sword. It may work as an adjuvant to amplify the immune reaction, but it can also lead to inhibition of cap-dependent translation, a decrease in further RNA uptake by cells, and acceleration of transcript degradation, all of which can lead to a decrease in protein expression. Which of these apparently opposing forces that are activated when unmodified RNA is introduced into cells (decreased transcript expression or an increase in cell self-adjuvant activity) do the authors think is most important in determining transcript expression and immune response in their constructs?\nSupplementary Tables (sequences, Table S1)\nThe sequences in Table S1 and S2 are a little confusing and difficult to navigate and might be easier to follow if presented as the complete mRNA sequences rather than partial sequences of the DNA template.\nThe UX1 sequence is listed as being 88 nucleotides long in the manuscript but in the supplemental data, table S1, it only contains 80 nucleotides. Can the authors clarify.\nThe UPA protective sequence is described in the manuscript as being comprised of the UX1 and PABP-binding motif. In the Supplemental Data, Table S1, the 5' end of the UPA sequence contains 4 nucleotides (AATT) in the 5' terminus that are not present in the UX1 sequence. Can the authors clarify."}, "author_response": "Proposed point-by-point responses to reviewers’ comments\nNCOMMS-25-65642-T \"Engineering Linear Cap-independent mRNA Vaccines with Intrinsic\nAdjuvanticity for Potent Cancer Immunotherapy\"\nRESPONSE TO REVIEWERS’ COMMENTS\nReviewer #1 (Remarks to the Author):\nYu et al describes an mRNA platform in which the translation of mRNA is cap-dependent\nand such mRNA is capable of immune system activation through a combination of PRRs.\nMajor comments:\n- in figure 2F and 2G, authors mention clearly more potent in vivo effiency of their platform\ncompared to especially capped mRNA with modification (mCAP) which is not directly evident\nfrom the data shown. authors need to either show more quantitative data (like AUC) or change\ntheir title for this session\nResponse:\nWe appreciate the reviewer's insightful suggestions. We calculated the area under the\ncurve (AUC) for in vivo luminescence signals in mice over the 48 to 120 hours period\nfollowing mRNA administration and performed statistical analysis. This analysis\nrevealed that the 2UPA group demonstrated a significantly higher AUC during this\ninterval than the m1Ψ-modified capped mRNA (mCAP) group (p < 0.05) (Figure 2H).\nThese findings substantiate our assertion that engineered LciRNA exhibits superior in\nvivo persistence compared to m1Ψ-modified capped mRNA.\n- in Figure 4a and espeically in 4B, it is astonishing to see that unmodified but capped mRNA is\nnot activating BMDCs which is contrary to existing literature. Can the authors comment on that?\nResponse:\nWe thank the reviewer’s insightful inquiry. Notably, our data indicate that unmodified,\ncapped mRNA elicits detectable innate immune activation in bone marrow-derived\ndendritic cells (BMDCs), as evidenced by significant upregulation of IL-6 and IFN-β\nexpression post-transfection (Fig. 4A, S4A, S4C). However, at equivalent doses,\nLciRNA induces substantially broader and more potent activation of antigen\npresentation machinery, type I interferon responses, and RIG-I/TLR\npathway-associated genes in BMDCs (Fig. 4A–C, S4A–H). These findings\ndemonstrate that LciRNA possesses superior innate immune-stimulatory capacity\ncompared to unmodified capped mRNA.\nCurrent literature establishes that cytosolic and endosomal pattern recognition\nreceptors (PRRs) detect distinct molecular signatures of exogenous RNA: RIG-I\nrecognizes 5′-triphosphate termini and double-stranded RNA (dsRNA) motifs; TLR3,\nMDA5, and PKR sense dsRNA structures; while TLR7, TLR8, and TRIM25 are\nactivated by unmodified uridine residues (Curr Opin Immunol., 2022, PMID:\n36334350；Science, 2025, PMID: 40179174). The modest immunostimulatory activity\nof unmodified capped mRNA is thus likely attributable to recognition of unmodified\nuridines. In contrast, LciRNA integrates multiple molecular features recognizable by\nintracellular RNA sensors—specifically, unmodified uridine residues, exposed\n5′-triphosphate groups, highly structured viral RNA motifs, and a UPA motif\nidentified herein to engage PKR.\n- In the tumor experiments, a proper control to their mRNA platform is indeed unmodified capped\nmRNA and not modified capped mRNA. do the authors have data in one of the tumor experiments\nfor this comparison?\nResponse:\nWe appreciate the reviewer’s constructive suggestion. Consistent with our data,\nm1Ψ-modified capped mRNA exhibits markedly stronger in vivo protein expression\nthan unmodified capped mRNA (Figure 2G), whereas the latter induces modestly\nenhanced innate immune activation (Figure 4A, S4A). Therefore, it is reasonable to\ninclude unmodified capped mRNA in the animal models. In light of this, we repeated\nthe tumor model studies described in the manuscript, incorporating direct\ncomparisons between m1Ψ-modified capped mRNA, unmodified capped mRNA and\nLciRNA. Our results demonstrate that LciRNA incorporating dual UPA motifs (2UPA)\nconsistently elicited the most potent antitumor immunity and achieved the greatest\ntumor suppression efficacy—across both murine melanoma (B16F10-OVA) and HPV\nE6E7-expressing TC-1 tumor models (Figure 5 & 6). Notably, unmodified capped\nmRNA showed comparable antigen-specific T cell responses and therapeutic efficacy\nto m1Ψ-modified mRNA. This equivalence likely stems from its modest innate\nimmunostimulation failing to fully compensate for significantly reduced antigen\nexpression kinetics.\n- Can the authors provide data on the purity of the mRNAs used (e.g. double stranded mRNA\ncontent, etc)?\nResponse:\nWe thank the reviewer for the valuable suggestion. Indeed, mRNA purity and residual\ndouble-stranded RNA (dsRNA) contaminants critically determine immunostimulatory\nprofiles and protein expression kinetics. In this study, all mRNA constructs were\nsynthesized using an established, rigorously optimized in vitro transcription (IVT)\nplatform. Denaturing gel electrophoresis confirmed high purity for all the mRNAs\nused in BMDC activation assays (Fig. S4I). Moreover, all the IVT template DNAs\nwere amplified with primers featuring 2’-O-methyl modifications at the 3’-terminal\nnucleotides—a validated strategy to minimize dsRNA byproducts (Science, 2025,\nPMID: 40179174). As a result, dsRNA ELISA analysis showed dsRNA levels were\nconsistently <0.1% of total RNA mass across all transcripts (Fig. S4J). Collectively,\nthese stringent quality controls support the conclusion that the enhanced\nimmunostimulatory properties of LciRNA reflect its intrinsic molecular design rather\nthan artifacts arising from dsRNA contamination.\n- Can the authors comment on the immunostimulatory properties of the lipids used in their vaccine\nplatform?\nResponse:\nWe thank the reviewer’s insightful inquiry. We formulated and manufactured lipid\nnanoparticles (LNPs) using the same composition and process as Moderna’s\nmRNA-1273 (i.e., SM102, cholesterol, DSPC, and PEG2000). The ionizable cationic\nlipid, DSPC, and PEG components of LNPs have each been implicated in\npro-inflammatory activity and, in some contexts, in antibody-mediated immune\nresponse (J Nanobiotechnology., 2022, PMID: 35701851). A recent study further\ndemonstrated that empty SM102 LNPs (without mRNA) can activate immune\nsignaling via TLR4 (NPJ Vaccines., 2025, PMID: 40246950). To exclude LNP\ncomposition as a confounding contributor to the immunostimulatory profile, we\nobserved for our vaccines, we therefore prepared empty SM102 LNPs and compared\ntheir ability to activate BMDCs with that of the complete vaccine formulations. While\nempty LNPs did induce low levels of inflammatory cytokines and T-cell\nco-stimulatory molecules, the magnitude of activation was substantially lower than\nthat elicited by the full LciRNA vaccine (Figure. S4A-H).\n- Have the authors investigated the antigen-specific immune responses that could be elicited\nagainst the viral motifs they introduced to the mRNa backbone?\nResponse:\nWe thank the reviewer’s insightful inquiry. Although antigen-specific immune\nresponses directed against viral RNA motifs are theoretically conceivable, there is\ncurrently limited experimental evidence to support their occurrence in vivo. Adaptive\nantigen recognition is mediated principally by T cells via the T cell receptor (TCR),\nwhich recognizes peptide antigens presented on major histocompatibility complex\nmolecules, and by B cells via the B cell receptor (BCR), which can bind a broad\nspectrum of antigen types including proteins, polysaccharides, lipids, and nucleic\nacids (Cellular and Molecular Immunology, 10th ed.). However, reports in which\nexogenous mRNA itself — rather than the protein it encodes — functions as an\nimmunogen that elicits sequence-specific B cell responses are scarce. Antibodies\nreported to target viral RNA sequences have predominantly been generated by in vitro\nselection approaches (e.g., phage display) rather than arising spontaneously after\nexposure to viral RNA (Nucleic Acids Res., 2025, PMID: 39739875; Nat Commun.,\n2019, PMID: 31399592).\nMoreover, exogenously delivered mRNA is typically encapsulated in LNPs or viral\nparticles and released into the cytosol. To our knowledge, there is no established\ncellular pathway that presents cytosolic mRNA on the cell surface in a configuration\naccessible to BCR recognition. For these reasons, we conclude that current evidence\ndoes not indicate that viral RNA motifs per se induce antigen-specific adaptive\nimmune responses. In contrast, multiple studies have demonstrated that viral RNA\nmotifs are potent activators of innate immune pathways (Mol Cell., 2017, PMID:\n28625551; Mol Cell., 2022, PMID: 34951963).\nReviewer #2 (Remarks to the Author):\nThe authors have submitted a manuscript aiming to optimize an mRNA vaccination\napproach by developing a linear cap-independent mRNA with a goal to improve the stability\nand translatability of the mRNA construct. This is done with modifications of every aspect of\nthe non-translated regions. The approach is comprehensive, the data clearly presented, and\nthe results encouraging. Ultimately, the culmination of “proof of concept” in vivo testing of\nthis approach is performed in three murine models, the first is a non-tumor model which\nshows that demonstrates superiority of driving immune responsiveness with UPA-FLUC and\n2UPA-FLUC constructs over control, and then two tumor models, one B16-OVA melanoma\nand HPV positive tumor model, both of which demonstrate potent efficacy compared to\nmCAP-FLUC and mCAP-OVA. There are a few issues to be addressed.\nFirstly, the initial construct selected UX1 as sfRNA and then all modifications were built around\nthis. Where other sfRNAs selected for optimization as well? For example, were modifications to a\nconstruct starting with sfRNA of ZX1 or DX1 made showing that similar approach with selection\nof PABP motif over polyC or polyT or optimization of 3’ and 5’ UTRs were similarly beneficial?\nMainly, it would be helpful to know if these approaches generally improve the performance\nbroadly or if this is just a function of UX1 as sfRNA.\nResponse:\nWe thank the reviewer for the valuable suggestion. Whether the PABP motif and the\noptimized 5′and 3′UTR sequences are equally effective when incorporated into\nLciRNA vectors containing alternative xrRNAs is indeed an important question. To\naddress this, we evaluated these sequence elements in a DX1-based LciRNA. The\nresults show that the PABP motif together with the β-globin 5’ and 3’ UTRs remain\nthe most favorable configurations (Fig. S1A). The broad enhancement conferred by\nthe PABP motif likely reflects its capacity to recruit PABP, thereby stabilizing the\nRNA and promoting translation, whereas the benefit of the β-globin UTRs may derive\nfrom their synergy with EV-A-S1 to facilitate translation initiation and increase\nLciRNA stability. Taken together, these data indicate that the enhancing effects of\nthese three sequence elements on LciRNA performance are not strictly dependent on\ntheir combination with the UX1 sequence.\nSecondly, please explain why you are using GLUC-mRNA constructs for in vitro and\nFLUC-constructs in vivo.\nResponse:\nWe thank the reviewer’s insightful inquiry. Gaussia luciferase (GLUC) is a secreted\nluciferase that contains an N-terminal signal peptide; consequently, assessment of\nexpression from GLUC-encoding mRNA in vitro obviates cell-lysis steps and is both\nsimpler and more reproducible. Quantification of GLUC activity in culture\nsupernatants also permits longitudinal monitoring of mRNA expression over extended\ntime courses. By contrast, for in vivo bioluminescence imaging a non-secreted\nreporter such as firefly luciferase (FLUC) is preferable because secreted reporters like\nGLUC diffuse systemically and therefore do not permit straightforward, spatially\nresolved imaging of tissue-localized mRNA expression.\nThirdly, given the robust PPR activity of UPA-FLUC and 2UPA-FLUC, it would be helpful to\nunderstand the difference in outcomes between a non-translating mRNA and translating RNA\nagainst OVA or E6/E7 in context of UPA/2UPA constructs. For example, what is the influence of\nthe PPR activity of UPA/2UPA-FLUC compared to UPA/2UPA-OVA or UPA/2UPA-E6E7?\nResponse:\nWe thank the reviewer’s insightful inquiry. We directly compared the capacity of\nLciRNA and capped mRNA encoding either OVA or FLUC to activate innate immune\nresponses in BMDCs. UPA- and 2UPA-derived OVA and FLUC constructs produced\ncomparable levels of BMDC activation, and both induced responses that were\nsignificantly greater than those elicited by capped mRNA vaccines or by empty LNPs\n(Figure S4A). These data indicate that the immunostimulatory properties of LciRNA\nare not dependent on the specific coding sequence but rather arise from its noncoding\nelements and intrinsic molecular features.\nReviewer #3 (Remarks to the Author):\nThe manuscript ‘Engineering Linear Cap-independent mRNA vaccines with intrinsic\nadjuvanticity for potent cancer immunotherapy’ describes the construction of linear mRNA\ntranscripts that can function without a 5’ cap or modified nucleotides. The construct initiates\ntranslation from an Enterovirus A internal ribosomal entry site (IRES). To protect the 5’ end\nfrom exonucleases, the authors tested several Flavivirus subgenomic RNAs that contain\nstructured exoribonuclease-resistant RNA, settling on an 88nt sequence from the Usutu\nvirus.\nThe manuscript has three main parts, the description of how this capless mRNA was\nconstructed, characterized, and tested against canonical (capped) mRNA (Figures 1, 2, 3), its\neffect on gene expression when transfected into dendritic cells in vitro and mice in vivo\n(Figure 4), and testing its efficacy as a cancer vaccine in two mouse models (Figures 5,6).\nGeneral Comments:\nOverall, this is a well-written paper. The experiments are well designed and executed. The\nmethodology is clear and easy to follow. The characterization of the mRNA construct is thorough\nand the experiments using siRNA to assess the relative importance of the different RNA binding\nproteins on the constructs in Figure 3 are very well done. The cancer vaccine studies in Figures 5\nand 6 are also well done and demonstrate that mRNA vaccines can activate cell mediated\nimmunity sufficiently to cause tumor regression.\nThe major concern is that the principal concept described in this manuscript, that it is possible to\nconstruct a functional linear capless mRNA comprised of unmodified nucleotides whose 5’ end is\nprotected by secondary structures and whose translation is driven by an IRES, is not novel. This\nwas originally described in 2023 (PMID: 36828937) in cells and its efficacy as a protective\nvaccine against a lethal dose of Influenza A confirmed in mice in 2025 (PMID: 39988620) by the\nsame group. While the specific structure of the 5’ terminal protective sequences (hairpins) in those\npapers differ from the terminal protective sequences described in this manuscript (the 88\nnucleotide UX1), they are similar in length and have been clearly shown to protect the 5’ terminus\nsufficiently to allow protein expression in vitro and in vivo.\nIn the introduction, the authors cite three published paper that describe linear capless mRNA\ntranscripts. These constructs are dismissed as not likely to be very effective, however, because\nthey generally fail to provide sufficient protection to the 5’ end (lines 83-85). The first cited\nmanuscript (PMID: 39195827) describes a mRNA transcript that lacks a 5’ cap and initiates\ntranslation of the envelope protein E2 of the bovine viral diarrhea virus-1 using the coxsackievirus\nB3 IRES. This construct did not contain structured elements in the 5’ terminus but was able to\ngenerate essentially equivalent neutralizing antibodies as capped mRNA containing modified\nnucleotides in rodents and guinea pigs. It was not tested in a live viral challenge infection.\nThe second cited paper (PMID: 39648715) describes capless mRNA constructs lacking modified\nnucleotides that initiated translation through the EMCV IRES. These constructs also lacked\nstructured sequences in the 5’ terminus but effectively expressed various proteins/antigens (eGFP,\nluciferase, SARS-CoV Delta spike, etc.) to generate neutralizing antibodies in vivo. This mRNA\nconstruct was not tested in a live animal challenge.\nThe third cited paper (also referenced above ((PMID: 39988620)) describes a linear capless\nmRNA without modified nucleotides containing the EMCV IRES to express the full-length\nInfluenza A surface protein hemagglutinin. This construct provided essentially full protection\nagainst a lethal influenza A infection when used as a vaccine in mice. In contrast to the constructs\nin the other two papers, this construct did contain structured sequences (hairpins) in the 5’\nterminus and thus is most similar to the construct described in this paper.\nWhile it is possible (although not demonstrated) that the mRNA transcript described in this\nmanuscript is more stable and more effective at protein expression than those used in the above\npapers, there is no disputing that the capless mRNA constructs described in those papers are\neffective at generating neutralizing antibodies in vivo. Furthermore, there is no single universal\n\"Xrn1 stop sequence\"; instead, Xrn1 stalls at specific structural features within RNA molecules,\nsuch as G-rich regions forming G-quadruplexes and stem-loop (SL) and dumbbell (DB) structures.\nThe structures must be stable and \"rigid\" enough to physically block Xrn1's progression. Technically,\nhairpins on the 5’ end are, in fact, a Xrn1 stop sequence. So, the novelty of this system is not clear.\nA difference in degree perhaps, but not a substantive difference.\nIf the authors have evidence that the constructs described in these three papers fail to provide\nsufficient protection to the mRNA 5’ end, it would be helpful to include it.\nResponse:\nWe sincerely appreciate the reviewer’s thoughtful review and constructive feedback\non our manuscript. We appreciate the acknowledgment of the rigorous experimental\ndesign and execution. Below, we address the primary concern regarding the novelty of\nthe LciRNA platform, with a focus on its innovative integration of\nimmuno-translation coupling and superior efficacy compared to existing platforms.\nFirst, LciRNA achieves unprecedented stability and expression efficacy through\nits uniquely engineered UPA architecture. In contrast to earlier approaches (Vet Sci.,\n2024, PMID: 39195827; Ann Med., 2025, PMID: 39648715; Gene Ther., 2025, PMID:\n39988620), which left the RNA 5′ end unprotected or relied on hairpin structures to\nprotect cap-independent mRNAs, our systematic screening revealed UX1—a\nflaviviral xrRNA—as the superior element for preventing exonucleolytic degradation.\nCritically, fusing UX1 with a PABP-binding motif (forming UPA) and deploying it in\ntandem repeats (2UPA) enabled LciRNA to outperform not only prior hairpin designs\nbut even standard capped/modified mRNAs. Direct comparisons in Figure 2D\ndemonstrate that 2UPA-LciRNA exhibits 10.5-fold higher expression than IRES-only\ncontrols and 5.6-fold higher than PABP motif-IRES constructs. Moreover, we\nsynthesized mRNAs bearing the exact hairpin structures from the study (Gene Ther.,\n2025, PMID: 39988620) (2HP/double-hairpin; 3HP/triple-hairpin) and confirmed\ntheir significantly weaker stability and expression relative to UPA-LciRNA (Figure\nS2B). This optimization allows LciRNA to match or exceed the in vivo persistence of\ncapped mRNAs (Figure 2E, 2H), overcoming a key limitation of prior\ncap-independent platforms.\nSecond, LciRNA uniquely couples robust immune-stimulation with\nuncompromised translation—resolving the \"two-edged sword\" dilemma\ninherent to unmodified RNA vaccines. As noted by the reviewer, unmodified RNAs\nface a trade-off: strong immune activation inhibits cap-dependent translation and\naccelerates RNA decay. LciRNA breaks this paradigm through two synergistic\nmechanisms: (i) Its EV-A-S1 IRES drives cap-independent translation, evading\nimmune-mediated translational suppression; (ii) The UPA structure recruits\nRNA-stabilizing RBPs (PABP, IGF2BP1, YBX1; Figure 3) to resist degradation even\namid inflammation (Figure 2G–H). Concurrently, LciRNA’s unmodified nucleotides,\nexposed 5′-triphosphate terminus, viral-derived structured motifs, and UPA-PKR\ninteractions (Figure 4A-C, S3A-B, S4A-H) enable broad activation of\npattern-recognition receptors. This triggers dendritic cell maturation, proinflammatory\ncytokine release (IL-12, IFN-α), and T-cell priming—far exceeding the mild\nadjuvanticity of unmodified capped mRNA (which relies solely on uridine sensing).\nThus, LciRNA transforms immune activation from a translational inhibitor into a\nsynergistic partner.\nThird, LciRNA demonstrates transformative efficacy in cancer models. While\nprior studies achieved neutralizing antibodies against pathogens (e.g., influenza),\nnone validated robust T-cell responses or tumor control. In contrast, LciRNA vaccines\nachieved significant tumor growth suppression in B16F10 melanoma and HPV\nassociated tumor models (Figures 5–6), correlating with expanded tumor-infiltrating\nCD8+ T cells. This therapeutic synergy—enabled by immuno-translation\ncoupling—establishes LciRNA as the first cap-independent platform to cure\nestablished tumors without chemical modifications or capping. Its streamlined\nproduction further underscores its translational value.\nIn summary, LciRNA’s novelty lies not merely in structural optimization but in\nits paradigm-shifting resolution of immuno-translation antagonism, unlocking\npotent cancer immunotherapy inaccessible to prior platforms. We have clarified\nthese advances in the revised manuscript.\nSpecific comments/Questions:\nFigure 3I and 3J: Knocking down ILF2 and ILF3 decreases GLuc activity in all three constructs\n(UPA, IRES, mCAP). If ILF2 and ILF3 are specifically binding and stabilizing UPA and not the\nother constructs tested (IRES and mCAP), then why does siRNA knockdown decrease GLuc\nactivity in all three constructs? (In contrast, knocking down IGF2BP1 and YBX1 seems to\nselectively decrease GLuc activity in the UPA construct).\nResponse:\nWe thank the reviewer’s insightful inquiry. LF2 and ILF3 typically form a\nheterodimer in cells and perform diverse functions, including regulation of apoptosis,\nmitosis, and ribosome biogenesis (Mol Cell Biol., 2008, Cell Rep., 2020, PMID:\n18458058; Cell Rep., 2020, PMID: 32433969; J Mol Neurosci., 2021, PMID:\n32748330; Mol Cell Biol., 2015, PMID: 26240280). Knockdown of ILF2 or ILF3\nmay induce global translational repression and trigger apoptosis, which would be\nexpected to result in a broad reduction in expression of all GLUC-encoding mRNAs.\nFigures 4A and 4B: Figure 4A indicates that UPA and 2UPA increase IL6, IFN-a, and IFN-b to a\nsignificantly greater degree than the capped mRNA without modified nucleotides (CAP-FLUC).\nThe prevailing theory since the Kariko paper in 2005 is that it is the unmodified nucleotides that\ntend to activate the inflammatory/interferon pathways in dendritic cells. Therefore, gene activation\nshould be similar in any construct that contains unmodified nucleotides. Is the difference in\nactivation due to the number of unmodified nucleotides present in the UPA/2UPA constructs\ncompared to the CAP-FLUC construct or is it due to something else?\nResponse:\nWe thank the reviewer’s insightful inquiry. The proportion of uridine (U) in the\nLciRNA-specific region (2UPA + EV-A-S1) and in the three coding sequences\n(FLUC, OVA, E6E7) was 20.2%, 14.2%, 24.0%, and 21.1%, respectively. Thus, the U\ncontent of the 2UPA + EV-A-S1 region is not the highest. According to these\ncalculations, the overall U content of the LciRNA exceeds that of capped mRNA only\nwhen the CDS is FLUC, whereas when the CDS is OVA or E6E7 the U content is\nlower than that of the capped mRNA. However, LciRNA encoding OVA nevertheless\nelicited significantly stronger immunostimulatory activity than the corresponding\ncapped mRNA. Collectively, these observations indicate that the immunostimulatory\nproperties of LciRNA are not determined by the relative proportion of uridine.\n(Number of U / all nucleotides: 178/880 for 2UPA + EV-A-S1; 235/1653 for FLUC;\n284/1185 for OVA; 163/771 for E6E7.)\nThis same issue also relates to Figure 4B. The heat map indicates that the capped mRNA construct\nwithout modified nucleotides (CAP-FLUC) does not upregulate inflammatory/type 1 interferon\ngenes when transfected into mouse dendritic cells in contrast to the UPA/2UPA constructs that do.\nWhat specifically is activating the dendritic cells when they are transfected with UPA and 2UPA in\nFigure 4B? If it is the presence of unmodified nucleotides, why does the CAP-FLUC not activate\nthe dendritic cells. Is it another component of UPA/2UPA?\nResponse:\nWe thank the reviewer’s insightful inquiry. Notably, our qPCR data indicate that\nunmodified, capped mRNA elicits detectable innate immune activation in bone\nmarrow-derived dendritic cells (BMDCs), as evidenced by significant upregulation of\nIL6 and IFN-β expression post-transfection (Fig. 4A, S4A, S4C). However, at\nequivalent doses, LciRNA induces substantially broader and more potent activation of\nantigen presentation machinery, type I interferon responses, and RIG-I/TLR\npathway-associated genes in BMDCs (Fig. 4A–C, S4A–H). These findings\ndemonstrate that LciRNA possesses superior innate immune-stimulatory capacity\ncompared to unmodified capped mRNA.\nCurrent literature establishes that cytosolic and endosomal pattern recognition\nreceptors (PRRs) detect distinct molecular signatures of exogenous RNA: RIG-I\nrecognizes 5′-triphosphate termini and double-stranded RNA (dsRNA) motifs; TLR3,\nMDA5, and PKR sense dsRNA structures; while TLR7, TLR8, and TRIM25 are\nactivated by unmodified uridine residues (Curr Opin Immunol., 2022, PMID:\n36334350; Science, 2025, PMID: 40179174). The modest immunostimulatory activity\nof unmodified capped mRNA is thus likely attributable to recognition of unmodified\nuridines. In contrast, LciRNA integrates multiple molecular features recognizable by\nintracellular RNA sensors—specifically, unmodified uridine residues, exposed\n5′-triphosphate groups, highly structured viral RNA motifs, and a UPA motif\nidentified herein to engage PKR.\nFigure 5B and 6B: ‘volumes’ is misspelled in the body of the graph.\nResponse:\nWe thank the reviewer for identifying this typographical error; it has been corrected in\nthe figures.\nLine 128: ….we extracted sfRNA sequences from five flaviviruses:\nIs ‘extracted’ the best word here? Perhaps, ‘ we generated DNA templates that contained the\nsequences from five flaviviruses’\nResponse:\nWe thank the reviewer for this valuable suggestion. The corresponding wording in the\nmanuscript has been revised accordingly.\nLine 179: Notably, the 2UPA LciRNA achieved the highest expression:\nRNA expression and stability depend in part, on the size of the 5’UTR. If a long sequence\nupstream of IRES is present mRNA tends to degrade slower. A better comparison would be to\ncompare UPA + a random sequence of the same length to 2UPA. This will confirm that stability of\nthe 2UPA is specific to the sequence and not a side effect of increased length.\nResponse:\nWe thank the reviewer for this valuable suggestion. To exclude potential confounding\neffects of transcript length, we generated an LciRNA variant in which the second UPA\nelement within 2UPA was rendered nonfunctional while the first UPA remained intact\n(UPA+mUPA), thereby preserving the overall length of the 2UPA construct. We then\ncompared GLUC expression from 1 to 5 days post-transfection. The UPA+mUPA\nvariant exhibited expression levels and durability similar to those of the single-UPA\nLciRNA and markedly lower than those of the intact 2UPA construct （Figure S2B）.\nThese results demonstrate that the enhanced activity of 2UPA relative to UPA is not\nattributable to increased transcript length.\nLine 249…initiate innate immune pathways, conferring a self-adjuvant effect that can amplify\nadaptive antitumor immunity:\nDo the authors believe that inducing local intracellular inflammation by using unmodified\nnucleotides helps develop a better immune response even if this strategy (using unmodified RNA)\ncan result in lower antigen expression? If so, then using capped mRNA with unmodified\nnucleotides should induce a better immune response than capped mRNA with modified\nnucleotides, but it does not.\nThe activation of the intracellular inflammatory/interferon 1 pathways by unmodified nucleotides\nis a two-edged sword. It may work as an adjuvant to amplify the immune reaction, but it can also\nlead to inhibition of cap-dependent translation, a decrease in further RNA uptake by cells, and\nacceleration of transcript degradation, all of which can lead to a decrease in protein expression.\nWhich of these apparently opposing forces that are activated when unmodified RNA is introduced\ninto cells (decreased transcript expression or an increase in cell self-adjuvant activity) do the\nauthors think is most important in determining transcript expression and immune response in their\nconstructs?\nResponse:\nWe thank the reviewer for this insightful question. Previous studies by our group and\nothers have demonstrated that both enhanced antigen expression and stronger\nadjuvant activity can improve vaccine efficacy (Theranostics, 2025, PMID: 39816687;\nSci Transl Med., 2023, PMID: 36867683; Proc Natl Acad Sci U S A., 2023, PMID:\n37155869; Nat Biomed Eng., 2025, PMID: 39920212). However, as the reviewer\nnoted, achieving both simultaneously is inherently challenging. The LciRNA platform\nmay help reconcile this trade-off by enabling spatiotemporal coordination between\nantigen expression and immune activation. On one hand, the unique terminal\narchitecture of LciRNA, together with its incorporated viral RNA elements, elicits\nbroad and robust innate immune stimulation (Figure 4A–C, S4A–H). On the other\nhand, its cap-independent, IRES-driven translation initiation mitigates the impact of\ninnate immune–induced translational repression, while the UPA sequence slows\nXRN1-mediated RNA decay, thereby promoting more sustained antigen expression\ncompared with capped, modified mRNA (Figure 2G, 2H). Through the combined\neffects of these properties, LciRNA effectively transforms the classical “two-edged\nsword” of immune activation versus protein expression into a coordinated mechanism\nthat synergistically enhances antigen-specific immunity.\nSupplementary Tables (sequences, Table S1)\nThe sequences in Table S1 and S2 are a little confusing and difficult to navigate and might be\neasier to follow if presented as the complete mRNA sequences rather than partial sequences of the\nDNA template.\nResponse:\nWe thank the reviewer for the suggestion. The complete mRNA sequence has now\nbeen provided.\nThe UX1 sequence is listed as being 88 nucleotides long in the manuscript but in the supplemental\ndata, table S1, it only contains 80 nucleotides. Can the authors clarify.\nResponse：\nWe thank the reviewer for pointing out this issue. A copy-and-paste error occurred\nwhen collecting the sequences; the correct sequence is 88 nt in length and has now\nbeen corrected.\nThe UPA protective sequence is described in the manuscript as being comprised of the UX1 and\nPABP-binding motif. In the Supplemental Data, Table S1, the 5' end of the UPA sequence contains\n4 nucleotides (AATT) in the 5' terminus that are not present in the UX1 sequence. Can the authors\nclarify.\nResponse：\nWe thank the reviewer for identifying this issue. The inconsistency arose for the same\nreason as noted in the previous comment and has now been corrected."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors addressed my points in a sufficient manner", "reviewer_2": "The authors have responded comprehensively to the reviewers. The manuscript has improved in scope and breadth. Well done.", "reviewer_3": "1. The authors have revised and clarified the supplemental data which is greatly appreciated.\n2. The authors have added an additional experiment to demonstrate that the increased expression of the transcript with the 2UPA compared to that of UPA structure sequence at the 5’ end is not due solely to the increased length of the transcript since a random nucleotide sequence of similar length did not increase expression to the same degree that 2UPA did relative to UPA.\n3. The authors have also added experiments comparing mRNA gaussia luciferase (GLuc)-expressing constructs containing the protective 5’ secondary structures UPA and 2UPA to mRNA GLuc-expressing constructs that contain the protective 5’ secondary structures (double and triple hairpins) published in 2023 (PMID: 36828937) and 2025 (PMID: 39988620) that first demonstrated the in vitro and in vivo efficacy of a linear capless mRNA transcript with unmodified nucleotides driven by an IRES. Although the supplemental figure (Sup Figure 2B) should have included the same 5’ unprotected capless IRES-containing transcript that was used in Figure 2D, when comparing these two figures (Figure 2D and Supp Figure 2B), it is clear that including either the UPA/2UPA secondary structures or the double/triple hairpin secondary structures at the 5’ end, markedly improves GLuc expression (by about 2 logs) above the unprotected IRES construct. The UPA/2UPA constructs demonstrate a 20-40% increase in GLuc activity over the original double hairpin construct at 24 hours. It is not clear that this 20-40% increase represents a ‘substantially greater stability’ (lines 196-197) or only a modest increase in stability without clinical relevance since they were not compared in vivo, only in cells.\nNonetheless, these are important results because they confirm that linear capless mRNA can effectively express proteins in vitro and in vivo using an IRES as long as the 5’ end is protected by a secondary structure that slows exonuclease degradation.\n4. In summary, this manuscript describes a slightly better-expressing version of a linear capless IRES-containing mRNA transcript without modified nucleotides that protects its 5’ end by using secondary structures than the construct that was previously described by others to be effective in cells and animals. The strength of this paper is that it demonstrates that this type of linear capless mRNA construct generates a strong CD8 response which can induce regression of established tumors in mice, whereas the previous group that originally described this type of construct only demonstrated its efficacy in inducing a strong humoral immune response capable of protecting animals against a live viral infection.\nThere is some concern that the manuscript does not fairly characterize the positive results of prior publications on capless linear mRNA. The paper that first described a functional linear capless IRES-containing mRNA with protective 5’ secondary structures (PMID: 36828937) is not cited and while subsequent papers on this topic are cited, the manuscript states that all of these linear capless mRNA constructs ‘generally fail to provide sufficient protection to the mRNA’s 5’ end and, therefore, are unlikely to serve as viable substitutes for traditional capped mRNA’ (lines 86-87). A careful review of those papers reveals a different conclusion as was stated in the previous review of this manuscript. The UPA/2UPA sequences at the 5’ end of this construct may have different sequences and modestly better expression in vitro than the previously described hairpins that have been shown to be functional in vitro and in vivo, but the general concept, placing nucleotide sequences at the 5’ end that spontaneously form secondary structures to protect the capless transcript against exonuclease degradation, is the same."}, "author_response": "Proposed point-by-point responses to reviewers’ comments\nNCOMMS-25-65642A \" An Engineered Linear Cap-Independent mRNA Vaccine with Intrinsic\nAdjuvanticity Induces Potent Anti-Tumor Immunity \"\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors addressed my points in a sufficient manner\nResponse: We sincerely appreciate your recognition of our revisions.\nReviewer #2 (Remarks to the Author):\nThe authors have responded comprehensively to the reviewers. The manuscript has improved in\nscope and breadth. Well done.\nResponse: We thank the reviewer for acknowledging that we have adequately addressed your\nvaluable comments.\nReviewer #3 (Remarks to the Author):\nComments:\n1. The authors have revised and clarified the supplemental data which is greatly appreciated.\nResponse: We appreciate the reviewer's recognition of our efforts in revising and clarifying the\nsupplementary data.\n2. The authors have added an additional experiment to demonstrate that the increased expression\nof the transcript with the 2UPA compared to that of UPA structure sequence at the 5’ end is not due\nsolely to the increased length of the transcript since a random nucleotide sequence of similar\nlength did not increase expression to the same degree that 2UPA did relative to UPA.\nResponse: We thank the reviewer for their attention to our newly added experiments.\n3. The authors have also added experiments comparing mRNA gaussia luciferase\n(GLuc)-expressing constructs containing the protective 5’ secondary structures UPA and 2UPA to\nmRNA GLuc-expressing constructs that contain the protective 5’ secondary structures (double and\ntriple hairpins) published in 2023 (PMID: 36828937) and 2025 (PMID: 39988620) that first\ndemonstrated the in vitro and in vivo efficacy of a linear capless mRNA transcript with\nunmodified nucleotides driven by an IRES. Although the supplemental figure (Sup Figure 2B)\nshould have included the same 5’ unprotected capless IRES-containing transcript that was used in\nFigure 2D, when comparing these two figures (Figure 2D and Supp Figure 2B), it is clear that\nincluding either the UPA/2UPA secondary structures or the double/triple hairpin secondary\nstructures at the 5’ end, markedly improves GLuc expression (by about 2 logs) above the\nunprotected IRES construct. The UPA/2UPA constructs demonstrate a 20-40% increase in GLuc\nactivity over the original double hairpin construct at 24 hours. It is not clear that this 20-40%\nincrease represents a ‘substantially greater stability’ (lines 196-197) or only a modest increase in\nstability without clinical relevance since they were not compared in vivo, only in cells.\nNonetheless, these are important results because they confirm that linear capless mRNA can\neffectively express proteins in vitro and in vivo using an IRES as long as the 5’ end is protected by\na secondary structure that slows exonuclease degradation.\nResponse: We appreciate the reviewer's recognition of our work confirming the in vitro and in\nvivo efficacy of properly protected LciRNA, and we understand the reviewer's concern regarding\nthe degree of performance improvement of UPA/2UPA elements compared to hairpin sequences,\nparticularly regarding in vivo performance. As shown in Supplementary Figure 2B, at 24 hours,\nthe Gluc activity of the 2UPA group was approximately 40% higher than that of the double hairpin\ngroup (2HP). However, the advantage of 2UPA increased over time: at 48, 72, 96 and 120 h the\n2UPA group reached 2.4-, 5.2-, 29.9- and 22.2-fold the activity of the 2HP group, respectively.\nThese results indicate that transcripts containing the 2UPA element maintain effective expression\nfor significantly longer durations compared to those with 2HP elements. Specifically, at 72h and\n96h post-transfection, the 2UPA transcript retained approximately 36% and 20% of its 24h\nexpression level, whereas the 2HP transcript only retained about 9.5% and 0.9% of its 24h level,\ncorresponding to approximately 1/5 and 1/30 of the 2UPA level at those time points.\nFigures 2E, 2G and 2H further show that inclusion of the 2UPA permits uncapped transcripts to\nachieve in-cell stability comparable to Cap-1 m1Ψ-modified mRNA, and even achieves more\npersistent expression in mice. Given that the in vitro stability of the 2HP transcript is markedly\nlower than that of UPA and 2UPA, it is unlikely that 2HP would attain comparable in vivo stability.\nConsistent with these stability data, the mouse tumor model demonstrates that the 2UPA LciRNA\nvaccine—by virtue of its greater expression stability—induces stronger anti-tumor immune\nresponses and superior therapeutic efficacy compared with the UPA LciRNA vaccine. This\nindicates that the improvement in expression stability provided by the 2UPA element has clinical\nrelevance. Collectively, these results indicate that the RNA-stabilizing effect of 2UPA exceeds that\nof Cap-1 structures in vivo, thereby removing the principal stability disadvantage of uncapped\ntranscripts and substantially advancing the translational potential of cost-effective uncapped\nLciRNA cancer vaccines.\n4. In summary, this manuscript describes a slightly better-expressing version of a linear capless\nIRES-containing mRNA transcript without modified nucleotides that protects its 5’ end by using\nsecondary structures than the construct that was previously described by others to be effective in\ncells and animals. The strength of this paper is that it demonstrates that this type of linear capless\nmRNA construct generates a strong CD8 response which can induce regression of established\ntumors in mice, whereas the previous group that originally described this type of construct only\ndemonstrated its efficacy in inducing a strong humoral immune response capable of protecting\nanimals against a live viral infection.\nThere is some concern that the manuscript does not fairly characterize the positive results of prior\npublications on capless linear mRNA. The paper that first described a functional linear capless\nIRES-containing mRNA with protective 5’ secondary structures (PMID: 36828937) is not cited\nand while subsequent papers on this topic are cited, the manuscript states that all of these linear\ncapless mRNA constructs ‘generally fail to provide sufficient protection to the mRNA’s 5’ end and,\ntherefore, are unlikely to serve as viable substitutes for traditional capped mRNA’ (lines 86-87). A\ncareful review of those papers reveals a different conclusion as was stated in the previous review\nof this manuscript. The UPA/2UPA sequences at the 5’ end of this construct may have different\nsequences and modestly better expression in vitro than the previously described hairpins that have\nbeen shown to be functional in vitro and in vivo, but the general concept, placing nucleotide\nsequences at the 5’ end that spontaneously form secondary structures to protect the capless\ntranscript against exonuclease degradation, is the same.\nResponse: We thank the reviewer for their insightful comments and acknowledge the important\ncontributions of Solodushko et al. and Zheng et al. to the development of uncapped linear mRNA.\nOur manuscript does not claim to be the first to propose uncapped IRES-driven transcripts; rather,\nit establishes through systematic comparison that an uncapped LciRNA incorporating the\nengineered 2UPA element demonstrates significantly enhanced in vivo expression stability relative\nto m1Ψ-modified Cap-1 mRNA. Furthermore, the unique structural and sequence features of\nLciRNA concurrently confer potent innate immune activation, yielding superior anti-tumor\nimmunity compared to both unmodified and m1Ψ-modified capped mRNA platforms.\nIn response to the reviewer’s feedback, we have revised the manuscript to accurately\ncontextualize prior studies (including newly cited work, PMID: 36828937) and clarify the\ndistinctive attributes of our platform for cancer immunotherapy. Specifically, we have refined our\ndiscussion to state \"While uncapped linear mRNA designs have been previously explored, existing\nevidence did not establish their equivalence to capped mRNA in therapeutic efficacy. Furthermore,\nthe potential of uncapped mRNA vaccines in cancer immunotherapy remains uncharacterized.\",\ninstead of saying \" generally fail to provide sufficient protection,\". The core innovation of this\nstudy lies in the 2UPA-protected LciRNA platform, which achieves spatiotemporal coordination\nof antigen expression and immune stimulation via two synergistic properties: expression stability\nexceeding that of capped mRNA, and intrinsic self-adjuvanting activity. This dual functionality\ndrives robust CD8⁺ T-cell infiltration and activation within the tumor microenvironment,\nestablishing LciRNA as a therapeutically competitive, and cost-effective alternative to\nconventional mRNA vaccine architectures."}]

Decision letter